CN113045498A - 1, 5-diaryl pyrazole derivative, synthesis method and application - Google Patents
1, 5-diaryl pyrazole derivative, synthesis method and application Download PDFInfo
- Publication number
- CN113045498A CN113045498A CN202110312501.3A CN202110312501A CN113045498A CN 113045498 A CN113045498 A CN 113045498A CN 202110312501 A CN202110312501 A CN 202110312501A CN 113045498 A CN113045498 A CN 113045498A
- Authority
- CN
- China
- Prior art keywords
- pyrazole derivative
- celecoxib
- diaryl pyrazole
- och
- diaryl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
- C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
- C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D231/12—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Communicable Diseases (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to a 1, 5-diaryl pyrazole derivative and antibacterial application thereof, wherein the 1, 5-diaryl pyrazole derivative is obtained by carrying out structural modification on 1, 5-diaryl based on a celecoxib structure, and antibacterial activity in vitro and in vivo research is carried out after a synthesized target molecule is confirmed by the structure. The 1, 5-diaryl pyrazole derivative has good antibacterial activity, can be used for anti-infection treatment of bacteria, and provides more medicament options for clinical bacterial infection.
Description
Technical Field
The invention belongs to the field of medicinal chemistry, and particularly relates to a 1, 5-diaryl pyrazole derivative, a synthetic method and an application thereof in antibiosis.
Background
According to the latest clinical data, the phenomenon of multidrug resistance of pathogenic bacteria shows a rapid growth trend, including gram-positive bacteria such as methicillin-resistant staphylococcus aureus (MRSA), gram-negative bacteria such as pseudomonas aeruginosa and acinetobacter baumannii. This is a major health threat in the world of bacterial infections and there is therefore a pressing clinical need for new antibiotics and alternative therapies for the treatment of multi-drug resistant pathogenic bacteria.
Despite much effort by researchers, only a small fraction of antibiotics have entered the clinic in the last two decades. The reasons for failure of new drug discovery are multifactorial, limited on the one hand by inadequate understanding of the permeability of the compound through the membrane, and also by chemical diversity, among other reasons.
The relocation strategy provides a new strategy for antimicrobial discovery, which can be divided into four categories: 1) relocation of drugs (new use of old drugs); 2) target repositioning; 3) lead compound relocation and 4) phenotype and target based relocation. Antibacterial drugs exhibit physicochemical properties that are significantly different from those of other drugs, one antibacterial drug is often found to be combined with multiple targets, and the first 3 strategies of relocation strategies for discovery of novel antibacterial drugs are often not suitable. The fourth strategy, which is generally a phenotype-based screen that prioritizes antimicrobial activity over screening for specific enzymes and receptors, allows high quality lead compounds to be discovered and further developed, and is therefore well-suited for the discovery of new antimicrobial drugs, while also meeting the multi-target and pressing clinical medical needs of antimicrobial drugs.
Celecoxib is a COX-2 selective non-steroidal anti-inflammatory drug used to treat pain and inflammation. Celecoxib and its derivatives have been found to have antibacterial activity and Chiu et al have found that celecoxib and its derivatives exhibit activity against the gram-negative bacterium Francisella tularensis and, therefore, celecoxib has been relocated to a topical broad-spectrum antibacterial drug. The antibacterial activity mechanism of celecoxib mainly can inhibit DNA, RNA and protein synthesis and reverse the multi-drug resistance phenomenon of MRSA. These results indicate that celecoxib may be a better choice for targeted relocation of antimicrobial drug discovery. The structure of celecoxib can be modified, especially when a large group is connected to the 5-position of the benzene ring, the activity of celecoxib can be enhanced, but the toxicity is enhanced.
Therefore, the celecoxib derivatives with stronger antibacterial activity need to be found in the field, and a new, safe and effective drug selection is provided for people.
Disclosure of Invention
The invention aims to solve the technical problems mentioned in the background technology and provide a novel antibacterial compound, in particular to a 1, 5-diaryl pyrazole derivative, a synthetic method and application thereof in antibiosis.
In one aspect, the present invention provides a 1, 5-diaryl pyrazole derivative represented by the general formula (I):
wherein R is1The substituent is-SO2NH2，-NHSO2NH2，-CONH2；
R2、R3、R4、R5、R6Each independently selected from-H, -CH3，-CH2CH3，-F，-Cl，-Br，-I，-OCH3，-OCH2CH3，-NO2，CN，COOH；
The 1, 5-diaryl pyrazole derivative is not celecoxib.
As a further preferable embodiment of the present invention, R in the 1, 5-diaryl pyrazole derivative1The substituent is-SO2NH2；R2-R6The substituents are independently selected from-H, -CH3，-CH2CH3，-F，-Cl，-OCH3，-OCH2CH3。
In a further preferred embodiment of the present invention, the 1, 5-diaryl pyrazole derivative has the following structure:
in another aspect, the invention provides an application of the 1, 5-diaryl pyrazole derivative shown in the structural formula (I) in preparing a medicament for treating gram-positive bacteria or gram-negative bacteria infection.
As a further preferred embodiment of the present invention, the gram-positive bacterium is staphylococcus aureus or methicillin-resistant staphylococcus aureus; the gram-negative bacteria are acinetobacter baumannii or pseudomonas aeruginosa.
In another aspect, the present invention provides a pharmaceutical composition for treating gram-negative bacterial infections, said pharmaceutical composition comprising a 1, 5-diaryl pyrazole derivative of formula (I) and polymyxin B.
In another aspect, the present invention provides the use of a 1, 5-diaryl pyrazole derivative of the formula (I) for the preparation of a pharmaceutical composition for the treatment of gram-negative bacterial infections, said pharmaceutical composition comprising polymyxin B.
In another aspect, the present invention provides a method for preparing a 1, 5-diaryl pyrazole derivative, which is characterized by comprising the following steps:
wherein R is2-R6The substituents are each independently selected from-H, -CH3，-CH2CH3，-F，-Cl，-Br，-I，-OCH3，-OCH2CH3，-NO2，CN，COOH。
In another aspect, the present invention provides a method for preparing a 1, 5-diaryl pyrazole derivative, which is characterized by comprising the following steps:
wherein R is2-R6The substituents are each independently selected from-H, -CH3，-CH2CH3，-F，-Cl，-Br，-I，-OCH3，-OCH2CH3，-NO2，CN，COOH。
It is a further object of the invention to provide treatment of infections by drug-resistant bacteria by using such derivatives. The purpose of the invention is realized by the following technical scheme: the invention provides a synthesis method of 1, 5-diaryl pyrazole derivatives, which adopts substituted acetophenone as a starting material to be condensed with ethyl trifluoroacetate to form 1, 3-diketone substituted by benzene ring, wherein a sulfonamide compound is prepared by reacting 1, 3-diketone with p-hydrazino benzenesulfonyl amine hydrochloride to generate the 1, 5-diaryl pyrazole sulfonamide derivatives, and sulfonylurea derivatives are prepared by condensing 1, 3-diketone with p-nitrophenylhydrazine hydrochloride to obtain 1, 5-diaryl pyrazole parent nucleus and then SnCl2Reducing nitro into amino, condensing with chlorosulfonyl to obtain 1, 5-diaryl sulfonylurea derivative, separating and purifying to obtain pure product, and verifying the structure by nuclear magnetism and mass spectrum, wherein the specific compound is shown in Table 1 below.
Table 1 list of compounds of the invention
The antibacterial activity of the 1, 5-diaryl pyrazole derivatives on different kinds of bacteria is measured.
The invention discloses a synthesis method and an antibacterial application of a 1, 5-diaryl pyrazole derivative, wherein the 1, 5-diaryl is respectively subjected to structural modification on the basis of a celecoxib structure to obtain the 1, 5-diaryl pyrazole derivative, and a target molecule obtained by synthesis is subjected to in-vitro and in-vivo research on antibacterial activity after the structure is confirmed. The 1, 5-diaryl pyrazole derivative has good antibacterial activity, can be used for anti-infection treatment of bacteria, and provides more medicament options for clinical bacterial infection.
In addition, when the 1, 5-diaryl pyrazole derivative and polymyxin B are used in combination, the antibacterial effect is remarkably improved.
Drawings
FIG. 1 is the results of the Time-kill experiment of the effect of celecoxib and its derivatives on MRSA strains.
Fig. 2 is a graph showing the survival rate of mice after intraperitoneal injection in the results of the measurement of the antibacterial activity in vivo of celecoxib derivatives in mice.
FIG. 3 shows the serum TNF-alpha and IL-10 levels of mice in the blank group and the experimental group in the determination result of the in vivo antibacterial activity of celecoxib derivative mice.
Fig. 4 is a representative tissue section view in mouse tissues of blank and experimental groups in the assay of the antibacterial activity of celecoxib derivatives in vivo.
Figure 5 is a picture of the inhibition gel of DNA helicase and topoisomerase of celecoxib derivatives.
Detailed Description
In order to make the technical solution and advantages of the present invention more comprehensible, a detailed description is given below by way of specific examples. Unless defined otherwise, technical and scientific terms used herein have the same meaning as those in the technical field to which this application belongs.
Example 1
2, 6-dimethyl celecoxib, the synthesis steps are as follows:
the synthesis process comprises the following steps: dissolving 1g of 2, 6-dimethylacetophenone in 30mL of methanol to prepare a2, 6-dimethylacetophenone/methanol solution. 17.5mL of ethyl trifluoroacetate was dissolved in 20mL of methanol to prepare an ethyl trifluoroacetate/methanol solution.
Dissolving 3.5g of sodium methoxide in 20mL of methanol from a three-mouth bottle, cooling in an ice bath, stirring, controlling the temperature to be 4-5 ℃, slowly dropwise adding ethyl trifluoroacetate/methanol solution (controlling the flow rate to be 1 drop/s), dropwise adding 2, 6-dimethylacetophenone/methanol solution (controlling the flow rate to be 1 drop/s) after dropwise adding is finished, heating to 80 ℃ for reaction after the solution is completely dropwise added, detecting the reaction progress by TLC during the reaction, and displaying the end of the reaction by a dot plate. After drying by evaporation, 200mL of deionized water was added and the pH was adjusted to between 6 and 7 using concentrated HCl. Extraction was performed with ethyl acetate (50ml x 3), and the ethyl acetate layer was collected and dried over anhydrous sodium sulfate. The extract was again rotary evaporated and weighed to give 1.074g of product.
A250 ml round bottom flask equipped with a reflux device was charged with 1.074g of the product of the previous step, dissolved in 50ml of anhydrous ethanol, added with 1.8g of p-hydrazinylbenzenesulfonamide hydrochloride, heated under reflux for 12 hours (overnight reaction), and after completion of the reaction by TLC (petroleum ether: ethyl acetate 2:1), the heating was stopped, and the mixture was stirred and cooled to room temperature. Filtration, collecting the filter cake and filtrate separately, and detecting by TLC (petroleum ether: ethyl acetate 2:1), the product is mostly in the filtrate. The filtrate was spin-dried, dissolved in 100ml of ethyl acetate, washed with deionized water (50 ml. times.3), and the organic layer was separated and dried over anhydrous sodium sulfate. The extract was again rotary evaporated to give 1.02g of product. Recrystallization from ethanol/water gave the product. The structural information results are as follows:1H NMR(DMSO-d6,500MHz)δ(ppm)7.83(d,J＝8.6Hz,2H),7.42-7.47(m,4H),7.32(d,J＝7.6Hz),7.18(d,2H),7.13(s,1H),2.01(s,6H)；13C NMR(DMSO-d6,500MHz)δ(ppm)144.12,143.54,143.04,142.74,141.34,137.77,133.46,130.40,129.77,128.62,128.45,128.28,127.94,127.27,124.11,113.36,107.65,40.54,40.37,40.21,39.87,39.71,39.54；EI/mass(m/z):calculated 395.09,found[M-H]-394.08。
example 2
example 3
example 4
example 5
example 6
example 7
The synthesis steps of the 2, 4-dimethylsulfonylurea celecoxib are as follows:
the synthesis process comprises the following steps: 2g of 2, 4-dimethylacetophenone was dissolved in 30mL of anhydrous ethanol. Cooling to 4-5 ℃ in ice bath. 5g of sodium ethoxide was dissolved in 20mL of anhydrous ethanol. Slowly dropwise adding an ethanol solution of sodium ethoxide at a controlled temperature, controlling the dropwise adding temperature to be not more than 10 ℃, keeping the temperature and stirring for 30min after the dropwise adding is finished, dissolving 30mL of ethyl trifluoroacetate in 10mL of anhydrous ethanol, then adding a new dropping funnel, controlling the temperature to be about 10 ℃, slowly dropwise adding, heating until the reflux reaction is carried out for 2h after the dropwise adding is finished, detecting by TLC (petroleum ether: ethyl acetate 5:1) until the reaction is not finished, and supplementing 1g of sodium ethoxide and 3mL of ethyl trifluoroacetate into a three-neck flask. After 2h, the reaction is completed, the reaction solution is dried by spinning, 100mL of water is added, concentrated hydrochloric acid is added dropwise to adjust the pH value to 6-7, ethyl acetate is extracted for three times by 50mL of ethyl acetate, an ethyl acetate layer is collected, dried by anhydrous sodium sulfate, and weighed by spinning, so that 3.5g of 1, 3-diketone intermediate is obtained.
A250 mL round bottom flask equipped with a reflux device was charged with 3.5g of 1, 3-dione intermediate, dissolved in 40mL of anhydrous ethanol, and 2.711g of p-nitrophenylhydrazine hydrochloride was added, and the mixture was heated under reflux for 4-5 hours for TLC detection (petroleum ether: ethyl acetate 5: 1). After the reaction was completed, heating was stopped, the mixture was cooled to room temperature with stirring, and the filtrate and the cake were collected by filtration, respectively, and the filtrate and the cake were checked by TLC (petroleum ether: ethyl acetate 2:1), and the product was added to the filtrate, collected and dried by spinning, ethyl acetate (50mL) and water (50mL) were added and extracted three times, the organic phases were combined, dried with anhydrous sodium sulfate and spun to obtain a crude product, which was purified by column chromatography (developer: petroleum ether) to obtain 2.7g of 3-trifluoromethyl-1- (4-nitrophenyl) -5- (2 ', 4' -dimethylphenyl) pyrazole.
6.75g of SnCl2 2H2O was dissolved in 50mL of ethanol at room temperature, 2.7g of 3-trifluoromethyl-1- (4-nitrophenyl) -5- (2 ', 4' -dimethylphenyl) pyrazole was added, the mixture was refluxed for 3 hours, and then the reaction was detected by TLC (petroleum ether: ethyl acetate 5:1), and 1.68g of SnCl was added to the mixture to find that the reaction was not completed2 2H21.68g of SnCl is continuously added after 1h after O, the reaction is not finished and the TLC detection is carried out22H2O, after 1 hour, the reaction is finished, the reaction solution is dried in a spinning mode, 50mL of 1mol/L HCl is added to adjust the pH value to about 2, the solubility is low, hydrochloric acid salt solution cannot be completely formed, so that 1mol/L NaOH solution is added to adjust the pH value to 11, a large amount of milky solid precipitate is formed, ethyl acetate is added to the milky solid precipitate for suction filtration, the filter cake is transferred to a flask after the suction filtration, the ethyl acetate is added to the flask for stirring and washing, and the mixture is fed into the flask againAnd carrying out suction filtration to obtain filtrate, extracting the filtrate for three times by using ethyl acetate and water to obtain an organic layer, drying the organic layer by using anhydrous sodium sulfate, and carrying out rotary evaporation on the organic layer to obtain 2.2g of a reduction product.
2.2g of the reduced product was dissolved in 50mL of dichloromethane, and after stirring at 0 ℃ for 10min, a solution of 0.85g of sulfamoyl chloride in 5mL of dichloromethane and 1mL of triethylamine were added dropwise. After the dropwise addition is finished, the reaction is carried out at normal temperature, and TLC detection shows that the reaction is finished. Then washed with water and the pH adjusted to neutral. The organic layer was dried over anhydrous sodium sulfate, then concentrated by rotary evaporation to give the crude product, which was purified by column chromatography. The structural information results are as follows:1H NMR(DMSO-d6,500MHz)δ(ppm)7.10(t,2H),7.03(d,J＝7.65Hz,2H),6.91(t,3H),6.51(d,J＝8.5Hz,2H),5.37(s,2H),2.29(s,3H),2.00(s,3H)；13C NMR(DMSO-d6,500MHz)δ(ppm)149.31,144.14,141.01,140.71,139.19,136.94,131.26,130.99,128.08,126.88,126.63,126.08,123.19,121.06,113.76,106.05,60.18,40.51,40.35,40.18,39.85,39.68,39.51,21.14,19.91,14.45；EI/mass(m/z):calculated 410.10,found[M-H]-409.09。
experimental example 1 sensitivity assay of celecoxib derivatives to Standard and clinical strains
The strain and the reagent for experiment are commercially available, such as MH broth (CAMHB drug sensitive culture) from culture broth of Shanghai Majia biological Limited; strain information: s.aureus, ATCC 29213; MRSA, ATCC 1026; pneumoniae, ATCC 49619; e.faecalis, ATCC 29212; coli, ATCC 25922; baumann ii, clinical strain; aeruginosa, ATCC 27853; pneumoniae, clinical strain.
The sensitivity of the celecoxib derivative to 8 different types of standard strains and not less than 15 clinical drug-resistant strains (MRSA and A. baumann ii) is measured, and the result is expressed by MIC value, and the specific steps are as follows:
1) preparation of celecoxib derivative drug sensitive plate
Adding the diluted 7-13 solutions of celecoxib derivatives with different concentrations into a sterile 96-well plate respectively, freeze-drying, sealing, and storing at-20 ℃ for later use;
2) test strains
Staphylococcus aureus ATCC29213, MRSA (standard strain ATCC1026 and clinical strains of no less than 20 strains), streptococcus pneumoniae ATCC49619, enterococcus faecalis ATCC29212, escherichia coli ATCC25922, acinetobacter baumannii (clinical strains that are all sensitive to antibacterial agents), multidrug-resistant acinetobacter baumannii (no less than 20 clinical strains), pseudomonas aeruginosa ATCC27853, klebsiella pneumoniae (clinical strains);
3) preparation of bacterial suspension
Activating and culturing the test strain for 18-24h, and preparing bacterial suspension (concentration of about 1.5 × 10) with concentration equivalent to 0.5 McLeod's turbidimetric standard by using normal saline direct suspension method8CFU/mL), the bacterial suspension was diluted with MH broth to a final bacterial suspension concentration of 5 × 105CFU/mL, 150. mu.L of diluted bacterial solution was added to each well, 150. mu.L of medium was added to the blank control, 150. mu.L of the mixture of bacterial suspension and medium was added to the negative control, inoculation was completed within 15 minutes, and each experiment was repeated three times. And (5) incubating at 35 ℃ for 16-24h to judge the result.
4) Result judgment
The lowest drug concentration that completely inhibited bacterial growth in the wells was the MIC (. mu.g/mL). The test is only significant when there is significant growth of bacteria in the positive control wells (i.e. no antibiotic), and when a single jump occurs in the broth dilution method, the highest concentration of drug that inhibits bacterial growth should be recorded and the negative control wells inoculated with bacteria in a blood plate to determine whether the strain is contaminated. The results were judged according to the criteria for human antibiotic judgment (M45-A2) provided by CLSI.
5) MIC results for celecoxib derivatives
Experimental results show that the compound 12(2, 4-dimethyl celecoxib) has a good inhibiting effect on S.aureus, methicillin-resistant staphylococcus aureus (MRSA), acinetobacter baumannii and MRSA clinical strains.
TABLE 2 results of antibacterial Activity of celecoxib derivatives against gram-positive bacteria
TABLE 3 results of antibacterial Activity of celecoxib derivatives against gram-negative bacteria
TABLE 4 measurement of the antibacterial Activity of celecoxib analogs on clinically isolated MRSA resistant strains
Experimental example 2 determination of sensitivity of celecoxib derivatives to conventional antibacterial drugs
A sub-inhibitory concentration (1/2MIC) of a conventional antibiotic (including vancomycin, levofloxacin, linezolid, polymyxin B) solution and a dilution gradient solution of celecoxib derivative in a multiple ratio (0.125-64 μ g/mL) were added to a sterile 96-well plate in sequence, freeze-dried and stored at-20 ℃, MRSA, a. baumannii, p. aeruginosa, k. pneumoniae were added in the above steps, three duplicate wells were set for each concentration, and the MIC values of the experimental results were recorded, with the results shown in table 5.
The results show that the celecoxib derivative shows better inhibition effect on gram-negative bacteria when polymyxin B with 1/2MIC concentration is added, and especially the inhibition effect of the compound 12 is most obvious.
TABLE 5 results of the antibacterial activity of celecoxib and derivatives in combination with polymyxin B on G-bacteria
EXAMPLE 3 Time-kill experiment of celecoxib derivatives on bacteria
According to the MIC result measured by the celecoxib derivative, preferred compound solution with the concentration of 2MIC, 4MIC and 8MIC is added into a sterile 96-well plate, the plate is placed at-20 ℃ for storage after freeze drying, then MRSA bacterial liquid is added in the steps, three multiple wells are arranged at each concentration, incubation is carried out at 35 ℃ for 24h, 1 mu L of each well is sampled and inoculated into a blood plate at the time points of incubation of 0h, 4h, 8h, 12h, 16h and 24h, the colony number in the plate is counted after the culture of 24h, when the colony number remaining in the 24h is less than 0.01 percent of the original colony number, the plate is regarded as a bactericide, otherwise, the plate is regarded as a bacteriostatic agent, and the result is shown in the attached figure 1 of the.
EXAMPLE 4 growth inhibition assay of celecoxib derivatives in human mammalian cells
1) Digesting and counting the cells to prepare a cell suspension of 6.0 × 104Each well of a 96-well cell culture plate is added with 100 mu L of cell suspension;
2) the 96-well cell culture plate is placed at 37 ℃ and 5% CO2Culturing for 24h in an incubator;
3) diluting the drug with culture medium to required working solution concentration, adding 100 μ L corresponding drug-containing culture medium into each well, and setting up negative control group;
4) culturing 96-well cell culture plates in a 5% CO2 incubator at 37 deg.C for 48 hr;
5) MTT staining of 96-well plates, λ 490nm, and OD determination;
6) add 20. mu.L MTT (5mg/mL) per well and continue culturing in the incubator for 4 hours;
7) discarding the supernatant, adding 150 μ L DMSO into each well, and gently mixing by shaking for 10 min; λ 490nm, OD value per well read by microplate reader, and inhibition was calculated, and the results are shown in table 6.
And calculating the inhibition rate of each group.
TABLE 6 results of experiments on the inhibition of the growth of HepG-2 by celecoxib and derivatives
The results of the experiments show that compound 12 is most selective between eukaryotic and prokaryotic bacteria.
Experimental example 5 measurement of in vivo antibacterial Activity of celecoxib and derivatives thereof
1) ICR mice are bred adaptively for one week, are fed with water normally and are illuminated day and night;
2) the test strain is activated and cultured for 18-24h before the experiment, MRSA colony is picked out and placed in physiological saline to prepare the concentration of about 2.4 × 109CFU/mL bacterial suspension;
3) purchasing mucin in intestines and stomach of pigs, preparing a solution with the concentration of 10% (W/V) by using normal saline, heating and stirring for 40min, and adjusting the pH value to be alkaline by using sodium bicarbonate to form a suspension;
4) mixing the bacterial suspension and the mucin solution in equal volume to obtain a final concentration of 1.2 × 109CFU/mL, and the final concentration of mucin is 5%;
5) the preparation method of the celecoxib and the derivatives thereof comprises the following steps of preparing a solution of celecoxib and the derivatives thereof, wherein the solution is prepared by adopting ethanol: tween 80: the medicine is suspended in water in a ratio of 1:1:8, the positive medicine vancomycin is good in water solubility and is dissolved in water, the concentration of celecoxib and derivatives thereof is set to be 20mg/kg, and the dose of vancomycin is 30 mg/kg;
5) after weighing the mice, 0.5mL of mixed solution (bacterial suspension and mucin) is injected into the abdominal cavity of the mice, the medicines are respectively injected into the abdominal cavity after half an hour and eight hours of infection, and the physiological state, the weight change, the body temperature change and the survival rate of the mice within 48 hours are observed. Blood was collected from the eyeball at 2h and 24h after infection, and serum was isolated and the contents of inflammatory factors and chemical factors were measured. Dissecting the mouse 24h after infection, separating the tissue, rinsing half of the mouse by using sterile normal saline, placing the mouse in the sterile normal saline for homogenation to obtain tissue homogenate, timely sampling, inoculating the tissue homogenate into a blood plate, culturing for 24h, and then counting colonies. And the other half is rinsed in normal saline and then placed in formalin, wax block sections are made after H & E staining, and histopathological changes are observed microscopically, and the results are shown in the accompanying drawing 2, the accompanying drawing 3 and the accompanying drawing 4 of the specification.
In the attached figure 2 of the specification, A is the survival rate after intraperitoneal injection 11, B is the survival rate after intraperitoneal injection 12, and C is the colony count after collecting liver, spleen, kidney, homogenizing and inoculating tissues. The results show that: intraperitoneal injection of compound 12 can improve survival in the acute peritonitis model in MRSA-infected mice.
TABLE 7 results of scoring of degree of tissue lesions for each experimental group
Acute peritonitis models were developed for ICR mouse infection using MRSA standard strains. Results are expressed as mean ± SD (6 mice per group); xp <0.01 has significant differences when compared to the negative control group, # p <0.05 and # pp <0.01 have very significant differences when compared to MRSA infected mice, and the results show: the survival rate of the animals in the model group within 24 hours is 0, the survival rate of the animals in the administration group within 24 hours is 20%, and the pathological changes of the model group in the tissue are mainly spleen macrophage hyperplasia, phagocytosis phenomenon is enhanced, and a large amount of nuclear fragments are phagocytosed. Renal tubular epithelial cells are severely edematous. After the application of the medicine, the spleen and kidney lesion degree is obviously reduced, the compound 12 administration group is the lightest, and the vancomycin administration group is the second, and the spleen and kidney lesion degree of mice in the compound 11 administration group is also obviously reduced compared with that in the model group.
EXAMPLE 6 enzyme inhibition assay of celecoxib and derivatives thereof
Based on the molecular docking of celecoxib and derivatives thereof with various enzymes from bacteria in the early stage, the results show that the celecoxib and derivatives thereof have the possibility of action with dihydrofolate reductase, DNA helicase and topoisomerase, and therefore, the results of in vitro inhibition experiments using staphylococcus aureus dihydrofolate reductase, DNA helicase and topoisomerase (bacterial culture, escherichia coli transfection and enzyme extraction) are as follows:
TABLE 8 inhibition Activity results of celecoxib and its derivatives on dihydrofolate reductase and DNA helicase in Staphylococcus aureus
The figure of the gel picture of the inhibition of DNA helicase and topoisomerase of the celecoxib derivative is shown in the attached figure 5 of the specification, and the result shows that the compound 12 and the celecoxib have inhibition effects on both dihydrofolate reductase and DNA helicase, wherein 2, 4-dimethyl celecoxib has IC (integrated Circuit) of the dihydrofolate reductase50105.1. mu.g/mL, inhibition IC of DNA helicase50It was 122.8. mu.g/mL.
It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations encompassed by the claims. Various modifications and changes may also be made on the basis of the above embodiments without departing from the scope of the present disclosure. Likewise, various features of the above embodiments may be arbitrarily combined to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above examples only represent some embodiments of the present invention, and do not limit the scope of the present invention.
Claims (9)
1. A 1, 5-diaryl pyrazole derivative or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, metabolite or prodrug thereof, characterized by being represented by the general structure (I):
wherein R is1Is selected from-SO2NH2，-NHSO2NH2，-CONH2；
R2、R3、R4、R5、R6Each independently selected from-H, -CH3，-CH2CH3，-F，-Cl，-Br，-I，-OCH3，-OCH2CH3，-NO2，CN，COOH；
The 1, 5-diaryl pyrazole derivative is not celecoxib.
2. The 1, 5-diaryl pyrazole derivative according to claim 1,
wherein R is1The substituent is-SO2NH2；
R2、R3、R4、R5、R6Each independently selected from-H, -CH3，-CH2CH3，-F，-Cl，-OCH3，-OCH2CH3。
3. The 1, 5-diaryl pyrazole derivative according to claim 1, which is a compound according to any one of the formulas 8, 9, 10 and 12,
4. use of a 1, 5-diaryl pyrazole derivative or celecoxib or pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, metabolite or prodrug thereof according to claims 1-3 for the preparation of a medicament for the treatment of gram-positive or gram-negative bacterial infections.
5. Use according to claim 4, characterized in that the gram-positive bacterium is Staphylococcus aureus or methicillin-resistant Staphylococcus aureus; the gram-negative bacteria are acinetobacter baumannii or pseudomonas aeruginosa.
6. A pharmaceutical composition for the treatment of gram-negative bacterial infections comprising a 1, 5-diaryl pyrazole derivative or celecoxib or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, metabolite or prodrug thereof according to any one of claims 1 to 3, wherein the pharmaceutical composition further comprises polymyxin B.
7. Use of a 1, 5-diaryl pyrazole derivative according to claims 1 to 3 for the preparation of a pharmaceutical composition for the treatment of gram-negative bacterial infections, said pharmaceutical composition comprising polymyxin B, said pharmaceutical composition further comprising a 1, 5-diaryl pyrazole derivative or celecoxib, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, metabolite or prodrug thereof.
8. A preparation method of a 1, 5-diaryl pyrazole derivative is characterized by comprising the following preparation steps:
wherein R is2、R3、R4、R5、R6Each independently selected from-H, -CH3，-CH2CH3，-F，-Cl，-Br，-I，-OCH3，-OCH2CH3，-NO2，CN，COOH。
9. A preparation method of a 1, 5-diaryl pyrazole derivative is characterized by comprising the following preparation steps:
wherein R is2、R3、R4、R5、R6Each independently selected from-H, -CH3，-CH2CH3，-F，-Cl，-Br，-I，-OCH3，-OCH2CH3，-NO2，CN，COOH。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110312501.3A CN113045498B (en) | 2021-03-24 | 2021-03-24 | 1, 5-diaryl pyrazole derivative, synthesis method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110312501.3A CN113045498B (en) | 2021-03-24 | 2021-03-24 | 1, 5-diaryl pyrazole derivative, synthesis method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113045498A true CN113045498A (en) | 2021-06-29 |
| CN113045498B CN113045498B (en) | 2023-01-24 |
Family
ID=76514832
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110312501.3A Active CN113045498B (en) | 2021-03-24 | 2021-03-24 | 1, 5-diaryl pyrazole derivative, synthesis method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113045498B (en) |
Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5466823A (en) * | 1993-11-30 | 1995-11-14 | G.D. Searle & Co. | Substituted pyrazolyl benzenesulfonamides |
| CN1826140A (en) * | 2002-01-23 | 2006-08-30 | 法玛西雅厄普约翰美国公司 | Combination therapy for the treatment of bacterial infections |
| WO2010036958A1 (en) * | 2008-09-25 | 2010-04-01 | Cedars-Sinai Medical Center | Methods of treatment for mrsa infections |
| US20120108823A1 (en) * | 2009-04-22 | 2012-05-03 | The Ohio State University Research Foundation | Anti-francisella agents |
| WO2012061260A1 (en) * | 2010-11-01 | 2012-05-10 | The Ohio State University Research Foundation | Anti-staphylococcal celecoxib derivatives |
| CN102871996A (en) * | 2012-09-10 | 2013-01-16 | 中国医学科学院医药生物技术研究所 | Antibiotic composition and application thereof |
| CN103570622A (en) * | 2012-09-07 | 2014-02-12 | 北京京卫燕康药物研究所有限公司 | Preparation method of celecoxib |
| CN107987020A (en) * | 2018-01-12 | 2018-05-04 | 山东大学 | 3,5- diaryl pyrazoles or 3,4- diaryl pyrazoles azole derivative and its application |
| WO2019155442A1 (en) * | 2018-02-12 | 2019-08-15 | Vyome Therapeutics Limited | Treatment of gram-negative folliculitis or an inflammation thereof with besifloxacin |
| WO2019222801A1 (en) * | 2018-05-21 | 2019-11-28 | Telethon Kids Institute | Promotion of epithelial repair via the administration of activators of pi3k/akt signalling |
| CN110652512A (en) * | 2018-12-28 | 2020-01-07 | 暨南大学 | Application of crizotinib in preparation of anti-gram-positive-bacteria drugs |
| WO2020069211A1 (en) * | 2018-09-27 | 2020-04-02 | Decoy Biosystems, Inc. | Methods of treatment of infections using bacteria |
| CN111714453A (en) * | 2020-06-29 | 2020-09-29 | 瑞希(重庆)生物科技有限公司 | Antibacterial micelle and preparation method thereof |
-
2021
- 2021-03-24 CN CN202110312501.3A patent/CN113045498B/en active Active
Patent Citations (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5466823A (en) * | 1993-11-30 | 1995-11-14 | G.D. Searle & Co. | Substituted pyrazolyl benzenesulfonamides |
| CN1826140A (en) * | 2002-01-23 | 2006-08-30 | 法玛西雅厄普约翰美国公司 | Combination therapy for the treatment of bacterial infections |
| WO2010036958A1 (en) * | 2008-09-25 | 2010-04-01 | Cedars-Sinai Medical Center | Methods of treatment for mrsa infections |
| US20110251162A1 (en) * | 2008-09-25 | 2011-10-13 | Cedars-Sinai Medical Center | Methods of treatment for mrsa infections |
| US20120108823A1 (en) * | 2009-04-22 | 2012-05-03 | The Ohio State University Research Foundation | Anti-francisella agents |
| US20130289004A1 (en) * | 2010-11-01 | 2013-10-31 | The Ohio State University Research Foundation | Anti-staphylococcal celecoxib derivatives |
| WO2012061260A1 (en) * | 2010-11-01 | 2012-05-10 | The Ohio State University Research Foundation | Anti-staphylococcal celecoxib derivatives |
| CN103570622A (en) * | 2012-09-07 | 2014-02-12 | 北京京卫燕康药物研究所有限公司 | Preparation method of celecoxib |
| CN102871996A (en) * | 2012-09-10 | 2013-01-16 | 中国医学科学院医药生物技术研究所 | Antibiotic composition and application thereof |
| CN107987020A (en) * | 2018-01-12 | 2018-05-04 | 山东大学 | 3,5- diaryl pyrazoles or 3,4- diaryl pyrazoles azole derivative and its application |
| WO2019155442A1 (en) * | 2018-02-12 | 2019-08-15 | Vyome Therapeutics Limited | Treatment of gram-negative folliculitis or an inflammation thereof with besifloxacin |
| WO2019222801A1 (en) * | 2018-05-21 | 2019-11-28 | Telethon Kids Institute | Promotion of epithelial repair via the administration of activators of pi3k/akt signalling |
| US20210196687A1 (en) * | 2018-05-21 | 2021-07-01 | Telethon Kids Institute | Promotion of Epithelial Repair |
| WO2020069211A1 (en) * | 2018-09-27 | 2020-04-02 | Decoy Biosystems, Inc. | Methods of treatment of infections using bacteria |
| US20200101119A1 (en) * | 2018-09-27 | 2020-04-02 | Decoy Biosystems, Inc. | Methods of treatment of infections using bacteria |
| CN110652512A (en) * | 2018-12-28 | 2020-01-07 | 暨南大学 | Application of crizotinib in preparation of anti-gram-positive-bacteria drugs |
| CN111714453A (en) * | 2020-06-29 | 2020-09-29 | 瑞希(重庆)生物科技有限公司 | Antibacterial micelle and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| VINAY KUMAR STHALAM: "《An Integrated Continuous Flow Micro-Total Ultrafast Process System (μ-TUFPS) for the Synthesis of Celecoxib and Other Cyclooxygenase Inhibitors》", 《ORG. PROCESS RES. DEV》 * |
| WILLIAM T. BARKER等: "《Repurposing Eukaryotic Kinase Inhibitors as Colistin Adjuvants in Gram-Negative Bacteria》", 《ACS INFECT. DIS》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113045498B (en) | 2023-01-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6050763B2 (en) | Antibacterial compounds / adjuvant compounds and methods | |
| TWI422372B (en) | Rifamycin derivatives | |
| CN102180890A (en) | Cefathiamidine hydrate and preparation method and application thereof | |
| WO2018177218A1 (en) | Preparing method for and uses of 3,5-disubstituted methylpyrazolo[1,5-a] pyrimidin-7-phenolate analogues and derivatives | |
| CN110312722A (en) | Antimicrobial and its preparation and application | |
| CN114213433B (en) | Process for the preparation of C-3 tetrahydrofuran substituted cephalosporin-siderophore conjugates | |
| CN113045498B (en) | 1, 5-diaryl pyrazole derivative, synthesis method and application | |
| EP2524922B1 (en) | Preparation method of optically active s-(-)-prulifloxacin | |
| CN113354561B (en) | Biguanide derivatives and their use and formulations | |
| KR100332280B1 (en) | Anti-Helicobacter Philolyze | |
| WO2014161412A1 (en) | Tricyclic quinolone derivative and preparation method and use thereof | |
| CN105209458B (en) | It is novel that there is the active 1,2,4- oxadiazole compound of resisting gram-positive pathogen | |
| CN109851611B (en) | Sulfadiazine compound and preparation method and application thereof | |
| CN102603738B (en) | Stable moxifloxacin hydrochloride compound | |
| CN110981888A (en) | N-aryl dithiopyrryl ketonuria and amino ester derivatives, preparation and application thereof | |
| CN101838277A (en) | Cefotetan disodium hydrate and preparation method and application thereof | |
| CN107567458A (en) | The compound of squalamine analogues as antiseptic | |
| CN110845473B (en) | Inhibitor Lo-ethyl acetate for resisting staphylococcus aureus virulence and biofilm formation and application thereof | |
| CN112194631B (en) | Schiff base bridged sulfaimidazole compound and preparation method and application thereof | |
| US20100137388A1 (en) | Therapeutic Pro-Antibiotic Agents and Methods of Use Thereof | |
| CN115260162B (en) | 3-Hydroxy-4-pyridone-ciprofloxacin coupling, preparation method and application thereof | |
| CN116082280B (en) | 4-aryl-5-methylene-2 (5H) -furanone derivative and preparation method and application thereof | |
| CA2497410A1 (en) | Antibacterial pyrazole carboxylic acid hydrazides | |
| JP3796612B2 (en) | Antibacterial agent | |
| JP2004137185A (en) | Antimicrobial agent containing thiophene skeleton |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20210629 Assignee: Jiangsu chengkai traditional Chinese Medicine Co.,Ltd. Assignor: JIANGSU FOOD & PHARMACEUTICAL SCIENCE COLLEGE Contract record no.: X2023980034087 Denomination of invention: A 1,5-diarylpyrazole derivative, synthesis method, and use Granted publication date: 20230124 License type: Common License Record date: 20230324 |
|
| EE01 | Entry into force of recordation of patent licensing contract | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20210629 Assignee: Jiangsu Boda Testing Technology Co.,Ltd. Assignor: JIANGSU FOOD & PHARMACEUTICAL SCIENCE COLLEGE Contract record no.: X2023980041890 Denomination of invention: A 1,5-diarylpyrazole derivative, synthesis method, and use Granted publication date: 20230124 License type: Common License Record date: 20230919 |
|
| EE01 | Entry into force of recordation of patent licensing contract |