A macrocyclic amphiphilic calixarene antibacterial agent with both bacterial membrane disruption and quorum sensing interference

Abstract

The present application relates to a kind of cationic amphiphilic calixarene by the destruction of bacterial membrane and the realization of effective removal of multi-drug resistant bacteria by interference of quorum sensing. Based on the easy modification of calixarene and its unique skeleton, as well as assembly and recognition properties, a macrocyclic amphiphilic antibacterial agent is designed, which can both enhance the ability to destroy bacterial membrane and interfere with bacterial quorum sensing. By modifying quaternary ammonium salt groups on the upper edge of calixarene and hydrophobic alkyl chains on the lower edge, a quaternary ammonium salt amphiphilic calixarene with antibacterial ability is obtained. Compared with traditional quaternary ammonium salt modified single alkyl chain antibacterial agent, the calixarene skeleton significantly improves the ability to destroy bacterial membrane. The hydrophobic cavity of calixarene can recognize a wide variety of guest molecules, and has moderate intensity binding to signal molecules released in the quorum sensing of gram-negative bacteria, thereby interfering with the production of virulence factors, biofilm formation and other biological functions, killing multi-drug resistant gram-negative bacteria.

Description

[0001] This research is supported by the National Natural Science Foundation of China Youth Science Foundation Project (No: 22201299). TECHNICAL FIELD

[0002] The present application belongs to the field of new antibacterial materials, and relates to a synthesis method of a macrocyclic amphiphilic antibacterial agent based on a calixarene skeleton and having the functions of destroying bacterial membranes and interfering with bacterial quorum sensing, and application thereof in killing multi-drug resistant Gram-negative bacteria. Based on the unique skeleton structure of the amphiphilic calixarene, the destruction ability of the calixarene to bacterial membranes is enhanced, and the inhibitory effect on multi-drug resistant bacteria is still significant. At the same time, the hydrophobic cavity of the calixarene can bind various Gram-negative bacterial quorum sensing signal molecules, thereby interfering with the normal life activities of bacteria, and further increasing the application prospect of the calixarene in inhibiting multi-drug resistant bacteria. BACKGROUND

[0003] Bacterial infection has always been one of the global problems threatening human health. Since the discovery of penicillin, the era of antibiotics has begun to fight bacterial infection. However, the abuse of antibiotics has led to the emergence and rapid development of bacterial drug resistance, and multi-drug resistant superbugs have made antibiotic therapy ineffective. According to the prediction of WHO, drug-resistant bacteria will become the number one killer threatening human life safety in 2050, and the number of people dying from bacterial infection worldwide each year will exceed one million. People have been trying to find alternative antibacterial therapies, such as cationic surfactants, for combating multi-drug resistant bacterial infections.

[0004] Cationic surfactants mainly kill bacteria by destroying bacterial membranes through electrostatic interaction of positive charge head groups and hydrophobic interaction of hydrophobic tail chains. Bacteria are difficult to bypass this non-specific membrane destruction mechanism, so it is difficult to develop resistance to surfactants. Cationic surfactants have the advantages of clear structure, simple synthesis, easy modification, and easy mass production, making them a widely used disinfecting and sterilizing means.

[0005] Studies have shown that bacteria communicate with each other through quorum sensing. Quorum sensing is a mechanism of intercellular communication in bacteria, based on the diffusion of signal molecules, so that bacterial populations can regulate global behaviors such as virulence factor production and biofilm formation according to population density. Quorum sensing is an important regulatory mechanism for bacterial populations to adapt to environmental changes, and also provides an opportunity for new antibacterial strategies to interfere with bacterial physiological functions through molecular recognition strategies.

[0006] Calixarene is a kind of cyclic oligomer formed by several phenol units connected by methylene bridge, and the hydrophobic cavity of calixarene can adapt to different size guest molecules, which is the advantage of calixarene as a supramolecular macrocycle host. The modification site of calixarene is rich, and it is convenient to modify the amphiphilic calixarene to obtain macrocyclic amphiphilic calixarene as a new type of cationic surfactant. The skeleton, assembly and recognition properties of calixarene make it have a unique advantage in the field of antibacterial. SUMMARY

[0007] The application prepares macrocyclic amphiphilic antibacterial agent based on calixarene skeleton, compared with single chain, the oligomeric cyclic skeleton enhances the destruction ability to bacterial membrane, and can kill multi-drug resistant bacteria more effectively. At the same time, the hydrophobic cavity of calixarene can combine with various gram-negative bacterial quorum sensing signal molecules, interfere with bacterial quorum sensing, and affect the normal biological function of bacteria. This “two-pronged” strategy provides a new idea for the difficult problem of multi-drug resistant bacteria infection.

[0008] To achieve the above purpose, the application adopts the following technical scheme:

[0009] A macrocyclic amphiphilic calixarene antibacterial agent with the characteristics of destroying bacterial membrane and interfering with quorum sensing, characterized in that a supramolecular host calixarene is used as a basic skeleton, a quaternary ammonium salt group is modified on the upper edge of the calixarene, and different length alkyl chains are modified on the lower edge, and the structure is as follows:

[0010]

[0011] The calixarene skeleton is a macrocyclic compound formed by several modified phenol units connected by methylene, represented by the structure in the figure. The structure contained in the bracket part is the structural unit of the macrocyclic amphiphilic calixarene antibacterial agent, the cyclic dotted line represents that the bracket part structure is connected to form a ring, and n represents the number of bracket part structure units in the ring structure, usually n = 4, 5, 6, 8; X- is a counterion, X is a halogen atom, usually chlorine, bromine, iodine; The R group modified on the lower edge is a straight chain alkyl chain of different lengths, which refers to an alkyl group with a structure formula of CmH2m+1, wherein m is the number of carbon atoms.

[0012] The macrocyclic amphiphilic calixarene antibacterial agent with the characteristics of destroying bacterial membrane and interfering with quorum sensing, the typical compound is as follows:

[0013]

[0014] The disclosed calixarene antibacterial agent with both membrane disruption and quorum sensing interference has a unique cyclic skeleton, which greatly improves the membrane disruption ability compared with single-chain antibacterial agents with the same number of active units. The reason is that the hydrophobic cavity of the calixarene skeleton can bind to various signal molecules in the quorum sensing of gram-negative bacteria, and can interfere with quorum sensing through molecular recognition strategy to affect the normal biological function of bacteria. Another feature is that the new macrocyclic amphiphilic antibacterial agent can achieve the purpose of killing bacteria and interfering with quorum sensing in a short time after being added to the bacterial culture solution and simply mixed.

[0015] The application further discloses a preparation method of the macrocyclic amphiphilic calixarene antibacterial agent with both membrane disruption and quorum sensing interference, and has the characteristics that the following steps are performed:

[0016] (1) a certain amount of calix[n]arene, a base and a solvent are added to a reaction bottle, and after stirring, 1-bromoalkane is added, heated to a certain temperature, reacted for a certain time, and the reaction is monitored by thin layer chromatography; after the reaction is completed, the excess base is neutralized by adding dilute hydrochloric acid, extracted with dichloromethane, and the crude product is obtained by reduced pressure distillation, and then recrystallized with dichloromethane / methanol; the ratio of the calix[n]arene, the base and the bromoalkane is 1:10:10 (molar ratio); the reaction temperature is 80-100 °C; the reaction time is 24-36 h; the dilute hydrochloric acid used in the post-treatment usually has a concentration of 1 M; and the solvent is a strong polar aprotic solvent, and acetonitrile, N,N-dimethylformamide or dimethyl sulfoxide is usually used;

[0017] (2) the product prepared in (1), excess sodium nitrate and trifluoroacetic acid are added to a reaction bottle to dissolve, and stirred at room temperature for a period of time; after the reaction is completed, a large amount of water is slowly added, and the reaction is extracted and separated with dichloromethane; the feeding ratio of the product in (1), sodium nitrate and trifluoroacetic acid is 1:40:80 (molar ratio); the reaction time is 8-10 h; the volume of water added for quenching the reaction is usually greater than 2 times the volume of the reactants; and the organic phase is washed with saturated sodium carbonate for 2-3 times;

[0018] (3) the product prepared in (2) is added to a reaction bottle, dissolved with a solvent, then hydrazine hydrate and palladium carbon powder are added, stirred uniformly, heated to a certain temperature, and reacted for a period of time; after the reaction is completed, the reaction is filtered and treated; the ratio of the product in (2), hydrazine hydrate and palladium carbon is 1:40:2 (molar ratio); the solvent used is ethyl acetate:ethanol = 1:1 (volume ratio); the heating temperature is 80-90 °C; the reaction time is 8-12 h; and the post-treatment is performed;

[0019] (4) Add the product obtained in (3), potassium carbonate, and iodomethane to the reaction flask, and add anhydrous tetrahydrofuran as a solvent; seal the tube and heat to (100-110 °C) for 72-80 h; perform post-treatment, dissolve the filter cake in hot water, add ammonium hexafluorophosphate, and filter to obtain the precipitate; dissolve the filter cake in acetonitrile, add tetrabutylammonium chloride, and filter to obtain the solid, which is the target macrocyclic amphiphilic antibacterial agent; the ratio of the product obtained in (3), potassium carbonate, and iodomethane is 1:20:100 (molar ratio).

[0020] This invention further discloses the application of macrocyclic amphiphilic calixarane antibacterial agents, which possess both the ability to disrupt bacterial membranes and interfere with quorum sensing, in the preparation of antibacterial agents for killing drug-resistant Gram-negative bacteria. The Gram-negative bacteria referred to are *Pseudomonas aeruginosa*. Experimental results show that macrocyclic amphiphilic calixarane antibacterial agents can kill *P. aeruginosa* more efficiently and can interfere with the quorum sensing process of *P. aeruginosa* by capturing quorum sensing signal molecules, inhibiting various biological functions including biofilm formation and pyocyanin production.

[0021] The present invention is described in more detail below:

[0022] A macrocyclic amphiphilic antibacterial agent based on a calixarene skeleton has the following general structural formula: [in calix...] n The upper edge of the aromatic skeleton ( n = 4, 5, 6, 8) Modification of quaternary ammonium salt head groups, X − This is a counter ion. X represents a halogen atom, including chlorine, bromine, and iodine. The R groups modified at the lower edge are alkyl chains of varying lengths.

[0023]

[0024] Furthermore, the present invention provides a method for preparing this type of macrocyclic amphiphilic antibacterial agent, which mainly includes the following steps:

[0025] Step 1: Add a certain amount of [unspecified substance] to the reaction flask. n Aromatic hydrocarbons and a suitable amount of alkali were added to a solvent, stirred until homogeneous, and then 1-bromoalkane was added. The mixture was heated to a certain temperature and reacted for a certain time. The reaction was monitored by thin-layer chromatography. After the reaction was completed, dilute hydrochloric acid was added to neutralize the excess alkali. The mixture was extracted with dichloromethane, and the crude product was obtained by vacuum distillation. The crude product was then recrystallized from dichloromethane / methanol. n The ratio of aromatic hydrocarbons, alkali, and bromoalkane is 1:10:10 (molar ratio); the reaction temperature is typically 80-100 °C; the reaction time is 24-36 h; the dilute hydrochloric acid used for post-treatment is typically 1 M. The solvent is generally acetonitrile. N , N - Polar aprotic solvents such as dimethylformamide.

[0026] Step two: Add the product prepared in step one, excess sodium nitrate into a reaction bottle and dissolve with trifluoroacetic acid as solvent, stir at room temperature for a period of time. After the reaction is completed, add a large amount of water slowly, and extract with dichloromethane. Wash the organic phase with saturated sodium carbonate solution, and then dry with anhydrous sodium sulfate. After filtering the drying agent, remove the solvent under reduced pressure, and recrystallize with dichloromethane / methanol. The feeding ratio of the product prepared in step one, sodium nitrate and trifluoroacetic acid is generally 1:40:80 (molar ratio); the reaction time is usually 8-10 h; the volume of water added to quench the reaction is usually more than 2 times the volume of the reactants; the number of times of washing the organic phase with saturated sodium carbonate is usually 2-3 times.

[0027] Step three: Add the product prepared in step two, add an appropriate amount of solvent to dissolve, then add an appropriate amount of hydrazine hydrate and palladium carbon powder, stir uniformly, and heat to a certain temperature for a period of time. After the reaction is completed, filter, and distill the filtrate under reduced pressure to obtain the crude product, and finally recrystallize with dichloromethane / methanol. The ratio of the product prepared in step two, hydrazine hydrate and palladium carbon is 1:40:2 (molar ratio); the solvent used is usually ethyl acetate: ethanol = 1:1 (volume ratio); the heating temperature is usually 80-90 °C; the reaction time is usually 8-12 h; when filtering, a layer of diatomite should be placed on the filter paper, and the filter residue should not be dried too much to avoid fire. The amino compound is slowly oxidized in the air, and the product should be prepared as soon as possible for the next step.

[0028] Step four: Add the product prepared in step three, potassium carbonate, iodomethane into a reaction bottle, and add an appropriate amount of anhydrous tetrahydrofuran as solvent. Seal the tube and heat to a higher temperature for a long time. After the reaction is completed, filter, and wash the filter cake with ethanol, dichloromethane and cold water. Dissolve the filter cake in hot water, add an appropriate amount of ammonium hexafluorophosphate, and filter to obtain the precipitate. Dissolve the filter cake in acetonitrile, add an appropriate amount of tetrabutylammonium chloride, and filter the precipitated solid to obtain the target macrocyclic amphiphilic antibacterial agent. The ratio of the product prepared in step three, potassium carbonate and iodomethane is 1:20:100 (molar ratio); the reaction temperature is usually 110 °C; the reaction time is usually 72 h.

[0029] The application provides an application of a calixarene-based macrocyclic amphiphilic antibacterial agent in the field of antibacterial agents. The prepared macrocyclic amphiphilic antibacterial agent is added into a bacterial culture solution, and shaken uniformly, so that the double-mode antibacterial purpose of destroying bacterial membranes and interfering with quorum sensing to affect the normal biological functions of bacteria is achieved.

[0030] The macrocyclic amphiphilic calixarene antibacterial agent disclosed in the application has the advantages and beneficial effects of destroying bacterial membranes and interfering with quorum sensing.

[0031] (1) This invention provides a macrocyclic amphiphilic antibacterial agent based on a calixarene skeleton. Compared with single-chain cationic surfactant antibacterial agents with the same number of quaternary ammonium salt head groups, the calixarene skeleton improves the antibacterial agent's ability to destroy bacterial membranes and exhibits excellent antibacterial performance at low concentrations. In addition, this product has good water solubility and has good application prospects in the fields of medical, hygiene and personal care products.

[0032] (2) This invention also demonstrates a method to recognize and capture quorum sensing signal molecules of Gram-negative bacteria through host-guest interaction, and to a certain extent affects the normal biological functions of bacteria such as the production of virulence factors and the formation of biofilms, providing a new approach to antibacterial activity based on molecular recognition strategies.

[0033] (3) This invention utilizes calixarenes, which have unique skeletal, assembly, and recognition properties, to cleverly combine two antibacterial strategies: enhancing bacterial membrane disruption and interfering with quorum sensing, making bacteria more susceptible to resistance to this type of novel antibacterial agent. At the same time, thanks to the ease of modification of calixarenes, it is possible to achieve the mass production and diverse modification of this type of macrocyclic amphiphilic antibacterial agent. Attached Figure Description

[0034] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the description of the embodiments or prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without additional creative effort.

[0035] Figure 1 The 1H NMR spectrum of 25,26,27,28-tetraoctyl-quaternary ammonium calix[4] aromatic hydrocarbon (QAC4A-8C) is shown in CD3OD, 400 MHz, 25 ℃.

[0036] Figure 2 for N,N,N - Trimethyl-4-octyloxyaniline (QA-Ph-8C) 1H NMR spectrum (DMSO- d 6, 400MHz, 25℃);

[0037] Figure 3 The 1H NMR spectrum (DMSO-) of 25,26,27,28-tetradodecyl-quaternary ammonium calix[4] aromatic hydrocarbon (QAC4A-12C) d 6, 400 MHz, 25 °C);

[0038] Figure 4Flitration of QAC4A-8C to Fl (A: Fluorescence titration of Fl (0.8 μΜ) with QAC4A-8C in HEPES (10 mM, pH = 7.4); B: Fitting plot according to 1:1 binding model);

[0039] Figure 5 Flitration of QAC4A-8C to the quorum sensing signal molecule 3-oxo-dodecanoyl homoserine lactone (3OC 12 HSL) (A: Fluorescence titration of the quorum sensing signal molecule 3OC 12 HSL with Fl @ QAC4A-8C (0.5 μΜ:0.5 μΜ) in HEPES (10 mM, pH = 7.4); B: Fitting plot according to 1:1 competitive binding model);

[0040] Figure 6 Bactericidal effect of QAC4A-8C and control monomer against P. aeruginosa (CFU: Colony Forming Unit) tested by standard plate count method,

[0041] wherein Fig. A is the bactericidal effect of QAC4A-8C at different concentrations against P. aeruginosa, and Fig. B is the bactericidal effect of control monomer at different concentrations against P. aeruginosa;

[0042] Figure 7 Determination of the damage of control monomer and QAC4A-8C to bacterial cell membrane by 1-anilinonaphthalene (NPN) fluorescence experiment (fluorescence intensity in the figure is the fluorescence intensity at 420 nm, and the monomer concentration in the figure is 4 times the concentration of QAC4A-8C);

[0043] Figure 8 Growth curve of P. aeruginosa when co-incubated with QAC4A-12C at different concentrations;

[0044] Figure 9 Inhibition effect of QAC4A-12C at different concentrations on the secretion of pyocyanin by P. aeruginosa;

[0045] Figure 10 Inhibition effect of QAC4A-12C at different concentrations on the formation of biofilm by P. aeruginosa. DETAILED DESCRIPTION

[0046] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings. The embodiments mentioned below are implemented on the premise of the technical solutions of the present application, and detailed implementation processes have been given, but it must be declared that the protection scope of the present application is not limited to the following embodiments. The sources used are as follows:

[0047] 4-tert-butylphenol, diphenyl ether, formaldehyde aqueous solution, ethyl acetate, ethanol, glacial acetic acid, 4-dimethylaminophenol, 1-bromon-octane, trifluoroacetic acid, sodium nitrate, Pd / C, hydrazine hydrate, iodomethane, potassium carbonate, tetrahydrofuran, ammonium hexafluorophosphate, tetrabutylammonium chloride (TBAC), 1-bromododecane, sodium hydroxide, acetonitrile, N,N-dimethylformamide, sodium fluorescein, 1-anilinonaphthalene (NPN) were purchased from Shanghai Biotechmed Co., Ltd. and used without special treatment before use.

[0048] Proteose peptone, yeast extract, crystal violet, 3-oxododecanoyl homoserine lactone (3OC 12 HSL) were purchased from Shanghai Yuan Ye Biotechnology Co., Ltd.

[0049] t -Bu-C4A is a calixarene starting material, which is obtained by reacting p-tert-butylphenol and formaldehyde aqueous solution. The specific reaction steps are referred to the literature: Organic Syntheses, Coll. Vol. 8, p. 75 (1993); Vol. 68, p. 234 (1990) This reaction is very mature, so it is not described here.

[0050] Example 1: Synthesis of macrocyclic amphiphilic antibacterial agent: 25, 26, 27, 28-tetraoctyl-quaternary ammonium salt calix[4]arene (QAC4A-8C)

[0051]

[0052] Step one: t Synthesis of -Bu-C4A-8C

[0053] Into the reaction bottle were added t -Bu-C4A (1.00 g, 1.5 mmol), 1-bromon-octane (2.95 g, 15.3 mmol), sodium hydroxide (0.61 g, 15.3 mmol), acetonitrile (50 mL). After mixing well, reflux for 24 hours. After the reaction was completed, it was cooled to room temperature, and 1 M dilute hydrochloric acid was slowly added under stirring to neutralize the excess sodium hydroxide. Then dichloromethane was added for extraction, and the organic phase was dried over anhydrous Na2SO4, filtered, and the filtrate was rotary evaporated to obtain the crude product, which was recrystallized with dichloromethane / methanol to obtain white solid t -Bu-C4A-8C, 1.07 g, yield 65%.

[0054] Step two: Synthesis of NO2-C4A-8C

[0055] Into the reaction bottle were added tBu-C4A-8C (1.00 g, 0.91 mmol), trifluoroacetic acid (8.3 g, 72.8 mmol) and sodium nitrate (3.85 g, 36.4 mmol). After mixing well, the reaction was carried out at room temperature overnight. After the reaction was completed, 70 mL of water was added and stirred vigorously. 50 mL of dichloromethane was added to extract the solution, and the organic phase was washed twice with saturated sodium carbonate solution and then dried with anhydrous Na2SO4. After suction filtration, the crude product was obtained by distillation under reduced pressure, and recrystallized with dichloromethane / methanol to obtain a light yellow solid NO2-C4A-8C 0.86 g, yield 90%.

[0056] Step three: synthesis of NH2-C4A-8C

[0057] In the reaction bottle, NO2-C4A-8C (0.8 g, 0.54 mmol), Pd / C (5%, 0.15 g), hydrazine hydrate (2 mL) were added, 30 mL of ethyl acetate: ethanol = 1:1 (volume ratio) mixed solvent was added, and refluxed overnight. After the reaction was completed and cooled to room temperature, suction filtration (filter paper plus diatomite), after distillation under reduced pressure, the crude product was recrystallized with dichloromethane / methanol to obtain nearly white solid NH2-C4A-8C 0.62 g, yield 91.4%.

[0058] Step four: synthesis of QAC4A-8C

[0059] In the bottle, NH2-C4A-8C (0.5 g, 0.53 mmol), potassium carbonate (1.5 g, 10.8 mmol), 40 mL of anhydrous tetrahydrofuran were added, stirred for 3 h, then 6 mL of methyl iodide was added, and the reaction was carried out in a sealed tube at 110 °C for 3 days. After the reaction was completed and cooled to room temperature, the sealed tube was opened, suction filtered, and the filter cake was dissolved in water. 0.3 g of ammonium hexafluorophosphate was added with stirring, and a large amount of white flocculent precipitate appeared. After suction filtration, the obtained solid was dissolved in acetonitrile, 0.5 g of tetrabutylammonium chloride was added with heating and stirring, a large amount of white flocculent precipitate was precipitated, and the stirring was continued for 1 h, and the refrigerator was refrigerated and placed. After suction filtration, white solid QAC4A-8C 67 mg was finally obtained, yield 10%.

[0060] Step five: structural characterization

[0061] As shown in the following Figure 1 , the structure of compound QAC4A-8C is 1 H NMR (400 MHz, MeOH- d 4, d): 7.66 (s, 8H); 4.51 (d, 4H); 3.93 (t, 8H); 3.53 (d, 4H); 3.48 (s, 36H); 1.97 (t, 8H); 1.26 (m, 40H); 0.84 (t, 12H).

[0062] Example 2: N,N,N Synthesis of 4-trimethyl-4-octyloxyaniline (QA-Ph-8C)

[0063]

[0064] Step 1: Synthesis of DMA-Ph-8C

[0065] 4-Dimethylaminophenol (1.00 g; 7.28 mmol), 1-bromo-n-octane (1.41 g; 7.28 mmol), and sodium hydroxide (0.60 g; 15.0 mmol) were added to a reaction flask and dissolved in 30 mL of acetonitrile. The mixture was refluxed overnight. After cooling to room temperature, excess alkali was neutralized with 1 M dilute hydrochloric acid, and the mixture was extracted with dichloromethane. The organic phase was dried and then evaporated to dryness to obtain the crude product. Column chromatography (petroleum ether:ethyl acetate = 1:1) yielded a brown solid, totaling 0.98 g, with a yield of 54.1%.

[0066] Step 2: Synthesis of QA-Ph-8C

[0067] Following the synthesis of QAC4A-8C, approximately 600 mg of the target product was finally obtained, with a yield of 49.9%.

[0068] Step 3: Structural Characterization

[0069] As attached Figure 2 As shown, QA-Ph-8C 1 H NMR (400 MHz, DMSO-) d 6, d ): 7.87 (d, 2H); 7.12(d, 2H); 4.03 (t, 2H); 3.58 (s, 9H); 1.71 (t, 2H); 1.34 (m, 10H); 0.86 (t, 3H).

[0070] Example 3: Synthesis of macrocyclic amphiphilic antibacterial agent 25,26,27,28-tetradecyl-quaternary ammonium calix[4] aromatic hydrocarbon (QAC4A-12C)

[0071]

[0072] Step 1: tSynthesis of -Bu-C4A-12C

[0073] Add to the reaction flask t -Bu-C4A (1.0 g, 1.53 mmol), 1-bromo-n-dodecane (3.81 g, 15.3 mmol), sodium hydroxide (0.61 g, 15.3 mmol), and acetonitrile (50 mL) were mixed thoroughly and refluxed for 24 h. After the reaction, the product was handled according to the synthesis method of QAC4A-8C. t -Bu-CA4-12C total 1.2 g, yield 59.1%.

[0074] Step 2: Synthesis of NO2-C4A-12C

[0075] The specific method is the same as that used in the synthesis of NO2-C4A-8C. A total of 0.95 g of NO2-C4A-12C was finally obtained, with a yield of 81.5%.

[0076] Step 3: Synthesis of NH2-C4A-12C

[0077] The specific method is the same as that used in the synthesis of NH2-C4A-8C. A total of 0.68 g of NH2-C4A-12C was finally obtained, with a yield of 94.4%.

[0078] Step 4: Synthesis of QAC4A-12C

[0079] The specific method is the same as that used in the synthesis of QAC4A-8C. A total of 180 mg of QAC4A-12C was finally obtained, with a yield of 28.3%.

[0080] Step 5: Structural Characterization

[0081] As attached Figure 3 As shown, compound QAC4A-12C 1 H NMR (400 MHz, DMSO-) d 6, d ): 7.74 (s, 8H); 4.39 (d, 4H); 3.87 (t, 8H); 3.44 (s, 40H); 1.98 (t, 8H); 1.32 (m, 52H); 0.85 (t, 12H).

[0082] Example 4: Determination of the binding constant of QAC4A-8C with quorum sensing signal molecules

[0083] Considering the fact that the calixarene host is modified with quaternary ammonium salt groups, which are positively charged, a negatively charged fluorescent dye was chosen as the fluorescent probe. QAC4A-8C can encapsulate the fluorescent dye, quenching its fluorescence. When a competing guest molecule (such as a quorum sensing signal molecule) binds to QAC4A-8C, the fluorescent dye is displaced from the cavity of QAC4A-8C, and the fluorescence is recovered.

[0084] Step one: determination of the binding constant of QAC4A-8C and fluorescein (Fl)

[0085] A solution of 0.5 μΜ Fl (solvent: HEPES buffer solution, pH = 7.4) was added to a quartz cuvette. A solution containing a certain concentration of QAC4A-8C (solvent: HEPES buffer solution, pH = 7.4) was also prepared. The solution was added dropwise to the quartz cuvette, and the fluorescence change before and after each addition was recorded (excitation wavelength: 490 nm). The formula for direct titration was used for fitting. The binding constant of the model host-guest pair QAC4A-8C@Fl was obtained K a = (4.76 ± 0.63) × 10 7 M −1 Figure 4 .

[0086] Step two: determination of the binding constant of QAC4A-8C and quorum sensing signal molecules

[0087] A solution of a known concentration of fluorescein and QAC4A-8C was added to a quartz cuvette. A larger concentration of a methanol solution of a quorum sensing signal molecule was also prepared (the concentration of the methanol solution was as large as possible to reduce the impact of the addition of methanol on the fluorescence signal of the dye). The methanol solution of the signal molecule was added dropwise to the quartz cuvette, and the fluorescence change before and after each addition was recorded (excitation wavelength: 490 nm). The formula for competitive titration was used for fitting. The binding constant of QAC4A-8C and 3-oxododecanoyl homoserine lactone (3OC 12 HSL) was obtained K a = (1.5 ± 0.18) × 10 5 M −1 Figure 5 .

[0088] Example 5: Practical example of typical antibacterial agent QAC4A-8C versus single-chain antibacterial agent in killing multidrug-resistant gram-negative bacteria (Pseudomonas aeruginosa).

[0089] Method: standard plate counting method

[0090] Steps:

[0091] ​​Multidrug-resistant *Pseudomonas aeruginosa* was incubated overnight in LB broth (specifically, 1.00 g sodium chloride, 0.5 g peptone, and 1 g yeast extract per 100 mL of water), centrifuged at 5000 rpm for 5 min, and the supernatant was discarded. The bacterial cell resuspended in sterile PBS, and the absorbance (OD) at 600 nm was measured using UV spectroscopy. 600 (Optical density of bacterial suspension). Gradually dilute with PBS to adjust OD. 600 = 2.0. Mix equal volumes of the prepared bacterial solution and pre-prepared antibacterial agents of different concentrations (the concentration of the single-chain antibacterial agent is equal to that of the macrocyclic amphiphilic antibacterial agent). n times, of which n (Equal to the number of phenol units in calixarene). Shake the mixture well and incubate at 37 °C for 30 min. After incubation, dilute with sterile PBS. 4 Take 100 μL and spread it evenly on LB agar solid medium plates (solid medium is made by adding 2.0 g of agar powder to every 100 mL of liquid medium). Incubate overnight at 37 °C, then remove the plates and count the colonies. Sterilization rate = [(number of colonies on PBS plate – number of colonies on antimicrobial agent plate) / number of colonies on PBS plate] × 100%.

[0092] in conclusion:

[0093] Experimental results show that macrocyclic amphiphilic antibacterial agents exhibit a higher bactericidal rate compared to single-chain antibacterial agents. Figure 6 Only a concentration of 31.2 μM is needed to reduce the number of Pseudomonas aeruginosa colonies by two orders of magnitude.

[0094] Example 6: Determination of the ability of macrocyclic amphiphilic and single-chain antibacterial agents to disrupt bacterial membranes by fluorescence assay of 1-anilinenaphthalene (NPN).

[0095] Intact bacterial membranes prevent the insertion of hydrophobic NPN molecules. When the bacterial membrane is disrupted, NPN molecules can insert into it, producing fluorescence. Therefore, the stronger the NPN fluorescence intensity, the greater the degree of disruption to the bacterial membrane, and the stronger the antibacterial agent's ability to destroy the bacterial membrane. The specific procedure is as follows: Prepare a bacterial suspension (1 mL, 1 × 10⁻⁶) 6 CFU / mL) was mixed with an equal volume of different pre-adjusted concentrations of antibacterial agents and incubated at 37 °C for 3 h. After incubation, the mixture was centrifuged at 5000 rpm for 2 min, the supernatant was discarded, and the bacterial cells at the bottom were resuspended in sterile PBS. 10 μL of NPN ethanol solution (10 μM) was added, and the mixture was incubated at room temperature for 10 min. The fluorescence intensity of each group was measured (excitation wavelength 350 nm). The results showed that compared with single-chain antibacterial agents, macrocyclic amphiphilic antibacterial agents significantly improved the ability to disrupt bacterial membranes. Figure 7 ).

[0096] Example 7: Interference of macrocyclic amphiphilic antibacterial agent to quorum sensing of Pseudomonas aeruginosa

[0097] A control compound QAC4A-12C was prepared as in Example 3, which has the same cavity as QAC4A-8C, but due to the longer alkyl chain, its assembly density is much higher than QAC4A-8C. Therefore, QAC4A-12C has no bactericidal activity and incubation with Pseudomonas aeruginosa does not affect the normal growth of the bacteria Figure 8 ), making it a compound for verifying the recognition of calixarene cavity to quorum sensing signal molecules and interference with the function of bacterial quorum sensing.

[0098] Pseudomonas aeruginosa secretes pyocyanin, which is one of its exotoxins, and the secretion of pyocyanin is controlled by the quorum sensing pathway mediated by acyl homoserine lactone, a type of quorum sensing signal molecule. Moreover, pyocyanin itself has a characteristic absorption at 695 nm, which is convenient for detection. Therefore, the amount of pyocyanin secretion was used as a verification means to verify the interference of macrocyclic amphiphilic antibacterial agents with quorum sensing. The specific implementation is as follows: Pseudomonas aeruginosa wild type PA O1 ) was cultured overnight, 200 μL of LB liquid medium was added to each well of a 96-well plate. Then 2 μL of the incubated bacterial solution was added to each well, and 100 μL was removed. QAC4A-12C solution diluted to the required concentration with LB liquid medium was added to each well as required, and incubated at 37 °C for 24 h. Centrifugation was performed at 5000 rpm for 10 min, and the supernatant was transferred to a new 96-well plate. The absorbance at 695 nm was measured using a microplate reader. The results showed that the secretion of Pseudomonas aeruginosa pyocyanin could be reduced to some extent Figure 9 ).

[0099] In addition, the quorum sensing mechanism is also closely related to the formation of bacterial biofilm. The inhibition of biofilm formation by macrocyclic amphiphilic antibacterial agents was measured using crystal violet staining to illustrate the interference with bacterial quorum sensing. A higher concentration of antibacterial agent solution was diluted to the required concentration with LB liquid medium, and the bacterial solution with OD 600 = 0.2 and the antibacterial agent solution were mixed in equal volumes in a 96-well plate (100 μL of bacterial solution and 100 μL of antibacterial agent solution), and incubated at 37 °C for 48 h. After incubation, the bacterial solution was removed, and the biofilm was fixed with methanol for 30 min. Then crystal violet solution (1%) was added for staining for 10 min. After staining, the excess free dye was washed off by repeatedly washing with PBS, and 200 μL of 30% acetic acid solution was added to dissolve the crystal violet bound to the biofilm. The absorbance at 590 nm was measured using a microplate reader.

[0100] The results show that the macrocyclic amphiphilic antibacterial agent can inhibit the formation of P. aeruginosa biofilm without affecting the normal growth of bacteria. Figure 10 ).

[0101] The above merely describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are all within the protection scope of the present application.

Claims

1. A macrocyclic amphiphilic antibacterial agent based on a calixarene skeleton, 25,26,27,28-tetraoctyl-quaternary ammonium salted calixarene [4] (QAC4A-8C), characterized in that... It has the following structure: .

2. The application of the macrocyclic amphiphilic antibacterial agent based on the calixarane skeleton as described in claim 1 in the preparation of an antibacterial agent for killing drug-resistant Gram-negative bacteria; wherein the Gram-negative bacteria refers to Pseudomonas aeruginosa.

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