阅读说明：本技术 具有抗耐药性的抗菌脒类低聚物及其制作方法和用途 (Antibacterial amidine oligomer with anti-drug resistance and preparation method and application thereof ) 是由 冯欣欣 白玉罡 王建雪 陈先会 陈亚杰 于 2021-08-02 设计创作，主要内容包括：本发明公开了一种具有抗耐药性的抗菌脒类低聚物及其制作方法、用途、抗菌机理以及在胞内感染模型和偶发分枝杆菌感染的斑马鱼模型中杀菌的应用,脒类低聚物的抗菌种类包括大肠杆菌、粪肠球菌、金黄色葡萄球菌、肺炎克雷伯氏菌、鲍曼不动杆菌、铜绿假单胞菌、耻垢分枝杆菌、偶发分枝杆菌等。本发明公开的结构具有破坏细菌细胞膜和结合其染色体DNA的双重抗菌机制、具有快速杀菌和抗耐药性的特征,并且呈现出抗菌广谱性。此外,该结构对血细胞有较低的毒性,在血液中有较高的安全性。同时该结构能够应用于耻垢分枝杆菌和偶发分枝杆菌感染的Raw264.7细胞模型中,该结构对MRSA和偶发分枝杆菌感染的斑马鱼模型也有较好的治疗效果。(The invention discloses an antibacterial amidine oligomer with drug resistance, a preparation method, application, an antibacterial mechanism and application of the amidine oligomer in sterilization of an intracellular infection model and a zebra fish model infected by mycobacterium fortuitum, wherein the antibacterial species of the amidine oligomer comprise escherichia coli, enterococcus faecalis, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa, mycobacterium smegmatis, mycobacterium fortuitum and the like. The structure disclosed by the invention has a dual antibacterial mechanism of destroying bacterial cell membranes and combining with the chromosomal DNA of the bacterial cells, has the characteristics of quick sterilization and drug resistance, and shows broad antibacterial spectrum. In addition, the structure has low toxicity to blood cells and high safety in blood. Meanwhile, the structure can be applied to a Raw264.7 cell model infected by mycobacterium smegmatis and mycobacterium fortuitum, and has a good treatment effect on MRSA and mycobacterium fortuitum infected zebra fish models.)

1. An antibacterial amidine oligomer having resistance to drugs, wherein the amidine oligomer has the following formula:

wherein n is more than or equal to 5 and less than or equal to 8, and the molecular formula of R1 is one or more of formula (I), formula (II), formula (III), formula (IV), formula (V), formula (VI), formula (VII), formula (VIII) and formula (IX):

wherein, the molecular formula of R2 is one or more of formula (a), formula (b), formula (c), formula (d), formula (e), formula (f), formula (g), formula (h) and formula (i):

2. the oligomer of claim 1 wherein R1 is any one of formula (I), formula (II), formula (III), or formula (IV) and R2 is formula (a).

3. The oligomer of claim 2 wherein R1 is of formula (II) and R2 is of formula (a).

4. The oligomer of claim 1, wherein R1 is any one of formula (II) and formula (III), and R2 is formula (b).

5. A preparation method for synthesizing antibacterial amidine oligomer with drug resistance is characterized by comprising the following steps:

s1 preparing a compound monomer having the following formula:

s2: reacting said compound monomer with H₂N-R₂-NH₂Reacting anhydrous N, N-Dimethylformamide (DMF) and N, N-Diisopropylethylamine (DIPEA) to obtain the amidine oligomer;

wherein the molecular formula of R1 includes one or more of formula (I), formula (II), formula (III), formula (IV), formula (V), formula (VI), formula (VII), formula (VIII) and formula (IX):

wherein the molecular formula of R2 includes one or more of formula (a), formula (b), formula (c), formula (d), formula (e), formula (f), formula (g), formula (h) and formula (i):

6. the manufacturing method according to claim 5, wherein the step S1 includes the following steps:

s11: weighing 5-cyanoindole or 6-cyanoindole and di-tert-butyl dicarbonate, adding dichloromethane, uniformly mixing, adding a catalytic amount of 4-Dimethylaminopyridine (DMAP), stirring at 2-6 ℃ for half an hour, then recovering to the room temperature of 20-25 ℃, stirring overnight, filtering and washing the mixture, then carrying out spin drying by using a rotary evaporator to obtain a solid product, and then purifying the product by using a silica gel column to obtain a white solid;

s12: weighing the compound obtained in S11, adding tetrahydrofuran, and mixing well in N₂Add 2M LDA solution under protection. Stirring at-20 deg.C for a while, adding trimethyl tin chloride solid, stirring at room temperature overnight, spin-drying the mixture on a rotary evaporator, washing with saturated saline solution, drying, and purifying by column chromatography to obtain white solid product;

s13: weighing the organotin compound and the 4-bromobenzonitrile compound obtained in the step S12, adding anhydrous dioxane, uniformly mixing, adding a tetratriphenylphosphine palladium catalyst under the protection of nitrogen, stirring for 12 hours at the temperature of 105-.

7. The manufacturing method according to claim 5, wherein the step S2 includes the following steps:

s21: reacting said compound monomer with H₂N-R₂-NH₂Mixing with anhydrous N, N-Dimethylformamide (DMF),adding N, N-Diisopropylethylamine (DIPEA), and stirring at 45-55 deg.C for 96-144h under the protection of inert gas to obtain a mixture;

s22: adjusting the pH of the mixture obtained in S21 to 1-2, then intercepting to obtain a substance with molecular weight of more than 1KDa, and freeze-drying to obtain white solid, namely the amidine oligomer.

8. Use of the oligomer of claim 1 wherein the amidine oligomer is used as an antibacterial agent.

9. The use of the anti-drug-resistant antibacterial amidine oligomer according to claim 8, wherein the amidine oligomer is used for antibacterial purposes, and the antibacterial species of the amidine oligomer include Escherichia coli, enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Mycobacterium smegmatis, Mycobacterium fortuitum and the like.

Technical Field

The invention relates to the field of pharmaceutical and chemical materials, in particular to an antibacterial amidine oligomer with drug resistance and a preparation method and application thereof.

Background

The abuse of antibiotics in the antibacterial field has accelerated the emergence of drug-resistant strains, multi-drug resistant strains, and bacterial drug resistance has become a widespread and serious health hazard to humans. Therefore, the development of novel antibacterial agents which can rapidly kill drug-resistant bacteria without causing the development of bacterial drug resistance has been urgently needed. Recently, the concept of an anti-drug resistant antibacterial agent has been established, aiming to solve the problem of drug resistance of bacteria by designing a cationic amidine type antibacterial oligomer having a dual antibacterial mechanism.

Amidine anti-drug resistant antibacterials with a dual antibacterial mechanism should have the following characteristics: 1) has good killing effect on various pathogenic bacteria including multidrug resistant pathogens and has better antibacterial broad spectrum. 2) Can effectively kill drug-resistant bacteria in the using process, and the rate of drug resistance of the bacteria to the drug-resistant bacteria is low. 3) Has low toxicity to mammalian cells and can be used in the treatment of in vivo models of bacterial infections. It is well known that the root cause of antibiotic resistance is mutation of its target, rendering drugs of various mechanisms of action ineffective. Thus, if a drug can be targeted to multiple targets, or its target involves a complex biological process, the probability of developing drug resistance will be greatly reduced. Antibacterial polymers this type of compound conforms to the concept of resistance to drugs, but traditional high molecular weight antibacterial polymers have the disadvantage of being highly toxic. The polymerization is carried out in a polycondensation mode, the molecular weight of the polymer can be effectively controlled, the antibacterial agent with lower molecular weight can be obtained, and the toxicity to mammalian cells can be effectively reduced. Meanwhile, a module capable of combining bacterial DNA is introduced into a molecular main chain, so that a second antibacterial mechanism is obtained, namely, the aim of resisting bacteria is fulfilled by combining the chromosomal DNA of the bacteria and blocking important life processes such as replication and translation of the chromosomal DNA.

Disclosure of Invention

In order to solve the problem of high toxicity of the traditional high molecular weight antibacterial polymer, the invention discloses an antibacterial amidine oligomer with drug resistance and a preparation method and application thereof.

An antibacterial amidine oligomer with drug resistance, application and a preparation method thereof, wherein the molecular formula of the amidine oligomer is as follows:

wherein n is more than or equal to 5 and less than or equal to 8, and the molecular formula of R1 is one or more of formula (I), formula (II), formula (III), formula (IV), formula (V), formula (VI), formula (VII), formula (VIII) and formula (IX):

wherein, the molecular formula of R2 is one or more of formula (a), formula (b), formula (c), formula (d), formula (e), formula (f), formula (g), formula (h) and formula (i):

the structure disclosed by the invention has a dual antibacterial mechanism of destroying a bacterial cell membrane and combining with a chromosome DNA of the bacterial cell membrane, has the characteristics of quick sterilization and drug resistance, and shows broad-spectrum antibacterial activity. Meanwhile, the structure has low toxicity to blood cells and higher therapeutic index. In addition, the structure can effectively kill bacteria infected in mammalian cells and pathogenic bacteria infected in zebra fish bodies.

Drawings

FIGS. 1A and 1B are schematic reaction diagrams according to the present invention;

FIG. 2 is a flow experiment, which proves that P2 has strong membrane rupture ability;

FIG. 3 is an SEM image showing that the surface of the P2-treated M.smegmatis cell membrane was shrunk to various degrees;

FIG. 4 is a graph showing the results of a dynamic light scattering experiment in which P2 has the ability to bind to bacterial genomic DNA;

FIG. 5 is a graph showing the results of Confocal that P2 can bind to the DNA of bacteria and fluoresce;

FIG. 6 is a graph showing sensitization of P2 to rifampin, a common antibiotic used in clinical treatment of M.tuberculosis infection;

FIG. 7 is a graph comparing the rate of resistance development after treatment of M.smegmatis for 210 passages with the sub-minimum inhibitory concentrations of rifampicin and P2, respectively;

FIG. 8 is a Confocal plot of Raw264.7 cells infected with M.smegmatis;

FIG. 9 is a Confocal plot of the bactericidal effect of P2 in a Raw264.7 cell model of M.smegmatis infection;

FIGS. 10a and 10b are graphs showing the intracellular bactericidal effect of P2 in Raw264.7 cell model of M.smegmatis and M.fortuitum infection, respectively;

FIG. 11 is a graph of the results of P2 experiments to improve survival of MRSA-infected zebrafish;

FIG. 12 is a graph showing the results of P2 experiments to improve survival of Mycobacteria fortuitum-infected zebrafish;

FIGS. 13-1 to 13-36 are nuclear magnetic spectra of the compounds of examples 1 to 9.

Detailed Description

The technical solution of the present invention is described in detail below by means of specific embodiments and with reference to the attached drawings, and the components or devices in the following embodiments are all general standard components or components known to those skilled in the art, and the structure and principle thereof can be known to those skilled in the art through technical manuals or through routine experiments.

Description of the drawings: in the following examples of the invention, R1 represents a compound of any one of the formulae (I), (II), (III), (IV), (V), (VI), (VII), (VIII) or (IX):

r2 represents a compound having a molecular formula of any one of the formulae (a), (b), (c), (d), (e), (f), (g), (h) and (i):

example one

The test shows that the antibacterial amidine oligomer with broad-spectrum antibacterial property has the chemical formula as follows:

wherein n is 5. ltoreq. n.ltoreq.8, and R1 and R2 have the structures described above. As can be seen from the above structural formula, in which 81 compounds can be obtained by different combinations of R1 and R2, for the convenience of analysis and description of these compounds, 60 compounds are labeled in the following table 1, and it should be noted that, for those skilled in the art, compounds that are not labeled can also be synthesized, and the properties and synthesis manner of these compounds that are not labeled are similar to those of the labeled 60 compounds, so that they are not further analyzed and described in the present invention.

The structural formulas and antibacterial activities and biocompatibility of 60 compounds are listed in tables 1 and 2, and it can be seen from table 2 that the therapeutic indexes (IC50/MIC) of P1, P2, P3, P4, P6 and P7 are all greater than 200, and the better therapeutic effect is achieved, especially the therapeutic index of P2 is 354.2, which is the best therapeutic effect.

TABLE 1 structural formula List of P1-60

TABLE 2P 1-P60 list of antibacterial activity and biocompatibility

^aGram positive bacteria.^bGram negtive bacteria.^cSelectivity index＝IC₅₀/MIC(M.s.).IC₅₀:P1-P20,cell line is HEK293T cell；P21-P60,cell line is NIH 3T3 cell.S.a,Staphylococcus aureus；K.p,Klebsiella pneumoniae,E.c,Escherichia coli,A.b,Acinetobacter baumannii,E.fa,Enterococcus faecalis.

Example two

As shown in fig. 1, the invention also discloses a preparation method for synthesizing the antibacterial amidine oligomer with the anti-drug resistance, which comprises the following steps:

s1 preparing a compound monomer having the following formula:

s2: and (3) reacting the compound monomer with H2N-R2-NH2, anhydrous N, N-Dimethylformamide (DMF) and N, N-Diisopropylethylamine (DIPEA) to obtain the amidine oligomer, wherein R1 and R2 have the structures in the description.

Wherein, the step S1 includes the following steps:

s11: weighing 5-cyanoindole or 6-cyanoindole and di-tert-butyl dicarbonate, adding dichloromethane, uniformly mixing, adding a catalytic amount of 4-Dimethylaminopyridine (DMAP), stirring at 2-6 ℃ for half an hour, then recovering to the room temperature of 20-25 ℃, stirring overnight, filtering and washing the mixture, then carrying out spin drying by using a rotary evaporator to obtain a solid product, and then purifying the product by using a silica gel column to obtain a white solid;

s12: conversion obtained by weighing S11Adding tetrahydrofuran, mixing, and adding N₂Add 2M LDA solution under protection. Stirring at-20 deg.C for a period of time, adding trimethyl tin chloride solid, stirring at room temperature overnight, spin-drying the mixture on a rotary evaporator, washing with saturated saline solution, drying, and purifying the obtained crude product by column chromatography to obtain white solid product;

s13: weighing the organotin compound and the 4-bromobenzonitrile compound obtained in the step S12, adding anhydrous dioxane, uniformly mixing, adding a tetratriphenylphosphine palladium catalyst under the protection of nitrogen, stirring at 105-115 ℃ for 12 hours to obtain a mixture, washing and extracting the mixture, then spin-drying, and purifying by using a column chromatography method to finally obtain the white or yellow compound monomer.

The step S2 includes the following steps:

s21: reacting said compound monomer with H₂N-R₂-NH₂Mixing with anhydrous N, N-Dimethylformamide (DMF), adding N, N-Diisopropylethylamine (DIPEA), and stirring at 45-55 deg.C for 96-144h under the protection of inert gas to obtain a mixture;

s22: adjusting the pH of the mixture obtained in S21 to 1-2, then intercepting to obtain a substance with molecular weight of more than 1KDa, and freeze-drying to obtain white solid, namely the amidine oligomer.

Experimental example 1

This experimental example is detailed with the compound P2:

materials and methods: all reagents were supplied by TCI (usa), Sigma-Aldrich, mclin, hadamard et al organic reagents and pecan biotechnology, all used without further purification (among others). Ultrapure water for the experiments was obtained from a Milli-Q purification instrument. The inert gas is nitrogen.

The synthetic synthesis procedure for P2 is shown in fig. 1. Firstly, synthesizing 4', 6-diimino ester-2-phenyl indole hydrochloride monomer. 6-cyanoindole (10g,70.3mmol) and di-tert-butyl dicarbonate (BOC) (35.36g,162mmol) are weighed, then 100mL of dichloromethane is added and mixed uniformly, then a catalytic amount of 4-Dimethylaminopyridine (DMAP) (0.86g,7.03mmol) is added, stirring is carried out at room temperature for 12-24h to obtain a mixture, the mixture is extracted and dried to obtain a solid mixture, and the solid mixture is subjected to column chromatography to obtain 13.6g of a white solid product with the yield of 80%. The BOC protected 6-cyanoindole compound (0.3g,1.238mmol) obtained in the previous step and trimethyltin chloride (0.32g, 1.606mmol) were weighed out and mixed well by adding 20mL of dry tetrahydrofuran solution. The temperature of the reaction mixture was maintained at-20 ℃ and then 2M LDA was added under nitrogen protection. After stirring at-20 ℃ for 30min, the reaction mixture was allowed to warm to room temperature for further 6h, and then 30mL of water was added to quench the reaction. After removal of the tetrahydrofuran solvent on a rotary evaporator, extraction was performed with ethyl acetate, and the product obtained after spin-drying was purified by column chromatography to give a white solid product (0.6g, 50%). The reaction product (3g, 7.406mmol) and 4-bromobenzonitrile (1.754g, 9.628mmol) from the previous step were weighed into 20mL of dry dioxane, and the reaction was heated to 110 ℃ under nitrogen protection and stirred under reflux for 12-24 h. The mixture was spin-dried on a rotary evaporator and the product mixture was purified by column chromatography to give 1.5g of the final product in 50% yield. In a 20mL vial, dry absolute ethanol was added and acetyl chloride was slowly added dropwise. Stirring at room temperature for 30 min. 100mg of the reaction product obtained in the previous step was weighed and dissolved in absolute ethanol, and then added dropwise to an ethanol solution of acetyl chloride. The bottle cap was closed, stirring was kept vigorously at room temperature for 4 days, and the product was washed by precipitation with anhydrous ether. The product was then spin dried on a rotary evaporator. 100mg of a yellow solid powder was obtained in 50% yield.

4', 6-diiminoate-2-phenylindole hydrochloride monomer (46mg, 0.14mmol) and 1, 6-hexanediamine (13.132mg, 0.14mmol) were weighed into a 7mL glass vial containing 1.5mL dry DMF. Then 75. mu.L of DIPEA was added and the bottle was closed. Then stirred for 4 days with heating at 50 ℃. After the reaction was complete, 2mL of 3M HCl was added to the mixture. The mixture was dialyzed with a dialysis bag having a molecular weight cut-off of 1kDa for 24-48h, and then lyophilized to give 12mg of a yellow solid.

P2 has broad-spectrum antibacterial activity compound P2 was tested for its antibacterial activity against a variety of bacteria (table 2). P2 showed good antibacterial activity, and the minimum inhibitory concentration was in the lower range (0.0625-8. mu.g/mL) for all tests. P2 has good antibacterial activity and selectivity to Mycobacterium smegmatis, and the minimum inhibitory concentration is below single digit and two orders of magnitude lower than that of other strains. The antibacterial activity data show that P2 not only has good antibacterial broad-spectrum property, but also has good potential antibacterial activity on mycobacterium pathogenic microorganisms represented by Mycobacterium smegmatis.

P2 has lower hemolytic toxicity Table 2 shows that the series of antibacterial oligomers have lower hemolytic toxicity, wherein the half hemolytic concentration of P2 is 1062.9 μ g/mL, and the lower hemolytic toxicity is shown. The P2 can effectively kill various pathogenic bacteria and drug-resistant pathogenic bacteria, simultaneously keeps lower hemolytic toxicity, has little damage to blood cells, and indicates that the biocompatibility of the P2 is higher.

Verification of antibacterial mechanism of targeted bacterial cell membranes the membrane rupture ability of P2 was evaluated by flow experiments. In flow experiments, Propidium Iodide (PI) is used as a fluorescent dye to assess the integrity of bacterial cell membranes. After centrifugation of overnight cultured M.smegmatis, the medium was discarded, washed three times with PBS, and then resuspended to OD with 50. mu.M PI in PBS_600nmAfter addition of P2 at the indicated concentration for 4h at 37 ℃, samples were analyzed using a BD Accuri C6 Plus flow cytometer, 0.1. As shown in FIG. 2, M.smegmatis treated with P2 had more PI accumulated and thus emitted more fluorescence. Untreated Mycobacterium smegmatis showed very low fluorescence.

Scanning electron microscopy was further used to demonstrate the membrane rupture ability of P2. As shown in FIG. 3, the bacteria treated with P2 all showed significant membrane disruption and shrinkage relative to the untreated Mycobacterium smegmatis control group.

Validation of the coupled bacterial DNA antibacterial mechanism the design of the oligomer was based mainly on a dual antibacterial mechanism of membrane targeting and DNA targeting. In addition to the demonstration of the ability of P2 to disrupt bacterial cell membranes, the oligomer was also demonstrated to have the ability to bind bacterial DNA. Dynamic Light Scattering (DLS) studies indicate that P2 can promote the aggregation of mycobacterium smegmatis genomic DNA. As shown in FIG. 4, the particle size of oligomer-DNA complex formed by binding P2 to DNA is much larger than that of P2 and that of DNA, demonstrating that P2 and DNA have stronger affinity to bind to bacterial DNA.

After determining that P2 has the capability of binding to DNA in vitro, a P2 compound is labeled with rhodamine, and is incubated with bacteria, and the binding of the compound to the bacterial DNA is observed. FIG. 5 shows the staining of M.smegmatis by P2, and the "P2-DNA complex" channel and rhodamine channel, which fluoresce after P2 binds to bacterial DNA, can well coincide, indicating that P2 can bind to bacterial DNA, further demonstrating the mechanism by which P2 can bind to bacterial DNA.

Sensitization of P2 to clinically common antibiotics of mycobacterium tuberculosis infection as described above, P2 not only targets the bacterial cell membrane but also causes rupture of the bacterial cell membrane. The combination of P2 and small-molecule antibiotics can promote the antibiotics to enter the bacteria, increase the accumulation of the antibiotics in the bacteria, improve the antibacterial activity of the bacteria and reduce the minimum inhibitory concentration of the bacteria. To verify the sensitizing effect of P2 on small molecule antibiotics, the antibiotics Ethambutol (EMB), Streptomycin (STR), Kanamycin (KAN), Isoniazid (INH), Rifampicin (RIF) were sensitized with P2 at the next lowest inhibitory concentration. As shown in fig. 6, the addition of P2 significantly reduced the minimum inhibitory concentration of rifampicin (by 8-fold).

Measurement of the rate of resistance to drug development of P2 introduces a dual antibacterial mechanism that reduces the rate at which bacteria develop resistance to compounds. The target of the cell membrane-targeted antibacterial polymer is a cell membrane, and due to the very complicated biosynthesis of the cell membrane, drug resistance can be generated only when a plurality of random mutations occur simultaneously and cause changes in the membrane structure. Therefore, the cell membrane targeting type polymer is considered as a low-drug-resistance antibacterial compound. As a cell membrane targeting polymer, P2 also has the characteristic of low drug resistance. In addition, P2 uses bacterial DNA as a secondary target, and therefore bacteria require both membrane and DNA structural mutations to develop resistance to P2. The rate of drug resistance generation of P2 was experimentally determined, as shown in fig. 7, after mycobacterium smegmatis was repeatedly treated with rifampicin, a standard antibiotic with the second lowest inhibitory concentration, the strain generated drug resistance mutation, which is shown as the lowest inhibitory concentration of rifampicin rising continuously, and the minimum inhibitory concentration of bacteria increased 32 times after 210 generations of replication. In contrast, P2 developed little resistance under the same conditions. The above results indicate that bacterial resistance is difficult to develop relative to the traditional antibiotic P2. Since P2 has the problem that it is not easy to cause resistance to bacterial cell membrane, the combination of P2 and rifampicin can effectively reduce the generation of rifampicin resistance, and fig. 7 shows that even after mycobacterium smegmatis 210 generations is treated with this combination, it hardly causes resistance to mycobacterium smegmatis.

Experimental example 2

5-cyanoindole or 6-cyanoindole (10g,70.3mmol) and di-tert-butyl dicarbonate (35.36g,162mmol) were weighed into 400mL of anhydrous dichloromethane. Thereafter, 4-dimethylaminopyridine (0.86g,7.03mmol) was added. And (3) putting the mixture into an ice water bath, stirring for 2 hours, and quenching the reaction by using ice water after the reaction is finished. The organic phase was extracted with dichloromethane, and the organic phases after extraction were combined and then spin-dried on a rotary evaporator. The crude product obtained is purified by column chromatography. The product was obtained as a white solid in 37% and 47% yields of BOC-protected 5-cyanoindole and BOC-protected 6-cyanoindole, respectively.

N-BOC-5-cyanoindoles¹H NMR(400MHz,CDCl₃):δ8.27(d,J＝8.6Hz,1H),7.92(s,1H),7.73(d,J＝2.9Hz,1H),7.57(d,J＝8.6Hz,1H),6.64(d,J＝2.9Hz,1H),1.71(s,9H).¹³C NMR(101MHz,CDCl₃):δ149.0,137.1,130.5,128.1,127.3,125.8,119.8,116.0,106.9,106.1,84.9,28.1.。

N-BOC-6-cyanoindoles¹H NMR(400MHz,CDCl₃):δ8.53(s,1H),7.79(d,J＝3.6Hz,1H),7.66(d,J＝8.1Hz,1H),7.51(d,J＝8.1,1H),6.66(d,J＝3.6Hz,1H),1.71(s,9H).¹³C NMR(101MHz,CDCl₃):δ149.0,134.2,133.8,129.3,125.8,121.7,120.0,119.8,107.2,107.1,85.0,28.1.。

Experimental example 3

A BOC-protected cyanoindole derivative (0.17g, 1.24mmol) and 0.32g of trimethyltin chloride were weighed into a two-necked flask containing 5mL of a dry tetrahydrofuran solution, and one neck of the flask was connected to a vacuum line. The mixed system is put at-20 ℃ for cooling. Ventilating the solution to fill the reaction bottle with N₂The protective atmosphere of (1). Under cooling, 0.81mL of lithium diisopropylamide (2M LDA in N-heptane-tetrahydrofuran-diethyl ether) was carefully aspirated through a syringe under a stream of N₂Slowly charged into the reaction flask under the protection of (1). After maintaining the reaction at low temperature for a period of time, the flask was moved to 20-25 ℃ to continue the reaction overnight. The reaction was terminated by adding 30mL of water to the solution, and then the tetrahydrofuran in the solution was evaporated off by rotary evaporation. The crude product was dissolved with ethyl acetate, and then the organic phase was washed with saturated brine. The organic phase was collected and dried over anhydrous magnesium sulfate. After the ethyl acetate was evaporated off, a solid mixture was obtained and purified by column chromatography using PE as eluent, DCM ═ 1: 1.

N-BOC-1-trimethylstannyl-5-cyanoindole:¹H NMR(400MHz,CDCl₃):δ8.03(d,J＝8.6Hz,1H),7.86(s,1H),7.50(d,J＝8.6Hz,1H),6.77(s,1H,coupling with 117Sn/119Sn could be observed),1.74(s,9H),0.36(s,9H,coupling with ¹¹⁷Sn/¹¹⁹Sn could be observed).¹³C NMR(101MHz,CDCl₃):δ151.5,146.6,139.2,132.2,126.5,124.8,120.0,117.6,115.9,105.5,85.3,28.1,-7.2。

N-BOC-1-trimethylstannyl-6-cyanoindole:¹H NMR(400MHz,CDCl₃):δ8.27(s,1H),7.59(d,J＝8.1Hz,1H),7.45(d,J＝8.1Hz,1H),6.80(s,1H,coupling with ¹¹⁷Sn/¹¹⁹Sn could be observed),1.73(s,9H),0.34(s,9H,coupling with ¹¹⁷Sn/¹¹⁹Sn could be observed).¹³C NMR(101MHz,CDCl₃):δ151.4,148.6,136.4,135.3,125.5,120.8,120.5,119.6,117.8,106.0,85.5,28.1,-7.2。

experimental example 4

The synthesis steps of the compound 3 series compounds are similar, and the synthesis of one of the compounds is exemplified below. N-BOC-1-trimethylstannyl-6-cyanoindole (3.0g, 7.4mmol) and 4-bromobenzonitrile (9.63mmol) were weighed into a two-necked flask containing 20mL of dried dioxane, after which the reaction flask was connected to a vacuum line, the dioxane solvent was degassed under vacuum and N was used₂Replacement is carried out to fill the reaction system with N₂. Tetrakis (triphenylphosphine) palladium catalyst (0.86g, 0.74mmol) was weighed out and added quickly to a solution containing 1mL of anhydrous dioxane. After that, the above solution was added dropwise to the reaction system with a syringe under a nitrogen blanket. The above reaction solution was heated to 110 ℃ in an oil bath and kept at this temperature for 24 hours. After the reaction was completed, the dioxane solvent was removed by rotary evaporation. The solid obtained after spin-drying was then dissolved in 200mL of ethyl acetate and the palladium complex was destroyed by adding 5mL of concentrated aqueous ammonia. And the organic phase was washed with deionized water, after which the organic phases were combined and spin dried. And separating and purifying the crude product by column chromatography. The eluent used was n-hexane: ethyl acetate 4: 1. The product was obtained as a white or yellow solid powder.

R1＝H：¹H NMR(400MHz,DMSO-d6):δ12.35(s,1H),8.15(s,1H),8.10(d,J＝8.3Hz,2H),7.97(d,J＝8.3Hz,2H),7.60(d,J＝8.5Hz,1H),7.51(d,J＝8.5Hz,1H),7.30(s,1H).¹³C NMR(101MHz,DMSO-d6):δ139.7,138.7,136.0,133.5,128.5,126.8,126.4,125.7,120.9,119.3,113.3,110.6,102.5,102.4。

R1＝H：¹H NMR(400MHz,DMSO-d6):δ12.37(s,1H),8.12(d,J＝8.0Hz,2H),7.99(d,J＝8.0Hz,2H),7.90(s,1H),7.77(d,J＝8.2Hz,1H),7.38(d,J＝8.2Hz,1H),7.31(s,1H).¹³C NMR(101MHz,DMSO-d6)δ140.1,136.8,135.8,133.5,131.9,126.6,122.9,122.2,120.9,119.2,116.8,110.9,104.2,102.5。

R1＝CH₃：¹H NMR(400MHz,DMSO-d6):δ12.08(s,1H),7.89(s,2H),7.86-7.74(m,3H),7.39(d,J＝8.2Hz,1H),6.94(s,1H),2.54(s,3H).¹³C NMR(101MHz,DMSO-d6):δ140.0,137.8,136.5,135.9,135.0,131.7,130.4,130.3,122.6,122.0,120.9,119.2,116.7,111.1,104.7,103.8,21.1。

R1＝OCH₃：¹H NMR(400MHz,DMSO-d6):δ11.92(s,1H),8.03(d,J＝8.0Hz,1H),7.91(s,1H),7.76(d,J＝8.2Hz,1H),7.69(s,1H),7.58(d,J＝8.0Hz,1H),7.36(d,J＝8.2Hz,1H),7.27(s,1H),4.04(s,3H).¹³C NMR(101MHz,DMSO-d6):δ156.6,137.3,136.0,131.4,129.3,125.3,124.7,122.5,122.0,121.0,119.1,116.8,116.2,111.8,104.8,103.8,56.9。

Experimental example 5

The synthesis of compound 4 is the same, as follows: 6mL of the dried acetyl chloride solution was slowly added dropwise to a bottle containing 10mL of anhydrous ethanol. The bottle cap was tightened and kept vigorously stirred at room temperature for 30 min. 100mg of the product obtained in the previous step was weighed and dissolved in absolute ethanol by ultrasound. And then quickly added dropwise into the reaction system. The bottle cap is quickly screwed down, and the bottle mouth is sealed by a sealing film. Keeping the reaction system to stir at normal temperature for 4 days, monitoring by a TLC method, and adding anhydrous ether into the reaction system after the reaction is finished. The reaction mixture was then transferred to a 50mL centrifuge tube for centrifugation and the supernatant discarded. After the reaction product was washed three times, it was dried overnight in a vacuum oven. Yellow solid powder is obtained, and the yield is about 60 percent.

4a：¹H NMR(400MHz,DMSO-d6):δ12.95(s,1H),8.55(s,1H),8.25(s,4H),7.93(d,J＝8.5,1H),7.68(d,J＝8.5Hz,1H),7.44(s,1H),4.67(m,4H),1.52(m,6H)。¹³C NMR(101MHz,DMSO-d6):δ171.5,170.2,141.4,138.6,137.4,130.0,128.0,125.7,124.8,124.0,122.8,117.2,112.4,103.3,69.7,69.3,13.7,13.6。

4b：¹H NMR(400MHz,DMSO-d6):δ12.97(s,1H),8.25-8.33(m,5H),7.81(s,2H),7.38(s,1H),4.67(m,4H),1.52(m,6H)。¹³C NMR(101MHz,DMSO-d6):δ171.9,170.6,141.4,137.6,137.1,133.8,130.4,126.5,125.7,121.5,120.1,119.2,114.5,102.8,70.1,69.7,14.1,14.0。

4c：¹H NMR(400MHz,DMSO-d6):δ12.60(s,1H),8.33(s,1H),8.21(s,1H),8.12(d,J=8.1Hz,1H),7.91(d,J=8.1Hz,1H),7.82(m,2H),6.99(s,1H),4.68(m,4H),2.62(s,3H),1.52(d,6H)。¹³C NMR(101MHz,DMSO-d6):δ171.9,170.7,141.4,138.0,137.3,136.2,133.6,132.1,130.2,127.2,125.8,121.3,119.9,118.7,114.4,105.0,70.2,69.8,21.6,14.1。

4d：¹H NMR(400MHz,DMSO-d6):δ12.38(s,1H),8.34(s,1H),8.22(d,J＝8.3Hz,1H),8.10(s,1H),7.77-7.87(m,3H),7.35(s,1H),4.68(m,4H),4.12(s,3H),1.52(m,6H)。¹³C NMR(101MHz,DMSO-d6):δ171.9,170.3,156.7,138.6,136.3,133.3,129.1,126.7,126.0,122.2,121.3,119.8,118.8,114.7,113.3,105.2,70.2,69.7,57.1,14.1。

Experimental example 6

Taking the synthesis example of P2, the method comprises the following steps: 4', 6-diimidate-2-phenylindole hydrochloride monomer (46mg,0.113mmol) and 1, 6-hexanediamine (13.132mg, 0.113mmol) were weighed into a 7mL glass vial containing 1.5mL dry DMF. Then 75. mu.L of DIPEA was added and the bottle was closed. Followed by stirring for 4 days with heating at 50 ℃. After the reaction was complete, 2mL of 3M HCl was added to the mixture. The mixture was dialyzed with a dialysis bag having a molecular weight cut-off of 1kDa for 24-48h, and then lyophilized to give 12mg of a yellow solid.

Experimental example 7

Taking the synthesis example of P16, the method comprises the following steps: 4', 6-diiminoate-2- (2-methoxy) -phenylindole hydrochloride monomer (46mg, 0.105mmol) and N, N-bis (3-aminopropyl) methylamine (15.25mg, 0.105mmol) were weighed into a 7mL glass vial containing 1.5mL dry DMF. Then 75. mu.L of DIPEA was added and the bottle was closed. Then stirred for 4 days with heating at 50 ℃. After the reaction was complete, 2mL of 3M HCl was added to the mixture. The mixture was dialyzed with a dialysis bag having a molecular weight cut-off of 1kDa for 24-48h and then lyophilized to give 25mg of a yellow solid.

Experimental example 8

Taking the synthesis example of P17, the method comprises the following steps: 4', 5-diiminoate-2-phenylindole hydrochloride monomer (46mg,0.113mmol) and 2,2' -oxydianeamine (11.77mg,0.113mmol) were weighed into a 7mL glass vial containing 1.5mL dry DMF. Then 75. mu.L of DIPEA was added and the bottle was closed. Then stirred for 4 days with heating at 50 ℃. After the reaction was complete, 2mL of 3M HCl was added to the mixture. The mixture was dialyzed with a dialysis bag having a molecular weight cut-off of 1kDa for 24-48h and then lyophilized to give 25mg of a yellow solid.

Experimental example 9

Synthesis of mono, monomers

Compound 1(10.00g, 1.9eq), Compound 2(10.25g, 1.0eq) and potassium carbonate (12.21g, 2.0eq) were transferred to a 250mL round bottom flask, with acetonitrile as the solvent, at 110 ℃ under reflux overnight. The mixture was then cooled, spun dry and a small amount of ethyl acetate was added to make a suspension. To the obtained suspension was added a 1M NaOH solution, and the mixture was stirred at room temperature for 10 minutes. The suspension was then filtered and washed repeatedly with water and ethyl acetate to remove impurities. The resulting solid was dried at 60 ℃ to give compound 1-2(9.50g, 69.75%).

1-2：¹H NMR(400MHz,CDCl₃)δ7.57(d,J＝8.2Hz,4H),6.95(d,J＝8.3Hz,4H),4.20–4.18(t,4H),3.95–3.93(t,4H).

Compound 1(10.00g, 1.9eq), compound 5(10.16g, 1.0eq) and potassium carbonate (12.21g, 2.0eq) were transferred to a 250mL round bottom flask, with acetonitrile as the solvent, at 110 ℃ under reflux overnight. The mixture was then cooled, spun dry and a small amount of ethyl acetate was added to make a suspension. To the obtained suspension was added a 1M NaOH solution, and the mixture was stirred at room temperature for 10 minutes. The suspension was then filtered and washed repeatedly with water and ethyl acetate to remove impurities. The resulting solid was dried at 60 ℃ to give compounds 1-5(9.60g, 70.90%).

1-5：¹H NMR(400MHz，CDCl₃)δ7.57(d，J＝8.4Hz，4H)，6.93(d，J＝8.4Hz，4H)，4.03(t，J＝6.2Hz，4H)，1.88(m，J＝14.1，6.8Hz，4H)，1.67(m，J＝7.1Hz，2H)。

Compound 2 (10.00g, 1.9eq), compound 8 (11.66 g, 1.0eq) and potassium carbonate (12.21g, 2.0eq) were transferred to a 250mL round bottom flask, with acetonitrile as the solvent, at 110 ℃ under reflux overnight. The mixture was then cooled, spun dry and a small amount of ethyl acetate was added to make a suspension. To the obtained suspension was added a 1M NaOH solution, and the mixture was stirred at room temperature for 10 minutes. The suspension was then filtered and washed repeatedly with water and ethyl acetate to remove impurities. The resulting solid was dried at 60 ℃ to give compounds 2-8(10.24g, 68.09%).

2-8:¹H NMR(400MHz，CDCl₃)δ7.46(s，4H)，7.38(q，J＝8.0Hz，2H)，7.27(s，2H)，7.21(d，J＝7.2Hz，4H)，5.10(s，4H)。

Compound 1-2(5.00g) was weighed into a 500mL two-necked flask, and a mixture of 120mL of anhydrous ethanol and 50mL of anhydrous chloroform was added thereto, and hydrogen chloride gas dried over anhydrous calcium chloride was continuously introduced into the flask to react at room temperature for 4-5 days until the reaction was completed (monitored by TLC). After the reaction was completed, the product was washed with anhydrous ether, and the obtained product was dried in vacuo to obtain 1-2-S (5.23g, 80.59%) as a white solid.

1-2-S：¹H NMR(400MHz，DMSO)δ8.14(d，J＝7.6Hz，4H)，7.18(d，J＝7.5Hz，4H)，4.60(t，J＝5.9Hz，4H)，4.27(t，4H)，3.86(t，4H)，1.47(q，6H)。

Compound 1-5(5.00g) was weighed into a 500mL two-necked flask, and a mixture of 120mL of anhydrous ethanol and 50mL of anhydrous chloroform was added thereto, and hydrogen chloride gas dried over anhydrous calcium chloride was continuously introduced into the flask, followed by reaction at room temperature for 4-5 days until the reaction was completed (monitored by TLC). After the reaction was completed, the product was washed with anhydrous ether, and the obtained product was dried in vacuo to obtain 1-5-S (5.56g, 85.67%) as a white solid.

1-5-S：¹H NMR(400MHz，DMSO)δ11.77(s，2H)，11.01(s，2H)，8.14(d，J＝8.6Hz，4H)，7.15(d，J＝8.6Hz，4H)，4.60(t，J＝6.8Hz，4H)，4.15(q，J＝6.1Hz，4H)，1.85–1.81(m，4H)，1.60(m，J＝6.9Hz，2H)，1.48(t，J＝6.9Hz，6H)。

Compound 2-8(5.00g) was weighed into a 500mL two-necked flask, and a mixture of 120mL of anhydrous ethanol and 50mL of anhydrous chloroform was added thereto, and hydrogen chloride gas dried over anhydrous calcium chloride was continuously introduced into the flask to react at room temperature for 4-5 days until the reaction was completed (monitored by TCL). After the reaction was completed, the product was washed with anhydrous ether, and the obtained product was dried in vacuo to obtain 2-8-S (5.05g, 79.53%) as a white solid.

2-8-S：¹H NMR(400MHz，DMSO)δ7.89(s，2H)，7.71(d，J＝7.6Hz，2H)，7.70–7.58(q，2H)，7.55(s，J＝18.2Hz，4H)，7.52–7.43(d，2H)，5.26(s，4H)，4.65(q，4H)，1.48(t，6H)。

Di, polymerization

The two monomers 1-2-S (0.4mmol, 1eq) and 2,2' -oxydiethylamine (0.4mmol, 1eq) were charged into a 20mL glass bottle and 4mL anhydrous DMF was added as solvent followed by N, N-diisopropylethylamine (264.5. mu.L, 1.6mmol, 4eq) and reacted at 50 ℃ for 4 days after sealing. After the reaction was completed, hydrochloric acid (3.0M, 2mL) was added to adjust to acidic conditions, and the solution was dialyzed against water (MWCO ═ 1kDa) for 24 hours. The purified solution was then lyophilized to give P24 as a pale yellow flocculent solid.

The two monomers 1-5-S (0.4mmol, 1eq) and 1, 4-butanediamine (0.4mmol, 1eq) were charged in a 20mL glass bottle and 4mL anhydrous DMF was added as solvent followed by N, N-diisopropylethylamine (264.5. mu.L, 1.6mmol, 4eq) and reacted at 50 ℃ for 4 days after sealing. After the reaction was completed, hydrochloric acid (3.0M, 2mL) was added to adjust to acidic conditions, and the solution was dialyzed against water (MWCO ═ 1kDa) for 24 hours. The purified solution was then lyophilized to give P46 as a white flocculent solid.

The two monomers 2-8-S (0.4mmol, 1eq) and 1, 4-butanediamine (0.4mmol, 1eq) were charged in a 20mL glass bottle and 4mL anhydrous DMF was added as solvent followed by N, N-diisopropylethylamine (264.5. mu.L, 1.6mmol, 4eq) and reacted at 50 ℃ for 4 days after sealing. After the reaction was completed, hydrochloric acid (3.0M, 2mL) was added to adjust to acidic conditions, and the solution was dialyzed against water (MWCO ═ 1kDa) for 24 hours. The purified solution was then lyophilized to give P54 as a white flocculent solid.

EXAMPLE III

To expand the utility of the antimicrobial amidine oligomers in intracellular bactericidal models, Raw264.7 cells infected with M.smegmatis and M.fortuitum were treated with P2 as exemplified by oligomer P2 to test their ability to kill intracellularly infected bacteria. The specific method is as follows.

Construction of intracellular infection model M.smegmatis was nucleated with 5. mu.g/mL of 4', 6-diamidino-2-phenylindole (DAPI) for 5 min. Then, Raw264.7 cells were infected with stained Mycobacterium smegmatis at an infection ratio (MOI) of 10, and after culturing in a cell incubator at 37 ℃ for 4 hours, extracellular Mycobacterium smegmatis was washed with PBS three times. PBS was then added and cells were imaged using confocal laser microscopy. As shown in FIG. 8 (20 μm scale), stained M.smegmatis were distributed in the cell matrix of Raw264.7 cells.

Confocal characterization of intracellular sterilization experiments to observe the effect of P2 intracellular sterilization, Confocal experiments of intracellular sterilization were performed. First, an intracellular sterilization model of M.smegmatis was constructed as described above, and then co-incubation with P2 and infected cells was performed while setting control experimental groups, a positive control group treated with the standard antibiotic rifampicin and a control group not treated with the drug. As shown in FIG. 9, the control group without drug treatment showed rapid proliferation of intracellular bacteria after 24 hours, which broke the intact cell membrane and exposed the cell nucleus. In the control group treated with rifampicin, there were still more bacteria in Raw264.7 cells, which affected the morphology of the cells. The experimental group treated by P2 can effectively kill the intracellular Mycobacterium smegmatis, so that the cells can maintain a better cell morphology, and the aim of saving the mammalian cells is fulfilled.

Evaluation of intracellular bactericidal capacity of P2 to test the minimum concentration of intracellular bactericidal of P2, experiments of intracellular bactericidal were performed in 96-well plates. Raw264.7 was infected with Mycobacterium smegmatis and Mycobacterium fortuitum, respectively, at the above infection rate (MOI 10), and extracellular bacteria were removed with gentamicin 4 hours after infection. Different concentrations of P2 and rifampicin, a standard antibiotic, as a control, were then added for 24h incubation with the cells. Thereafter, the cells were lysed, and then plated after 100-fold dilution with PBS, and colonies on agar plates were counted after 48 hours of incubation at 37 ℃. As shown in FIGS. 10a and 10b, P2 almost completely killed M.smegmatis and M.fortuitum in cells at 8. mu.g/mL and 16. mu.g/mL, respectively.

Application of P2 in MRSA and sporadic infection zebra fish models in order to evaluate bactericidal performance in P2 living bodies, MRSA and sporadic mycobacterium infection zebra fish models were respectively constructed.

Application of P2 in MRSA-infected zebrafish model 67-tailed zebrafish were divided into 4 groups: PBS group (18 tails); group P2 (18 tails); erythromycin (ERY) group (18 tails); methicillin (MET) group 13 tails. The infection was performed by intraperitoneal injection of MRSA into the abdominal cavity of zebrafish, followed by injection of P2 and various controls. And observing and recording the survival condition of the zebra fish. After 72 hours of continuous observation, statistical analysis was performed on the survival of zebrafish in each group. As shown in fig. 11, the experimental group injected with P2 had higher survival rates than the other control groups. The P2 is proved to have better killing effect on MRSA infected in zebra fish bodies and rescue effect on zebra fish infected with MRSA.

Application of P2 in a mycobacterium fortuitum-infected zebrafish model 30 tail zebrafish were divided into 3 groups: PBS group (10 tails); p2 group (10 tails); ethambutol (EMB) group (10 tails). Zebrafish infection was performed by intraperitoneal injection. PBS, P2 and Ethambutol (EMB) were then injected intraperitoneally. The survival of each group was initially observed. After continuous observation for about 140 hours, the abdominal part of the zebrafish infected with the adventitious mycobacteria is gradually developed with granuloma. The granuloma will grow larger and larger with time until the belly of the zebra fish breaks and dies. As shown in fig. 12, the survival rate of zebrafish in P2-injected experimental group was significantly higher than that in PBS group and standard antibiotic EMB group.

In summary, the oligomers can target both the cell membrane and chromosomal DNA of bacteria. Thanks to the double antibacterial mechanism, the oligomer retains all antibacterial advantages of the traditional antibacterial polymer, has antibacterial broad spectrum and faster bactericidal kinetics, and can kill drug-resistant pathogenic bacteria. The oligomer can effectively kill intracellular infected mycobacterium smegmatis and mycobacterium fortuitum, and has good bactericidal activity in MRSA and mycobacterium fortuitum infected zebra fish models, thereby remarkably improving the survival rate of zebra fish.