Pyrrole tripolymer and preparation and antibacterial application thereof

Abstract

The invention belongs to the technical field of medical antibacterial materials, and discloses a pyrrole tripolymer as well as preparation and antibacterial application thereof. The method comprises the following steps: oxidizing a compound of formula II; or oxidizing the compound shown in the formula III, and reacting with tertiary amine to obtain a pyrrole tripolymer; the structure of the pyrrole tripolymer is shown as a formula I in the specification. The pyrrole tripolymer disclosed by the invention has a relatively good antibacterial effect and relatively high antibacterial selectivity on multi-drug-resistant bacteria, does not induce the bacteria to generate drug resistance, and can effectively promote healing of infected wounds. The pyrrole tripolymer disclosed by the invention is mainly used for killing bacteria through a dual mechanism of destroying a bacterial membrane and DNA (Deoxyribose Nucleic Acid). The pyrrole tripolymer also has a broad-spectrum antibacterial effect and is used for preparing an antibacterial agent or an antibacterial drug. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of medical antibacterial materials, and specifically relates to a pyrrole trimer and its preparation and antibacterial application. Background Art

[0002] Due to the widespread use of antibiotics, the emergence of multidrug-resistant (MDR) bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), has caused serious deaths worldwide. To combat MDR bacteria, there is an urgent need to develop new antimicrobial agents while avoiding the further development of resistance. Compared with traditional antibiotics, antimicrobial oligomers have attracted great interest due to their advantages such as simple preparation and ease of modification. For example, oligo(p-phenylenevinylene), oligo(p-phenyleneethynylene), and oligo(thiopheneethynylene) with photodynamic activity have been used as antimicrobial agents to treat bacterial infections. Recently, positively charged oligomeric amidines and oligomeric guanidines with dual membrane and DNA targeting capabilities have been shown to effectively treat MDR bacterial infections. However, the preparation of antimicrobial oligomers is usually subject to problems such as uncontrollable oligomerization, cumbersome purification procedures, and low selectivity for bacteria. Therefore, it is very necessary to develop oligomers with precise structures and high antimicrobial selectivity against MDR bacteria. Summary of the invention

[0003] In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide a pyrrole trimer and a preparation method thereof.

[0004] Another object of the present invention is to provide applications of the pyrrole trimer.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A method for preparing a pyrrole trimer comprises the following steps: oxidizing a compound of formula II; or oxidizing a compound of formula III and reacting it with a tertiary amine to obtain a pyrrole trimer;

[0007]

[0008] In formula II, R 1 , R 2 , R 5 is independently hydrogen, halogen, alkyl, alkyloxy, alkylcarbonyl, alkylthio, aryl, cyano, or nitro; the aryl is phenyl, hydroxy-substituted phenyl, methoxy-substituted phenyl, amino-substituted phenyl, or ester-substituted phenyl;

[0009] In formula II, R 3 , R 4 is hydrogen;

[0010] In formula III, R' is -O-(CH2)n X-substituted phenyl, X is a halogen, and n is a positive integer;

[0011] In Formula III, R 2 , R 5 is independently hydrogen, halogen, alkyl, alkyloxy, alkylcarbonyl, alkylthio, aryl, cyano, or nitro; the aryl is phenyl, hydroxy-substituted phenyl, methoxy-substituted phenyl, amino-substituted phenyl, or ester-substituted phenyl;

[0012] In Formula III, R 3 , R 4 For hydrogen.

[0013] Preferably, R in Formula II and Formula III 2 , R 5 Each is an alkyl group, such as methyl, ethyl;

[0014] Preferably, the R 1 It is phenyl, hydroxy-substituted phenyl, methoxy-substituted phenyl, dimethylamino-substituted phenyl, COOCH2CH3-substituted phenyl; each substituent in the substituted phenyl is para-substituted.

[0015] Preferably, R' is -O-(CH2) n X-substituted phenyl, X is Cl, Br, I, n is 2 to 15; each substituent in the substituted phenyl is para-substituted.

[0016] Preferably, after oxidation of formula III, the tertiary amine is N(R″)3, where R″ is an alkyl group; specifically, trimethylamine or triethylamine.

[0017] The oxidation refers to oxidation using an oxidant; the oxidant is cupric perchlorate (containing crystalline water and / or not containing crystalline water), cupric chloride, ferric chloride, silver hexafluoroantimonate, H2O2 or ammonium cerium nitrate, preferably cupric perchlorate.

[0018] The oxidation is carried out in a solvent.

[0019] When the oxidant is copper perchlorate, silver hexafluoroantimonate or ferric chloride, the oxidation is carried out using an organic solvent as a reaction medium;

[0020] When the oxidant is cupric chloride, H2O2 or ammonium cerium nitrate, the oxidation is carried out using water as the reaction medium.

[0021] When the oxidant is copper perchlorate, the organic solvent is preferably acetonitrile; when the oxidant is silver hexafluoroantimonate, the organic solvent is preferably dichloromethane; when the oxidant is ferric chloride, the organic solvent is preferably methanol.

[0022] The oxidation temperature is 20-30°C, preferably room temperature;

[0023] The oxidation time is 0.2 to 8 hours;

[0024] The molar ratio of the compound of formula II or III to the oxidant is 1:0.2 to 1:2.

[0025] When reacting with a tertiary amine, the reaction temperature is 20-30° C., and the reaction time is 8-12 hours; the molar ratio of the tertiary amine to the compound of formula III is (1.1-4):1.

[0026] After the oxidation reaction is completed, the product is concentrated under reduced pressure and separated and purified by column chromatography. The eluent is dichloromethane / methanol in a volume ratio of (8-10):1.

[0027] After the reaction with the tertiary amine is completed, the product is concentrated under reduced pressure and washed with ether.

[0028] The structure of the pyrrole trimer is Formula I

[0029]

[0030] Y - is the anion corresponding to the oxidant, and R is R in Formula II 1 or the phenyl group substituted by the ammonium salt formed by R′ and the tertiary amine in formula III ( substituted phenyl, R″ is alkyl).

[0033] The pyrrole trimer of the present invention is used for preparing an antibacterial agent.

[0034] An antibacterial agent or antibacterial drug, comprising the above-mentioned pyrrole trimer.

[0035] The antibacterial bacteria may be Gram-positive or Gram-negative bacteria; the Gram-positive bacteria include multi-drug resistant (MDR) bacteria.

[0036] When R in the pyrrole trimer is preferably phenyl, hydroxy-substituted phenyl, methoxy-substituted phenyl, dimethylamino-substituted phenyl, or COOCH2CH3-substituted phenyl, the pyrrole trimer has a good antibacterial effect against Gram-positive bacteria;

[0038] In the pyrrole trimer, when R is preferably When the phenyl group is substituted, R″ is an alkyl group, and the pyrrole trimer has a good antibacterial effect against Gram-positive bacteria and Gram-negative bacteria.

[0039] The pyrrole trimer is used for preparing a preparation or a medicine that kills multi-drug resistant bacteria without having drug resistance; or is used for preparing a preparation or a medicine that kills multi-drug resistant bacteria and promotes wound healing.

[0040] The pyrrole trimers of the present invention kill bacteria through a dual mechanism of destroying bacterial membranes and DNA.

[0041] Taking copper perchlorate as an oxidant as an example, the reaction equation of the present invention is:

[0042]

[0043] Compared with the prior art, the present invention has the following advantages and effects:

[0044] 1. The present invention successfully prepared a pyrrole trimer, the preparation method of the pyrrole trimer is simple, and it is easy to modify a variety of functional groups.

[0045] 2. The pyrrole trimer of the present invention can quickly kill floating multidrug-resistant bacteria, inhibit the formation of multidrug-resistant bacterial biofilms and eradicate mature biofilms; it will not induce bacteria to develop drug resistance; it can quickly and effectively kill bacteria in wounds and promote wound healing; it can effectively improve the survival rate of mice in an acute peritonitis model and reduce the bacterial load in organ tissues, etc.

[0046] 3. The pyrrole trimer of the present invention kills bacteria based on a dual destruction mechanism of membrane and DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 is the H NMR spectrum of compound Ⅰ-1 in CD2Cl2;

[0048] Figure 2 is the C NMR spectrum of compound Ⅰ-1 in CD2Cl2;

[0049] Figure 3 is a high resolution mass spectrum of compound formula Ⅰ-1;

[0050] Figure 4 The single crystal structure of the compound dihydropyrrole trimer (Formula I-1-1);

[0051] Figure 5 is the normalized absorption spectrum and normalized emission spectrum of Formula I-1 to Formula I-5 in methanol (50 μg / mL), λ ex =420nm;

[0052] Figure 6 is the normalized absorption spectrum and normalized emission spectrum of Formula I-6 to Formula I-10 in methanol (50 μg / mL), λ ex =420nm;

[0053] Figure 7 is the absorption spectrum and emission spectrum of formula I-1 in glycerol-methanol mixture, the glycerol fraction increases from 10% to 90% (50 μg / mL), λ ex =420nm;

[0054] Figure 8Fluorescence imaging and antibacterial activity: (a) CLSM images of Staphylococcus aureus ATCC6538P and Escherichia coli ATCC8739 stained with Formula I-1, scale bar is 10 μm; (b, c) MIC and MBC values ​​of Formula I-1 to Formula I-5 against six MRSA strains; (d) bactericidal kinetics of Formula I-1 against MRSA (Staphylococcus aureus ATCC43300) at 2× and 5× MIC concentrations; (e) drug resistance study of Formula I-1 and antibiotic norfloxacin against MRSA; (f) cross-resistance study of Formula I-1 and antibiotics against MRSA at 1 / 2× MIC concentrations;

[0055] Fig. 9 The thermal stability and enzyme stability of the pyrrole trimer of formula I-1;

[0056] Fig.10 The inhibition and eradication of MRSA (Staphylococcus aureus ATCC43300) biofilm by formula I-1: (a, c) Schematic diagram of the inhibition of biofilm formation and eradication of mature biofilm by formula I-1; (b) Concentration-dependent inhibition of MRSA biofilm formation by formula I-1, vancomycin and norfloxacin; (d) Concentration-dependent eradication of MRSA mature biofilm by formula I-1, vancomycin and norfloxacin, n=3 per group, data are expressed as mean ± SD; (e) Representative 3D CLSM images of the control group and MRSA mature biofilms treated with formula I-1 (16×MIC), vancomycin (512×MIC) and norfloxacin (512×MIC), for fluorescein diacetate: λ ex =488nm,λ em =500-550nm; for propidium iodide: λ ex =543nm,λ em = 600-650 nm, scale bar 100 μm;

[0057] Fig.11 The antibacterial mechanism of formula I-1: (a) Depolarization effect of formula I-1 on the plasma membrane of Staphylococcus aureus at 2× and 5× MIC concentrations; (b) CLSM image of Staphylococcus aureus treated with formula I-1 and SYTOX; (c) CLSM image of Staphylococcus aureus treated with formula I-1 and FITC-dextran (500 kDa), SYTOX or FITC-dextran channel: λ ex =488nm,λ em =500-550nm; Formula I-1 channel: λ ex =405nm,λ em=500-550nm; scale bar is 10μm; (d, e) SEM and TEM characterization of Staphylococcus aureus treated with Formula I-1 at 5×MIC concentration for 2 hours; (f) Using Hoechst 33342 and ethidium bromide (EB) as dyes, the DNA dye complex was fluorescently quenched by adding Formula I-1, n=3 per group, data are expressed as mean±SD, ***p<0.001; (g) Schematic diagram of DNA competition binding; (h) ζ potential of Staphylococcus aureus genomic DNA treated with different concentrations of Formula I-1, n=3 per group, data are expressed as mean±SD; (i) Antibacterial activity of Formula I-1 in the presence of Staphylococcus aureus genomic DNA, n=3 per group, data are expressed as mean±SD, **p<0.01;

[0058] Fig.12 To study the in vivo antibacterial activity of pyrrole trimer Formula I-1 against MRSA using a mouse wound infection model: (a) Schematic diagram of the mouse skin wound MRSA infection model; (b, c) Colony photos and cell density on BP agar plates after 24 hours of treatment of infected tissues with saline, vancomycin and Formula I-1; (d) Photos of mouse skin wounds at specific time points after treatment with saline, vancomycin (32×MIC) and Formula I-1 (32×MIC), scale bar is 5 mm; (e) Schematic diagram of wound healing sites; (f) Changes in the size of mouse skin wounds after treatment; (g) Changes in mouse body weight after treatment; (h) H&E staining of wound tissue on the 15th day after treatment with saline, vancomycin and Formula I-1, green arrow: epidermis, orange arrow: hair follicle, scale bar is 400 μm; data are expressed as mean ± SD, n = 5 per group, *p < 0.05, ***p < 0.001, ns: no significant difference; day represents day;

[0059] Fig.13 CLSM images of Staphylococcus aureus ATCC43300 and Escherichia coli ATCC8739 stained with Formula I-6 to Formula I-10;

[0060] Fig.14 (a) the hemolysis rate of the pyrrole trimers of formula I-6 to formula I-10; (b) the selectivity index SI (HC) of the pyrrole trimers of formula I-6 to formula I-10 against Staphylococcus aureus and Escherichia coli 10 / MIC); (c) cell survival rate of pyrrole trimers of formula I-6 to formula I-10; (d) selectivity index SI (IC) of pyrrole trimers of formula I-6 to formula I-10 against Staphylococcus aureus and Escherichia coli 50 / MIC);

[0061] Fig.15 The thermal stability and enzyme stability of the pyrrole trimer of formula I-7;

[0062] Fig.16 The in vitro antibacterial activity of the pyrrole trimer formula I-7: (a, b) MIC values ​​and MBC values ​​of formula I-7 against Gram-positive (Gram+) and Gram-negative (Gram-) bacteria; (c) bactericidal kinetics of formula I-7 against Escherichia coli at 2× and 5× MIC concentrations; (d) drug resistance study of formula I-7 and antibiotic ampicillin against Escherichia coli; (e) cross-resistance study of formula I-7 and antibiotics against Escherichia coli at 1 / 2× MIC concentrations;

[0063] Fig.17 Inhibition and eradication of Escherichia coli biofilm by formula I-7: (a, c) Schematic diagram of inhibition of biofilm formation and eradication of mature biofilm by formula I-7; (b) Concentration-dependent inhibition of Escherichia coli biofilm formation by formula I-7 and ampicillin; (d) Concentration-dependent eradication of mature Escherichia coli biofilm by formula I-7 and ampicillin, n=3 per group, data are expressed as mean ± SD; (e) Representative 3D CLSM images of mature Escherichia coli biofilms of the control group and treated with formula I-7 (8×MIC) and ampicillin (64×MIC), for fluorescein diacetate: λ ex =488nm,λ em =500-550nm; for propidium iodide: λ ex =543nm,λ em = 600-650 nm, scale bar 100 μm;

[0064] Fig.18 The antibacterial mechanism of Formula I-7: (a) time-lapse confocal image of E. coli ATCC8739 in the presence of Formula I-7 (green, pseudo color, 5×MIC) and PI (red, 1.0 μg / mL); Formula I-7 channel: λ ex =405nm,λ em =550-650nm; PI channel: λ ex =543nm,λ em=550-700nm; scale bar is 2μm; (b, c, d) Fluorescence intensity distribution of formula I-7 and PI in E. coli cells at 304 seconds, 572 seconds and 1549 seconds; (e) Average fluorescence intensity curve of formula I-7 and PI in E. coli cells; (f) Formula I-7 depolarizes the cytoplasmic membrane of E. coli at 2× and 5× MIC concentrations; (g) Using Hoechst33342 and EB as dyes, the DNA dye complex was quenched by adding formula I-7, n=3 per group, data are expressed as mean ± SD, ***p<0.001; (h) Different concentrations of formula I-7 were used to treat E. coli genome D ζ potential of NA, n=3 for each group, data are expressed as mean ± SD; (i) antibacterial activity of formula I-7 in the presence of E. coli genomic DNA, n=3 for each group, data are expressed as mean ± SD, ***p<0.001; (j) fluorescence intensity of E. coli treated with PBS, formula I-7 and a mixture of formula I-7 and NAC in the presence of DCFH-DA, n=3 for each group, data are expressed as mean ± SD, **p<0.01; (k) MIC value of formula I-7 against E. coli in the presence or absence of NAC; (l) SEM and TEM characterization of E. coli after treatment with formula I-7 at a concentration of 5×MIC for 2 hours;

[0065] Fig.19 To study the in vivo antibacterial activity of pyrrole trimer Formula I-7 against Escherichia coli using the mouse acute peritonitis model: (a) Schematic diagram of the E. coli-induced mouse acute peritonitis model; (b) Survival rate of mice in the E. coli-induced peritonitis model (5 days); (c, d) CFU of bacteria in the main organs, blood and peritoneal fluid of mice after treatment with normal saline, ampicillin and Formula I-7 and representative EMB plate photos; (e) After treatment with normal saline, ampicillin and Formula I-7, H&E staining of heart, liver, spleen, lung and kidney sections of mice was performed, and the data are expressed as mean ± SD, n = 6 per group, *p < 0.05, **p < 0.01, ***p < 0.001, ns: no significant difference;

[0066] Fig. 20To study the in vivo antibacterial activity of pyrrole trimer Formula I-7 against MRSA using a mouse wound infection model: (a) Schematic diagram of the mouse skin MRSA infection model; (b, c) Photos of bacterial colonies and MRSA cell density on BP agar plates after 1 day of treatment of infected tissues with saline, vancomycin and Formula I-7; (d) Photos of MRSA-infected mouse skin wounds after treatment at specified time points, with a scale of 5 mm; (e) Schematic diagram of wound healing sites; (f) Changes in mouse skin wound size after treatment; (g) Changes in mouse body weight after treatment; (h, i) H&E and Masson staining of tissue sections on the 12th day after treatment with saline, vancomycin and Formula I-7, n=4 per group, data are expressed as mean ± SD, **p<0.01, ***p<0.001, ns: no significant difference; day represents day;

[0067] Fig.21 The toxicity study of pyrrole trimer in mice in Example 13: (a) body weight of normal mice after pyrrole trimer treatment; (bh) blood biochemical indexes of mice (ALT, AST, BUN, CREA, K) at 2 days and 14 days + 、Na + and Cl - ), n = 5 in each group, data are expressed as mean ± SD, ns: no significant difference;

[0068] Fig. 22 H&E staining was performed on the heart, liver, spleen, lung and kidney sections of the mice at 2 and 14 days for Example 13. DETAILED DESCRIPTION

[0069] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0070] Example 1

[0071] Pyrrole monomers with different substituents:

[0072] The following 2-hydroxy-1-(4-(2-nitro-2-yl)-2-nitropropene-1-yl)-2-nitropropene-1-yl groups with different N-aryl substituents (-Ph, -PhOH, -PhOMe, -PhNMe2 and -PhCO2Et, -O-(CH2)) were prepared by Paal-Knorr reaction. n -Br) (pyrroles with different substituents are prepared by existing methods), the structure is as follows:

[0073]

[0074] Example 2

[0075] Preparation of pyrrole trimer:

[0076] (1) Preparation of pyrrole trimers, i.e. compounds of formula I-1 to formula I-5:

[0077] Taking compound formula I-1 as an example, compound formula II-1 (86 mg, 0.5 mmol) was dissolved in MeCN (5 mL), and then Cu(ClO4)2·6H2O (185 mg, 0.5 mmol) was added and stirred at room temperature for 2 hours. Then, the mixture was concentrated under reduced pressure and further separated and purified by column chromatography (SiO2) using dichloromethane / methanol in a volume ratio of 9 / 1 as an eluent to obtain a brown-black pyrrole trimer compound formula I-1 (165 mg, yield 54%).

[0078] The synthesis steps of compounds of formula I-2 to I-5 are the same as those of the above compound of formula I-1, and the reaction conditions are shown in Table 1.

[0079] Table 1 Synthesis parameters of compounds of formula I-2 to formula I-5

[0080]

[0081]

[0082] (2) Preparation of pyrrole trimers, i.e. compounds of formula I-6 to I-10: Take compound I-6 as an example.

[0083] (2-1) Compound III-1 (154 mg, 0.5 mmol) was dissolved in MeCN (5 mL), and then Cu(ClO4)2·6H2O (93 mg, 0.25 mmol) was added and stirred at room temperature for 90 minutes. Then, the mixture was concentrated under reduced pressure and further separated and purified by column chromatography (SiO2), using dichloromethane / methanol volume ratio of 9 / 1 as eluent to obtain brown-black pyrrole trimer compound II-6 (179 mg, yield 36%). Compound III-2, compound III-3, compound III-4 and compound III-5 were used to prepare compound II-7, compound II-8, compound II-9 and compound II-10, respectively. The preparation steps were the same as compound II-6, except that the oxidant and reaction time were different. The specific synthesis parameters are shown in Table 2.

[0084] (2-2) Compound II-6 (100 mg) was dissolved in MeCN (5 mL), and then an excess of trimethylamine (30% ethanol solution) was added, and stirred at room temperature overnight. After the substrate was completely reacted by spot plate monitoring, the mixture was concentrated under reduced pressure and washed with ether to obtain an ionized alkyl-modified pyrrole trimer compound I-6. The synthesis steps of compounds I-7 to I-10 are the same as those of the above compound I-6.

[0085] Table 2 Synthesis parameters of compounds of formula II-7 to formula II-10

[0086]

[0087]

[0088] (3) Study the effect of reaction conditions on oxidative dehydrogenation reaction:

[0089] Compound II-1 cannot obtain pyrrole trimer in other solvents such as methanol, ethanol, petroleum ether, ethyl acetate, dichloromethane, etc. when copper perchlorate is used as an oxidant.

[0090] Compound II-1 can be used in methanol with ferric chloride as an oxidant to obtain a pyrrole trimer.

[0091] Compound II-1 in water with copper chloride as an oxidant can give a pyrrole trimer.

[0092] Compound II-1 can be treated in water with hydrogen peroxide as an oxidant to obtain a pyrrole trimer.

[0093] Compound II-1 can be used in water with ammonium cerium nitrate as an oxidant to obtain a pyrrole trimer.

[0094] Compound II-1 can obtain pyrrole trimer in dichloromethane with silver hexafluoroantimonate as oxidant.

[0095] Example 3

[0096] Structural characterization of pyrrole trimer:

[0097] Taking compound I-1 as an example, by H NMR spectrum ( Figure 1 )、Carbon Spectrum( Figure 2 ) and high-resolution mass spectrometry ( Figure 3 ) conducted a detailed structural verification, and the results are as follows: 1 H NMR (400MHz, CD2Cl2) δ (ppm): 7.57-7.47 (m, 11H), 7.24 (d, J = 8.4Hz, 2H), 6.99 (d, J = 6.8Hz, 2H), 6.94 (s, 1H ),5.91(s,1H),5.89(s,1H),2.50(s,3H),2.39(s,3H),1.98(s,3H),1.93(s,3H),1.91(s,3H),1.68(s,3H). 13C NMR(100MHz,CD2Cl2)δ(ppm):177.4,174.8,138.6,137.8,136.7,133.9,131.9,130.5,129.8,129.7,129.4,129.2,129.0,1 28.5,128.3,127.9,126.7,113.4,113.1,110.4,106.7,106.1,82.2,29.7,25.9,17.0,14.6,12.6,11.4.HRMS(ESI)m / z:[M] + Calculate for C 36 H 36 N3 + :510.2904,found:510.2838.

[0098] In order to further verify the structure of the pyrrole trimer, the compound of formula I-1 was reduced with NaBH4 to obtain the dihydropyrrole trimer (Formula I-1-1), and the single crystal X-ray diffraction was clearly verified ( Figure 4 ).

[0099] Reaction equation:

[0100]

[0101] Example 4

[0102] Characterization of photophysical properties of pyrrole trimer:

[0103] The photophysical properties of pyrrole trimers were studied. The maximum absorption and emission wavelengths of these pyrrole trimers (Formula I-1 to Formula I-10) in methanol were 420 nm and 520 nm, respectively. Figure 5 , Figure 6 ), which can be attributed to their intramolecular charge transfer properties, where the 3″-pyrrolyl group acts as an electron donor group and the 2'H-pyrrol-1'-ium part acts as an electron acceptor group. Taking compound I-1 as an example, in a methanol / glycerol mixture, the emission of compound I-1 gradually increases with increasing viscosity ( Figure 7 ), which can be attributed to the restriction of intramolecular motion in high viscosity media.

[0104] Example 5

[0105] In vitro antibacterial activity of pyrrole trimers of formula I-1 to formula I-5:

[0106] (1) The bacterial imaging ability of pyrrole trimers was studied under a confocal laser scanning microscope. Taking Formula I-1 as an example, it can effectively stain Gram-positive Staphylococcus aureus (S. aureus, ATCC6538P, Guangdong Microbiological Culture Collection Center) with a high signal-to-noise ratio, while Gram-negative Escherichia coli (E. coli, ATCC8739, Guangdong Microbiological Culture Collection Center) was only observed with a poor fluorescence signal ( Figure 8 a).

[0107] (2) The antibacterial activity of compounds of formula I-1 to formula I-5 was studied. For Gram-positive Staphylococcus aureus, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values ​​of formula I-1 with N-phenyl substituents were 3.13 μg / mL and 6.25 μg / mL, respectively (Table 3). At the same time, formula I-2 to formula I-5 with different N-phenyl substituents (4-OH-Ph, 4-OMe-Ph, 4-NMe2-Ph, 4-CO2Et-Ph) also showed lower MIC values ​​(0.78-6.25 μg / mL) and MBC values ​​(6.25-25 μg / mL) (Table 3). We further studied the antibacterial activity of Formula I-1 to Formula I-5 against six other MRSA strains (Staphylococcus aureus Mu50, Staphylococcus aureus USA300, Staphylococcus aureus ATCC700698, Staphylococcus aureus ATCC700788, Staphylococcus aureus ATCC33591 and Staphylococcus aureus ATCC43300 (all of the above strains were purchased)). The pyrrole trimers Formula I-1 to Formula I-5 all showed lower MIC and MBC values, in the range of 0.78-25 μg / mL and 3.13-50 μg / mL, respectively ( Figure 8 b, Figure 8 c), which suggests that they are promising for the treatment of MRSA infections. However, the antibacterial activity of Formula I-1 to Formula I-5 against Gram-negative Escherichia coli was poor (Table 3), which may be due to the fact that the interaction of the pyrrole trimers of Formula I-1 to Formula I-5 with Gram-negative bacteria is inhibited by the dense outer lipopolysaccharide (LPS) layer. In addition, the imino part in the pyrrole trimer skeleton is indispensable for its antibacterial activity, which is confirmed by the significant reduction in the antibacterial activity of the dihydropyrrole trimer Formula I-1-1 after reduction with NaBH4 (MIC>200μg / mL). It is worth noting that Formula I-1 to Formula I-5 exhibited lower hemolytic activity, and their HC 10 (Concentration causing 10% hemolysis) value is greater than 200 μg / mL. 10 The selectivity index (SI) calculated by MIC / I is greater than 32 (Table 3), which indicates that Formula I-1 to Formula I-5 have good biocompatibility and are expected to selectively ablate Gram-positive bacteria.

[0108] Table 3 Antibacterial and hemolytic activities of pyrrole trimers of formula I-1 to formula I-5

[0109]

[0110] (3) The kinetic antibacterial activity and resistance-inducing properties of the pyrrole trimer formula I-1 were studied. The time-dependent bacterial killing kinetics showed that formula I-1 could completely kill MRSA (Staphylococcus aureus ATCC43300) within 20 minutes at concentrations of 2× and 5× MIC ( Figure 8 d), indicating that Formula I-1 can act as a rapid bacterial killer. Since the emergence of resistance is a major challenge in the treatment of bacterial infections with antibiotics, we further investigated whether the pyrrole trimer would induce resistance to the antibiotic norfloxacin as a comparison. Serial passages of MRSA treated with Formula I-1 at sub-MIC doses (1 / 2×MIC) for 28 consecutive days did not induce resistance (MIC 28 / MIC0=1), while the MIC of norfloxacin increased 1024-fold ( Figure 8 e). In addition, MRSA treated with Formula I-1 did not develop cross-resistance to conventional antibiotics, while MRSA treated with norfloxacin showed varying degrees of cross-resistance to other antibiotics, for example, resistance to ampicillin increased by 512 times ( Figure 8 f). These results indicate that Formula I-1 can be used as an excellent antibacterial agent without causing drug resistance.

[0111] (4) The thermal stability and biological stability of the pyrrole trimer of formula I-1 were studied. The antibacterial activity of formula I-1 remained unchanged after exposure to high temperature (100°C) for 2 hours or treatment with proteinase K for 24 hours ( Fig. 9 ). These results further verified the antibacterial application prospects of pyrrole trimers.

[0112] Example 6

[0113] Inhibition and eradication ability of pyrrole trimer formula I-1 on MRSA biofilm:

[0114] Bacterial biofilms are often found on medical implant surfaces and are difficult to eradicate due to their resistance to conventional antibiotics and dense three-dimensional polymer networks. To address this challenge, we investigated whether the pyrrole trimer formula I-1 could inhibit MRSA biofilm formation and eradicate mature biofilms ( Fig.10 a, Fig.10 c) If Fig.10As shown in Figure b, Formula I-1 can effectively inhibit the formation of MRSA (Staphylococcus aureus ATCC43300) biofilm at a low concentration of 1×MIC, while antibiotics vancomycin and norfloxacin require high concentrations of 4×MIC and 16×MIC, respectively. In order to eradicate mature biofilms, which are more challenging than inhibiting biofilm formation, the concentration of Formula I-1 was further increased to 8×MIC, and the survival rate of mature MRSA biofilms was significantly reduced to 4.6% ( Fig.10 d). The excellent biofilm eradication ability of Formula I-1 was further verified by 3D CLSM images of biofilms stained with fluorescein diacetate (FDA) / propidium iodide (PI), which showed a significant reduction in bacterial density and biofilm thickness ( Fig.10 e). In contrast, mature biofilms treated with 512×MIC of vancomycin and 1024×MIC of norfloxacin showed high survival rates of 65.3% and 77.1%, respectively ( Fig.10 d), CLSM images further confirmed this ( Fig.10 e). These results demonstrate the great potential of pyrrole trimers in combating MRSA biofilms.

[0115] Example 7

[0116] Antibacterial mechanism of pyrrole trimer formula I-1:

[0117] Using formula I-1 as a model compound, its dose-dependent bacterial membrane depolarization ability was first studied. Using 3,3′-dipropylthiodicarbocyanine iodide (DiSC3(5)) dye as a membrane potential-sensitive fluorescent indicator, after treating Staphylococcus aureus with formula I-1, its fluorescence intensity increased by about 10 times at a concentration of 2×MIC and by about 20 times at a concentration of 5×MIC ( Fig.11 a), which indicates that Formula I-1 can effectively destroy the bacterial membrane potential. CLSM images show that bacteria treated with Formula I-1 can be well stained by SYTOX (a membrane-impermeable nucleic acid dye), indicating that the permeability of the bacterial membrane is destroyed ( Fig.11 b). The pore size of damaged bacterial membranes caused by Formula I-1 was evaluated by FITC-dextran (500 kDa, diameter ~29 nm). CLSM images showed that FITC-dextran macromolecules with 500 kDa were able to enter bacterial cells, indicating that the pore size induced by Formula I-1 was larger than 29 nm ( Fig.11 c). SEM and TEM also directly observed the damaged bacterial morphology induced by Formula I-1, showing that the bacterial membrane was obviously shrunk and the cytoplasmic contents flowed out ( Fig.11 d, Fig.11e). In order to study whether the positively charged Formula I-1 can bind to the negatively charged DNA, a DNA competition binding experiment was performed using Hoechst 33342 and ethidium bromide (EB) as indicators. After adding Formula I-1, the fluorescence quenching rates of Hoechst 33342 and EB were 73% and 59%, respectively ( Fig.11 f, Fig.11 g), which can be attributed to the higher binding affinity of Formula I-1 to DNA. The effective binding of positively charged Formula I-1 to negatively charged S. aureus genomic DNA was further verified by zeta potential measurement. As the concentration of Formula I-1 increased from 5 μg / mL to 20 μg / mL, the zeta potential gradually increased from -34.3 mV to +26.3 mV ( Fig.11 h). In addition, the addition of genomic DNA to the bacterial solution can inhibit the antibacterial activity of Formula I-1 ( Fig.11 i) These results indicate that Formula I-1 can bind to bacterial DNA and cause damage, which is crucial for its bactericidal ability.

[0118] Example 8

[0119] In vivo anti-MRSA infection performance of pyrrole trimer formula Ⅰ-1:

[0120] The in vivo therapeutic potential of the pyrrole trimer of formula Ⅰ-1 was evaluated using a MRSA-infected mouse skin wound model. Fig.12 a). Full-thickness wounds were created on the back skin of mice using an 8 mm biopsy punch and subsequently infected with MRSA (Staphylococcus aureus ATCC43300) for 12 hours. Formula I-1, vancomycin (positive control), and saline (negative control) were then topically applied to the wound every 4 hours for a total of three treatments. After 24 hours of treatment with Formula I-1, a 3.0 log reduction in colony forming units (CFU) was observed compared to the saline control group. Fig.12 b, Fig.12 c). On the 15th day after treatment, the group treated with Formula I-1 showed the most significant recovery, with only 10.4% of the original wound size remaining. In contrast, the wound sizes of the vancomycin and saline treatment groups were 15.2% and 17.5%, respectively ( Fig.12 d, Fig.12 e, Fig.12 f). In addition, to evaluate the biosafety of Formula Ⅰ-1, we monitored the relative body weight of infected mice over 15 days, and the results showed no significant difference in body weight ( Fig.12 g). We further evaluated the wound healing effects by hematoxylin and eosin (H&E) staining of skin wound tissue sections after treatment ( Fig.12h). The image shows that the intact epidermis (green arrow) and hair follicles (orange arrow) of the Formula I-1 and vancomycin treatment groups are clearly visible. In contrast, the saline treatment group showed epidermal thickening and reduced hair follicles. These results indicate that Formula I-1 has good biocompatibility and can effectively eradicate MRSA infection and promote the wound healing process.

[0121] Example 9

[0122] In vitro antibacterial activity of pyrrole trimers of formula I-6 to formula I-10:

[0123] With -(CH2) n -NMe3 + Pyrrole trimers of formula I-6 to formula I-10 were prepared by modifying the N-aryl ring with Br groups (n=3, 6, 8, 10, 12). CLSM images showed that formula I-6 could effectively stain Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli ( Fig.13 ). Then, the MIC and MBC values ​​of pyrrole trimers of formula I-6 to formula I-10 against Gram-negative Escherichia coli were tested, which were 1.56 to 3.13 μg / mL and 1.56 to 6.25 μg / mL, respectively (Table 4). In addition, they also showed excellent antibacterial activity against Gram-positive Staphylococcus aureus, with MIC and MBC values ​​of 1.56 to 3.13 μg / mL and 1.56 to 6.25 μg / mL, respectively (Table 4). Among these tripyrroles, formula I-7 (n=6) with a 1,6-hexene linker showed the lowest hemolytic and cytotoxic properties, HC 10 The value is 400 μg / mL, IC 50 The value is 300 μg / mL ( Fig.14 ). It also showed the highest antimicrobial selectivity index, HC 10 / MIC=64 and IC 50 / MIC=48 (Table 4). The thermal stability and biostability of Formula I-7 were measured at high temperature (100℃ for 2 hours) or in the presence of protease (24 hours), and its antibacterial activity remained good and the MIC value remained unchanged ( Fig.15 ), indicating its great potential for in vivo antimicrobial applications.

[0124] Table 4 Antibacterial and hemolytic activities of pyrrole trimers of formula I-6 to formula I-10

[0125]

[0126] Further, the antibacterial activity of pyrrole trimer formula I-7 against Gram-positive bacteria (Gram+) and Gram-negative bacteria (Gram-) was investigated, with 8 bacterial strains each, including drug-resistant strains (methicillin-resistant S. aureus USA300, S. aureusMu50, S. aureus ATCC43300, S. aureus ATCC33591, S. aureus ATCC700698, and S. aureusATCC700788; ampicillin and piperacillin-resistant E. coli ATCC25922; sulfamethoxazole and tetracycline-resistant P. aeruginosa PAO1), drug-sensitive strains (S. aureus ATCC6538P and S. epidermidis ATCC49134), and E. coli ATCC8739, E. coli ATCC35218, and S. coli BL21, E. hormaechei ATCC700323, S. typhimurium ATCC14028 and P. aeruginosa ATCC27853 (all of the above strains were purchased). Fig.16 a and Fig.16 As shown in Figure b, the pyrrole trimer formula I-7 has strong antibacterial activity against the tested bacteria, with MIC and MBC values ​​of 1.56-3.13 μg / mL for Gram-positive bacteria and 6.25-50 μg / mL for Gram-negative bacteria. The antibiotic norfloxacin (Gram+ control group) is insensitive to S. aureus Mu50, S. aureus ATCC33591 and S. aureus ATCC700788 (MIC>200 μg / mL); the antibiotic ampicillin (Gram- control group) is insensitive to E. coli ATCC35218, P. aeruginosa PAO1 and P. aeruginosa ATCC27853 (MIC and MBC>200 μg / mL). Time-kill kinetic studies show that at a concentration of 2×MIC, formula I-7 completely kills Escherichia coli ATCC8739 within 40 minutes ( Fig.16 c) Antimicrobial resistance studies have shown that in the presence of sub-MIC doses of Formula I-7, E. coli will not develop any resistance when continuously cultured for 28 days (MIC 28 / MIC0=1); however, after E. coli was treated with sub-MIC doses of ampicillin for 28 days, its MIC increased by 64 times ( Fig.16 d). In addition, we found that E. coli treated with formula I-7 did not develop cross-resistance to traditional antibiotics. In contrast, E. coli treated with ampicillin showed a 16-fold increase in resistance to levofloxacin and a 32-fold increase in resistance to kanamycin ( Fig.16 e).

[0127] Example 10

[0128] Inhibition and eradication ability of pyrrole trimer formula I-7 on Escherichia coli biofilm:

[0129] We further investigated the ability of the pyrrole trimer I-7 to inhibit E. coli biofilm formation and eradicate mature biofilms ( Fig.17 a, Fig.17 c). At a concentration of 1×MIC, Formula I-7 completely inhibited the formation of E. coli biofilm, while at 8×MIC, Formula I-7 effectively eradicated 92% of mature biofilm ( Fig.17 b, Fig.17 d). In contrast, ampicillin required a concentration of 2×MIC to inhibit E. coli biofilm formation and a concentration of 64×MIC to eradicate 90% of mature biofilms ( Fig.17 b, Fig.17 d). 3D CLSM images show that the bacterial activity was significantly reduced after treatment with Formula I-7 and ampicillin ( Fig.17 e). These results indicate that Formula I-7 has great potential in combating E. coli biofilm.

[0130] Embodiment 11

[0131] Antibacterial mechanism of pyrrole trimer formula I-7:

[0132] The antibacterial mechanism of the pyrrole trimer I-7 was further explored. CLSM was used to trace I-7 and PI, allowing us to monitor the death process of E. coli ( Fig.18 a). Initially, the green fluorescence of Formula I-7 was observed to be significantly enriched on the bacterial cell membrane. Then, this green fluorescence spread throughout the bacterial cell. After about 300 seconds, the red fluorescence of PI appeared in the cytoplasm and gradually accumulated ( Fig.18 b, Fig.18 c, Fig.18 d, Fig.18 e), indicating that the E. coli membrane is destroyed and the bacteria are dead. In addition, Formula I-7 causes a significant depolarization of the E. coli inner membrane ( Fig.18 f). The bacterial DNA binding ability of Formula I-7 was further studied and confirmed by DNA competition binding experiments and zeta potential measurements. After adding Formula I-7, the fluorescence quenching rates of Hoechst 33342 and EB were 72% and 64%, respectively ( Fig.18 g), the zeta potential gradually increased from -34.0 mV to +25.1 mV as the concentration of Formula I-7 increased ( Fig.18 h). In addition, in the presence of formula I-7 and DNA, the OD of Escherichia coli 600The value increased from 0.06 to 0.61 ( Fig.18 i), which indicates that Formula I-7 loses its antibacterial activity after binding to DNA. Using 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA) as a ROS indicator, after treatment with Formula I-7, the ROS level in Escherichia coli cells increased by 2.7 times compared with the control group ( Fig.18 j). In the presence of N-acetyl-L-cysteine ​​(NAC), a ROS quencher, the elevated ROS levels were completely suppressed to normal levels ( Fig.18 j). The decrease in ROS levels was associated with a decrease in anti-E. coli activity, with the MIC value increasing from 6.25 μg / mL to 25 μg / mL in the presence of NAC ( Fig.18 k). These findings suggest that the induction of intracellular ROS plays an important role in the antibacterial mechanism of Formula I-7. In addition, SEM and TEM images showed that after treatment with Formula I-7, the bacterial membrane was significantly shrunk, the membrane was significantly damaged, and the cytoplasmic content was reduced ( Fig.18 (Fig. 1).

[0133] Example 12

[0134] In vivo anti-E. coli infection performance of pyrrole trimer formula Ⅰ-7:

[0135] Furthermore, the therapeutic potential of Formula I-7 was evaluated in the context of systemic infection using a mouse acute peritonitis model ( Fig.19 a). Mice were infected with a lethal dose of E. coli ATCC25922 by intraperitoneal injection (100% mortality in the saline treatment group within 24 hours). In contrast, the survival rate of the Formula I-7 treatment group after 120 hours was 86%, while the survival rate of the ampicillin treatment group was only 50% ( Fig.19 b). Notably, at 48 hours, treatment with Formula I-7 effectively reduced the bacterial load in five representative organs (heart, liver, spleen, lung, and kidney) as well as blood and peritoneal fluid by 3.6-5.1 log ( Fig.19 c. Fig.19 d). Of note, ampicillin only resulted in a 2.4-3.8 log reduction in bacterial load ( Fig.19 c, Fig.19 d). We also performed histological analysis of major organs (heart, liver, spleen, lung, and kidney) using H&E staining, which revealed organ damage caused by bacterial infection in the saline group ( Fig.19 e). Treatment with formula I-7 or ampicillin effectively alleviated this organ damage. In addition, the pyrrole trimer formula I-7 showed excellent in vivo anti-infection efficacy in a mouse skin model infected with MRSA ( Fig. 20). It is noteworthy that, although many polymers and oligomers with promising antimicrobial properties have been reported in previous studies, most of them were used at significantly higher concentrations in in vivo applications than in vitro applications. Nevertheless, lowering the in vivo therapeutic concentrations could help mitigate the potential risks associated with these drugs, even if polymers and oligomers exhibited lower in vivo toxicity.

[0136] Example 13

[0137] In vivo toxicity study of pyrrole trimer:

[0138] The in vivo toxicity of pyrrole trimers (Formula I-1 and Formula I-7) was evaluated, because even some classic antibiotics, such as vancomycin, can be nephrotoxic. Pyrrole trimers were repeatedly administered by intraperitoneal injection, and no significant differences were observed between the mice in the administration group and the saline group within 14 days. This included changes in mouse behavior, survival rate, body weight ( Fig.21 a) and blood biochemical indicators ( Fig.21 b, Fig.21 c, Fig.21 d, Fig.21 e, Fig.21 f, Fig.21 g, Fig.21 h). The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice treated with Formula I-1 and Formula I-7 remained within the normal range, indicating no significant liver toxicity. In addition, blood urea nitrogen (BUN) and creatinine (CREA) levels were also normal, indicating no renal toxicity. + ), sodium (Na + ) and chloride (Cl - ) levels confirmed that no electrolyte imbalance occurred in the body. H&E staining of mouse organ tissue sections showed no obvious damage ( Fig. 22 The pyrrole trimer of the present invention has low in vivo toxicity and high antibacterial efficacy against MDR bacterial infection.

Claims

1. A method for preparing a pyrrole trimer, characterized in that: The following steps are involved: Oxidizing the compound of formula II; or oxidizing the compound of formula III and reacting it with a tertiary amine to obtain a pyrrole trimer; In formula II, R 1 , R 2 , R 5 is independently hydrogen, halogen, alkyl, alkyloxy, alkylcarbonyl, alkylthio, aryl, cyano, or nitro; the aryl is phenyl, hydroxy-substituted phenyl, methoxy-substituted phenyl, amino-substituted phenyl, or ester-substituted phenyl; In formula II, R 3 , R 4 is hydrogen; In formula III, R' is -O-(CH2) n X-substituted phenyl, X is a halogen, and n is a positive integer; In Formula III, R 2 , R 5 is independently hydrogen, halogen, alkyl, alkyloxy, alkylcarbonyl, alkylthio, aryl, cyano, or nitro; the aryl is phenyl, hydroxy-substituted phenyl, methoxy-substituted phenyl, amino-substituted phenyl, or ester-substituted phenyl; In Formula III, R 3 , R 4 For hydrogen.

2. The method for preparing the pyrrole trimer according to claim 1, characterized in that: R in Formula II and Formula III 2 , R 5 Each is a methyl group and an ethyl group; The R 1 It is phenyl, hydroxy-substituted phenyl, methoxy-substituted phenyl, dimethylamino-substituted phenyl, COOCH2CH3-substituted phenyl; each substituent in the substituted phenyl is para-substituted; R' is -O-(CH2) n X-substituted phenyl, X is Cl, Br, I, n is 2 to 15; each substituent in the substituted phenyl is para-substituted; After oxidation of formula III, the tertiary amine is N(R″)3, where R″ is an alkyl group; specifically, trimethylamine or triethylamine.

3. The method for preparing the pyrrole trimer according to claim 1, characterized in that: The oxidation refers to oxidation with an oxidant; the oxidant is copper perchlorate, copper chloride, ferric chloride, silver hexafluoroantimonate, H2O2 or ammonium cerium nitrate containing or not containing crystalline water; The oxidation is carried out in a solvent; When the oxidant is copper perchlorate, silver hexafluoroantimonate or ferric chloride, the oxidation is carried out using an organic solvent as a reaction medium; When the oxidant is cupric chloride, H2O2 or ammonium cerium nitrate, the oxidation is carried out with water as the reaction medium; When the oxidant is copper perchlorate, the organic solvent is acetonitrile; when the oxidant is silver hexafluoroantimonate, the organic solvent is dichloromethane; when the oxidant is ferric chloride, the organic solvent is methanol.

4. The method for preparing the pyrrole trimer according to claim 1, characterized in that: The oxidation temperature is 20-30°C; The oxidation time is 0.2 to 8 hours; The molar ratio of the compound of formula II or III to the oxidant is 1:0.2 to 1:2; When reacting with a tertiary amine, the reaction temperature is 20-30° C., and the reaction time is 8-12 hours; the molar ratio of the tertiary amine to the compound of formula III is (1.1-4):

1.

5. A pyrrole trimer obtained by the preparation method according to any one of claims 1 to 4, characterized in that: The structure of the pyrrole trimer is Formula I Y - is the anion corresponding to the oxidant; R, R 2 , R 5 is independently hydrogen, halogen, alkyl, alkyloxy, alkylcarbonyl, alkylthio, aryl, cyano, nitro; the aryl is phenyl, hydroxy-substituted + phenyl, methoxy-substituted phenyl, amino-substituted phenyl, ester-substituted phenyl, -O-(CH2) n -N(R″)3X - Substituted phenyl, wherein R" is alkyl, X is halogen, and n is an integer greater than 0.

6. The pyrrole trimer according to claim 5, characterized in that: R 2 , R 5 Each is a methyl group and an ethyl group; The R is phenyl, hydroxy substituted phenyl, methoxy substituted phenyl, dimethylamino substituted phenyl, -COOCH2CH3 substituted phenyl, Substituted phenyl, wherein X is Cl, Br, I, R″ is alkyl, and n is 2 to 15; each substituent in the substituted phenyl is para-substituted.

7. The use of the pyrrole trimer according to claim 5 or 6, characterized in that: The pyrrole trimer is used to prepare an antibacterial agent and / or an antibacterial drug; The antibacterial bacteria are Gram-positive bacteria and Gram-negative bacteria.

8. The use according to claim 7, characterized in that: When R in the pyrrole trimer is phenyl, hydroxy-substituted phenyl, methoxy-substituted phenyl, dimethylamino-substituted phenyl, or -COOCH2CH3-substituted phenyl, the bacteria are Gram-positive bacteria; In the pyrrole trimer, when R is When the phenyl group is substituted, the bacteria are Gram-positive bacteria and / or Gram-negative bacteria.

9. The use according to claim 7, characterized in that: The pyrrole trimer is used for preparing a preparation or a medicine that kills multi-drug resistant bacteria without having drug resistance; or is used for preparing a preparation or a medicine that kills multi-drug resistant bacteria and promotes wound healing.

10. The use according to claim 5 or 6, characterized in that: The pyrrole trimer is used to prepare a fluorescent imaging preparation for distinguishing Gram-positive bacteria from Gram-negative bacteria, and R in the pyrrole trimer is phenyl, hydroxy-substituted phenyl, methoxy-substituted phenyl, dimethylamino-substituted phenyl, or -COOCH2CH3-substituted phenyl.

Images