Cyclic ester peptide compound, producing strain of cyclic ester peptide compound, preparation method of cyclic ester peptide compound and application of cyclic ester peptide compound in preparation of antibacterial drugs

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a cyclic ester peptide compound, a producing strain thereof, a preparation method and application of the cyclic ester peptide compound in preparation of antibacterial medicines. The invention provides a cyclic ester peptide compound as shown in a formula I, a stereoisomer, a tautomer and an isotope derivative of the cyclic ester peptide compound, or a pharmaceutically acceptable salt of the cyclic ester peptide compound, a producing strain Streptomyces alboflavus sp.NX-12 of the cyclic ester peptide compound, and fermentation, separation and purification methods of the cyclic ester peptide compound and the stereoisomer, the tautomer and the isotope derivative of the cyclic ester peptide compound. The cyclic ester peptide compound has strong inhibitory activity on a variety of gram-positive bacteria, can be used for preparing antibacterial drugs, shows application potential in treatment of infection and other related diseases, and compared with the existing similar gram-positive bacterium-resistant glycopeptide antibiotic vancomycin, the cyclic ester peptide compound has the advantages that the cyclic ester peptide compound can be used for preparing the antibacterial drugs; the cyclic ester peptide compound disclosed by the invention has an equivalent antibacterial effect. The preparation method of the cyclic ester peptide compound is simple in process, short in time consumption, capable of greatly reducing the medicine cost and suitable for large-scale production. # imgabs0 #

Description

Technical Field

[0001] The present application belongs to the field of biomedicine technology, and specifically relates to a cyclic lipopeptide compound, its producing bacteria, preparation method and use in the preparation of antibacterial drugs. Background Art

[0002] With the widespread use and abuse of antibiotics, the problem of bacterial resistance is becoming increasingly serious, the efficacy of traditional antibiotics has significantly decreased, and there is an urgent need to develop new antibiotics.

[0003] Cyclic peptide compounds are a class of compounds with a cyclic structure formed by amino acids connected by peptide bonds, which have unique spatial conformation and chemical stability. Due to their structural peculiarity, cyclic peptide compounds show superior properties in drug development, such as high receptor selectivity and stability, as well as good oral bioavailability. In recent years, cyclic peptide compounds have made significant progress in the research of antibacterial, antiviral, and antitumor activities. Examples of cyclic peptide compounds that have been successfully used in clinical practice include antibiotics and antitumor drugs [1-5]. For example, vancomycin, which is effective against methicillin-resistant Staphylococcus aureus, has been widely used in the clinical treatment of severe bacterial infections [1]; daptomycin, a cyclic lipopeptide antibiotic, is mainly used to treat complicated skin and soft tissue infections caused by drug-resistant Gram-positive bacteria [2]; the immunosuppressant sirolimus (Rapamycin) is often used to prevent organ transplant rejection and has shown potential in anti-cancer research [3]. Cyclic peptide compounds usually exert their antibacterial effects by interfering with bacterial cell wall synthesis, membrane function and protein synthesis. These diverse mechanisms give them unique advantages in combating drug-resistant bacteria.

[0004] Advances in genomics have provided new opportunities for the discovery and development of cyclopeptide compounds. Through genomic methods, potential cyclopeptide synthesis gene clusters can be mined from microbial genomes, and then the production of new cyclopeptide compounds can be achieved through the study and optimization of biosynthetic pathways[6].

[0005] (1) Prediction of secondary metabolite gene clusters: By computationally analyzing secondary metabolite gene clusters in microbial genomes, we can predict gene clusters that may encode cyclic peptide compounds and verify their products through experiments. (2) Synthetic biology: Using synthetic biology methods, we can reconstruct and optimize the synthesis pathways of cyclic peptide compounds to improve their yield and activity. (3) Metabolic engineering: Through metabolic engineering, we can transform production strains to improve the production efficiency of cyclic peptide compounds and meet the needs of large-scale production. Therefore, we can use this effective method to discover new antibiotics with antibacterial activity from the secondary metabolites of actinomycetes.

[0006] Based on this, the present invention is proposed.

[0007] [References]

[0008] 1. Van Bambeke, F.; Van Laethem, Y.; Courvalin, P. Glycopeptide Antibiotics: From Conventional Molecules to New Derivatives. Chem. Rev. 2004, 104(12), 739–762.

[0009] 2. Steenbergen, J. N.; Alder, J.; Thorne, G. M.; Tally, F. P. Daptomycin: A Lipopeptide Antibiotic for the Treatment of Serious Gram-Positive Infections. J. Antimicrob. Chemother. 2005, 55(3), 283–288.

[0010] 3. Moreira, J. B.; de Oliveira, L. S.; Dias, R. O.; da Costa, R. M. G.; Dias, S. C.; Franco, O. L. Capreomycin and New Approaches to Overcome Microbial Resistance. J. Mol. Med. 2021, 99(4), 521–530.

[0011] 4. Sarbassov, D. D.; Sabatini, D. M. Sirolimus (Rapamycin): From the Soil of Easter Island to a Therapy for HCC. J. Hepatol. 2005, 42(4), 692–701.

[0012] 5. Walsh, C. T.; Fischbach, M. A. Natural Products Version 2.0: Connecting Genes to Molecules. J. Am. Chem. Soc. 2010, 132(8), 2469–2493.

[0013] 6. Zhao, Y.; Liu, H.; Zhang, Summary of the Invention

[0014] The present application provides a cyclic lipopeptide compound, its stereoisomers, tautomers, isotopic derivatives, or pharmaceutically acceptable salts thereof, its producing bacteria, preparation methods, and its use in the preparation of antibacterial drugs. The cyclic lipopeptide compound described in the present application has strong inhibitory activity against a variety of Gram-positive bacteria and can be used to prepare antibacterial drugs, showing potential for application in the treatment of infections and other related diseases. Compared with the existing similar anti-Gram-positive glycopeptide antibiotic vancomycin, the cyclic lipopeptide compound described in the present application has an equivalent antibacterial effect.

[0015] In a first aspect, the present application provides a cyclic ester peptide compound represented by formula I, its stereoisomers, tautomers, isotopic derivatives, or pharmaceutically acceptable salts thereof,

[0016]

[0017] In formula I, R1 is H, H, or H, OH, or =O, and R2 is hydrogen, hydroxy, methoxy, or halogen.

[0018] In one group of embodiments, R2 is chloro, fluoro or bromo.

[0019] In one embodiment, the cyclic ester peptide compound represented by formula I is zhengxinmycin A of formula II, zhengxinmycin B of formula III, zhengxinmycin C of formula IV, or zhengxinmycin D of formula V.

[0020]

[0021] In a second aspect, the present application provides a Streptomyces strain Streptomyces alboflavus sp. NX-12 for producing a cyclic ester peptide compound shown in Formula I, its stereoisomers, tautomers, isotopic derivatives, or pharmaceutically acceptable salts thereof, with a deposit number of CGMCC NO.33132 and a deposit date of December 20, 2024. The depository unit is the General Microbiology Center of the China Culture Collection Administration, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0022] In a third aspect, the present application provides a method for preparing a cyclic lipopeptide compound represented by Formula I, its stereoisomers, tautomers, isotopic derivatives, or pharmaceutically acceptable salts thereof, comprising the following steps: (1) fermentation: fermenting Streptomyces alboflavus sp. NX-12 by shaking culture in a liquid culture medium; (2) extraction: collecting the fermentation broth, extracting it with an organic solvent, and concentrating the organic phase to obtain an extract; (3) separation and purification: separating and purifying the cyclic lipopeptide compound using silica gel column chromatography, gel column chromatography, or HPLC.

[0023] In one embodiment, in step (1), the liquid culture medium is G2 medium. Specifically, Streptomyces alboflavus sp. NX-12 is fermented in G2 liquid culture medium at 26-32° C. and 180-250 rpm with shaking for 2-8 days, preferably at 28-30° C. and 200-220 rpm with shaking for 4-6 days.

[0024] In one group of embodiments, in the step (1), Streptomyces alboflavus sp.NX-12 is first shaken cultured in a liquid culture medium to obtain a seed solution, which is then inoculated into the liquid culture medium at a 5-15% inoculum (volume ratio, preferably 8-10%) for shaken culture and fermentation; preferably, the seed solution is obtained by shaking culture in a G2 liquid culture medium, more preferably, the seed solution is obtained by shaking culture at 26-32°C and 180-250 rpm for 1-4 days, and most preferably, the seed solution is obtained by shaking culture at 28-30°C and 200-220 rpm for 2-3 days.

[0025] In one set of embodiments, in step (1), Streptomyces alboflavus sp. NX-12 is cultured on a solid culture medium to produce spores, and then the spores are inoculated into a liquid culture medium and shaken to obtain a seed solution; preferably, the spores are cultured on MS solid culture medium, more preferably at 26-32° C. for 2-8 days, and most preferably at 28-30° C. for 3-5 days.

[0026] In one embodiment, in step (2), the organic solvent is selected from a mixed solvent of one or more of chloroform, dichloromethane, ethyl acetate, alcohols with a carbon chain of ≤4, acetone, and butanone. The alcohols with a carbon chain of ≤4 are selected from methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, and tert-butanol. The organic solvent is preferably ethyl acetate. Extraction is performed using 1-5 volumes of the organic solvent, preferably 2-3 volumes of the organic solvent, and the organic phase is concentrated under reduced pressure to obtain an extract.

[0027] In one embodiment, in step (3), silica gel column chromatography, gel column chromatography, and HPLC are sequentially performed for separation and purification.

[0028] In one embodiment, in step (3), the elution solvent used for silica gel column chromatography and gel column chromatography is selected from the group consisting of: a mixed solvent of one or more of chloroform, dichloromethane, ethyl acetate, alcohols with a carbon chain of ≤4, acetone, and butanone. The alcohols with a carbon chain of ≤4 are selected from methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, and tert-butanol. The silica gel column chromatography preferably uses dichloromethane and methanol for gradient elution; more preferably, an organic solvent gradient elution of dichloromethane:methanol = 1:0, 1:3, 1:1, and 0:1 (volume ratio) is used in sequence, and the eluate with dichloromethane:methanol = 1:3 is concentrated and then subjected to gel column chromatography, which can be dissolved in methanol after concentration and then subjected to gel column chromatography. The gel column chromatography preferably uses methanol for elution.

[0029] In the case of silica gel column chromatography or gel column chromatography, the collected fractions can be determined by HPLC, such as using C 18 The chromatographic column or phenyl bonded column has a detection wavelength of 220-240 nm and an elution solvent selected from 0.1% TFA aqueous solution and acetonitrile.

[0030] In one embodiment, in step (3), the HPLC column includes but is not limited to C 18 Chromatographic column and phenyl bonded column, the detection wavelength is 220-240nm, the elution solvent is selected from methanol-water, acetonitrile-water; preferably, C 18 The chromatographic column has a detection wavelength of 240 nm, and the elution solvent is a 78% methanol-22% water mixed solvent. Specifically, the purification can be carried out using an HPLC preparative column and a semi-preparative column in sequence.

[0031] The preparation method of the cyclic ester peptide compound described in the present application is simple in process, short in time, greatly reduces the production cost of the drug, and is suitable for large-scale production.

[0032] In a fourth aspect, the present application provides a Streptomyces alboflavus sp. NX-12 fermentation extract, which is the extract obtained through steps (1) and (2) of the third aspect.

[0033] In a fifth aspect, the present application provides a cyclic lipopeptide compound of Formula I, its stereoisomers, tautomers, isotopic derivatives, or pharmaceutically acceptable salts thereof for use in the preparation of antibacterial drugs. The present application also provides a cyclic lipopeptide compound of Formula I, its stereoisomers, tautomers, isotopic derivatives, or pharmaceutically acceptable salts thereof for use in the treatment or prevention of fungal infections.

[0034] In a sixth aspect, the present application provides the use of a fermentation extract of Streptomyces alboflavus sp. NX-12 in the preparation of an antibacterial drug. The present application also provides the use of a fermentation extract of Streptomyces alboflavus sp. NX-12 in the treatment or prevention of fungal infections.

[0035] In one set of embodiments, the antimicrobial drug is an antibacterial drug, preferably an anti-Gram-positive bacteria drug.

[0036] In one set of embodiments, the fungi are bacteria, preferably Gram-positive bacteria.

[0037] In one set of embodiments, the Gram-positive bacteria are selected from Staphylococcus aureus, more preferably Staphylococcus aureus 25923 and Staphylococcus aureus USA300.

[0038] In a seventh aspect, the present application provides a pharmaceutical composition comprising a cyclic ester peptide compound shown in Formula I, its stereoisomers, tautomers, isotopic derivatives, or pharmaceutically acceptable salts thereof, or a Streptomyces alboflavus sp. NX-12 fermentation extract, and pharmaceutically acceptable excipients.

[0039] In one set of embodiments, the pharmaceutical composition further comprises other active ingredients, preferably other antibacterial active ingredients.

[0040] After research, it was found that compared with the existing technology, this application has the following characteristics and significant advantages:

[0041] The cyclic lipopeptide compounds described herein have strong inhibitory activity against a variety of Gram-positive bacteria and can be used to prepare antibacterial drugs. Compared to vancomycin, an existing glycopeptide antibiotic for Gram-positive bacteria, the cyclic lipopeptide compounds described herein have equivalent antibacterial efficacy.

[0042] 2. The preparation method of the cyclic ester peptide compound described in the present application is simple in process, short in time, greatly reduces the cost of the drug, and is suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 HPLC chromatograms of fermentation products using different fermentation media.

[0044] Figure 2 The figure is a flow chart for the extraction and separation of compounds represented by formula II-V.

[0045] Figure 3 This is the high-resolution electrospray ionization mass spectrum of the compound represented by formula II.

[0046] Figure 4 The compound represented by formula II 1 H NMR spectrum.

[0047] Figure 5 The compound represented by formula II 13 C NMR spectrum.

[0048] Figure 6 This is the high-resolution electrospray mass spectrum of the compound represented by formula III.

[0049] Figure 7 The compound represented by formula III 1 H NMR spectrum.

[0050] Figure 8 The compound represented by formula III 13 C NMR spectrum.

[0051] Figure 9 This is the high-resolution electrospray mass spectrum of the compound represented by formula IV.

[0052] Figure 10 For the compound shown in formula IV 1 H NMR spectrum.

[0053] Figure 11 For the compound shown in formula IV 13 C NMR spectrum.

[0054] Figure 12 This is the high-resolution electrospray ionization mass spectrum of the compound represented by Formula V.

[0055] Figure 13 The compound represented by formula V 1 H NMR spectrum.

[0056] The deposit number of Streptomyces alboflavus sp.NX-12 is CGMCC NO.33132, the deposit date is December 20, 2024, the depository is the General Microbiology Center of China Culture Collection Administration, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0057] The following describes the specific embodiments of the present application in detail. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present application and are not intended to limit the present application.

[0058] The endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0059] Before describing this application in detail, it should be understood that the terminology used herein is intended only to describe specific embodiments and is not intended to limit the scope of this application, which is defined solely by the appended claims. In order to more fully understand the present application described herein, the following terms are used and their definitions are as follows. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art to which this application belongs.

[0060] Unless otherwise specified, the following terms used in this application have the following definitions.

[0061] The term "halogen" refers to F, Cl, Br, I.

[0062] The term "pharmaceutically acceptable salt" refers to salts that are suitable for use in contact with mammalian tissues, particularly human tissues, without excessive toxicity, irritation, or allergic reaction, and are commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment. If the compound is basic, pharmaceutically acceptable salts include salts prepared from inorganic acids and also include salts prepared from organic acids. If the compound is acidic, pharmaceutically acceptable salts include salts prepared from inorganic bases and / or organic bases.

[0063] The term "pharmaceutically acceptable excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavoring, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, emulsifier, disintegrant approved by the relevant governmental regulatory authorities as acceptable for human or veterinary use.

[0064] The pharmaceutical composition of the present application can be prepared into tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, solutions, suppositories, injections, inhalants, and gels.

[0065] Example 1: Screening of culture medium for strain Streptomyces alboflavus sp. NX-12

[0066] Use a cotton swab to dip Streptomyces alboflavus sp.NX-12, evenly spread it on a MS solid culture medium plate (agar 20g / L, mannitol 20g / L, soybean powder 20g / L), and incubate it upside down in a 28℃ incubator for 3-5 days until the spores cover the surface. 2 The agar blocks containing bacteria were inoculated into 100 mL of 11 culture media listed in Table 1 (the culture media used were G2, F1, F2, B1, B2, YMS, NR, MKB, Iron-depleted No. 5, ISP3, and GYM) and fermented in liquid form for 4 days (28°C, 220 rpm). The fermentation broths were collected, extracted with equal amounts of ethyl acetate, concentrated under reduced pressure using a rotary evaporator, and dissolved in chromatography-grade methanol. HPLC analysis was performed on each of the broths (HPLC analysis conditions: Agilent HPLC, diode array detector (DAD), C 18 Analytical column (5 μm, 250 × 4.6 mm), detection wavelengths 240 nm, 360 nm, injection volume 30 μL, flow rate 1 mL / min, room temperature. Elution system: Solution A: water (0.1% trifluoroacetic acid), Solution B: acetonitrile; elution conditions: 0-5 min, B%: 30%; 5-10 min, B%: 30%-40%; 10-13 min, B%: 40%-52%; 13-35 min, B%: 52%-65%; 35-40 min, B%: 65%-100%) Figure 1 HPLC-DAD-MS and the Antibase database were used to analyze and eliminate duplicates. Product variations were compared and their molecular weights were analyzed using LC-MS. Ultimately, G2 medium was selected.

[0067] Table 1: The composition and preparation method of the culture medium are as follows:

[0068]

[0069]

[0070] Example 2: Fermentation of Streptomyces

[0071] Use a cotton swab to dip Streptomyces alboflavus sp.NX-12, evenly spread it on a MS solid culture medium plate (agar 20g / L, mannitol 20g / L, soybean powder 20g / L), and incubate it upside down in a 28℃ incubator for 3-5 days until the spores cover the surface. 2 The agar block containing bacteria was inoculated into 100 mL of G2 liquid medium (20 g / L millet, 10 g / L glucose, 3.0 g / L peptone, 4.5 g / L sodium chloride, 1.0 g / L calcium carbonate) and cultured at 28° C., 220 rpm for 48 hours to prepare seed solution. The seed solution was inoculated into G2 fermentation medium (composition of ingredients similar to G2 liquid medium) at a 10% inoculum size and cultured at 28° C., 220 rpm for 4-6 days.

[0072] Example 3: Extraction of fermentation products

[0073] The fermentation broth of strain Streptomyces alboflavus sp. NX-12 was collected and extracted with 2 volumes of ethyl acetate overnight. The organic phase was collected and the ethyl acetate was recovered under reduced pressure using a rotary evaporator to obtain an ethyl acetate extract.

[0074] Example 4: Separation and purification of compounds represented by formula II and III

[0075] according to Figure 2 The flow chart shown is used to separate and purify the compounds shown in formula II and III.

[0076] 1) Silica gel column chromatography of ethyl acetate extract

[0077] The ethyl acetate extract was mixed with a small amount of silica gel (200-300 mesh) and then rapidly separated using a 100g pre-packed silica gel normal phase column. The mobile phase was a dichloromethane / methanol solvent system. Elution was performed using four solvent systems: dichloromethane, dichloromethane:methanol = 1:3, dichloromethane:methanol = 1:1, and methanol. Each fraction was collected and concentrated, dissolved in methanol, filtered, and then analyzed by HPLC. HPLC analysis conditions: Agilent HPLC, diode array detector (DAD), C 18Analytical column (5 μm, 250 × 4.6 mm), detection wavelengths at 240 nm and 360 nm, injection volume 30 μL, flow rate 1 mL / min, room temperature. Elution system: Solution A: water (0.1% trifluoroacetic acid), Solution B: acetonitrile; elution conditions: 0-5 min, B%: 30%; 5-10 min, B%: 30%-40%; 10-13 min, B%: 40%-52%; 13-35 min, B%: 52%-65%; 35-40 min, B%: 65%-100%). Eluates with similar compounds were combined according to HPLC chromatographic peaks to obtain four fractions: Fractions I-IV. The target compound was in Fraction II.

[0078] 2) Gel column chromatography of target fraction II

[0079] Concentrate fraction II to dryness, dissolve it in methanol to 2 mL, and slowly and evenly load it with a glass dropper. The mobile phase is methanol. Collect one tube every 20 minutes using a fraction collector. Take 1 mL and filter it before HPLC analysis. HPLC analysis conditions: Agilent HPLC, diode array detector (DAD), C 18 Analytical column (5 μm, 250 × 4.6 mm), detection wavelengths at 240 nm and 360 nm, injection volume 30 μL, flow rate 1 mL / min, room temperature. Elution system: Solution A: water (0.1% trifluoroacetic acid), Solution B: acetonitrile; elution conditions: 0-5 min, B%: 30%; 5-10 min, B%: 30%-40%; 10-13 min, B%: 40%-52%; 13-35 min, B%: 52%-65%; 35-40 min, B%: 65%-100%). Fractions with similar compositions were combined to obtain fractions AC. The target compound was found in fraction B.

[0080] 3) HPLC preliminary preparation of fraction B

[0081] The preparative column used was C 18 Column (250×20 mm, 5 μm), preparative liquid phase SHIMADZU LC-16P system, UV detector, detection wavelength range 190-600 nm, online detection wavelength at 240 nm; injection volume 800 μL, flow rate 8 mL / min, room temperature. Elution system: Solution A: water, Solution B: methanol; elution conditions: B%: 78% constant gradient, constant flow rate, collection t R The organic phase of the eluate at 42-50 min was removed by concentration under reduced pressure and then lyophilized for subsequent purification.

[0082] 4) HPLC purification of the compound of formula II and the compound of formula III

[0083] The semi-preparative column used was C 18 Column (250×10 mm, 5 μm), semi-preparative liquid phase Agilent 1260 Infinity LC system, UV detector, detection wavelength range 190-600 nm, online detection wavelength 240 nm; injection volume 300 μL, flow rate 2 mL / min, room temperature. The fraction collected in step 3) was eluted using the following elution system: solution A: water, solution B: methanol; elution conditions: B%: 78% constant gradient, constant flow rate elution to obtain compound II (t R =35min) and compound of formula III (t R =38min).

[0084] Example 5: Structural Identification of Zhengxinmycin A, a Compound of Formula II

[0085] The obtained compound of Formula II, zhengxinmycin A, is a white powdery solid that is soluble in acetone, ethyl acetate, methanol, chloroform, acetonitrile, etc., but insoluble in water. It is colorless in acetonitrile, pale yellow-green in methanol, and fluorescent yellow-green in DMSO. High-resolution electron spray mass spectrometry (HRESIMS) shows a quasi-molecular ion peak of m / z 842.3267 [M+H] + (like Figure 3 As shown), it suggests that its molecular composition is C 38 H 48 ClN9O 11 The UV spectrum of the compound represented by Formula II shows maximum absorption peaks at 233nm, 316nm and 353nm, revealing the presence of unsaturated structural units in the compound. 1 HNMR, 13 C NMR data (such as Figure 4-5 and Table 2) to determine its structural formula.

[0086] Example 6: Structural Identification of Zhengxinmycin B, a Compound of Formula III

[0087] The resulting compound, Formula III, zhengxinmycin B, is a white powdery solid that is soluble in acetone, ethyl acetate, methanol, chloroform, acetonitrile, and the like, but insoluble in water. It is colorless in acetonitrile, yellow-green in methanol, and fluorescent yellow-green in DMSO. High-resolution electron spray mass spectrometry (HRESIMS) revealed a quasi-molecular ion peak at m / z 828.3415 [M+H] + (like Figure 6 As shown), it suggests that its molecular composition is C 38 H 50 ClN9O 10The UV spectrum of the compound represented by formula III shows maximum absorption peaks at 233nm, 316nm and 353nm, revealing the presence of unsaturated structural units in the compound. 1 HNMR, 13 C NMR data (such as Figure 7-8 and Table 2) to determine its structural formula.

[0088] Table 2: NMR data of compounds of formula II and formula III

[0089]

[0090]

[0091]

[0092] Compound of formula II: 1 H NMR, 600 MHz, 13 C NMR, 150 MHz; Compound of formula III: 1 H NMR, 500 MHz, 13 CNMR, 125MHz.

[0093] Example 7: Fermentation, extraction, separation and purification of the compound of formula IV and the compound of formula V

[0094] Fermentation was carried out according to Example 2, except that sodium chloride in the G2 liquid medium and G2 fermentation medium [millet 20 g / L, glucose 10 g / L, peptone 3.0 g / L, sodium chloride 4.5 g / L, calcium carbonate 1.0 g / L] was replaced with sodium bromide, and other conditions and steps were the same.

[0095] The fermentation product was extracted, separated and purified according to Example 3-4. R =40min, compound of formula V t R =44min.

[0096] Example 8: Structural Identification of Compound IV Zhengxinmycin C

[0097] The resulting compound, formula IV, zhengxinmycin C, is a yellow-brown powdery solid that is soluble in acetone, ethyl acetate, methanol, chloroform, acetonitrile, and the like, but insoluble in water. It appears yellow in methanol and yellow-brown in DMSO. High-resolution electron spray mass spectrometry (HRESIMS) revealed a quasi-molecular ion peak at m / z 886.2768 [M+H]. + ( Figure 9 ), suggesting that its molecular composition is C 38 H48 BrN9O 11 The UV spectrum of the compound of formula IV shows maximum absorption peaks at 233nm, 316nm and 353nm, which is basically consistent with compounds II and III. 1 H NMR data and HR-MS 2 Cracking (such as Figure 10-11 and Table 3) to determine its structural formula.

[0098] Example 9: Structural Identification of Compound V Zhengxinmycin D

[0099] The obtained compound of formula V, zhengxinmycin D, is a white powdery solid that is soluble in acetone, ethyl acetate, methanol, chloroform, acetonitrile, etc., but insoluble in water. It is yellow in methanol and yellow-brown in DMSO. High-resolution electron spray mass spectrometry (HRESIMS) shows a quasi-molecular ion peak of m / z 872.2955 [M+H] + ( Figure 12 ), suggesting that its molecular composition is C 38 H 50 BrN9O 10 The UV spectrum of the compound of formula V shows maximum absorption peaks at 233nm, 316nm and 353nm, which is basically consistent with compounds II and III. 1 H NMR data and HR-MS 2 Cracking (such as Figure 13 and Table 3) to determine its structural formula.

[0100] Table 3: NMR data of compound IV and compound V

[0101]

[0102]

[0103] Compounds of formula IV and formula V: 1 H NMR, 500 MHz, 13 C NMR, 120 MHz.

[0104] Example 10: Antibacterial Activity Test of Compounds of Formula II, Formula III, Formula IV and Formula V

[0105] 1: Experimental Materials

[0106] 1 mg of each of the four cyclic ester peptide compounds was taken and dissolved in DMSO to prepare a 1 mg / mL stock solution.

[0107] The inhibitory activity against four strains of Gram-positive resistant bacteria, S. aureus ATCC 25923 and S. aureus USA300, and Gram-negative resistant bacteria, Klebsiella pneumoniae MGH78578 and Acinetobacter baumannii ATCC 19606, was evaluated.

[0108] Vancomycin and polymyxin B were selected as positive control drugs.

[0109] 2: Experimental Methods

[0110] Dilute the bacterial suspension of the four test active strains to 1×10 8 CFU / ml, add 100 μL / well bacterial solution to the 96-well plate; prepare the drug stock solution concentration to 16 μg / mL, dilute it doubly, add 100 μL of drug to each well, place the 96-well plate in a 37°C incubator and incubate for 18 hours, and use a microplate reader to measure the OD 600 value.

[0111] 3: Experimental results

[0112] Table 4 MIC values (μg / mL) of compounds of formula II, formula III, formula IV and formula V

[0113]

[0114] As can be seen, the compounds of Formula II, Formula III, Formula IV, and Formula V of the present application have strong inhibitory activity against a variety of Gram-positive bacteria and can be used to prepare drugs for antibacterial diseases. Compared with vancomycin, an existing glycopeptide antibiotic against Gram-positive bacteria, the cyclic lipopeptide compounds described in this application have equivalent antibacterial effects.

[0115] The scope of protection of this application is not limited to the above-described embodiments. Obviously, those skilled in the art may make various modifications and variations to this application without departing from the scope and spirit of the invention. If such modifications and variations fall within the scope of the claims of this application and their equivalents, the intention of this application also includes such modifications and variations.

[0116] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner unless there is any contradiction. In order to avoid unnecessary repetition, this application will not further describe various possible combinations.

[0117] In addition, the various implementation methods of the present application may be arbitrarily combined, and as long as they do not violate the concept of the present application, they should also be regarded as the contents disclosed in the present application.

Claims

1. A cyclic ester peptide compound represented by formula I, its stereoisomers, tautomers, isotopic derivatives, or pharmaceutically acceptable salts thereof, In formula I, R1 is H, H, or H, OH, or =O, and R2 is hydrogen, hydroxy, methoxy, or halogen.

2. The cyclic ester peptide compound of formula I according to claim 1, its stereoisomers, tautomers, isotopic derivatives, or pharmaceutically acceptable salts thereof, characterized in that: The cyclic lipopeptide compound represented by formula I is zhengxinmycin A, a compound of formula II, zhengxinmycin B, a compound of formula III, zhengxinmycin C, a compound of formula IV, or zhengxinmycin D, a compound of formula V; 3. A Streptomyces strain, Streptomyces alboflavus sp.NX-12, which was deposited in the General Microbiology Center of the China Culture Collection Administration on December 20, 2024, with the deposit number CGMCC No.33132, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

4. A method for preparing the cyclic ester peptide compound of formula I according to claim 1 or 2, its stereoisomers, tautomers, isotopic derivatives, or pharmaceutically acceptable salts thereof, comprising the following steps: (1) Fermentation: Streptomyces alboflavus sp. NX-12 was fermented by shaking culture in a liquid culture medium; (2) Extraction: The fermentation broth was collected, extracted with an organic solvent, and the organic phase was concentrated to obtain an extract; (3) Separation and purification: Silica gel column chromatography, gel column chromatography, and HPLC were used for separation and purification to obtain cyclic ester peptide compounds.

5. The preparation method according to claim 4, characterized in that In the step (1), Streptomyces alboflavus sp. NX-12 is fermented in a G2 liquid culture medium at 26-32° C. and 180-250 rpm for 2-8 days, preferably at 28-30° C. and 200-220 rpm for 4-6 days.

6. The preparation method according to claim 4 or 5, characterized in that In the step (1), Streptomyces alboflavus sp. NX-12 is first shaken cultured in a liquid culture medium to obtain a seed solution, which is then inoculated into the liquid culture medium at a 5-15% inoculum amount for shaken culture and fermentation; preferably, Streptomyces alboflavus sp. NX-12 is shaken cultured in a G2 liquid culture medium at 26-32° C. and 180-250 rpm for 1-4 days to obtain the seed solution, and more preferably, the seed solution is shaken cultured at 28-30° C. and 200-220 rpm for 2-3 days to obtain the seed solution.

7. The preparation method according to claim 6, characterized in that In the step (1), Streptomyces alboflavus sp. NX-12 is cultured on a solid culture medium to produce spores, and then the spores are inoculated into a liquid culture medium and shaken to obtain a seed solution; preferably, the spores are produced on a MS solid culture medium at 26-32° C. for 2-8 days; more preferably, the spores are produced on a MS solid culture medium at 28-30° C. for 3-5 days.

8. The preparation method according to claim 4 or 5, characterized in that In the step (2), the organic solvent is selected from: a mixed solvent of one or more of chloroform, dichloromethane, ethyl acetate, alcohols with a carbon chain of ≤4, acetone, and butanone.

9. The preparation method according to claim 4 or 5, characterized in that In the step (3), the elution solvent used for the silica gel column chromatography and the gel column chromatography is selected from: a mixed solvent of one or more of chloroform, dichloromethane, ethyl acetate, alcohols with a carbon chain ≤ 4, acetone, and butanone; preferably, the silica gel column chromatography uses dichloromethane and methanol for gradient elution, and the gel column chromatography uses methanol for elution.

10. The preparation method according to claim 9, characterized in that In the step (3), during silica gel column chromatography, organic solvent gradient elution is sequentially performed using dichloromethane:methanol = 1:0, 1:3, 1:1 and 0:1, and the eluate with dichloromethane:methanol = 1:3 is concentrated and then subjected to gel column chromatography.

11. The preparation method according to claim 4 or 5, characterized in that: In the step (3), the HPLC column is selected from C 18 Chromatographic column and phenyl bonded column, the detection wavelength is 220-240nm, and the elution solvent is selected from methanol-water and acetonitrile-water; preferably, C 18 The chromatographic column has a detection wavelength of 240 nm and an elution solvent of 78% methanol-22% water mixed solvent.

12. A Streptomyces alboflavus sp. NX-12 fermentation extract, which is obtained by steps (1) and (2) according to any one of claims 4 to 8.

13. Use of the cyclic ester peptide compound of formula I according to claim 1 or 2, its stereoisomers, tautomers, isotopic derivatives, or pharmaceutically acceptable salts thereof, or the Streptomyces alboflavus sp. NX-12 fermentation extract according to claim 12 in the preparation of antibacterial drugs.

14. The use according to claim 13, characterized in that The antimicrobial drug is an antibacterial drug, preferably a drug against Gram-positive bacteria.

15. A pharmaceutical composition comprising the cyclic ester peptide compound of formula I according to claim 1 or 2, its stereoisomers, tautomers, isotopic derivatives, or pharmaceutically acceptable salts thereof, or the Streptomyces alboflavus sp. NX-12 fermentation extract according to claim 12, as well as pharmaceutically acceptable excipients, and optionally other active ingredients.

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