Broad-spectrum macrocyclic polypeptide compound as well as preparation method and application thereof

Abstract

The invention provides a broad-spectrum macrocyclic polypeptide compound as well as a preparation method and application thereof, the structural formula of the broad-spectrum macrocyclic polypeptide compound is as shown in formula IX: # imgabs0 #. The broad-spectrum macrocyclic polypeptide compound disclosed by the invention has an antibacterial property, has obvious antibacterial activity on multiple bacteria such as staphylococcus aureus CMCC26003, escherichia coli ATCC8099, multi-drug-resistant acinetobacter baumannii and the like, and can be used for preparing a broad-spectrum macrocyclic polypeptide compound. And along with the increase of the sample concentration, the inhibition rate on bacteria is gradually improved.

Description

Technical Field

[0001] The present invention relates to the technical field of macrocyclic peptide compound synthesis, and in particular to a broad-spectrum macrocyclic peptide compound and a preparation method and application thereof. Background Art

[0002] As antibiotics are widely used in clinical practice, multidrug-resistant bacteria continue to emerge and increase. In recent years, the emergence of the most common multidrug-resistant Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii) and Gram-positive bacteria (Staphylococcus aureus) that cause hospital infections has become an increasingly serious problem and challenge worldwide. Faced with the increasingly serious problem of drug resistance, however, sufficient drugs have not yet been developed to solve this problem. Antimicrobial peptides (AMPs) are a class of small molecule peptides with antibacterial activity produced by organisms. They are widely present in nature, including humans, animals, plants and microorganisms. Antimicrobial peptides have a broad spectrum of antibacterial activity and can quickly detect and kill targets, including Gram-negative bacteria, Gram-positive bacteria, fungi, parasites, tumor cells, etc. Their mechanism of action mainly includes destroying the integrity of bacterial cell membranes, causing leakage of cell contents, and thus killing cells. In addition, antimicrobial peptides also have multiple biological functions such as anti-inflammatory, anti-tumor, antiviral and immune regulation.

[0003] Antimicrobial macrocyclic peptides are a class of biomacromolecules with significant antimicrobial activity, and they show great potential in combating multidrug-resistant bacteria. These macrocyclic peptides, through their unique macrocyclic structure, can effectively resist protease hydrolysis and maintain an active conformation to achieve effective target binding. They include nonribosomal peptides (NRPs) and ribosomal postsynthetic modified peptides (RiPPs), such as natamycin, luminol, and thiophanate-buprenorphine, which are used for antibiotic discovery. These macrocyclic peptides have antimicrobial activity against bacteria due to their structural rigidity and high target specificity, while having low toxicity to mammalian cells. The mechanism of action of antimicrobial macrocyclic peptides includes interaction with bacterial cell membranes, leading to cell membrane destruction or dysfunction, and inhibition of bacterial growth by interfering with bacterial cell wall synthesis. For example, some macrocyclic peptides are able to bind to bacterial lipid II, a key precursor for cell wall synthesis, thereby preventing cell wall synthesis. In addition, their antimicrobial efficacy can be enhanced through molecular engineering and self-assembly. In terms of clinical application, some antimicrobial macrocyclic peptides have entered the clinical trial stage, showing therapeutic potential for wound infections, etc. For example, PL-5 (peceleganan) spray has shown efficacy and safety in clinical trials for the treatment of wound infections. In addition, antimicrobial macrocyclic peptides can also enhance their stability and activity by methods such as combining with silver nanoparticles. In terms of new drug discovery, with the advancement of sequencing technology, a large amount of microbial genome data has provided new opportunities for the discovery of new peptide antibiotics through genome mining. For example, through genome mining strategies, researchers have successfully revealed previously unknown peptidases and post-modification enzymes involved in the biosynthesis of RiPP antibiotics, which helps to discover new antimicrobial macrocyclic peptides.

[0004] In general, antibacterial macrocyclic peptides have important application prospects in the development of new antibiotics to combat drug-resistant bacteria due to their unique structure and mechanism of action. With further research and development of these macrocyclic peptides, they are expected to become powerful tools for the treatment of bacterial infections in the future. Summary of the invention

[0005] In view of the shortcomings of the prior art described above, the purpose of the present invention is to provide a broad-spectrum macrocyclic peptide compound and a preparation method and application thereof. The broad-spectrum macrocyclic peptide compound provided by the present invention has broad-spectrum antibacterial properties and provides a research basis for the screening of new antibacterial drugs.

[0006] In order to achieve the above-mentioned and other related purposes, the present invention provides a broad-spectrum macrocyclic polypeptide compound, the structural formula of which is shown in Formula IX:

[0007] The broad-spectrum macrocyclic polypeptide compound of the present invention has antibacterial properties and has obvious antibacterial activity against Staphylococcus aureus CMCC26003, Escherichia coli ATCC8099, multidrug-resistant Acinetobacter baumannii and other bacteria, and its inhibition rate on bacteria gradually increases with the increase of sample concentration.

[0008] The present invention also provides a method for preparing the above broad-spectrum macrocyclic polypeptide compound, comprising: S1: Under room temperature, Fmoc-Phe-OH as shown in formula II and dichloro resin as shown in formula III are added to a dichloromethane solution and mixed, N,N-diisopropylethylamine is added, and after the substitution reaction is completed, the solution is washed with dimethylformamide to obtain a compound A as shown in formula IV; S2: Under room temperature, remove the Fmoc protecting group in compound A, and wash with dimethylformamide after the reaction to obtain compound B as shown in formula V; S3: using a solid phase synthesis method, Fmoc-Gly-OH, a peptide coupling reagent, and compound B are mixed in dimethylformamide, and then N,N-diisopropylethylamine is added; after removing the Fmoc protecting group; Boc-Trp(2-Py)-OH, a peptide coupling reagent, and compound B are mixed in dimethylformamide, and then N,N-diisopropylethylamine is added to synthesize a tripeptide chain as shown in formula VI; wherein the order of extension from the N-terminus to the C-terminus of the amino acids is: Boc-Trp(2-Py)-OH, Gly, Phe; S4: adding the tripeptide chain obtained in S3 to a mixed solution of hexafluoroisopropanol and dichloromethane to obtain a compound C as shown in Formula VII; S5: Under room temperature, compound C and 2-acryloylserine methyl ester hydrochloride as shown in formula I are added to dimethylformamide, and after mixing, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, and N,N-diisopropylethylamine are added to carry out condensation reaction to obtain compound D as shown in formula VIII; S6: adding compound D to dimethylformamide, adding copper acetate, silver hexafluoroantimonate, and dichloro(pentamethylcyclopentadienyl)rhodium dimer to carry out CH activation reaction to obtain a broad-spectrum macrocyclic polypeptide compound as shown in Formula IX;

[0009] The present invention uses a CH activation method to prepare a broad-spectrum macrocyclic polypeptide compound represented by Formula IX. First, Fmoc-Phe-OH (alanine containing a protective group) and dichlororesin are subjected to a substitution reaction in a dichloromethane solution under the action of N,N-diisopropylethylamine to generate a compound A as shown in Formula IV, and the amino acid is successfully linked to the resin carrier. The Fmoc protecting group is then removed, and the deprotection is to allow the subsequently added amino acid to be effectively coupled with the exposed amino group.

[0010] On the compound B shown in formula V, Fmoc-Gly-OH is first added in the order from the N-terminus to the C-terminus, and then Boc-Trp(2-Py)-OH is added. A peptide coupling reagent is used as a coupling agent, and N,N-diisopropylethylamine is used as a base. The tripeptide chain shown in formula VI is obtained by solid phase synthesis using a polypeptide resin. This step constructs a tripeptide chain of a specific sequence, which is the basis for the subsequent macrocyclization reaction. After the synthesis of the tripeptide chain is completed, the tripeptide compound is cut off from the resin, and the solid-phase synthesized tripeptide chain is released, making it free and ready for the next reaction. Subsequently, condensation is performed to prepare for the construction of a macrocyclic structure. Finally, the compound D shown in formula VIII is dissolved in dimethylformamide, and copper acetate, silver hexafluoroantimonate, and dichloro (pentamethylcyclopentadienyl) rhodium dimer are added. The compound is cyclized through a CH activation reaction, thereby obtaining the final target compound shown in formula IX, that is, a macrocyclic polypeptide compound with broad-spectrum antibacterial activity.

[0011] The overall scheme proposed in this invention aims to efficiently and accurately synthesize the target molecule while retaining and enhancing its antibacterial activity. Through reasonable selection of protecting groups, deprotection timing, and specific cyclization strategies, a macrocyclic polypeptide compound with excellent antibacterial properties is finally prepared.

[0012] Preferably, in step S1, the molar ratio of Fmoc-Phe-OH, dichlororesin and dichloromethane is (2-3):1:(3-4), more preferably 3:1:4.

[0013] Preferably, in step S3, the molar ratio of Fmoc-Gly-OH, peptide coupling reagent, N,N-diisopropylethylamine and compound B is (2-3):3:3:1, more preferably 3:3:3:1, and the molar ratio of Boc-Trp(2-Py)-OH, peptide coupling reagent, N,N-diisopropylethylamine and compound B is (2-3):3:3:1, more preferably 3:3:3:1.

[0014] Preferably, in steps S2 and S3, the Fmoc protecting group is removed by eluting the Fmoc protecting group with a mixed solution of piperidine and dimethylformamide.

[0015] Preferably, in the mixed solution of piperidine and dimethylformamide, the volume ratio of piperidine to dimethylformamide is 1:(2-3), more preferably 1:3.

[0016] Preferably, in step S4, in the mixed solution of hexafluoroisopropanol and dichloromethane, the volume ratio of hexafluoroisopropanol to dichloromethane is 1:(2-3), more preferably 1:3.

[0017] Preferably, in step S5, the molar ratio of compound C, 2-acryloylserine methyl ester hydrochloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, p-hydroxy 1-hydroxybenzotriazole, and N,N-diisopropylethylamine is 1:1:(1.5-2):1.5:3, more preferably 1:1:1.5:1.5:3; Preferably, in step S5, after the condensation reaction is completed, a separation and purification process is further included, and the separation and purification conditions are: column chromatography, using a mixed solution of dichloromethane:methanol in a volume ratio of (15-20):1 as the eluent.

[0018] Preferably, in step S6, the ratio of the amount of compound D, copper acetate, silver hexafluoroantimonate, and dichloro(pentamethylcyclopentadienyl)rhodium dimer is 1:(1-2):0.05:0.05, more preferably 1:2:0.05:0.05; the reaction solvent is dimethylformamide, and the reaction temperature is 35-37°C, more preferably 37°C.

[0019] Preferably, in step S6, after the activation reaction is completed, a separation and purification process is further included, and the separation and purification conditions are: column chromatography, using a mixed solution of dichloromethane:methanol in a volume ratio of (15-20):1 as the eluent.

[0020] The present invention also provides an application of the broad-spectrum macrocyclic polypeptide compound in the preparation of antibacterial drugs.

[0021] The broad-spectrum macrolide compound of the present invention has stronger bactericidal activity against multidrug-resistant strains of Acinetobacter baumannii than imipenem, and has a strong antibacterial effect against Staphylococcus aureus CMCC26003, Escherichia coli ATCC8099, and multidrug-resistant Acinetobacter baumannii. Based on the antibacterial properties of macrolide compounds, they can be used in many antibacterial scenarios, such as as antibacterial drugs to treat infections caused by Gram-positive bacteria and Gram-negative bacteria.

[0022] As described above, the present invention has the following beneficial effects: (1) The broad-spectrum macrocyclic polypeptide compound of the present invention has antibacterial properties and has obvious antibacterial activity against Staphylococcus aureus CMCC26003, Escherichia coli ATCC8099, multidrug-resistant Acinetobacter baumannii and other bacteria, and its inhibition rate on bacteria gradually increases with the increase of sample concentration; (2) The present invention introduces double bonds into the molecular structure of the polypeptide compound in advance, and then constructs a new broad-spectrum macrocyclic peptide compound in one step through a CH activation reaction. The preparation process is simple, the yield is high, the process conditions are easy to control, and it is easy to industrialize; (3) Based on the antibacterial properties of the macrolide compounds, the broad-spectrum macrolide compounds of the present invention can be used in many antibacterial scenarios, such as as antibacterial drugs to treat infections caused by Gram-positive bacteria and Gram-negative bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The H NMR spectrum of compound D represented by formula VIII is shown.

[0024] Figure 2 Shown is the NMR carbon spectrum of compound D represented by formula VIII.

[0025] Figure 3 is the H NMR spectrum of the broad-spectrum macrocyclic polypeptide compound represented by Formula IX; Figure 4 It is the NMR carbon spectrum of the broad-spectrum macrocyclic polypeptide compound represented by formula IX. DETAILED DESCRIPTION

[0026] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0027] It should be noted that the process equipment or devices not specifically specified in the following embodiments are all conventional equipment or devices in the art.

[0028] In addition, it should be understood that one or more method steps mentioned in the present invention do not exclude the existence of other method steps before or after the combination step or the insertion of other method steps between these explicitly mentioned steps, unless otherwise specified; it should also be understood that the combination connection relationship between one or more devices / apparatuses mentioned in the present invention does not exclude the existence of other devices / apparatuses before or after the combination device / apparatus or the insertion of other devices / apparatuses between these explicitly mentioned two devices / apparatuses, unless otherwise specified. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or the scope of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the present invention without substantially changing the technical content.

[0029] Example 1 The present application provides a method for preparing a broad-spectrum macrocyclic polypeptide compound, comprising the following steps: S1: Under room temperature, 3.48 g (9 mmol) of Fmoc-Phe-OH as shown in formula II and 3 g (3 mmol, degree of substitution 1.0 mmol / g) of dichlororesin as shown in formula III were placed in a solid phase synthesis reactor, 30 mL of dichloromethane was added, and finally 1.55 g (12 mmol) of N,N-diisopropylethylamine was added. After nitrogen was blown for reaction for 2 hours, 3 mL of methanol was added and reacted for 10 minutes. The reaction solution was washed with dimethylformamide for 3 times, 30 mL each time, to obtain compound A as shown in formula IV, which was directly used in the next step after washing; S2: Add 30 mL of a mixed solution of piperidine and dimethylformamide containing 25% piperidine by volume to the compound A of formula IV, deprotect for 30 minutes, and wash with dimethylformamide 4 times, 30 mL each time, to obtain a compound B of formula V, which is washed and used directly in the next step; S3: A solid phase synthesis method is used to synthesize a tripeptide chain as shown in Formula VI from a compound B as shown in Formula V. The operation steps are as follows: 3 mmol of the compound B as shown in Formula V, 2.8 g (9 mmol) of Fmoc-Gly-OH, and 3.42 g (9 mmol) of a peptide coupling reagent are added to 30 mL of dimethylformamide, and finally 1.16 g (9 mmol) of N,N-diisopropylethylamine is added to react for 1 hour. The ninhydrin detection solution shows that the reaction is colorless, indicating that the reaction is safe. The product is washed with dimethylformamide for 3 times, 30 mL each time, and directly used in the next step of the reaction; 30 mL of a mixed solution of piperidine and dimethylformamide containing 25% volume fraction of piperidine is added, and the product is deprotected for 30 minutes. The product is washed with dimethylformamide for 4 times, 30 mL each time, and directly used in the next step of the reaction; Add Boc-Trp(2-Py)-OH 3.4 g (9 mmol), peptide coupling reagent 3.42 g (9 mmol), dimethylformamide 30 mL, and finally add N,N-diisopropylethylamine 1.16 g (9 mmol) to react for 1 hour. The ninhydrin test solution shows colorless, indicating that the reaction is safe. Wash with dimethylformamide 3 times and methanol 3 times, each time 30 mL, to synthesize a tripeptide chain as shown in Formula VI, drain and use directly in the next reaction; S4: suspend the tripeptide chain shown in Formula VI in 50 mL of a mixed solution of hexafluoroisopropanol and dichloromethane containing 25% by volume of hexafluoroisopropanol, react for 2 hours, filter, wash the resin with dichloromethane 3 times, 50 mL each time, combine the organic phases, and spin-dry to obtain 1.4 g of a white foamy solid, which is Compound C shown in Formula VII; S5: accurately weigh 1.017 g (2 mmol) of compound C as shown in formula VII, 209 mg (2 mmol) of 2-acryloylserine methyl ester hydrochloride as shown in formula I, 298 mg (3 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 200 mg (3 mmol) of 1-hydroxybenzotriazole into a 100 mL round-bottom flask, add 20 mL of dimethylformamide solution, and finally add 774 mg (6 mmol) of N,N-diisopropylethylamine to react at room temperature for 3 hours. After the reaction is complete as monitored by TLC, dichloromethane and water are added to extract and collect the organic phase layer. The organic phase is washed once with 3% phosphoric acid aqueous solution, saturated sodium bicarbonate and saturated sodium chloride, each time 50 mL, and dried over anhydrous sodium sulfate; filtered, the solvent is removed by rotary evaporation under reduced pressure, and the concentrate is separated by silica gel column (dichloromethane: methanol = 20:1, v / v), and 650 mg of white foam compound is collected, which is compound D shown in formula VIII; The H NMR spectrum of compound D shown in formula VIII is as follows Figure 1 As shown: 1 H NMR (600MHz, DMSO) δ8.55(dd,J=4.8,1.2Hz,1H),8.46(d,J=7.7Hz,1H),8.41(t,J=5.6Hz,1H),8.36(d,J=8.3Hz,1H),8.16(d,J=7.8Hz,1H),7.9 9–7.95(m,1H),7.76(s,1H),7.65(d,J=7.8Hz,1H),7.62(d,J=8.3Hz,1H ),7.30–7.23(m,6H),7.22–7.15(m,2H),6.80(d,J=8.4Hz,1H),6.35(dd ,J=17.3,1.3Hz,1H),6.14(dd,J=17.3,10.4Hz,1H),5.97(dd,J=10.5,1.4Hz,1H),4.69(dd,J=12.5,5.2Hz,1H),4.59(dd,J=12.9,8.5Hz,1H),4 .40–4.33(m,2H),4.32–4.24(m,1H),3.80(d,J=3.6Hz,2H),3.66(s,3H) ,3.12–3.01(m,1H),2.96–2.89(m,1H),2.88–2.84(m,1H),1.26(s,9H).

[0030] The NMR carbon spectrum of compound D shown in formula VIII is as follows Figure 2 As shown: 13 C NMR (151MHz, DMSO) δ183.53,171.84,170.15,169.43,165.56,162.78,155.5 5,152.50,148.99,139.56,138.10,135.14,132.72,130.52,129.76,128.49, 128.23,126.74,124.91,123.44,121.26,120.40,119.51,115.08,114.38,114.06,78.68,63.53,52.76,51.55,42.08,38.09,36.24,31.24,28.49,28.02.

[0031] S6: Accurately weigh 148 mg (0.2 mmol) of compound D shown in formula VIII, copper acetate (72 mg, 0.4 mmol), silver hexafluoroantimonate (25 mg, 0.01 mmol), and dichloro(pentamethylcyclopentadienyl)rhodium dimer 6 mg (0.01 mmol) in a 100 mL round-bottom flask, and add 10 mL of dimethylformamide solution. Cover with a rubber stopper and incubate at 37°C for 5 hours. Add 40 mL of ethyl acetate and 20 mL of water to the reaction solution. The organic layer is washed with 20 mL of 1N hydrochloric acid, 20 mL of saturated sodium bicarbonate, and 20 mL of saturated sodium chloride solution, respectively, and dried with anhydrous sodium sulfate, filtered, and concentrated in vacuo. The concentrate is separated by silica gel column (dichloromethane: methanol = 20:1, v / v), and 80 mg of white foamy compound is collected, which is the broad-spectrum macrocyclic polypeptide compound shown in formula IX.

[0032]

[0033] The H NMR spectrum of the broad-spectrum macrocyclic polypeptide compound represented by Formula IX is as follows: Figure 3 As shown: 1 H NMR (600 MHz, DMSO) 1H NMR (600MHz, DMSO) δ8.71 (dd, J=4.8, 1.2Hz, 1H), 8.38 (t, J=6.1Hz, 1H), 8.24 (d, J=7 .1Hz,1H),8.09(td,J=7.8,1.9Hz,1H),7.98(d,J=8.0Hz,1H),7.94(d,J=8.7Hz,1H), 7.89(d,J=16.4Hz,1H),7.56(ddd,J=7.5,4.9,0.9Hz,1H),7.45(d,J=7.9Hz,1H),7.3 1(t,J=6.3Hz,1H),7.29–7.26(m,1H),7.21–7.18(m,1H),7.12(t,J=7.3Hz,1H),7.05 (t,J=7.5Hz,2H),6.98(d,J=7.3Hz,2H),5.52(d,J=16.3Hz,1H),4.94–4.86(m,1H), 4.40(dd,J=11.2,4.7Hz,1H),4.28–4.16(m,3H),3.97(dd,J=16.8,6.8Hz,1H),3.72( s,3H),3.60(dd,J=16.7,5.3Hz,1H),3.53(dd,J=14.4,5.3Hz,1H),3.24(dd,J=14.3, 8.0Hz, 1H), 3.05 (dd, J=13.8, 4.6Hz, 1H), 2.81 (dd, J=13.7, 9.7Hz, 1H), 1.25 (s, 9H).

[0034] The NMR carbon spectrum of the broad-spectrum macrocyclic polypeptide compound represented by Formula IX is as follows Figure 4 As shown: 13 C NMR(151MHz,DMSO)δ171.52,171.29,169.90,169.62,165.99,155.70,15 0.93,150.26,139.97,138.96,138.49,134.18,130.88,129.48,128.99,1 28.56,126.68,125.82,123.88,122.53,121.69,121.49,121.23,116.72 ,111.05,78.66,56.03,55.17,52.93,51.09,43.22,36.60,28.48,26.77.

[0035] From the above detection, it can be obtained that the structural formula of the broad-spectrum macrocyclic polypeptide compound prepared in this example is shown in Formula IX:

[0036] The present application also tested the bactericidal activity of the broad-spectrum macrocyclic peptide compound shown in Formula IX: The various strains used in the following examples were purchased from the China National Institute for the Control of Biological Products. The bactericidal activity of the cationic antibacterial cyclic peptide was tested by agar punching method, with the linear peptide shown in Formula VIII as a control, to evaluate the bactericidal activity of the broad-spectrum macrocyclic peptide compound shown in Formula IX of the present invention.

[0037] The bactericidal activity of antimicrobial peptides was determined according to the following steps: Bacterial recovery: Inoculate resistant Acinetobacter baumannii and streak it on NA nutrient agar medium, then culture it in a constant temperature incubator at 37°C for 16-20 hours.

[0038] Bacterial culture: Pick a single colony and place it in 100 mL MHB medium for culture. The culture temperature is also the optimal temperature for bacterial growth, 37°C. The shaker speed is 160 rpm and the culture is shaken (16-20 hours) until the bacterial growth state reaches the logarithmic phase.

[0039] Preparation of bacterial suspension: The concentration of bacteria is generally determined by McFarland turbidimetric tube: the turbidity of the McFarland turbidimetric tube is about 0.5 McFarland turbidity; at this time, the number of bacterial colonies is about 1.5x10 8 cfu / mL, and then diluted to 10 at 1:1000. 5 -10 6 cfu / mL of bacterial suspension.

[0040] Antibacterial experiment: The diluted bacterial suspension was evenly applied to 25mL NA culture medium at a volume of 0.1mL / plate; after the bacterial solution solidified, a hole (9mm in diameter) was punched. For the positive control, 50μL (0.4mg / mL, 0.2mg / mL, 0.1mg / mL) of imipenem was added; for the negative control, 50μL of deionized water was added; for the other holes, 50μL (0.4mg / mL, 0.2mg / mL, 0.1mg / mL) of peptide solution was added. The bacteria were cultured in a 37°C constant temperature incubator, and the size of the bacterial inhibition zone was measured after 16 hours, so that the antibacterial activity of the bacteria could be preliminarily determined. Three sets of parallel experiments were performed, and the test results are shown in Table 1: Table 1. Diameters of inhibition zones of different concentrations of broad-spectrum macrocyclic peptide compounds against multidrug-resistant strains of Acinetobacter baumannii (mm) From Table 1 above, it can be seen that the broad-spectrum macrocyclic peptide compound IX of the present invention has obvious bactericidal ability and is superior to the control linear peptide VIII, especially at a high concentration of 0.4 mg / mL, it is superior to imipenem.

[0041] The present application also tested the antibacterial activity of the broad-spectrum macrocyclic polypeptide compound shown in Formula IX: The various strains used in the following examples were purchased from the China National Institute for the Control of Biological Products. The minimum antibacterial capacity of the broad-spectrum macrolide compound was determined, and the linear peptide shown in Formula VIII before cyclization was used as a control to evaluate the antibacterial capacity of the broad-spectrum macrolide compound of the present invention.

[0042] The antibacterial activity of the broad-spectrum macrolide compound represented by Formula IX was determined according to the following steps: Bacteria growing in the logarithmic phase were collected and centrifuged at 8000 rpm for 2 min at 4°C. The mixture was washed three times with saline and fresh broth medium was added to make the bacterial suspension concentration 2.0 x 10 5 cfu / mL. Add 50 μL of bacterial suspension to the experimental wells of the 96-well cell culture plate (add 100 μL PBS to the surrounding wells), and then add 50 μL of peptide solution (imipenem solution) of different concentrations, so that the final concentration (μg / mL) of the peptide solution (imipenem solution) in each well in the horizontal row is: 512, 256, 128, 64, 32, 16, 12, 4. The same volume of PBS buffer was used as the growth control group, and three parallel groups were set up in each group. After the cell culture plate was covered, it was placed in a biochemical incubator at 37°C for 12 hours, and the bacterial growth in each well was determined by fully automatic enzyme labeling (OD600nm). The results are shown in Table 2: Table 2. Comparison of MICs of broad-spectrum macrocyclic peptide compounds represented by Formula IX against different bacteria The minimum inhibitory concentration (MIC) is defined as the peptide concentration at which bacterial growth is completely inhibited. The smaller the minimum inhibitory concentration value in Table 2, the stronger the antibacterial ability of the peptide.

[0043] As can be seen from Table 2, compared with the control straight-chain peptide, the broad-spectrum macrocyclic peptide compound represented by Formula IX of the present invention has a lower minimum inhibitory concentration, and the MIC is much smaller than that of the straight-chain peptide represented by Formula VIII, indicating that the antibacterial ability of the broad-spectrum macrocyclic peptide compound represented by Formula IX of the present invention is much stronger than that of the control straight-chain peptide represented by VIII.

[0044] The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and evolutions of the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A broad-spectrum macrocyclic polypeptide compound, characterized in that: Its structural formula is shown in Formula IX:

2. A method for preparing a broad-spectrum macrocyclic polypeptide compound as claimed in claim 1, characterized in that: include: S1: Under room temperature, Fmoc-Phe-OH as shown in formula II and dichloro resin as shown in formula III are added to a dichloromethane solution and mixed, N,N-diisopropylethylamine is added, and after the substitution reaction is completed, the solution is washed with dimethylformamide to obtain a compound A as shown in formula IV; S2: Under room temperature, remove the Fmoc protecting group in compound A, and wash with dimethylformamide after the reaction to obtain compound B as shown in formula V; S3: using a solid phase synthesis method, Fmoc-Gly-OH, a peptide coupling reagent, and compound B are mixed in dimethylformamide, and then N,N-diisopropylethylamine is added; after removing the Fmoc protecting group; Boc-Trp(2-Py)-OH, a peptide coupling reagent, and compound B are mixed in dimethylformamide, and then N,N-diisopropylethylamine is added to synthesize a tripeptide chain as shown in formula VI; wherein the order of extension from the N-terminus to the C-terminus of the amino acids is: Boc-Trp(2-Py)-OH, Gly, Phe; S4: adding the tripeptide chain obtained in S3 to a mixed solution of hexafluoroisopropanol and dichloromethane to obtain a compound C as shown in Formula VII; S5: Under room temperature, compound C and 2-acryloylserine methyl ester hydrochloride as shown in formula I are added to dimethylformamide, and after mixing, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, and N,N-diisopropylethylamine are added to carry out condensation reaction to obtain compound D as shown in formula VIII; S6: Compound D is added to dimethylformamide, and copper acetate, silver hexafluoroantimonate, and dichloro(pentamethylcyclopentadienyl)rhodium dimer are added to perform a CH activation reaction to obtain a broad-spectrum macrocyclic polypeptide compound as shown in Formula IX.

3. The preparation method according to claim 2, characterized in that: In step S1, the molar ratio of Fmoc-Phe-OH, dichlororesin and dichloromethane is (2-3):1:(3-4).

4. The preparation method according to claim 2, characterized in that: In step S3, the molar ratio of Fmoc-Gly-OH, peptide coupling reagent, N,N-diisopropylethylamine and compound B is (2-3):3:3:1, and the molar ratio of Boc-Trp(2-Py)-OH, peptide coupling reagent, N,N-diisopropylethylamine and compound B is (2-3):3:3:

1.

5. The preparation method according to claim 2, characterized in that: In steps S2 and S3, the Fmoc protecting group is removed by eluting the Fmoc protecting group with a mixed solution of piperidine and dimethylformamide.

6. The preparation method according to claim 5, characterized in that: In the piperidine and dimethylformamide mixed solution, the volume ratio of piperidine to dimethylformamide is 1:(2-3).

7. The preparation method according to claim 2, characterized in that: In step S4, in the mixed solution of hexafluoroisopropanol and dichloromethane, the volume ratio of hexafluoroisopropanol to dichloromethane is 1:(2-3).

8. The preparation method according to claim 2, characterized in that: In step S5, the molar ratio of compound C, 2-acryloylserine methyl ester hydrochloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, p-hydroxy 1-hydroxybenzotriazole, and N,N-diisopropylethylamine is 1:1:(1.5-2):1.5:3; After the condensation reaction is completed, a separation and purification step is also included. The separation and purification conditions are: column chromatography, using a mixed solution of dichloromethane: methanol in a volume ratio of (15-20):1 as an eluent.

9. The preparation method according to claim 2, characterized in that: In step S6, the ratio of the amount of compound D, copper acetate, silver hexafluoroantimonate, and dichloro(pentamethylcyclopentadienyl)rhodium dimer is 1:(1-2):0.05:0.05; the reaction solvent is dimethylformamide, and the reaction temperature is 35-37°C; after the activation reaction is completed, a separation and purification step is also included, and the conditions for the separation and purification are: column chromatography, with a mixed solution of dichloromethane: methanol in a volume ratio of (15-20):1 as the eluent.

10. Use of the broad-spectrum macrocyclic polypeptide compound as claimed in claim 1 in the preparation of antibacterial drugs.

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