Thirteen-peptide derivatives, methods of making and using the same

Abstract

The application discloses a decapeptide derivative, a preparation method and application thereof. The application protects a Paenibacillus polymyxa, that is, Paenibacillus polymyxa CPCC 101223, and the preservation registration number is CGMCC No. 22854. The application also protects a preparation method of the compound, which comprises the following steps: fermenting and culturing the Paenibacillus polymyxa CPCC 101223 to obtain the compound. The application also protects application of the compound or a pharmaceutically acceptable salt thereof in preparation of a bacterial inhibitor. The application also protects the bacterial inhibitor, which contains the compound or the pharmaceutically acceptable salt thereof. The compound of the application is derived from the Paenibacillus polymyxa, has a simple preparation process, has good activity against gram-negative drug-resistant bacteria, provides a theoretical basis for clarifying the structure-activity relationship of the decapeptide antibacterial activity, and is suitable for research on a gram-negative drug-resistant bacteria leading compound or preparation of a gram-negative drug-resistant bacteria drug.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to deca-peptide derivatives, their preparation methods, and applications. Background Technology

[0002] In recent years, the increasingly serious problem of bacterial resistance caused by various factors, such as the emergence of "superbugs," has made bacterial resistance and drug-resistant bacterial infections a huge challenge facing the global field of anti-infective treatment and antimicrobial drug development.

[0003] In 2017, the WHO released its first list of priority pathogens for which new antibiotics are urgently needed. Category 1 included carbapenem-resistant Acinetobacter baumannii (CRAB), Pseudomonas aeruginosa (CRPA), and Enterobacteriaceae (CRE). These "superbugs" are all Gram-negative drug-resistant bacteria. Currently, there are few effective drugs available for treating Gram-negative drug-resistant infections, and polymyxins are often used as a "last line of defense." In recent years, the polymyxin resistance gene mcr-1 has been discovered in Klebsiella pneumoniae and Escherichia coli, challenging the clinical effectiveness of polymyxins. Developing new drugs against Gram-negative drug-resistant bacteria or their lead compounds is therefore extremely urgent. Summary of the Invention

[0004] The purpose of this invention is to provide a decatheptide derivative, its preparation method, and its application.

[0005] This invention provides a strain of polymyxa, namely Paenibacillus polymyxa CPCC 101223, which was deposited on July 8, 2021, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with accession number CGMCC No. 22854.

[0006] The present invention also provides a method for preparing a general formula compound, comprising the following steps: fermenting and culturing Bacillus polymyxa CPCC 101223 to obtain the general formula compound.

[0007] Specifically, the preparation method includes the following steps: fermenting Bacillus polymyxa CPCC101223 in a culture medium to obtain a fermentation product, and obtaining the general formula compound from the fermentation product.

[0008] The culture medium may specifically be a microbial culture medium.

[0009] The culture medium may specifically be a fermentation culture medium.

[0010] The method for fermenting and culturing Bacillus polymyxa CPCC 101223 using a culture medium specifically includes the following steps:

[0011] (1) Inoculate Bacillus polymyxa CPCC 101223 into the fermentation medium and culture with shaking;

[0012] (2) After completing step (1), add macroporous adsorption resin to the system, shake and culture to obtain fermentation product.

[0013] The method for fermenting and culturing Bacillus polymyxa CPCC 101223 using a culture medium specifically includes the following steps:

[0014] (1) Inoculate Bacillus polymyxa CPCC 101223 into the fermentation medium and culture with shaking for 20 h;

[0015] (2) After completing step (1), add macroporous adsorption resin to the system and shake and culture for 4 hours to obtain fermentation product.

[0016] The method for fermenting and culturing Bacillus polymyxa CPCC 101223 using a culture medium specifically includes the following steps:

[0017] (1) Inoculate 100 mL of Bacillus polymyxa CPCC 101223 seed culture into 1 L of fermentation medium and culture with shaking for 20 h;

[0018] (2) After completing step (1), add 100 mL of macroporous adsorption resin to the system and shake and culture for 4 h to obtain the fermentation product.

[0019] The method for fermenting and culturing Bacillus polymyxa CPCC 101223 using a culture medium specifically includes the following steps:

[0020] (1) Inoculate 100 mL of Bacillus polymyxa CPCC 101223 seed culture into 1 L of fermentation medium and culture at 30 °C and 200 r / min for 20 h with shaking.

[0021] (2) After completing step (1), add 100 mL of macroporous adsorption resin to the system and culture at 30 °C and 200 r / min for 4 h to obtain the fermentation product.

[0022] The method for preparing any of the above-mentioned seed solutions includes the following steps: picking a single colony of Bacillus polymyxa CPCC101223, inoculating it into a seed culture medium, and culturing it with shaking to obtain the seed solution.

[0023] The method for preparing any of the above-mentioned seed liquids includes the following steps: picking a single colony of Bacillus polymyxa CPCC101223, inoculating it into a seed culture medium, and shaking it for 16 hours to obtain the seed liquid.

[0024] The method for preparing any of the above-mentioned seed liquids includes the following steps: picking a single colony of Bacillus polymyxa CPCC101223, inoculating it into 100 mL of seed culture medium, and culturing it at 30 °C and 200 r / min for 16 h with shaking to obtain the seed liquid.

[0025] Seed culture medium (TSB medium): contains 15 g / L tryptone, 5 g / L soybean peptone, 5 g / L sodium chloride, with the remainder being water; pH is 7.0-7.4.

[0026] Fermentation medium: containing 30 g / L corn flour, 10 g / L soluble starch, 1 g / L yeast powder, 5 g / L ammonium sulfate, 10 g / L calcium carbonate, with the remainder being water; pH 7.0-7.4.

[0027] The method for obtaining the general formula compound from the fermentation product includes the following steps:

[0028] (1) Take the fermentation product, collect the macroporous adsorption resin and transfer it to the chromatography column, then elute the chromatography column with deionized water, 40% isopropanol aqueous solution and 80% isopropanol aqueous solution in sequence, and collect the eluent after elution with 80% isopropanol aqueous solution.

[0029] (2) Take the column eluent obtained in step (1), load it onto a solid sample and perform chromatographic separation and purification to obtain Fr.4 and / or Fr.5;

[0030] (3) Take the Fr.4 obtained in step (2), load it onto a solid sample and perform chromatographic separation and purification to obtain Fr.4-4 and / or Fr.4-5 and / or Fr.4-6 and / or Fr.4-7 and / or Fr.4-8 and / or Fr.4-10 and / or Fr.4-11 and / or Fr.4-12 and / or Fr.4-13;

[0031] (4) Take Fr.5 and / or Fr.4-4 and / or Fr.4-5 and / or Fr.4-6 and / or Fr.4-7 and / or Fr.4-8 and / or Fr.4-10 and / or Fr.4-11 and / or Fr.4-12 and / or Fr.4-13, and purify them by liquid chromatography to obtain the compound of the general formula.

[0032] Step (1) of the method for obtaining the general formula compound from the fermentation product is as follows: Take 24L of the fermentation product, collect the macroporous adsorption resin and transfer it to a chromatography column (column volume 2L), then elute the chromatography column sequentially with 10L of deionized water, 20L of 40% isopropanol aqueous solution and 30L of 80% isopropanol aqueous solution, and collect the eluent after elution with 80% isopropanol aqueous solution.

[0033] In step (1), the elution flow rate is 2 times the column volume / h.

[0034] Step (2) of the method for obtaining the general formula compound from the fermentation product is as follows: take the eluent collected in step (1) after column chromatography, concentrate it, then add the swollen YMC ODS-AQ-HG packing material and mix well, then concentrate and evaporate to dryness to obtain a solid sample; fill the solid sample into the sample column, then connect the chromatographic column and separate and purify it using a chromatograph.

[0035] Step (2) of the method for obtaining the general formula compound from the fermentation product is as follows: take the eluent collected in step (1) after column chromatography, concentrate it to 100 ml using a rotary evaporator, then add 10 mL of swollen YMC ODS-AQ-HG packing material and mix well, then concentrate and evaporate to dryness to obtain a solid sample; fill the solid sample into a sample column, then connect the chromatographic column and separate and purify it using a chromatographic instrument.

[0036] In step (2) of the method for obtaining the general formula compound from the fermentation product, the chromatographic column is a FlashSpherical C18 20-35μm 100A 120g.

[0037] In step (2) of the method for obtaining the general formula compound from the fermentation product, the chromatograph is a TELEDYNE ISCO Combiflash Rf200 preparative chromatograph.

[0038] In step (2) of the method for obtaining the general formula compound from the fermentation product, the elution program is as follows: 0-20 min, the mobile phase consists of 20% mobile phase B and 80% mobile phase A; 20-35 min, the volume fraction of mobile phase B in the mobile phase increases linearly from 20% to 40%, and the corresponding volume fraction of mobile phase A in the mobile phase decreases linearly from 80% to 60%; 35-70 min, the mobile phase consists of 40% mobile phase B and 60% mobile phase A.

[0039] In step (2) of the method for obtaining the general formula compound from the fermentation product, the elution program is as follows: 0-20 min, the mobile phase consists of 20% mobile phase B and 80% mobile phase A; 20-35 min, the volume fraction of mobile phase B increases linearly from 20% to 40%, and the corresponding volume fraction of mobile phase A decreases linearly from 80% to 60%; 35-70 min, the mobile phase consists of 40% mobile phase B and 60% mobile phase A; 70-100 min, the volume fraction of mobile phase B increases linearly from 40% to 100%, and the corresponding volume fraction of mobile phase A decreases linearly from 60% to 0%; 100-120 min, the mobile phase consists entirely of mobile phase B.

[0040] Mobile phase A: composed of trifluoroacetic acid and water, with a trifluoroacetic acid concentration of 0.05%; Mobile phase B: composed of trifluoroacetic acid and acetonitrile, with a trifluoroacetic acid concentration of 0.05%.

[0041] In step (2) of the method for obtaining the general formula compound from the fermentation product, the mobile phase flow rate is 20 mL / min.

[0042] In step (2) of the method for obtaining the general formula compound from the fermentation product, the detection wavelength is 210 nm.

[0043] Collect the fractions, each fraction corresponding to a post-column solution of an elution peak, to obtain the following fractions: Fr.4 and / or Fr.5.

[0044] Fr.4 corresponds to retention time t R Elution peak at 25-40 min; Fr.5 corresponds to t R Elution peak at 40-45 min.

[0045] Step (3) of the method for obtaining the general formula compound from the fermentation product is as follows: take Fr.4, concentrate it, then add the swollen YMC ODS-AQ-HG packing material, then evaporate it to dryness to obtain a solid sample; fill the solid sample into the sample column, then connect the chromatographic column, and separate and purify it using a chromatograph.

[0046] Step (3) of the method for obtaining the general formula compound from the fermentation product is as follows: Take 300 mL of Fr.4, concentrate it to 10 mL using a rotary evaporator, then add 5 mL of swollen YMC ODS-AQ-HG packing material, and then evaporate it to dryness to obtain a solid sample; fill the solid sample into a sample column, then connect the chromatographic column, and separate and purify it using a chromatograph.

[0047] In step (3) of the method for obtaining the general formula compound from the fermentation product, the chromatographic column is a FlashSpherical C18 20-35μm 100A 120g.

[0048] In step (3) of the method for obtaining the general formula compound from the fermentation product, the chromatograph is a TELEDYNE ISCO Combiflash Rf200 preparative chromatograph.

[0049] In step (3) of the method for obtaining the general formula compound from the fermentation product, the elution program is as follows: 0-40 min, the mobile phase consists of 20% mobile phase B and 80% mobile phase A; 40-60 min, the volume fraction of mobile phase B increases linearly from 20% to 40%, and the corresponding volume fraction of mobile phase A decreases linearly from 80% to 60%; 60-90 min, the mobile phase consists of 40% mobile phase B and 60% mobile phase A; 90-120 min, the volume fraction of mobile phase B increases linearly from 40% to 100%, and the corresponding volume fraction of mobile phase A decreases linearly from 60% to 0%.

[0050] In step (3) of the method for obtaining the general formula compound from the fermentation product, the elution program is as follows: 0-40 min, the mobile phase consists of 20% mobile phase B and 80% mobile phase A; 40-60 min, the volume fraction of mobile phase B increases linearly from 20% to 40%, and the corresponding volume fraction of mobile phase A decreases linearly from 80% to 60%; 60-90 min, the mobile phase consists of 40% mobile phase B and 60% mobile phase A; 90-120 min, the volume fraction of mobile phase B increases linearly from 40% to 100%, and the corresponding volume fraction of mobile phase A decreases linearly from 60% to 0%; 120-140 min, the mobile phase consists entirely of mobile phase B.

[0051] Mobile phase A: composed of trifluoroacetic acid and water, with a trifluoroacetic acid concentration of 0.15%; Mobile phase B: composed of trifluoroacetic acid and acetonitrile, with a trifluoroacetic acid concentration of 0.15%.

[0052] In step (3) of the method for obtaining the general formula compound from the fermentation product, the mobile phase flow rate is 20 mL / min.

[0053] In step (3) of the method for obtaining the general formula compound from the fermentation product, the detection wavelength is 210 nm.

[0054] Collect fractions, each fraction corresponding to a single elution peak after column chromatography, to obtain the following fractions: Fr.4-4 and / or Fr.4-5 and / or Fr.4-6 and / or Fr.4-7 and / or Fr.4-8 and / or Fr.4-10 and / or Fr.4-11 and / or Fr.4-12 and / or Fr.4-13.

[0055] Fr.4-4 corresponds to retention time t R Elution peak at 27-30 min. Fr.4-5 corresponds to retention time t. R Elution peak at 30-46 min. Fr.4-6 corresponds to retention time t. RElution peak at 46-68 min. Fr. 4-7 corresponds to retention time t. R Elution peak at 68-72 min. Fr.4-8 corresponds to retention time t. R The elution peak is observed at 72-74 min. Fr. 4-10 corresponds to retention times t. R Elution peak at 78-85 min. Fr. 4-11 corresponds to retention time t. R Elution peak at 85-92 min. Fr. 4-12 corresponds to retention time t. R Elution peak at 92-102 min. Fr. 4-13 corresponds to retention time t. R Elution peak at 102-110 min.

[0056] In step (4) of the method for obtaining the general formula compound from the fermentation product, the chromatograph is a Shimadzu LC-20 liquid chromatograph (LC-20AD).

[0057] In step (4) of the method for obtaining the general formula compound from the fermentation product, the chromatographic column is a YMC-Pack ODS-AQ, 10mm*250mm, with a pore size of 5μm.

[0058] In step (4) of the method for obtaining the general formula compound from the fermentation product, the mobile phase flow rate is 4 mL / min.

[0059] In step (4) of the method for obtaining the general formula compound from the fermentation product, the detection wavelength is 210 nm.

[0060] Take Fr.5 and / or Fr.4-4 and / or Fr.4-5 and / or Fr.4-6 and / or Fr.4-7 and / or Fr.4-8 and / or Fr.4-10 and / or Fr.4-11 and / or Fr.4-12 and / or Fr.4-13, concentrate and evaporate to dryness, and dissolve in 40% acetonitrile aqueous solution to obtain the concentrated solution.

[0061] The concentrated solution of Fr.4-4 was loaded (the specific loading volume can be 3 mL); the mobile phase consisted of trifluoroacetic acid, acetonitrile and water, with a trifluoroacetic acid concentration of 0.15% and an acetonitrile concentration of 31%; the eluent corresponding to the elution peak with a retention time of 8.21 min was collected, concentrated by rotary evaporator, and then freeze-dried to obtain compound 1.

[0062] The concentrated solution of Fr.4-5 was loaded (the specific loading volume can be 3 mL); the mobile phase consisted of trifluoroacetic acid, acetonitrile and water, with a trifluoroacetic acid concentration of 0.15% and an acetonitrile concentration of 33%; the eluent corresponding to the elution peak with a retention time of 8.37 min was collected, concentrated by rotary evaporator, and then freeze-dried to obtain compound 2.

[0063] The concentrated solution of Fr.4-5 was loaded (the specific loading volume can be 3 mL); the mobile phase consisted of trifluoroacetic acid, acetonitrile and water, with a trifluoroacetic acid concentration of 0.15% and an acetonitrile concentration of 33%; the eluent corresponding to the elution peak with a retention time of 9.24 min was collected, concentrated by rotary evaporator, and then freeze-dried to obtain compound 3.

[0064] The concentrated solution of Fr.4-5 was loaded (the specific loading volume can be 3 mL); the mobile phase consisted of trifluoroacetic acid, acetonitrile and water, with a trifluoroacetic acid concentration of 0.15% and an acetonitrile concentration of 33%; the eluent corresponding to the elution peak with a retention time of 8.37 min was collected, concentrated using a rotary evaporator, and then freeze-dried to obtain compound 2; the eluent corresponding to the elution peak with a retention time of 9.24 min was collected, concentrated using a rotary evaporator, and then freeze-dried to obtain compound 3.

[0065] The concentrated solution of Fr.4-6 was loaded (the specific loading volume can be 3 mL); the mobile phase consisted of trifluoroacetic acid, acetonitrile and water, with a trifluoroacetic acid concentration of 0.15% and an acetonitrile concentration of 34%; the eluent corresponding to the elution peak with a retention time of 9.54 min was collected, concentrated by rotary evaporator, and then freeze-dried to obtain compound 4.

[0066] The concentrated solution of Fr.4-7 was loaded (the specific loading volume can be 3 mL); the mobile phase consisted of trifluoroacetic acid, acetonitrile and water, with a trifluoroacetic acid concentration of 0.15% and an acetonitrile concentration of 35%; the eluent corresponding to the elution peak with a retention time of 9.76 min was collected, concentrated by rotary evaporator, and then freeze-dried to obtain compound 5.

[0067] The concentrated solution of Fr.4-8 was loaded (the specific loading volume can be 3 mL); the mobile phase consisted of trifluoroacetic acid, acetonitrile and water, with a trifluoroacetic acid concentration of 0.15% and an acetonitrile concentration of 39%; the eluent corresponding to the elution peak with a retention time of 7.83 min was collected, concentrated by rotary evaporator, and then freeze-dried to obtain compound 6.

[0068] The concentrated solution of Fr.4-10 was loaded (the specific loading volume can be 3 mL); the mobile phase consisted of trifluoroacetic acid, acetonitrile and water, with a trifluoroacetic acid concentration of 0.15% and an acetonitrile concentration of 40%; the eluent corresponding to the elution peak with a retention time of 8.11 min was collected, concentrated by rotary evaporator, and then freeze-dried to obtain compound 7.

[0069] The concentrated solution of Fr.4-11 was loaded (the specific loading volume can be 2 mL); the mobile phase consisted of trifluoroacetic acid, acetonitrile and water, with a trifluoroacetic acid concentration of 0.15% and an acetonitrile concentration of 40%; the eluent corresponding to the elution peak with a retention time of 8.41 min was collected, concentrated by rotary evaporator, and then freeze-dried to obtain compound 8.

[0070] The concentrated solution of Fr.4-12 was loaded (the specific loading volume can be 2 mL); the mobile phase consisted of trifluoroacetic acid, acetonitrile and water, with a trifluoroacetic acid concentration of 0.15% and an acetonitrile concentration of 40%; the eluent corresponding to the elution peak with a retention time of 8.92 min was collected, concentrated by rotary evaporator, and then freeze-dried to obtain compound 9.

[0071] The concentrated solution of Fr.4-13 was loaded (the specific loading volume can be 2 mL); the mobile phase consisted of trifluoroacetic acid, acetonitrile and water, with a trifluoroacetic acid concentration of 0.15% and an acetonitrile concentration of 41%; the eluent corresponding to the elution peak with a retention time of 9.82 min was collected, concentrated by rotary evaporator, and then freeze-dried to obtain compound 10.

[0072] The concentrated solution of Fr.5 was loaded (the specific loading volume can be 2 mL); the mobile phase consisted of trifluoroacetic acid, acetonitrile and water, with a trifluoroacetic acid concentration of 0.15% and an acetonitrile concentration of 42%; the eluent corresponding to the elution peak with a retention time of 10.37 min was collected, concentrated by rotary evaporator, and then freeze-dried to obtain compound 11.

[0073] Unless otherwise specified, all percentages refer to volume percentages.

[0074] The macroporous adsorption resins mentioned above are all non-polar macroporous adsorption resins.

[0075] The macroporous adsorption resin mentioned above is DIAION HP20 macroporous adsorption resin.

[0076] The macroporous adsorption resin described above shall satisfy any one or any combination of the following indicators: water content (%) 55-65, wet apparent density (g / LR) 680, pore volume (ml / g) 1.3, specific surface area (m²) 2 / g) is 590, good frequency radius The particle size distribution is ≥90%, the effective particle size (mm) is ≥0.25, the uniformity coefficient is ≤1.6, and the operating temperature (°C) is ≤130.

[0077] The present invention also provides compounds of the general formula.

[0078] This invention also protects the use of *Bacillus polymyxa* CPCC 101223 in the preparation of general formula compounds.

[0079] This invention also protects the use of compounds of the general formula or pharmaceutically acceptable salts thereof in the preparation of bacterial inhibitors.

[0080] This invention also protects bacterial inhibitors containing compounds of general formula or pharmaceutically acceptable salts thereof.

[0081] This invention also protects a culture of *Bacillus polymyxa* CPCC 101223, which is a substance obtained by culturing *Bacillus polymyxa* CPCC101223. Specifically, the culturing can be performed using a culture medium. The culture medium can be a microbial culture medium. Specifically, the culture medium can be a fermentation culture medium. Specifically, the fermentation culture medium can be the aforementioned fermentation culture medium. The culture can be the fermentation product obtained by fermenting *Bacillus polymyxa* CPCC101223 using the aforementioned culture medium.

[0082] The present invention also protects the use of Bacillus polymyxa CPCC 101223 or / and cultures of Bacillus polymyxa CPCC 101223 in the preparation of bacterial inhibitors.

[0083] The present invention also protects a bacterial inhibitor containing a culture of Bacillus polymyxa CPCC 101223 or / and Bacillus polymyxa CPCC 101223.

[0084] The present invention also protects a method for preparing a bacterial inhibitor, comprising the following steps: using a culture of Bacillus polymyxa CPCC 101223 or / and Bacillus polymyxa CPCC 101223 as a component of the bacterial inhibitor to obtain the bacterial inhibitor.

[0085] Any of the above-mentioned general formula compounds are compounds of the following general formula:

[0086] X1-X2-Dab-Gly-Ser-Trp-Ser-Dab-Dab-X3-Glu-Val-X4-Ala;

[0087] X1 is 3-hydroxy-6-methyloctanoic acid, 6-methylheptanoic acid, 6-methyloctanoic acid, 3-hydroxy-8-methylnonanoic acid, 3-hydroxydecanoic acid or 3-hydroxy-8-methyldecanoic acid;

[0088] X2 is either Gly or Val;

[0089] X3 can be Val, Ile, or Trp;

[0090] X4 is either Val or Ile.

[0091] Specifically, the general formula compound may be compound 7, compound 9, compound 11, compound 10, compound 8, compound 4, compound 5, compound 6, compound 1, compound 2, or compound 3.

[0092] In compound 1, X1 is 3-hydroxy-6-methyloctanoic acid, X2 is Gly, X3 is Val, and X4 is Val.

[0093] In compound 2, X1 is 6-methylheptanoic acid, X2 is Gly, X3 is Val, and X4 is Val.

[0094] Compound 3, X1 is 6-methylheptanoic acid, X2 is Gly, X3 is Ile, and X4 is Val.

[0095] Compound 4, X1 is 6-methyloctanoic acid, X2 is Gly, X3 is Val, and X4 is Val.

[0096] Compound 5 has X1 being 6-methyloctanoic acid, X2 being Gly, X3 being Ile, and X4 being Val.

[0097] In compound 6, X1 is 6-methyloctanoic acid, X2 is Gly, X3 is Val, and X4 is Ile.

[0098] In compound 7, X1 is 3-hydroxy-8-methylnonanoic acid, X2 is Val, X3 is Trp, and X4 is Val.

[0099] In compound 8, X1 is 3-hydroxydecanoic acid, X2 is Val, X3 is Trp, and X4 is Val.

[0100] In compound 9, X1 is 3-hydroxy-8-methylnonanoic acid, X2 is Val, X3 is Trp, and X4 is Ile.

[0101] In compound 10, X1 is 3-hydroxy-8-methyldecanoic acid, X2 is Val, X3 is Trp, and X4 is Ile.

[0102] In compound 11, X1 is 3-hydroxy-8-methyldecanoic acid, X2 is Val, X3 is Trp, and X4 is Val.

[0103] Gly, Val, Ile, and Trp are all amino acids.

[0104] Gly stands for glycine. Val stands for valine. Ile stands for isoleucine. Trp stands for tryptophan.

[0105] The structural formula of compound 1 is as follows:

[0106]

[0107] The structural formula of compound 2 is as follows:

[0108]

[0109] The structural formula of compound 3 is as follows:

[0110]

[0111] The structural formula of compound 4 is as follows:

[0112]

[0113] The structural formula of compound 5 is as follows:

[0114]

[0115] The structural formula of compound 6 is as follows:

[0116]

[0117] The structural formula of compound 7 is as follows:

[0118]

[0119] The structural formula of compound 8 is as follows:

[0120]

[0121] The structural formula of compound 9 is as follows:

[0122]

[0123] The structural formula of compound 10 is as follows:

[0124]

[0125] The structural formula of compound 11 is as follows:

[0126]

[0127] Any of the bacteria mentioned above may be Gram-negative or Gram-positive.

[0128] The Gram-negative bacteria include, but are not limited to: Escherichia coli, Klebsiella pneumoniae, Pseudomonas syringae, Acinetobacter baumannii, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Providencia radiata, Stenotrophomonas maltophilia, or Shigella flexneri.

[0129] The Gram-positive bacteria include, but are not limited to: Staphylococcus aureus or Enterococcus faecalis.

[0130] The bacteria mentioned above can specifically be any of the bacteria listed in Table 5.

[0131] Any of the bacteria mentioned above can be either susceptible or resistant.

[0132] The drug resistance can be any one or any combination of the following: methicillin resistance, vancomycin resistance, resistance caused by extended-spectrum β-lactamase, resistance caused by New Delhi metallo-β-lactamase 1, and resistance caused by polymyxin resistance genes.

[0133] In the above text, the bacterial inhibitor, in addition to containing compounds of the general formula or their pharmaceutically acceptable salts, may also contain suitable carriers or excipients. The carrier materials here include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Water-soluble carrier materials are preferred. Using these materials, various dosage forms can be formulated, including but not limited to tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, lyophilized powder injections, etc. These can be conventional formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems. To formulate unit-dose dosage forms into tablets, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and aluminum silicate; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and polyvinylpyrrolidone; and disintegrants. Examples of carriers include dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose, and ethylcellulose; disintegration inhibitors include sucrose, tristearate, cocoa butter, and hydrogenated oil; absorption enhancers include quaternary ammonium salts and sodium dodecyl sulfate; and lubricants include talc, silica, corn starch, stearates, boric acid, liquid paraffin, and polyethylene glycol. Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into pills. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, gelucire, kaolin, talc, etc.; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste, etc.; and disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methylcellulose, ethylcellulose, etc. To formulate unit-dose dosage forms into suppositories, a wide variety of carriers known in the art can be used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc. To formulate unit-dose dosage forms into injectable formulations, such as solutions, emulsions, lyophilized powders for injection, and suspensions, all diluents commonly used in the art can be used, such as water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc.In addition, to prepare isotonic injection solutions, appropriate amounts of sodium chloride, glucose, or glycerol can be added to the injectable formulation. Furthermore, conventional solubilizers, buffers, pH adjusters, etc., can also be added. Furthermore, if necessary, colorants, preservatives, flavorings, tasters, sweeteners, or other materials can be added to the pharmaceutical formulation. The above dosage forms can be used for administration via injection, including subcutaneous, intravenous, intramuscular, and intracavitary injections; cavity administration, such as rectal and vaginal; respiratory administration, such as nasal administration; and mucosal administration.

[0134] This invention isolated and obtained *Paenibacillus polymyxa* CPCC 101223, and derived 11 novel decadecapeptide products from its fermentation products. These products exhibit good antibacterial activity (including against Gram-positive and Gram-negative bacteria), particularly against Gram-negative drug-resistant bacteria. The structural differences between compounds 1-11 significantly affect their antibacterial activity. The compounds of this invention are derived from *Paenibacillus polymyxa*, have a simple preparation process, and exhibit good activity against Gram-negative drug-resistant bacteria. They provide a theoretical basis for elucidating the structure-activity relationship of decadecapeptide antibacterial activity and are suitable for the research of lead compounds against Gram-negative drug-resistant bacteria or the preparation of drugs against Gram-negative drug-resistant bacteria. Attached Figure Description

[0135] Figure 1 The image shows the UV spectrum of compound 7 in a 50% acetonitrile aqueous solution.

[0136] Figure 2 The peak representing the second charge of compound 8 is [M+2H]. 2+ 796.0 secondary mass spectrum.

[0137] Figure 3 The peak representing the second charge of compound 10 is [M+2H]. 2+ 809.9 secondary mass spectrum. Detailed Implementation

[0138] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention. The embodiments provided below can serve as a guide for those skilled in the art to make further improvements and do not constitute a limitation on the present invention in any way. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments can be obtained commercially. The macroporous adsorption resin used in the embodiments is DIAION HP20 macroporous adsorption resin (product of Mitsubishi Chemical Corporation, Japan); DIAION HP20 macroporous adsorption resin is a non-polar macroporous adsorption resin with a water content (%) of 55-65, a wet apparent density (g / LR) of 680, a pore volume (ml / g) of 1.3, and a specific surface area (m²). 2 / g) is 590, good frequency radius The particle size distribution is ≥90% (≥0.25mm), effective particle size (mm) ≥0.25, uniformity coefficient ≤1.6, and operating temperature (°C) ≤130. YMC ODS-AQ-HG packing material: a product of YMC Corporation, Japan, item number AQG12S50. Unless otherwise specified, the quantitative tests in the following examples were performed in triplicate, and the results were averaged. Unless otherwise specified, % in the examples refer to volume percentage.

[0139] Example 1: Acquisition, identification, and preservation of Bacillus polymyxa CPCC 101223

[0140] I. Isolation of bacterial strains

[0141] The strain was isolated from the stem tissue of the medicinal plant *Eucommia ulmoides*. Plant samples were collected in July 2011 from Cangshan World Geopark, Dali City, Dali Prefecture, Yunnan Province. The surface-sterilized plant tissue was aseptically pulverized and inoculated onto starch-inorganic salt separation medium, and incubated at 28°C for 21 days. Colonies were picked from the separation medium and streaked for purification, yielding pure cultures of the strain, one of which was named strain CPCC 101223.

[0142] II. Identification of Strains

[0143] Morphological and physiological-biochemical characteristics were identified according to "Bergey's Manual of Systematic Bacteriology" (Garrity, 2001) and "Commonly Used Identification Methods in General Bacteriology" (Dong Xiuzhu et al., 2001), following conventional methods. Morphological characteristics of strain CPCC 101223: colonies are smooth, viscous, raised, and milky white, adhering to the surface of the culture medium, without soluble pigments; under a microscope, the cells are rod-shaped, (2.4-3.2) × (0.8-1.0) μm, and the spores are spindle-shaped, 3.4 × 1.6 μm; Gram staining is positive. Physiological and biochemical characteristics of strain CPCC101223: it can decompose lactose, galactose, maltose, mannitol, and mannose, producing acid and gas; it decomposes glucose, producing acid but not gas.

[0144] Cells of strain CPCC 101223 were collected, genomic DNA was extracted, and the 16S rRNA gene was amplified by PCR. The amplified product was recovered, purified, and sequenced, and then sequenced in the GenBank database. The amplified product, as shown in Sequence 1 of the sequence listing, showed 99.80% similarity to Paenibacillus polymyxa ATCC 842 (AFOX01000032).

[0145] Based on morphological characteristics, physiological and biochemical characteristics, and homology analysis of the 16S rRNA gene sequence, strain CPCC101223 belongs to Paenibacillus polymyxa.

[0146] III. Preservation of bacterial strains

[0147] Paenibacillus polymyxa CPCC 101223 was deposited on July 8, 2021, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with accession number CGMCC No. 22854.

[0148] Example 2: Preparation of compounds by fermentation of Bacillus polymyxa CPCC 101223

[0149] I. Cultivation and Fermentation

[0150] Seed culture medium (TSB medium): containing 15 g / L tryptone, 5 g / L soybean peptone, 5 g / L sodium chloride, with the remainder being water; pH 7.0-7.4; sterilized by steam at 121℃ for 15 min.

[0151] Fermentation medium: containing 30 g / L corn flour, 10 g / L soluble starch, 1 g / L yeast powder, 5 g / L ammonium sulfate, 10 g / L calcium carbonate, with the remainder being water; pH 7.0-7.4, sterilized by steam at 121℃ for 15 min.

[0152] 1. Pick a single colony of Bacillus polymyxa CPCC 101223 and inoculate it into a 500mL Erlenmeyer flask containing 100mL of seed culture medium. Incubate at 30℃ and 200r / min for 16h with shaking to obtain the seed culture.

[0153] 2. Inoculate 100 mL of the seed culture obtained in step 1 into a 5 L Erlenmeyer flask containing 1 L of fermentation medium, and culture at 30 °C and 200 r / min for 20 h with shaking.

[0154] 3. After completing step 2, add 100 mL of macroporous adsorption resin that has been sterilized by moist heat to the system, and incubate at 30℃ and 200 r / min for 4 h to obtain the fermentation product.

[0155] Multiple repeated treatments were performed simultaneously, yielding a total of 24 L of fermentation product.

[0156] II. Isolation and Purification of Compounds

[0157] 1. Take 24 L of the fermentation product from step one, pass it through a 60-mesh sieve, and rinse the sieve repeatedly with water until the washing liquid is clear. Then, transfer the macroporous adsorption resin to a chromatography column (column volume 2 L). Next, elute the column sequentially with 10 L of deionized water, 20 L of 40% isopropanol aqueous solution, 30 L of 80% isopropanol aqueous solution, and 10 L of isopropanol, at a flow rate of 2 column volumes / h. Collect the eluent (30 L) obtained during the 80% isopropanol aqueous solution elution.

[0158] 2. Take the eluent obtained in step 1, load it onto a solid sample and perform chromatographic separation and purification.

[0159] Specific method: Take the eluent collected in step 1 after column chromatography, concentrate it to 100 ml using a rotary evaporator, then add 10 mL of swollen YMC ODS-AQ-HG packing material and mix well, then concentrate and evaporate to dryness to obtain a solid sample; fill the solid sample into a sample column, then connect the chromatographic column, and use a chromatograph for separation and purification.

[0160] The chromatographic column was a Flash Spherical C18 20-35μm 100A 120g.

[0161] The chromatograph used was a TELEDYNE ISCO Combiflash RF200 preparative chromatograph.

[0162] Mobile phase A: composed of trifluoroacetic acid and water, with a trifluoroacetic acid concentration of 0.05%; Mobile phase B: composed of trifluoroacetic acid and acetonitrile, with a trifluoroacetic acid concentration of 0.05%. Mobile phase flow rate: 20 mL / min. Detection wavelength: 210 nm.

[0163] Elution program: 0-20 min, the mobile phase consists of 20% mobile phase B and 80% mobile phase A; 20-35 min, the volume fraction of mobile phase B increases linearly from 20% to 40%, and the corresponding volume fraction of mobile phase A decreases linearly from 80% to 60%; 35-70 min, the mobile phase consists of 40% mobile phase B and 60% mobile phase A; 70-100 min, the volume fraction of mobile phase B increases linearly from 40% to 100%, and the corresponding volume fraction of mobile phase A decreases linearly from 60% to 0%; 100-120 min, the mobile phase consists entirely of mobile phase B.

[0164] The entire elution process yielded 9 fractions (each fraction corresponds to the post-column solution of one elution peak, and the 9 fractions are named Fr.1 to Fr.9 respectively), where Fr.4 corresponds to retention time t. R Elution peak at 25-40 min, Fr.5 corresponds to t R Elution peak at 40-45 min.

[0165] 3. Take the Fr.4 obtained in step 3, load it onto a solid sample and perform chromatographic separation and purification.

[0166] Specific method: Take 300 mL of Fr.4 obtained in step 2, concentrate it to 10 mL using a rotary evaporator, then add 5 mL of swollen YMC ODS-AQ-HG packing material, and then evaporate to dryness to obtain a solid sample; fill the solid sample into a sample column, then connect the chromatographic column, and use a chromatograph for separation and purification.

[0167] The chromatographic column was a Flash Spherical C18 20-35μm 100A 120g.

[0168] The chromatograph used was a TELEDYNE ISCO Combiflash RF200 preparative chromatograph.

[0169] Mobile phase A: composed of trifluoroacetic acid and water, with a trifluoroacetic acid concentration of 0.15%; Mobile phase B: composed of trifluoroacetic acid and acetonitrile, with a trifluoroacetic acid concentration of 0.15%. Mobile phase flow rate: 20 mL / min. Detection wavelength: 210 nm.

[0170] Elution program: 0-40 min, the mobile phase consists of 20% mobile phase B and 80% mobile phase A; 40-60 min, the volume fraction of mobile phase B increases linearly from 20% to 40%, and the corresponding volume fraction of mobile phase A decreases linearly from 80% to 60%; 60-90 min, the mobile phase consists of 40% mobile phase B and 60% mobile phase A; 90-120 min, the volume fraction of mobile phase B increases linearly from 40% to 100%, and the corresponding volume fraction of mobile phase A decreases linearly from 60% to 0%; 120-140 min, the mobile phase consists entirely of mobile phase B.

[0171] The entire elution process yielded 13 fractions (each fraction corresponds to the post-column solution of an elution peak, and the 13 fractions are named Fr.4-1 to Fr.4-13, respectively), where Fr.4-4 corresponds to a retention time t. R The elution peak is observed at 27-30 min, with Fr. 4-5 corresponding to retention times t. R The elution peak is observed at 30-46 min, with Fr. 4-6 corresponding to retention times t. R The elution peak is at 46-68 min, and Fr. 4-7 corresponds to retention times t. R The elution peak is at 68-72 min, and Fr. 4-8 corresponds to the retention time t. R The elution peak is at 72-74 min, and Fr. 4-10 corresponds to the retention time t. R The elution peak is at 78-85 min, and Fr. 4-11 corresponds to the retention time t. R The elution peak is at 85-92 min, and Fr. 4-12 corresponds to the retention time t. R The elution peak is between 92 and 102 min, and Fr. 4-13 corresponds to the retention time t. R Elution peak at 102-110 min.

[0172] Each fraction was concentrated and dried using a rotary evaporator, and then dissolved in an appropriate amount of 40% acetonitrile aqueous solution to obtain the concentrated solution of each fraction.

[0173] 4. Obtaining compounds

[0174] Chromatograph: Shimadzu LC-20 liquid chromatograph (LC-20AD).

[0175] Chromatographic column: YMC-Pack ODS-AQ, 10mm*250mm, pore size 5μm, YMC Corporation, Japan.

[0176] The mobile phase flow rate was 4 mL / min. The detection wavelength was 210 nm.

[0177] 3 mL of the concentrated Fr.4-4 obtained in step 5 was loaded. The mobile phase consisted of trifluoroacetic acid, acetonitrile, and water, with a trifluoroacetic acid concentration of 0.15% and an acetonitrile concentration of 31%. The eluent corresponding to the elution peak with a retention time of 8.21 min was collected, concentrated using a rotary evaporator, and then freeze-dried to obtain compound 1.

[0178] 3 mL of the concentrated Fr.4-5 obtained in step 5 was loaded. The mobile phase consisted of trifluoroacetic acid, acetonitrile, and water, with a trifluoroacetic acid concentration of 0.15% and an acetonitrile concentration of 33%. The eluent corresponding to the retention time of 8.37 min was collected, concentrated using a rotary evaporator, and then freeze-dried to obtain compound 2. The eluent corresponding to the retention time of 9.24 min was collected, concentrated using a rotary evaporator, and then freeze-dried to obtain compound 3.

[0179] 3 mL of the concentrated Fr.4-6 obtained in step 5 was loaded. The mobile phase consisted of trifluoroacetic acid, acetonitrile, and water, with a trifluoroacetic acid concentration of 0.15% and an acetonitrile concentration of 34%. The eluent corresponding to the elution peak with a retention time of 9.54 min was collected, concentrated using a rotary evaporator, and then freeze-dried to obtain compound 4.

[0180] 3 mL of the concentrated Fr.4-7 obtained in step 5 was loaded. The mobile phase consisted of trifluoroacetic acid, acetonitrile, and water, with a trifluoroacetic acid concentration of 0.15% and an acetonitrile concentration of 35%. The eluent corresponding to the elution peak with a retention time of 9.76 min was collected, concentrated using a rotary evaporator, and then freeze-dried to obtain compound 5.

[0181] 3 mL of the concentrated Fr.4-8 obtained in step 5 was loaded. The mobile phase consisted of trifluoroacetic acid, acetonitrile, and water, with a trifluoroacetic acid concentration of 0.15% and an acetonitrile concentration of 39%. The eluent corresponding to the elution peak with a retention time of 7.83 min was collected, concentrated using a rotary evaporator, and then freeze-dried to obtain compound 6.

[0182] 3 mL of the concentrated Fr.4-10 obtained in step 5 was loaded. The mobile phase consisted of trifluoroacetic acid, acetonitrile, and water, with a trifluoroacetic acid concentration of 0.15% and an acetonitrile concentration of 40%. The eluent corresponding to the elution peak with a retention time of 8.11 min was collected, concentrated using a rotary evaporator, and then freeze-dried to obtain compound 7.

[0183] 2 mL of the concentrated Fr.4-11 obtained in step 5 was loaded. The mobile phase consisted of trifluoroacetic acid, acetonitrile, and water, with a trifluoroacetic acid concentration of 0.15% and an acetonitrile concentration of 40%. The eluent corresponding to the elution peak with a retention time of 8.41 min was collected, concentrated using a rotary evaporator, and then freeze-dried to obtain compound 8.

[0184] 2 mL of the concentrated Fr.4-12 obtained in step 5 was loaded. The mobile phase consisted of trifluoroacetic acid, acetonitrile, and water, with a trifluoroacetic acid concentration of 0.15% and an acetonitrile concentration of 40%. The eluent corresponding to the elution peak with a retention time of 8.92 min was collected, concentrated using a rotary evaporator, and then freeze-dried to obtain compound 9.

[0185] 2 mL of the concentrated Fr.4-13 obtained in step 5 was loaded. The mobile phase consisted of trifluoroacetic acid, acetonitrile, and water, with a trifluoroacetic acid concentration of 0.15% and an acetonitrile concentration of 41%. The eluent corresponding to the elution peak with a retention time of 9.82 min was collected, concentrated using a rotary evaporator, and then freeze-dried to obtain compound 10.

[0186] Take 100 mL of Fr.5 obtained in step 2, concentrate and evaporate to dryness using a rotary evaporator, then dissolve in 2 mL of 40% acetonitrile aqueous solution, and then load the sample. The mobile phase consists of trifluoroacetic acid, acetonitrile, and water, with a trifluoroacetic acid concentration of 0.15% and an acetonitrile concentration of 42%. Collect the eluent corresponding to the elution peak with a retention time of 10.37 min, concentrate it using a rotary evaporator, and then freeze-dry it to obtain compound 11.

[0187] All 11 compounds (compound 1 to compound 11) prepared were in powder form and were readily soluble in 40% acetonitrile aqueous solution.

[0188] Fr.4-4 prepared 7.2 mg of compound 1, Fr.4-5 prepared 11.4 mg of compound 2 and 5.8 mg of compound 3, Fr.4-6 prepared 13.3 mg of compound 4, Fr.4-7 prepared 5.7 mg of compound 5, Fr.4-8 prepared 7.6 mg of compound 6, Fr.4-10 prepared 10.0 mg of compound 7, Fr.4-11 prepared 4.1 mg of compound 8, Fr.4-12 prepared 6.1 mg of compound 9, Fr.4-13 prepared 4.3 mg of compound 10, and Fr.5 prepared 11.9 mg of compound 11.

[0189] The UV spectra of the 11 compounds in a 50% acetonitrile aqueous solution were similar, all exhibiting the following characteristics: maximum absorption peaks at 197 nm, 219 nm, 280 nm, and 288 nm. For example, the UV spectrum of compound 7 in a 50% acetonitrile aqueous solution is shown below. Figure 1 .

[0190] III. Identification of Compounds

[0191] The 11 compounds prepared in step two were dissolved in DMSO-d6 and then subjected to 600 MHz chromatography. 1 H-NMR, 13 C-NMR, 1 H- 1 H COSY, NOESY, HSQC and HMBC nuclear magnetic resonance spectroscopy analysis, 13 C-NMR and 1 The peak assignments for each H-NMR spectrum are shown in Tables 1, 2, 3, and 4.

[0192] Compounds 8 and 10, due to partial NMR signal loss, underwent LC-ESI-MS / MS analysis using an Ultimate 3000-LTQ XL instrument to verify the amino acid residue composition and sequence. Mobile phase A: acetonitrile; Mobile phase B: composed of formic acid and water, with a formic acid concentration of 0.15%. Mobile phase flow rate: 0.3 mL / min. Elution program (30 min): Initially, the mobile phase consisted of 10% mobile phase A and 90% mobile phase B; during elution, the volume fraction of mobile phase A increased linearly; at the end of elution, the mobile phase consisted entirely of mobile phase A.

[0193] Secondary mass spectra of compounds 8 and 10 are shown below. Figure 2 and Figure 3 .

[0194] Table 1. Compounds 1, 2, and 4 13 C-NMR and 1 Peak assignments in the H-NMR spectrum (DMSO-d6, 600MHz)

[0195]

[0196]

[0197]

[0198] Compounds 3, 5, and 6 in Table 2 13 C-NMR and 1 Peak assignments in the H-NMR spectrum (DMSO-d6, 600MHz)

[0199]

[0200]

[0201]

[0202] Table 3 Compounds 7, 8 and 11 13 C-NMR and 1 Peak assignments in the H-NMR spectrum (DMSO-d6, 600MHz)

[0203]

[0204]

[0205]

[0206] Table 4 Compounds 9 and 10 13 C-NMR and 1 Peak assignments in the H-NMR spectrum (DMSO-d6, 600MHz)

[0207]

[0208]

[0209]

[0210] Upon identification, the 11 compounds prepared in step two were all novel lipopeptide compounds, consisting of a fatty acid and thirteen amino acids forming a linear molecule, with the following general structural formula:

[0211] X1-X2-Dab-Gly-Ser-Trp-Ser-Dab-Dab-X3-Glu-Val-X4-Ala.

[0212] In compound 1, X1 is 3-hydroxy-6-methyloctanoic acid, X2 is Gly, X3 is Val, and X4 is Val;

[0213] In compound 2, X1 is 6-methylheptanoic acid, X2 is Gly, X3 is Val, and X4 is Val;

[0214] In compound 3, X1 is 6-methylheptanoic acid, X2 is Gly, X3 is Ile, and X4 is Val;

[0215] In compound 4, X1 is 6-methyloctanoic acid, X2 is Gly, X3 is Val, and X4 is Val;

[0216] In compound 5, X1 is 6-methyloctanoic acid, X2 is Gly, X3 is Ile, and X4 is Val;

[0217] In compound 6, X1 is 6-methyloctanoic acid, X2 is Gly, X3 is Val, and X4 is Ile;

[0218] In compound 7, X1 is 3-hydroxy-8-methylnonanoic acid, X2 is Val, X3 is Trp, and X4 is Val;

[0219] In compound 8, X1 is 3-hydroxydecanoic acid, X2 is Val, X3 is Trp, and X4 is Val;

[0220] In compound 9, X1 is 3-hydroxy-8-methylnonanoic acid, X2 is Val, X3 is Trp, and X4 is Ile;

[0221] In compound 10, X1 is 3-hydroxy-8-methyldecanoic acid, X2 is Val, X3 is Trp, and X4 is Ile;

[0222] In compound 11, X1 is 3-hydroxy-8-methyldecanoic acid, X2 is Val, X3 is Trp, and X4 is Val.

[0223] The structural formulas of the 11 compounds prepared in step two were identified as follows:

[0224]

[0225]

[0226] Example 3: In vitro antibacterial activity of the compound

[0227] Test compounds: 11 compounds prepared in step two of Example 2 and the positive control drug (colistin).

[0228] Colistin, used as a positive control drug, was purchased from the National Drug Standard Material Inquiry and Ordering Platform of the China National Institutes for Food and Drug Control, item number 130327.

[0229] Test bacteria: Internationally recognized standard strains of drug-resistant bacteria and representative drug-resistant strains isolated clinically in recent years, as detailed in Tables 5 and 6. Among the test bacteria, Staphylococcus aureus and Enterococcus faecalis are Gram-positive bacteria, while the others are Gram-negative bacteria.

[0230] Escherichia coli 08-85, Escherichia coli 13-43, Escherichia coli 13-68, and Klebsiella pneumoniae 09-20 are all described in the following literature: Low Prevalence of mcr-1 Among Clinical Enterobacteriaceae Isolates and Co-transfer of mcr-1 and blaNDM-1 from Separate Donors; MICROBIALDRUG RESISTANCE Volume 00, Number 00, 2020, DOI:10.1089 / mdr.2020.0212.

[0231] Enterobacter cloacae 1000654 is described in the following literature: Rasheed JK, Kitchel B, Zhu W, et al. New Delhi metallo-β-lactamase-producing Enterobacteriaceae, United States. Emerg Infect Dis. 2013; 19(6):870-878. doi:10.3201 / eid1906.121515.

[0232] *Pseudomonas syringae* CPCC 101099 was deposited on September 26, 2021, at the China Pharmaceutical Culture Collection (CPCC, No. 1, Tiantan Xili, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Beijing 100050, China). This strain has been available to the public from the CPCC since the date of deposit. The CPCC strain ordering website is http: / / www.cpcc.ac.cn / index.php?id=104.

[0233] The drug susceptibility test of the test compound was performed using the plate dilution method.

[0234] The test compound was serially diluted twofold (1 ml) with MH broth medium. 1 ml of the diluted solution was added to a petri dish, followed by 14 ml of melted MH agar medium. The mixture was then allowed to stand at room temperature until plates formed. The test bacteria were then inoculated (multiple inoculations can be made on each plate, ensuring even distribution to prevent interference; the inoculation density is approximately 10). 4 (cfu / point), incubate at 35℃ for 18 hours, and then observe the results. No sterile growth indicates that the sample is active.

[0235] The results are shown in Tables 5 and 6. The compound concentrations in Tables 5 and 6 refer to the concentrations of the compounds in the system, also known as working concentrations. All 11 compounds exhibited some antibacterial activity against the tested bacteria. Compounds 7-11 showed significantly stronger antibacterial activity against various Gram-negative drug-resistant bacteria than compounds 1-6, and stronger antibacterial activity against drug-resistant strains containing the mcr-1 resistance gene and other drug-resistant strains than the control drug colistin. Compound 7 showed the best activity.

[0236] Table 5. Results of in vitro antibacterial activity test of compound 7-11 (MIC agar method, unit μg / ml)

[0237]

[0238]

[0239] Table 6. Results of in vitro antibacterial activity tests of compounds 1-6 (MIC agar method, unit μg / ml)

[0240]

[0241] MRSA: Methicillin-resistant Staphylococcus aureus; VRE: Vancomycin-resistant Enterococcus; ESBLs: Extended-spectrum β-lactamases; NDM-1: New Delhi metallo-β-lactamase 1; mcr-1: Polymyxin resistance gene; + indicates presence of resistance enzyme or gene, - indicates absence of resistance enzyme or gene. ND: Not tested.

[0242] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. sequence list <110> Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences <120> Tridecapeptide Derivatives, Their Preparation Methods and Applications <130> GNCYX212008 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1542 <212> DNA <213> Paenibacillus polymyxa <400> 1 agagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60 ggggttaatt agaagcttgc ttctaattaa cctagcggcg gacgggtgag taacacgtag 120 gcaacctgcc cacaagacag ggataactac cggaaacggt agctaatacc cgatacatcc 180 ttttcctgca tgggagaagg aggaaaggcg gagcaatctg tcacttgtgg atgggcctgc 240 ggcgcattag ctagttggtg gggtaaaggc ctaccaaggc gacgatgcgt agccgacctg 300 agagggtgat cggccacact gggactgaga cacggcccag actcctacgg gaggcagcag 360 tagggaatct tccgcaatgg gcgaaagcct gacggagcaa cgccgcgtga gtgatgaagg 420 ttttcggatc gtaaagctct gttgccaggg aagaacgtct tgtagagtaa ctgctacaag 480 agtgacggta cctgagaaga aagccccggc taactacgtg ccagcagccg cggtaatacg 540 tagggggcaa gcgttgtccg gaattattgg gcgtaaagcg cgcgcaggcg gctctttaag 600 tctggtgttt aatcccgagg ctcaacttcg ggtcgcactg gaaactgggg agcttgagtg 660 cagaagagga gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg tggaggaaca 720 ccagtggcga aggcgactct ctgggctgta actgacgctg aggcgcgaaa gcgtggggag 780 caaacaggat tagataccct ggtagtccac gccgtaaacg atgaatgcta ggtgttaggg 840 gtttcgatac ccttggtgcc gaagttaaca cattaagcat tccgcctggg gagtacggtc 900 gcaagactga aactcaaagg aattgacggg gacccgcaca agcagtggag tatgtggttt 960 aattcgaagc aacgcgaaga accttaccag gtcttgacat ccctttgacc ggtctagaga 1020 tagacctttc cttcgggaca gaggagacag gtggtgcatg gttgtcgtca gctcgtgtcg 1080 tgagatgttg ggttaagtcc cgcaacgagc gcaaccctta tgcttagttg ccagcaggtc 1140 aagctgggca ctctaagcag actgccggtg acaaaccgga ggaaggtggg gatgacgtca 1200 aatcatcatg ccccttatga cctgggctac acacgtacta caatggccgg tacaacggga 1260 agcgaaggag cgatctggag ccaatcctag aaaagccggt ctcagttcgg attgtaggct 1320 gcaactcgcc tacatgaagt cggaattgct agtaatcgcg gatcagcatg ccgcggtgaa 1380 tacgttcccg ggtcttgtac acaccgcccg tcacaccacg agagtttaca acacccgaag 1440 tcggtgaggt aaccgcaagg agccagccgc cgaaggtggg gtagatgatt ggggtgaagt 1500 cgtaacaagg tagccgtatc ggaaggtgcg gctggatcac ct 1542

Claims

1. A method for preparing the compound, comprising the following steps: fermenting and culturing Bacillus polymyxa CPCC 101223 to obtain the compound; The compound is the following compound: X1-X2-Dab-Gly-Ser-Trp-Ser-Dab-Dab-X3-Glu-Val-X4-Ala; X1 is 3-hydroxy-8-methylnonanoic acid, X2 is Val, X3 is Trp, and X4 is Val; Polymyxobacterium CPCC 101223 is also known as Polymyxobacterium ( Paenibacillus polymyxa (CPCC101223, its accession number is CGMCC No. 22854.) 2. Application of *Bacillus polymyxa* CPCC 101223 in the preparation of compounds; The compound is the following compound: X1-X2-Dab-Gly-Ser-Trp-Ser-Dab-Dab-X3-Glu-Val-X4-Ala; X1 is 3-hydroxy-8-methylnonanoic acid, X2 is Val, X3 is Trp, and X4 is Val; Polymyxobacterium CPCC 101223 is also known as Polymyxobacterium ( Paenibacillus polymyxa (CPCC101223, its accession number is CGMCC No. 22854.) 3. A compound, characterized in that: The compound is the following compound: X1-X2-Dab-Gly-Ser-Trp-Ser-Dab-Dab-X3-Glu-Val-X4-Ala; X1 is 3-hydroxy-8-methylnonanoic acid, X2 is Val, X3 is Trp, and X4 is Val.

4. The use of the compound of claim 3 or a pharmaceutically acceptable salt thereof in the preparation of a bacterial inhibitor; wherein the bacteria are Gram-negative or Gram-positive bacteria; wherein the Gram-negative bacteria are Escherichia coli, Klebsiella pneumoniae, Pseudomonas syringae, Acinetobacter baumannii, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Prof. retinoicus, Stenotrophomonas maltophilia, or Shigella flexneri; wherein the Gram-positive bacteria are Staphylococcus aureus or Enterococcus faecalis.

5. A bacterial inhibitor comprising the compound of claim 3 or a pharmaceutically acceptable salt thereof; wherein the bacteria are Gram-negative or Gram-positive; wherein the Gram-negative bacteria are Escherichia coli, Klebsiella pneumoniae, Pseudomonas syringae, Acinetobacter baumannii, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Professorium retinoicum, Stenotrophomonas maltophilia, or Shigella flexneri; and wherein the Gram-positive bacteria are Staphylococcus aureus or Enterococcus faecalis.

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