CN113912626B - Broad spectrum resistant beta-lactam and cephalosporin antibiotics pathogen probe and synthetic method and application thereof - Google Patents

Broad spectrum resistant beta-lactam and cephalosporin antibiotics pathogen probe and synthetic method and application thereof Download PDF

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CN113912626B
CN113912626B CN202110975090.6A CN202110975090A CN113912626B CN 113912626 B CN113912626 B CN 113912626B CN 202110975090 A CN202110975090 A CN 202110975090A CN 113912626 B CN113912626 B CN 113912626B
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CN113912626A (en
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刘定斌
李文帅
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Jingqianshan Technology (Beijing) Co.,Ltd.
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    • C07D501/00—Heterocyclic compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
    • C07D501/14—Compounds having a nitrogen atom directly attached in position 7
    • C07D501/16—Compounds having a nitrogen atom directly attached in position 7 with a double bond between positions 2 and 3
    • C07D501/20—7-Acylaminocephalosporanic or substituted 7-acylaminocephalosporanic acids in which the acyl radicals are derived from carboxylic acids
    • C07D501/24—7-Acylaminocephalosporanic or substituted 7-acylaminocephalosporanic acids in which the acyl radicals are derived from carboxylic acids with hydrocarbon radicals, substituted by hetero atoms or hetero rings, attached in position 3
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    • C07D—HETEROCYCLIC COMPOUNDS
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    • C07D501/14—Compounds having a nitrogen atom directly attached in position 7
    • C07D501/16—Compounds having a nitrogen atom directly attached in position 7 with a double bond between positions 2 and 3
    • C07D501/20—7-Acylaminocephalosporanic or substituted 7-acylaminocephalosporanic acids in which the acyl radicals are derived from carboxylic acids
    • C07D501/22—7-Acylaminocephalosporanic or substituted 7-acylaminocephalosporanic acids in which the acyl radicals are derived from carboxylic acids with radicals containing only hydrogen and carbon atoms, attached in position 3
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Abstract

The invention discloses a probe compound for ultra-broad spectrum beta-lactam and cephalosporin antibiotics pathogenic bacteria, which is a compound with the following structure:
Figure DDA0003226998660000011
the invention takes the national important requirement of rapid detection of bacterial drug resistance as a research target, develops a resonance Raman probe with an off-on switch effect, and realizes the specific detection of the drug resistance of the ultra-broad spectrum beta-lactam antibiotics and the cephalosporin antibiotics at the single bacterial level.

Description

Ultra-broad spectrum resistant beta-lactam and cephalosporin antibiotics pathogen probe and synthetic method and application thereof
Technical Field
The invention belongs to the technical field of compounds, and particularly relates to a probe compound for high-sensitivity ultra-broad spectrum beta-lactam and cephalosporin antibiotics pathogenic bacteria, a synthetic method and an application thereof.
Background
Since the discovery of penicillin by fleming in 1929, antibiotics saved countless lives and became the defenders of human health. However, due to the large-scale use and even abuse of antibiotics, bacterial gene mutation is aggravated, and a large number of drug-resistant strains are caused to appear. Especially in recent years more and more superbugs have been found, which have caused great harm to human life health. Currently, drug-resistant bacterial infections cause 70 million deaths worldwide each year, with the majority occurring in developing countries, with an estimated 1000 million people by 2050. China promulgates' national action plan for restraining bacterial drug resistance in 2016-2020, and aims to restrain bacterial drug resistance and maintain the health of people. Therefore, the development of a rapid bacterial drug resistance detection method has very important value for guiding the accurate clinical use of antibiotics and reducing the abuse of antibiotics.
Beta-lactam antibiotics are the antibiotics which are most widely used in clinical anti-infection treatment at present. The production of beta-lactamase (beta-lactamase) is the main reason for the drug resistance of more than 80% of pathogenic bacteria. The number of beta-lactamase is over 200, and is often involved in multiple drug resistance of bacteria. Extended-Spectrum beta-Lactamases (ESBL) are a class of beta-Lactamases which can hydrolyze penicillins, cephalosporins and monocyclic antibiotics and can be inhibited by inhibitors such as clavulanic acid, sulbactam and tazobactam. ESBL positive means bacteria capable of producing ESBL, mainly including Escherichia coli, klebsiella pneumoniae, acinetobacter baumannii, enterobacter cloacae, serratia marcescens, citrobacter fraudonii, and Pseudomonas aeruginosa. The widespread use of third generation cephalosporins and monobactams is a major factor contributing to the emergence and spread of ESBLs-producing strains. AmpC enzyme is also called cephalosporin enzyme, is by Enterobacteriaceae bacteria or and Pseudomonas aeruginosa chromosome or plasmid mediated generation of a class of beta lactamase, which acts on cephalosporin, and not by clavulanic acid inhibition. Commonly found in Enterobacter, citrobacter, serratia, pseudomonas aeruginosa, and Hafnia alvei.
The detection method aiming at the beta-lactam antibiotic drug-resistant bacteria mainly comprises three methods: phenotypic method, genetic method and enzymatic hydrolysis method. Phenotypic methods generally detect the susceptibility of bacteria to beta-lactam antibiotics. The method needs to separate and purify pathogenic bacteria from a complex clinical environment, then culture the bacteria, and judge the drug sensitivity according to the growth state of the bacteria to be detected, the whole process takes several days, and the method lacks specificity and sensitivity, and cannot timely and accurately provide experimental basis for antibiotic selection for bacterial infectious diseases; although the genetic method can quickly and accurately detect whether bacteria contain drug resistance genes, the genetic method can only detect sites with elucidated drug resistance mechanisms, but cannot detect drug resistance genes with unknown mechanisms. In addition, the method cannot determine whether a strain containing a drug-resistant gene is in a drug-resistant state, cannot provide an MIC value, and cannot guide clinical medication dosage; the enzyme hydrolysis method utilizes the characteristic that beta-lactamase generated by drug-resistant bacteria can rapidly hydrolyze beta-lactam antibiotic, and drug-resistant bacteria can be detected by comparing the substrate colors of the beta-lactamase before and after hydrolysis. But the color change is slow, the sensitivity is low, and the interference of the color of the sample greatly limits the application. It can be seen that the phenotypic, genetic and enzymatic hydrolysis methods rely on complex sample processing and do not allow for in situ detection of related active substances produced by drug-resistant bacteria. In addition, these methods can only obtain the overall behavior of the evaluation of the drug resistance of the group bacteria, and cannot reflect the drug resistance difference of different individuals in the same batch of bacteria. Therefore, accurate, rapid and real-time detection of bacterial resistance, particularly carbapenem resistance, at a single bacterial level is helpful for guiding clinical timely and accurate use of antibiotics. The single bacterium imaging technology has important significance for researching the action mechanism of related active substances in the bacterial drug resistance generation process. In recent years, fluorescence imaging methods have been used to monitor changes in beta-lactamases in drug-resistant bacteria at the single bacteria level. The fluorescence method generally needs to design a specific fluorescent probe substrate, and the specific fluorescent probe substrate is subjected to specific hydrolysis reaction with the beta-lactamase in bacteria, so that the beta-amido bond is broken, the fluorescence signal is changed, and the detection of the beta-lactamase in the bacteria is realized. However, the fluorescent signal is easy to generate self-quenching phenomenon in complex physiological environment, and photobleaching is easy to generate under multiple times of irradiation of exciting light, so that the expression of the beta-lactamase in bacteria is difficult to continuously monitor for a long time. The development of a novel single-bacterium imaging method has important significance for the real-time dynamic study of drug resistance. Raman spectroscopy is an important means for analyzing molecular structure information in situ without damage, and is widely applied to the fields of food safety, medical diagnosis, cultural relic identification, petrochemical industry and the like. Compared with the classical fluorescence analysis method, the Raman spectrum has no photobleaching phenomenon, the signal generation is not influenced by complex physiological environment, and the sample pretreatment is not needed, so the method is very suitable for in-situ, dynamic and real-time analysis.
Through search, the following two patent publications related to the patent application of the present invention are found:
1. the fluorescent probe for resisting carbapenem antibiotic bacteria and the synthesis method and the application thereof (CN 106811192A), the structural general formula of the fluorescent probe is as follows: in the formula: x is a carbon atom or a sulfur atom; when X is CH, R1 is methyl, capable of being in the R or S configuration; or X is CH 2 Or S; the dye is boron dipyrrole, naphthalimide, coumarin,Fluorescein or rhodamine. The synthesis method of the fluorescent probe comprises the following steps: preparing a first compound 3; preparing a compound 4; and preparing the fluorescent probe CVB-1. The fluorescent probe can be made into test paper, a kit or a detection chip and can be applied to detecting carbapenemase and carbapenem-containing drug-resistant bacteria, the carbapenemase can be detected or distinguished by the phenomenon that the fluorescent probe changes in fluorescence intensity or color, and then pathogenic drug-resistant bacteria expressed by the carbapenemase can be rapidly detected, and antibiotics can be guided to be reasonably used in treatment or clinic, so that the fluorescent probe has important significance for using no or less antibiotics.
2. 6,7-trans cephalosporin-based probes (CN 106061949 a) for the detection of bacteria expressing metallo-beta-lactamases, the present disclosure includes embodiments of probes useful for the selective detection of metallo-beta-lactamases, in particular carbapenemases, to distinguish those species of bacteria that are carbapenem-resistant from those that are sensitive. Cephalosporin-based probes with the 6,7r,r configuration are susceptible to cleavage by beta-lactamases, but are indistinguishable from cleavage by metallo-beta-lactamases from other beta-lactamases. By modifying the side groups of the cephalosporin, selectivity can be introduced which allows the probe to distinguish between the various types of metallo-beta-lactamases and thus more narrowly define the strain of the bacterium and the type of metallo-beta-carbapenemase produced.
By contrast, the present patent application is substantially different from the above patent publications.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a probe compound for high-sensitivity beta-lactam and cephalosporin antibiotics pathogenic bacteria, a synthetic method and an application thereof.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a probe compound for ultra-broad spectrum beta-lactam and cephalosporin antibiotics germs is a compound with the following structure:
Figure GDA0004071546720000031
further, the probe compound is:
Figure GDA0004071546720000032
or, it is:
Figure GDA0004071546720000033
or, it is:
Figure GDA0004071546720000034
Figure GDA0004071546720000041
the preparation method of the probe compound comprises the following steps:
Figure GDA0004071546720000042
to an eggplant type reaction flask was added 1eq Pd (OAc) in sequence under argon protection 2 And 1eq P (o-Tol) 3 Repeatedly freezing and thawing for 3 times, stirring at room temperature for 1h to activate the catalyst; then sequentially adding 2.5eq cefdinir, 10eq of an electroabsorptive halogenated compound, DMF and triethylamine (10 1), carrying out 3 times of freezing and degassing operation, and finally heating the reaction to 80 ℃ for reaction for 12 hours; the reaction was terminated, ethyl acetate was added to dilute the solution, followed by 0.2mol/LHCl solution and NaHCO 3 Washing with saturated aqueous solution and saturated brine, anhydrous MgSO 4 Drying; the organic phase was concentrated and purified by silica gel column, wherein dichloromethane: the volume ratio of methanol is 5:1, obtaining a probe compound;
wherein, pd (OAc) 2 ：P(o-Tol) 3 : cefdinir: small molecule compounds: DMF: triethylamine: ethyl acetate:0.2mol/LHCl solution: naHCO 2 3 Saturated aqueous solution: proportion mmol of saturated saline solution: mmol: mmol: mmol: mL: mL: mL: mL: mL: mL is 2:2:5:20:30:3:100:100:100:100.
the probe compound is applied to the aspect of detecting the broad-spectrum beta-lactam and cephalosporin antibiotics pathogenic bacteria as the probe.
Further, the detection includes qualitative and quantitative detection.
Further, the qualitative detection comprises the following steps:
(1) Dissolving a probe compound in a solvent to form a solution, and forming a mixture with a sample to be detected;
(2) The compound solution was visually observed for change before and after mixing.
Further, the quantitative detection comprises the following steps:
(1) Dissolving a probe compound in a solvent to form a solution, and forming a mixture with a sample to be detected;
(2) And measuring the change of the optical signal of the compound through Raman spectroscopy, thereby determining the content or concentration of the extended-spectrum beta-lactamase and the cephalosporin enzyme or the bacteria containing the extended-spectrum beta-lactam and the cephalosporin drug resistance in the sample to be detected.
A kit comprising a probe compound as described above.
The invention has the advantages and positive effects that:
1. the invention takes the national important requirement of rapid detection of bacterial drug resistance as a research target, develops a resonance Raman probe with an off-on switch effect, and realizes the specific detection of the drug resistance of the ultra-broad spectrum beta-lactam and cephalosporin antibiotics at the single bacterial level.
2. The probe of the invention has no photobleaching phenomenon, sample pretreatment is not needed before detection, the detection condition is not influenced by complex physiological environment, and the detection result can be quantitatively analyzed, thus the method is an in-situ, real-time and dynamic quantitative detection method for bacterial drug resistance.
3. It has been unexpectedly discovered that when an aromatic ring is attached to an electron withdrawing group in the molecular structure of the present invention, there is a better spectral response than for an electron donating group.
4. The in-situ, real-time, dynamic, nondestructive and rapid detection and imaging of the extended-spectrum beta-lactamase and the cephalosporins are realized at the single bacteria level, and a rapid and effective method is provided for drug resistance evaluation and antibiotic selection.
5. The compound is a visual probe, can be used for detecting the extended-spectrum beta-lactamase and the cephalosporins enzyme in clinical samples such as blood, sputum, ascites and the like within 20 minutes, does not need to use large instruments and equipment, and is simple and convenient to operate and low in price.
6. The probe of the invention is matched with a beta-lactamase inhibitor clavulanic acid, so that the detection and the differentiation of the extended-spectrum beta-lactamase and the cephalosporin enzyme are realized.
7. The probe provided by the invention realizes detection of extended-spectrum beta-lactamase (BSBL), cephalosporinase (AmpC) and pathogenic bacteria thereof within 20 min. And the characteristics that clavulanic acid can inhibit ESBL and sulbactam can inhibit ESBL and AmpC are utilized to realize the distinction of extended-spectrum beta-lactamase (BSBL) and cephalosporinase (AmpC). High sensitivity by Raman detection down to 10 2 The drug-resistant bacteria of CFU/mL can reach 10 by only visual observation 3 CFU/mL. The detection method has the advantages of high sensitivity, short time consumption, simple and convenient operation, low price, no need of expensive large-scale instruments and no need of complex pretreatment on samples, and can realize bedside detection.
Drawings
FIG. 1 is a graph showing color change and ultraviolet absorption spectrum before and after hydrolysis of a probe according to the present invention;
FIG. 2 is a Raman spectrum of the probe of the present invention before and after hydrolysis;
FIG. 3 is a diagram showing the specificity of visual detection of extended-spectrum beta-lactamase and cephalosporinase in the present invention;
FIG. 4 is a diagram showing the specificity of the visual detection of extended spectrum beta-lactamase and cephalosporin enzyme resistant bacteria in the present invention;
FIG. 5 is a graph showing the sensitivity of Raman detection of extended spectrum beta-lactamase and cephalosporin enzyme resistant bacteria in accordance with the present invention;
FIG. 6 is a sensitivity chart for visually detecting extended-spectrum beta-lactamase and cephalosporin enzyme resistant bacteria in the invention.
Detailed Description
The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.
The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.
When no preparative route is involved, the starting materials and reagents used in the present invention are known products, and can be synthesized according to methods known in the art, or can be obtained by purchasing commercially available products. None of the commercially available reagents used were further purified.
The room temperature is 20-30 ℃.
The structure of the compounds of the invention is determined by Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS). NMR shift (. Delta.) of 10 -6 The units in (ppm) are given. NMR was measured by (Bruker ascend) TM Type 500) NMR spectrometer with solvent of deuterated dimethyl sulfoxide (DMSO-d 6) and deuterated chloroform (CDCl) 3 ) Deuterated methanol (CD) 3 OD), internal standard Tetramethylsilane (TMS). The following abbreviations are used for multiplicity of NMR signals: s = singlet, brs = broad, d = doublet, t = triplet, m = multiplet. Coupling constants are listed as J values, measured in Hz.
LC-MS was determined using a Thermo liquid chromatograph-mass spectrometer (UlltiMate 3000+ MSQ PLUS). HPLC was performed using Thermo high pressure liquid chromatography (Ultimate 3000). Reverse phase preparative chromatography a Thermo (UltiMate 3000) reverse phase preparative chromatograph was used. The flash column chromatography uses Ai Jieer (FS-9200T) automatic column-passing machine, and the silica gel pre-column uses tritai
Figure GDA0004071546720000061
The column is pre-packed. The thin layer chromatography silica gel plate is a tobacco yellow sea HSGF254 or Qingdao GF254 silica gel plate, and the specification of the thin layer chromatography separation and purification product is 0.4 mm-0.5 mm.
A probe compound for ultra-broad spectrum beta-lactam and cephalosporin antibiotics germs, which is a compound with the following structure:
Figure GDA0004071546720000062
Figure GDA0004071546720000071
Figure GDA0004071546720000072
or, it is:
Figure GDA0004071546720000073
or, it is:
Figure GDA0004071546720000074
or, it is:
Figure GDA0004071546720000075
or, it is:
Figure GDA0004071546720000076
or, it is:
Figure GDA0004071546720000077
the preparation method of the probe compound comprises the following steps:
Figure GDA0004071546720000078
to an eggplant type reaction flask was added 1eq Pd (OAc) in sequence under argon protection 2 And 1eq P (o-Tol) 3 Repeatedly freezing and thawing for 3 times, and stirring at room temperature for 1h to activate the catalyst; then 2.5eq cefdinir, 10eq of an electroattractive halogenated compound, DMF and triethylamine (10Freezing and degassing, and finally heating the reaction to 80 ℃ for reaction for 12 hours; the reaction was terminated, and 100mL of ethyl acetate was added to dilute the mixture, followed by 100mL of a 0.2mol/LHCl solution and 100mL of NaHCO 3 The mixture was washed with a saturated aqueous solution and 100mL of saturated brine, and dried over anhydrous MgSO 4 Drying; the organic phase was concentrated and purified on a silica gel column, where dichloromethane: the volume ratio of methanol is 5:1, obtaining the probe compound.
The probe compound is applied to the aspect of detecting the broad-spectrum beta-lactam and cephalosporin antibiotics germs as the probe.
Preferably, the detection comprises both qualitative and quantitative detection.
Preferably, the qualitative detection comprises the steps of:
(1) Dissolving a probe compound in a solvent to form a solution, and forming a mixture with a sample to be detected;
(2) The compound solution was visually observed for change before and after mixing.
Further, the quantitative detection comprises the following steps:
(1) Dissolving a probe compound in a solvent to form a solution, and forming a mixture with a sample to be detected;
(2) And measuring the change of the optical signal of the compound through Raman spectroscopy, thereby determining the content or concentration of the extended-spectrum beta-lactamase and the cephalosporin enzyme or the extended-spectrum beta-lactam and cephalosporin drug-resistant bacteria in the sample to be detected.
A kit comprising a probe compound as described above.
Specifically, the preparation and detection are as follows:
example 1
Figure GDA0004071546720000081
The above reaction formula is a structural change of the probe before and after the enzymatic hydrolysis.
As shown in FIGS. 1 and 2, it can be seen from FIGS. 1 and 2 that the probe amide ring can be hydrolyzed by extended spectrum beta-lactamase (ESBL) and cephalosporinase (AmpC) enzymes.
Figure GDA0004071546720000082
4-bromo-N, N-dimethylaniline (1g, 5 mmol) and methyl bromide (1.5 g, 15mmol) were added to a single-neck flask containing 20mL of ETOH under an argon atmosphere, stirred at room temperature for 12h and then heated under reflux for 1h until a large amount of a pale yellow solid was produced. A portion of ETOH was rotary evaporated, and recrystallized after refrigeration at-20 deg.C, and the resulting precipitate was washed successively with purified water and ether to obtain Compound 1 (1.02 g, 95% yield) as a white solid. Characterization data for compounds: 1H NMR (400MHz, meOD) delta 7.97 (d, J =8Hz, 2H), 7.88 (d, J =16Hz, 2H), 3.65 (s, 9H). 13C NMR (101MHz, meOD) delta
146.91,133.23,123.73,56.93.HRMS(ESI)m/z calcd for C9H13BrN+[M+H]+215.1135,found215.1119.
Figure GDA0004071546720000091
Sequentially adding Pd (OAc) into an eggplant-shaped reaction bottle under the protection of argon 2 (446.66mg, 2mmol) and P (o-Tol) 3 (608.89mg, 2mmol), repeated freeze-thawing for 3 times, and stirring at room temperature for 1h to activate the catalyst. Then cefdinir (1.98g, 5 mmol), compound 2 (4.56g, 20mmol), 30mL of DMMF and 3mL of triethylamine were added in sequence, and the reaction was cooled and degassed for 3 times, and finally the temperature was raised to 80 ℃ for 12 hours. The reaction was terminated, and 100mL of ethyl acetate was added to dilute the mixture, followed by 100mL of a 0.2mol/LHCl solution and 100mL of NaHCO 3 The mixture was washed with a saturated aqueous solution and 100mL of saturated brine, and dried over anhydrous MgSO 4 And (5) drying. The organic phase was concentrated and purified by silica gel column (dichloromethane: methanol = 5:1) to give compound 3 as a tan solid (1.19g, 45%). Characterization data for compounds: 1H NMR (400mhz, meod) δ 7.66 (d, J =12hz, 2h), 7.52 (d, J =16hz, 2h), 7.38 (d, J =8hz, 2h), 7.16 (s, 1H), 6.14 (s, 2H), 5.69 (s, 1H), 3.78 (s, 12H), 3.63 (d, J =8hz, 1h), 3.49 (d, J =8hz, 1h), 2.82 (s, 1H), 13C NMR (101mhz, meod) δ 168.79,164.48,163.42,161.57,150.99,141.63,141.29,132.70,129.36,128.18,124.94,119.93,113.42,59.33,57.40,55.78,25.67.HRMS(ESI)m/z calcd for C 23 H 25 N 6 O 5 S 2 + [M+H] + 529.6095,found 529.6082.
Example 2
Figure GDA0004071546720000092
Sequentially adding Pd (OAc) into an eggplant-shaped reaction bottle under the protection of argon 2 (446.66mg, 2mmol) and P (o-Tol) 3 (608.89mg, 2mmol), repeated freeze-thawing for 3 times, and stirring at room temperature for 1h to activate the catalyst. Then cefdinir (1.98g, 5 mmol), 2,4-dinitroiodobenzene (5.88g, 20mmol), 30mLDMF and 3mL triethylamine are added in sequence, the freezing and degassing operation is carried out for 3 times, and finally the reaction is heated to 80 ℃ for reaction for 12 hours. The reaction was terminated, and 100mL of ethyl acetate was added to dilute the mixture, followed by 100mL of a 0.2mol/LHCl solution and 100mL of NaHCO 3 The mixture was washed with a saturated aqueous solution and 100mL of saturated brine, and dried over anhydrous MgSO 4 And (5) drying. The organic phase was concentrated and purified by silica gel column (dichloromethane: methanol = 5:1) to give compound 3 as a tan solid (0.87g, 31%).
Example 3
Figure GDA0004071546720000101
Pd (OAc) is added into the eggplant type reaction bottle in turn under the protection of argon 2 (446.66mg, 2mmol) and P (o-Tol) 3 (608.89mg, 2mmol), repeated freeze-thawing for 3 times, and stirring at room temperature for 1h to activate the catalyst. Then cefdinir (1.98g, 5 mmol), 4-iodotrifluorotoluene (5.44g, 20mmol), 30mL of DMF and 3mL of triethylamine were added in sequence, and the reaction was cooled and degassed for 3 times, and finally the temperature was raised to 80 ℃ for 12 hours. The reaction was terminated, and 100mL of ethyl acetate was added to dilute the mixture, followed by 100mL of a 0.2mol/LHCl solution and 100mL of NaHCO 3 The mixture was washed with a saturated aqueous solution and 100mL of saturated brine, and dried over anhydrous MgSO 4 And (5) drying. The organic phase was concentrated and purified by silica gel column (dichloromethane: methanol = 5:1) to give compound 4 as a tan solid (1.05g, 39%)。
Example 4
Figure GDA0004071546720000102
Sequentially adding Pd (OAc) into an eggplant-shaped reaction bottle under the protection of argon 2 (446.66mg, 2mmol) and P (o-Tol) 3 (608.89mg, 2mmol), repeated freeze-thawing for 3 times, and stirring at room temperature for 1h to activate the catalyst. Then cefdinir (1.98g, 5 mmol), 4-iodocyanobenzene (4.58g, 20mmol), 30mL of DMMF and 3mL of triethylamine were added in sequence, the operation of freezing and degassing was performed for 3 times, and finally the reaction was heated to 80 ℃ for reaction for 12 hours. The reaction was terminated, and 100mL of ethyl acetate was added to dilute the mixture, followed by 100mL of a 0.2mol/LHCl solution and 100mL of NaHCO 3 The mixture was washed with a saturated aqueous solution and 100mL of saturated brine, and dried over anhydrous MgSO 4 And (5) drying. The organic phase was concentrated and purified over a silica gel column (dichloromethane: methanol = 5:1) to yield compound 5 as a tan solid (1.19g, 48%).
Example 5
Figure GDA0004071546720000111
Sequentially adding Pd (OAc) into an eggplant-shaped reaction bottle under the protection of argon 2 (446.66mg, 2mmol) and P (o-Tol) 3
(608.89mg, 2mmol), repeated freeze-thawing for 3 times, and stirring at room temperature for 1h to activate the catalyst. Then cefdinir is added in turn
(1.98g, 5 mmol), 4-iodophenylacetylene (4.56g, 20mmol), 30mL of DMF and 3mL of triethylamine were subjected to a freezing degassing operation for 3 times, and finally the temperature of the reaction was raised to 80 ℃ to react for 12 hours. The reaction was terminated, and 100mL of ethyl acetate was added to dilute the mixture, followed by 100mL of a 0.2mol/LHCl solution and 100mL of NaHCO 3 The mixture was washed with a saturated aqueous solution and 100mL of saturated brine, and dried over anhydrous MgSO 4 And (5) drying. The organic phase was concentrated and purified by silica gel column (dichloromethane: methanol = 5:1) to give compound 6 as a tan solid (0.94g, 48%).
Example 6
Figure GDA0004071546720000112
Sequentially adding Pd (OAc) into an eggplant-shaped reaction bottle under the protection of argon 2 (446.66mg, 2mmol) and P (o-Tol) 3 (608.89mg, 2mmol), repeated freeze-thawing for 3 times, and stirring at room temperature for 1h to activate the catalyst. Then cefdinir (1.98g, 5 mmol), 4-nitroiodobenzene (4.98g, 20mmol), 30mLDMF and 3mL of triethylamine are added in sequence, the freezing and degassing operations are carried out for 3 times, and finally the reaction is heated to 80 ℃ for reaction for 12 hours. The reaction was terminated, and 100mL of ethyl acetate was added to dilute the mixture, followed by 100mL of a 0.2mol/LHCl solution and 100mL of NaHCO 3 The mixture was washed with a saturated aqueous solution and 100mL of saturated brine and dried over anhydrous MgSO 4 And (5) drying. The organic phase was concentrated and purified by silica gel column (dichloromethane: methanol = 5:1) to give compound 7 as a tan solid (1.11g, 43%).
The correlation test of the present invention is as follows:
5.29mg of the compound was dissolved in 100. Mu.L of dimethyl sulfoxide solution to prepare a 0.1mol/L probe stock solution (stored in a brown bottle at 4 ℃). The probe stock solution was diluted with a phosphate buffer solution having a pH of 7.5 to prepare an assay concentration of 0.1 mmol/L.
Firstly, performing low-temperature ultrasonic wall breaking on 1mL of clinical samples (sputum, alveolar lavage fluid, pleural effusion, urine and serum), centrifuging for 5min at 8000r/min, adding 200 mu L of supernatant into 800 mu L of 0.1mmol/L probe solution, incubating for 10min at 37 ℃, observing the color change of the solution, and performing Raman spectrum detection.
The Raman spectrum testing method comprises the following steps:
the Raman instrument of the subject group is a Renysha Raman spectrometer, a capillary tube with the diameter of 0.3mm is used for sucking a sample to be detected, and Raman detection is carried out under a 50 Xeyepiece, wherein the laser wavelength is 633nm, the laser power is 20mW, and the exposure time is 1s.
1. Detection of various beta-lactamases (self-synthesized)
The required beta-lactamase gene sequence was downloaded from NCBI (https:// www.ncbi.nlm.nih.gov /), and whole gene synthesis and plasmid construction were performed by Shanghai Producer. The plasmid is transferred into an expression vector Top10 escherichia coli for protease synthesis. Finally, 11 beta-lactamase enzymes were synthesized, including 1 broad-spectrum beta-lactamase (BSBL), 3 extended-spectrum beta-lactamase (ESBL), 2 cephalosporinase (AmpC) and 5 carbapenemases (Carbapenemase). 50. Mu.L of 0.1mmol/L probe solution was added with 1. Mu.L of the enzyme solution, incubated at 37 ℃ for 10min, and color change was observed.
The results are shown in fig. 3, from which it can be seen that clavulanic acid can inhibit ESBL and sulbactam can inhibit ESBL and AmpC.
2. Detection of clinical strains resistant to beta-lactamase (containing beta-lactamase)
11 beta-lactamase-resistant bacteria were purchased from Nanjing Lexus biosciences, including 1 broad-spectrum beta-lactamase (BSBL) resistant bacteria (ATCC 35218), 3 extended-spectrum beta-lactamase (ESBL) resistant bacteria (NCTC 13464, NCTC 13351, ATCC 700603), 2 cephalosporinase (AmpC) resistant bacteria (ATCC 25830, ATCC 29544), and 5 Carbapenemase (Carbapenemase) resistant bacteria (NCTC 13442, ATCC BAA2146, NCTC 13440, ATCC BAA 1605, ATCC 700721). 50 μ L of 0.1mmol/L probe solution was added to 10 μ L of bacterial solution, incubated at 37 ℃ for 10min, and color change was observed.
The results are shown in fig. 4, from which it can be seen that clavulanic acid can inhibit ESBL and sulbactam can inhibit ESBL and AmpC.
3. Detection limit for Raman detection of beta-lactamase drug-resistant bacteria
50 mu L of 0.1mmol/L probe solution is added with bacterial liquid with different concentrations, incubated at 37 ℃ for 20min, and subjected to Raman spectrum detection. The results are shown in FIG. 5.
4. Visual detection limit for detecting beta-lactamase drug-resistant bacteria
50 mu L of 0.1mmol/L probe solution is added with bacterial liquid with different concentrations, incubated at 37 ℃ for 20min, and color change is observed. The results are shown in FIG. 6.
The probe provided by the invention realizes detection of extended-spectrum beta-lactamase (BSBL), cephalosporinase (AmpC) and pathogenic bacteria thereof within 20 min. And the characteristics that clavulanic acid can inhibit ESBL and sulbactam can inhibit ESBL and AmpC are utilized to realize super-broadSpectrum beta-lactamase (BSBL) and cephalosporinase (AmpC). High sensitivity by Raman detection down to 10 2 The drug-resistant bacteria of CFU/mL can reach 10 by only visual observation 3 CFU/mL. The detection method has the advantages of high sensitivity, short time consumption, simple and convenient operation, low price, no need of expensive large-scale instruments and no need of complex pretreatment on samples, and can realize bedside detection.
Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the embodiments disclosed.

Claims (1)

1. The probe compound is applied to the aspect of detecting the broad spectrum beta-lactam and cephalosporin antibiotics germs as a probe, wherein the probe compound has the following structure:
Figure FDA0004058953180000011
the application comprises the following steps:
(1) Dissolving a probe compound in a solvent to form a solution, and forming a mixture with a sample to be detected;
(2) And measuring the change of the optical signal of the probe compound through Raman spectroscopy so as to determine the content or concentration of the extended-spectrum beta-lactamase and the cephalosporin enzyme or the drug-resistant bacteria containing the extended-spectrum beta-lactam and the cephalosporin in the sample to be detected.
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