A strain of Nocardia sp. YINM00009 and its application in the preparation of cyclic lipopeptide compounds

Abstract

The present invention discloses a strain of Nocardia YINM00009 and its application in the preparation of cyclic lipopeptide compounds, belonging to the field of microbial technology. The Nocardia YINM00009 is an endophyte of Polygonatum daturae in Kunming, Yunnan Province, and can metabolize a large number of novel cyclic lipopeptide compounds after fermentation; the fermentation step mainly includes: fermenting the strain under suitable culture conditions to obtain a fermentation broth, after solvent extraction and concentration, combined with column chromatography, 9 cyclic lipopeptide compounds can be prepared, the total concentration of the 9 cyclic lipopeptide compounds in the fermentation broth can reach 10.2~16.5mg / L, and the purity after extraction and separation can reach more than 99%, of which 2 cyclic lipopeptide compounds have strong anti-tumor cytotoxic activity. The method is low in cost, simple in process, and environmentally friendly, and can be used for the mass production and preparation of such cyclic lipopeptide compounds, which not only meets the needs of a low-carbon economy, but also lays the foundation for the production of cyclic lipopeptide compounds and the development of related products in the later stage.

Description

Technical Field

[0001] The invention relates to a Nocardia sp. YINM00009 and application thereof in preparing cyclic lipopeptide compounds, belonging to the technical field of microorganisms. Background Art

[0002] In recent years, microbial fermentation has attracted increasing attention as a method for preparing target active compounds due to its advantages such as short metabolic cycles, mild reaction conditions, few byproducts, and strong stereoselectivity. In actual production, there are already numerous applications where microbial fermentation has been used to produce compounds with medicinal or economic value.

[0003] Cyclic lipopeptides are amphiphilic cyclic molecules formed by ester or amide bonds between fatty acids and peptide fragments. Derived primarily from microbial secondary metabolites, cyclic lipopeptides exhibit environmentally friendly properties such as low toxicity, minimal irritation, and biodegradability. Furthermore, most cyclic lipopeptides possess unique biological activities, including antibacterial and antitumor properties, attracting considerable attention in recent years. Daptomycin, a novel cyclic lipopeptide antibiotic derived from the fermentation broth of Streptomyces reseosporus, was discovered by Eli Lilly and Company in the 1980s and successfully developed by Cubist Pharmaceuticals in 1997. Daptomycin possesses not only a novel chemical structure but also a mode of action distinct from any previously approved antibiotic: it disrupts amino acid transport across the cell membrane, thereby hindering the biosynthesis of peptidoglycan in the bacterial cell wall and altering the properties of the cytoplasmic membrane. Daptomycin disrupts bacterial cell membrane function in multiple ways, rapidly killing Gram-positive bacteria. In addition to its ability to act against most clinically relevant Gram-positive bacteria, daptomycin also exhibits potent in vitro activity against isolates resistant to methicillin, vancomycin, and other drugs. This property is of great clinical significance for patients with critically ill infections. Currently, cyclic lipopeptides are primarily obtained through the separation and purification of microbial fermentation metabolites. However, the strains capable of metabolizing cyclic lipopeptides are mostly plant- or soil-derived actinomycetes, which produce low yields of cyclic lipopeptides. This low yield has hindered the development and utilization of cyclic lipopeptides.

[0004] Nocardia Nocardia sungurluensis YINM00009 is an endophytic actinomycete obtained from Polygonatum sibiricum in Kunming, Yunnan. Analysis of its secondary metabolites revealed that it has a high yield of novel cyclic lipopeptide compounds and is easy to separate and purify. Therefore, this strain has the potential to be developed as an engineered bacterium to produce novel cyclic lipopeptide compounds. Summary of the Invention

[0005] One of the objects of the present invention is to provide a strain of Nocardia sp. YINM00009, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on October 28, 2024. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with a deposit number of GDMCC No: 65367 and a taxonomic name: Nocardia sungurluensis .

[0006] The second object of the present invention is to provide an application of Nocardia sp. YINM00009 in the preparation of novel cyclic lipopeptide compounds.

[0007] There are 9 novel cyclolipopeptide compounds prepared by the present invention, among which the structure of the first compound is shown in Formula I;

[0008] Formula I.

[0009] The structure of the second compound is shown in Formula II;

[0010] Formula II.

[0011] The structure of the third compound is shown in Formula III;

[0012] Formula III.

[0013] The structure of the fourth compound is shown in Formula IV;

[0014] Formula IV.

[0015] The structure of the fifth compound is shown in Formula V;

[0016] Formula V.

[0017] The structure of the sixth compound is shown in Formula VI;

[0018] Formula VI.

[0019] The structure of the seventh compound is shown in Formula VII;

[0020] Formula VII.

[0021] The structure of the eighth compound is shown in Formula VIII;

[0022] Formula VIII.

[0023] The structure of the ninth compound is shown in Formula IX;

[0024] Formula IX.

[0025] The third object of the present invention is to provide a method for preparing novel cyclic lipopeptide compounds by Nocardia YINM00009, which mainly comprises the following steps:

[0026] (1) Bacterial activation: Nocardia YINM00009 was inoculated into the strain activation medium for activation and used for later use.

[0027] (2) Preparation of bacterial strains: The activated bacterial cells in step (1) are inoculated into a seed culture medium for cultivation to obtain bacterial strains.

[0028] (3) Fermentation process: The strain prepared in step (2) is inoculated into a fermentation medium for fermentation to obtain a fermentation liquid.

[0029] (4) After the fermentation, the fermentation broth was extracted with an equal volume of ethyl acetate and concentrated to obtain a crude extract. The crude extract was dissolved in dichloromethane-methanol and eluted by gel column chromatography using dichloromethane-methanol eluent to obtain 200 tubes of eluent. After TLC plate color development, tubes 1-27, 28-32, 33-47, 48-54, 55-70, 71-86, 87-100, 101-115, 116-135, and 136-200 were combined to obtain 10 eluents.

[0030] (5) The eluates from tubes 28-32, 33-47, 48-54, and 55-70 obtained in step (4) were selected for further separation, and gradient elution was performed on a silica gel column using petroleum ether-ethyl acetate as eluent. Finally, semi-preparative high performance liquid chromatography was used for purification to obtain the compounds represented by Formula I to Formula IX.

[0031] Preferably, the components of the activation medium in step (1) are: yeast extract 4.0±0.5 g / L, glucose 4.0±0.5 g / L, malt extract 10.0±0.5 g / L, agar 15.0±0.5 g / L, pH 7.0±0.2, the culture temperature is 28±0.5°C, and the culture time is 5~7 days.

[0032] Preferably, the composition of the seed culture medium in step (2) is: yeast extract 4.0±0.5 g / L, glucose 4.0±0.5 g / L, malt extract 10.0±0.5 g / L, pH 7.0±0.2; the culture temperature is 28±0.2°C, and the culture time is 3~5 days.

[0033] Preferably, the composition of the fermentation medium in step (3) is: sucrose 100±5 g / L, glucose 10±0.5 g / L, acid hydrolyzed casein 0.12±0.1 g / L, yeast extract 5 g±0.5 / L, 3-(N-morpholino)propanesulfonic acid 21±0.5 g / L, trace elements 1.0 mL, K2SO4 0.25±0.1 g / L, MgCl2·6H2O 10 g±0.5 / L, pH=7.0±0.2; the inoculum size of the strain is 10% of the volume of the fermentation medium; the culture temperature is 28±0.2°C, and the culture time is 10-20 days.

[0034] Preferably, in step (4), ethyl acetate extraction is performed three times; the volume ratio of dichloromethane to methane in the dichloromethane-methanol solution used to dissolve the crude extract is 1:2; and the gel column chromatography filler used is hydroxypropyl dextran gel (Sephadex LH-20).

[0035] Preferably, the volume ratio of dichloromethane to methanol in the dichloromethane-methanol eluent in the gel column chromatography elution in step (4) is 1:1.

[0036] Preferably, the particle size of the silica gel used in step (5) is 300-400 mesh.

[0037] Preferably, the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate eluent used in the forward silica gel column chromatography gradient elution in step (5) is 30:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:5, 1:10, wherein the volume ratio of petroleum ether to ethyl acetate eluent in tubes 28-32 is 1:1, containing the compounds of formula I and formula II; wherein the volume ratio of petroleum ether to ethyl acetate eluent in tubes 33-47 is 1:1, containing the compounds of formula I and formula II; The eluent of petroleum ether-ethyl acetate with a volume ratio of 1:1 contains compounds represented by formula IV and formula VIII; the eluent of petroleum ether-ethyl acetate with a volume ratio of 2:1 in tubes 48-54 contains compounds represented by formula III and formula VII; the eluent of petroleum ether-ethyl acetate with a volume ratio of 1:2 in tubes 55-70 contains compounds represented by formula V and formula VI; the eluent of petroleum ether-ethyl acetate with a volume ratio of 5:1 in tubes 33-47 contains the compound represented by formula IX.

[0038] The fourth object of the present invention is to provide the use of 9 novel cyclic lipopeptide compounds in the preparation of drugs for treating lung cancer, liver cancer, breast cancer or colon cancer.

[0039] Beneficial effects of the present invention

[0040] (1) The present invention provides a Nocardia strain capable of producing high levels of cyclic lipopeptides. Nocardia sungurluensis YINM00009, this strain can produce 9 new cyclic lipopeptides with high yield.

[0041] (2) This invention obtained 9 new cyclolipopeptides for the first time, enriching the types of cyclolipopeptides.

[0042] (3) The raw materials used in the method of the present invention are cheap and easily available, the reaction conditions are mild, the process is simple, the equipment requirements are simple, and the environment is pollution-free, which is suitable for the scale-up production of cyclic lipopeptide compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The crystal structure of the compound of formula V provided by the present invention.

[0044] Figure 2 The present invention provides the crystal structure of the compound of formula VI.

[0045] Figure 3 The present invention provides the crystal structure of the compound of formula IX. DETAILED DESCRIPTION

[0046] The technical solutions of the present invention are further described below with reference to the accompanying drawings and through specific embodiments. However, the following embodiments are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is subject to the claims. Technical operations not specifically described are well known to those skilled in the art.

[0047] The surface of Polygonatum sibiricum collected from Kunming, Yunnan was sterilized by soaking in sodium hypochlorite solution (effective chlorine concentration 5.0%), 75% ethanol and washing with sterile water. The plant tissue was then processed into small pieces and implanted into an activation medium sterilized at 121°C. After culturing at 28°C for 7 days, the endophytic bacteria were continuously purified using the activation medium sterilized at 121°C. Nocardia sungurluensis YINM00009, deposit number is GDMCC No.65367.

[0048] The Nocardia sp. YINM00009 described in the present invention is a rare actinomycete with a smooth surface, no spores, and no aerial hyphae on ISP2 medium. The 16s RNA sequence of Nocardia sp. YINM00009 is shown in SEQ ID No. 1:

[0049]

[0050] The culture medium used in the examples is as follows: All culture media need to be sterilized at 121°C for 20 min before use.

[0051] (1) The composition of the activation medium is: yeast extract 4.0 g / L, glucose 4.0 g / L, malt extract 10.0 g / L, agar 15.0 g / L, pH 7.0.

[0052] (2) The composition of the seed culture medium is: yeast extract 4.0 g / L, glucose 4.0 g / L, malt extract 10.0 g / L, pH 7.0.

[0053] (3) The composition of the fermentation medium was as follows: sucrose 100 g / L, glucose 10 g / L, acid hydrolyzed casein 0.12 g / L, yeast extract 5 g / L, 3-(N-morpholino)propanesulfonic acid 21 g / L, trace elements 1.0 mL, K2SO4 0.25 g / L, MgCl2·6H2O 10 g / L, pH = 7.0.

[0054] Example 1

[0055] Nocardia YINM00009 is used to prepare a novel cyclic lipopeptide compound, which specifically includes the following steps:

[0056] (1) Bacterial activation: Nocardia YINM00009 was inoculated into the activation medium, cultured in a constant temperature incubator at 28°C for 5 days, and then placed in a refrigerator at 4°C for later use.

[0057] (2) Preparation of bacterial strains: The activated bacterial cells were inoculated into the seed culture medium and fermented at 28°C and 200 rpm for 3 days.

[0058] (3) Fermentation process: The strain prepared in step (2) was added into 20 L fermentation medium (sucrose 100 g / L, glucose 10 g / L, acid hydrolyzed casein 0.12 g / L, yeast extract 5 g / L, 3-(N-morpholino)propanesulfonic acid 21 g / L, trace elements 1.0 mL, K2SO4 0.25 g / L, MgCl2·6H2O 10 g / L, pH = 7.0) at a volume fraction of 10%), and fermented at 28°C and 200 rpm for 15 days to obtain a fermentation broth.

[0059] (4) After the fermentation, the fermentation liquid was extracted three times with an equal volume of ethyl acetate and concentrated in vacuum to obtain a crude extract (9.8 g). The crude extract was detected by high performance liquid chromatography, and the concentrated crude extract was dissolved in an appropriate amount of dichloromethane-methanol mixed solution (the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixed solution was 1:2) to obtain a crude extract solution; the prepared crude extract solution was slowly added to the gel column filled with filler using a dropper, and the gel column chromatography was eluted using an eluent with a volume ratio of dichloromethane to methanol of 1:1. Every 3 minutes, 1 tube (volume of 10.0 mL) of eluent was connected with an automatic sampler, and a total of 200 tubes of eluent were obtained. After TLC spotting, development and color development, the color of the spot and R f The values ​​of tubes 1-27, 28-32, 33-47, 48-54, 55-70, 71-86, 87-100, 101-115, 116-135, and 136-200 were merged to obtain 10 eluates, which were expressed as Fr.1-Fr.10.

[0060] (5) Take 10 aliquots of the eluate for TLC spot plate and HPLC analysis respectively. Tubes 28-32, 33-47, 48-54 and 55-70 were developed by TLC spot plate (developing solvent: dichloromethane / methanol volume ratio of 15 / 1) and found to have many spots. At the same time, combined with HPLC analysis (chromatographic gradient elution conditions: mobile phase is methanol and water, 0 min, 30% methanol / 70% water; 30 min, 100% methanol; 35 min, 100% methanol) has many peaks, so the eluates of fractions Fr.2, Fr.3, Fr.4 and Fr.5 were selected for further separation, and the eluates were slowly added dropwise to the prepared 300-400 mesh silica gel filler with a dropper and stirred until the solvent was completely evaporated. After loading, the forward silica gel column (300-400 mesh) was chromatographed on an eluent with a petroleum ether:ethyl acetate volume ratio of 30:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:5, and 1:10 to obtain the eluate.

[0061] According to the results of TLC spot plate and HPLC analysis, the eluent of petroleum ether-ethyl acetate volume ratio of 1:1 in fraction Fr.2 was selected and purified by semi-preparative HPLC (semi-preparative gradient elution method: 0 min, 95% methanol; 5 min, 100% methanol; 15 min, 100% methanol) to obtain compound I (210.0 mg) and compound II (190 mg). HPLC analysis of compound I (retention time t R =33.5min) and compound II (retention time t R=32.5min) with a purity of over 99% (analytical gradient elution method: 0 min, 30% methanol; 30 min, 100% methanol).

[0062] According to the results of TLC spot plate and HPLC analysis, the eluent of petroleum ether-ethyl acetate volume ratio of 1:1 in fraction Fr.3 was selected and purified by semi-preparative HPLC (semi-preparative gradient elution method: 0 min, 95% methanol; 5 min, 100% methanol; 15 min, 100% methanol) to obtain compound IV (205.0 mg) and compound VIII (198 mg). HPLC analysis of compound IV (retention time t R =32.7min) and compound VIII (retention time t R =33.7min) with a purity of over 99% (analytical gradient elution method: 0 min, 30% methanol; 30 min, 100% methanol).

[0063] According to the results of TLC spot plate and HPLC analysis, the eluent of petroleum ether-ethyl acetate volume ratio of 2:1 in fraction Fr.4 was selected and purified by semi-preparative HPLC (semi-preparative gradient elution method: 0 min, 95% methanol; 5 min, 100% methanol; 15 min, 100% methanol) to obtain compound III (180.0 mg) and compound VII (160 mg). HPLC analysis of compound III (retention time t R =33.0min) and compound VII (retention time t R =34.1min) with a purity of over 99% (analytical gradient elution method: 0 min, 30% methanol; 30 min, 100% methanol).

[0064] According to the results of TLC spot plate and HPLC analysis, the eluent of petroleum ether-ethyl acetate volume ratio of 1:2 in fraction Fr.5 was selected and purified by semi-preparative HPLC (semi-preparative gradient elution method: 0 min, 95% methanol; 5 min, 100% methanol; 15 min, 100% methanol) to obtain compound V (150.0 mg) and compound VI (165 mg). HPLC analysis of compound V (retention time t R =32.2min) and compound VI (retention time t R =33.2min) with a purity of over 99% (analytical gradient elution method: 0 min, 30% methanol; 30 min, 100% methanol).

[0065] Based on the results of TLC spot plate and HPLC analysis, the eluent of petroleum ether-ethyl acetate volume ratio of 5:1 in fraction Fr.2 was selected and purified by semi-preparative HPLC (semi-preparative gradient elution method: 0 min, 95% methanol; 5 min, 100% methanol; 15 min, 100% methanol) to obtain compound IX (280.0 mg). Compound IX was analyzed by HPLC (retention time t R =34.5min) with a purity of more than 99% (analytical gradient elution method: 0 min, 30% methanol; 30 min, 100% methanol).

[0066] Finally, after high performance liquid chromatography analysis, the concentration of compound I in the fermentation broth was 10.5 mg / L; the concentration of compound II was 9.5 mg / L; the concentration of compound III was 9.0 mg / L; the concentration of compound IV was 10.25 mg / L; the concentration of compound V was 7.5 mg / L; the concentration of compound VI was 8.25 mg / L; the concentration of compound VII was 8.0 mg / L; the concentration of compound VIII was 9.9 mg / L; and the concentration of compound IX was 14.0 mg / L.

[0067] Example 2

[0068] Identification of the structure of the obtained novel cyclic lipopeptide compound

[0069] The compound I obtained in Example 1 was separated and characterized by one-dimensional nuclear magnetic resonance spectroscopy (1D-NMR), two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR), high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and secondary mass spectrometry (Q-TOF-MS). 2 ) to identify its structure.

[0070] From the HSQC spectrum combined with the carbon spectrum, the chemical shift δ assignments of H and the C connected to compound I are shown in Table 1:

[0071] Table 1 shows the isolated compound I obtained in Example 1. 1 H (600MHz) and 13 C (150 MHz) NMR data, DMSO-d6 as solvent

[0072]

[0073]

[0074] HR-ESI-MS m / z of compound Ⅰ: [M+H] + The quasi-molecular ion peak is 1049.7373 (C 58 H 97 N8O9[M+H] +, calc m / z: 1049.7373) indicates that its molecular formula is C 58 H 96 N8O9, contains 15 degrees of unsaturation. 1 H-NMR, 13 C-NMR and HSQC spectra analysis showed that compound Ⅰ contained 11 methyl groups [δ H 0.74 (δ C 11.5), δ H 1.34 (δ C 17.3), δ H 0.83 (δ C 23.3), δ H 0.85 (δ C 22.9), δ H 0.84 (δ C 14.0), δ H 0.71 (δ C 15.1), δ H 0.82 (δ C 21.9), δ H 0.82 (δ C 21.3), δ H 0.82 (δ C 20.9), δ H 0.81 (δ C 21.3), δ H 1.21 (δ C 15.2)], 19 methylene groups [δ H 1.67,1.48(δ C 37.8), 3.07, 3.03 (δ C 27.0), δ H 1.37 (δ C 41.5), δ H 1.45 (δ C 40.4), δ H 1.37 (δ C 41.5), δ H 1.21 (δ C 15.2), δ H 2.30,2.23(δ C 35.0), δ H 1.53 (δ C 25.5), δ H 1.22 (δ C 28.5), δ H 1.22 (δ C28.7), δ H 1.22 (δ C 28.7), δ H 1.22 (δ C 28.8), δ H 1.22 (δ C 28.8), δ H 1.22 (δ C 28.9), δ H 1.22 (δ C 28.9), δ H 1.22 (δ C 29.0), δ H 1.22 (δ C 29.0), δ H 1.22 (δ C 29.0), δ H 1.22 (δ C 31.3), δ H 1.24 (δ C 22.1)], 17 methylenes [δ H 4.26 (δ C 56.8), δ H 4.85 (δ C 68.8), δ H 4.30 (δ C 50.9), δ H 1.34 (δ C 24.3), δ H 4.43 (δ C 54.2), δ H 7.18 (δ C 123.6), δ H 7.59 (δ C 118.2), δ H 6.96 (δ C 118.3), δ H 7.06 (δ C 121.1), δ H 7.33 (δ C 111.5), δ H 3.87 (δ C 50.9), δ H 1.53 (δ C 24.6), δ H 4.33 (δ C 51.4), δ H 1.38 (δ C 24.3), δH 4.29 (δ C 53.2), δ H 1.48 (δ C 24.1), δ H 4.23 (δ C 48.3)] and 11 quaternary carbons [δ C 175.3, δ C 168.3, δ C 170.8, δ C 109.3, δ C 126.9, δ C 136.2, δ C 171.9, δ C 170.8, δ C 170.4, δ C 170.9, δ C 168.3].

[0075] Based on the above 1D, 2D NMR and high-resolution mass spectrometry data, the presence of amide carbon, α-C / α-H and β-C / β-H and saturated fatty alkyl chains were found. Preliminary analysis showed that the substance was a macrocyclic lipopeptide compound. In order to determine the type and connection order of the amino acid residues in the macrocyclic lipopeptide compound, Q-TOF-MS was tested. 2 Secondary mass spectrometry and fragment ion analysis preliminarily determined the planar structure of the macrocyclic lipopeptide. Searching relevant databases and related literature revealed that the molecule is a new, previously unreported cyclic lipopeptide compound, named nocacyclomycin Ⅰ.

[0076] To determine the absolute configuration of amino acids, 1.0 mg of nocacyclomycin I was weighed and acid-hydrolyzed with 6 M HCl (1 mL) at 110 °C for 24 h. The amino acid residues after acid hydrolysis were then reacted with Marfey's reagent L-FDAA. At the same time, amino acid standards (L- / D-) were reacted with L-FDAA under the same conditions. Finally, the absolute configuration of the amino acid residues in the sample was analyzed by LC-MS and was L-Thr, L-Leu, and L-Thr. 1 , D-Trp, L-Leu 2 , L-Leu 3 , L-Leu 4 , D-Ala.

[0077] In summary, it can be determined that the structural formula of Compound I isolated in Example 1 is:

[0078]

[0079] The compound II obtained in Example 1 was separated and characterized by one-dimensional nuclear magnetic resonance spectroscopy (1D-NMR), two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR), high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), and secondary mass spectrometry (Q-TOF-MS). 2 ) to identify its structure.

[0080] From the HSQC spectrum combined with the carbon spectrum, it can be obtained that the chemical shift δ assignments of H and the C connected to compound II are shown in Table 2:

[0081] Table 2 Compound II isolated in Example 1 1 H (600MHz) and 13 C (150 MHz) NMR data, DMSO-d6 as solvent

[0082]

[0083]

[0084] HR-ESI-MS m / z of compound II: [M+H] + The quasi-molecular ion peak is 1021.7062 (C 56 H 93 N8O9[M+H] + , calc m / z: 1021.7060) indicates that its molecular formula is C 56 H 92 N8O9, contains 15 degrees of unsaturation. According to HR-ESI-MS, 1 H-NMR and 13 Comparison of C-NMR with compound nocacyclomycin Ⅱ showed that compound Ⅱ had one less -CH2CH2- fragment than compound Ⅰ, so this compound was named nocacyclomycin Ⅱ.

[0085] To determine the absolute configuration of amino acids, 1.0 mg of nocacyclomycin II was weighed and acid-hydrolyzed with 6 M HCl (1 mL) at 110 °C for 24 h. The amino acid residues after acid hydrolysis were then reacted with Marfey's reagent L-FDAA. At the same time, amino acid standards (L- / D-) were reacted with L-FDAA under the same conditions. Finally, the absolute configuration of the amino acid residues in the sample was analyzed by LC-MS, which was L-Thr, L-Leu 1 , D-Trp, L-Leu 2 , L-Leu 3 , L-Leu 4 , D-Ala.

[0086] In summary, it can be determined that the structural formula of Compound II isolated in Example 1 is:

[0087]

[0088] The compound III isolated in Example 1 was characterized by one-dimensional nuclear magnetic resonance spectroscopy (1D-NMR), two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR), high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), and secondary mass spectrometry (Q-TOF-MS). 2 ) to identify its structure.

[0089] From the HSQC spectrum combined with the carbon spectrum, it can be obtained that the chemical shift δ assignments of H and the C connected to compound III are shown in Table 3:

[0090] Table 3 Compound III isolated in Example 1 1 H (600MHz) and 13 C (150 MHz) NMR data, chloroform-d as solvent

[0091]

[0092]

[0093] HR-ESI-MS m / z of compound III: [M+H] + The quasi-molecular ion peak is 982.6951 (C 54 H 92 N7O9[M+H] + , calc m / z: 982.6951) indicates that its molecular formula is C 54 H 91 N7O9, contains 13 degrees of unsaturation. According to HR-ESI-MS, Q-TOF-MS 2 、 1 H-NMR and 13 Comparison of C-NMR with compound nocacyclomycin Ⅱ showed that the phenylpropionic acid (Phe) residue of compound III replaced the tryptophan (Trp) residue in compound II, and compound III was named nocacyclomycin Ⅲ.

[0094] To determine the absolute configuration of amino acids, 1.0 mg of nocacyclomycin III was weighed and acid-hydrolyzed with 6 M HCl (1 mL) at 110 °C for 24 h. The amino acid residues after acid hydrolysis were then reacted with Marfey's reagent L-FDAA. At the same time, amino acid standards (L- / D-) were reacted with L-FDAA under the same conditions. Finally, the absolute configuration of the amino acid residues in the sample was analyzed by LC-MS, which was L-Thr, L-Leu, and L-Thr.1 , L-Phe, L-Leu 2 , L-Leu 3 , L-Leu 4 , D-Ala.

[0095] In summary, the structural formula of the compound isolated in Example 1 can be determined to be:

[0096]

[0097] The compound IV obtained in Example 1 was separated and characterized by one-dimensional nuclear magnetic resonance spectroscopy (1D-NMR), two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR), high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and secondary mass spectrometry MS. 2 Identify its structure.

[0098] From the HSQC spectrum combined with the carbon spectrum, it can be obtained that the chemical shift δ assignments of H and the C connected to compound IV are shown in Table 4:

[0099] Table 4 Compound D isolated from Example 1 1 H (600MHz) and 13 C (150 MHz) NMR data, chloroform-d as solvent

[0100]

[0101]

[0102] HR-ESI-MS m / z of compound IV: [M+H] + The quasi-molecular ion peak is 968.6796 (C 53 H 90 N7O9[M+H] + , calc m / z: 968.6795) indicates that its molecular formula is C 53 H 89 N7O9, contains 13 degrees of unsaturation. According to HR-ESI-MS, 1 H-NMR, 13 C-NMR and Q-TOF-MS 2 Compared with compound nocacyclomycin Ⅲ, the second leucine (Leu 2 ) was changed to valine (Val), and compound IV was named nocacyclomycin Ⅳ.

[0103] To determine the absolute configuration of amino acids, 1.0 mg of nocacyclomycin IV was weighed and acid-hydrolyzed with 6 M HCl (1 mL) at 110 °C for 24 h. The amino acid residues after acid hydrolysis were then reacted with Marfey's reagent L-FDAA. At the same time, amino acid standards (L- / D-) were reacted with L-FDAA under the same conditions. Finally, the absolute configuration of the amino acid residues in the sample was analyzed by LC-MS, which was L-Thr, L-Leu 1 , L-Phe, D-Val, L-Leu 2 , L-Leu 3 , D-Ala.

[0104] In summary, it can be determined that the structural formula of compound IV isolated in Example 1 is:

[0105]

[0106] The compound V isolated in Example 1 was characterized by one-dimensional nuclear magnetic resonance spectroscopy (1D-NMR), two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR), high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and secondary mass spectrometry MS. 2 Identify its structure.

[0107] From the HSQC spectrum combined with the carbon spectrum, the chemical shift δ assignments of H and the C connected to compound V are shown in Table 5:

[0108] Table 5 Compound D isolated from Example 1 1 H (600MHz) and 13 C (150 MHz) NMR data, DMSO-d6 as solvent

[0109]

[0110]

[0111] HR-ESI-MS m / z of compound V: [M+H] + The quasi-molecular ion peak is 1007.6909 (C 55 H 91 N8O9[M+H] + , calc m / z: 1007.6904) indicates that its molecular formula is C 55 H 90 N8O9, contains 15 degrees of unsaturation. According to HR-ESI-MS, 1 H-NMR, 13 C-NMR and Q-TOF-MS 2Compared with compound nocacyclomycin Ⅱ, the second leucine (Leu 2 ) was changed to valine (Val), and compound V was named nocacyclomycin V.

[0112] To determine the absolute configuration of amino acids, 1.0 mg of nocacyclomycin V was weighed and acid-hydrolyzed with 6 M HCl (1 mL) at 110 °C for 24 h. The amino acid residues after acid hydrolysis were then reacted with Marfey's reagent L-FDAA. At the same time, amino acid standards (L- / D-) were reacted with L-FDAA under the same conditions. Finally, the absolute configuration of the amino acid residues in the sample was analyzed by LC-MS, which was L-Thr, L-Leu 1 , D-Trp, D-Val, L-Leu 2 , L-Leu 3 , D-Ala.

[0113] In summary, it can be determined that the structural formula of compound V isolated in Example 1 is:

[0114]

[0115] The compound VI obtained in Example 1 was separated and characterized by one-dimensional nuclear magnetic resonance spectroscopy (1D-NMR), two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR), high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and secondary mass spectrometry MS. 2 Identify its structure.

[0116] From the HSQC spectrum combined with the carbon spectrum, the chemical shift δ assignments of H and the C connected to compound VI are shown in Table 6:

[0117] Table 6 Compound VI isolated in Example 1 1 H (600MHz) and 13 C (150 MHz) NMR data, methanol-d4 as solvent

[0118]

[0119]

[0120] HR-ESI-MS m / z of compound VI: [M+H] + The quasi-molecular ion peak is 1035.7218 (C 57 H 95 N8O9[M+H] + , calc m / z: 1035.7217) indicates that its molecular formula is C57 H 94 N8O9, contains 15 degrees of unsaturation. According to HR-ESI-MS, 1 H-NMR, 13 C-NMR and Q-TOF-MS 2 Compared with compound nocacyclomycin Ⅴ, compound nocacyclomycin Ⅵ has an additional -CH2CH2- fragment on the alkyl chain, so compound Ⅵ was named nocacyclomycin Ⅵ.

[0121] To determine the absolute configuration of amino acids, 1.0 mg of nocacyclomycin VI was weighed and acid-hydrolyzed with 6 M HCl (1 mL) at 110 °C for 24 h. The amino acid residues after acid hydrolysis were then reacted with Marfey's reagent L-FDAA. At the same time, amino acid standards (L- / D-) were reacted with L-FDAA under the same conditions. Finally, the absolute configuration of the amino acid residues in the sample was analyzed by LC-MS and was L-Thr, L-Leu, and L-Thr. 1 , D-Trp, D-Val, L-Leu 2 , L-Leu 3 , D-Ala.

[0122] In summary, it can be determined that the structural formula of compound VI isolated in Example 1 is:

[0123]

[0124] The compound VII isolated in Example 1 was characterized by one-dimensional nuclear magnetic resonance spectroscopy (1D-NMR), two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR), high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and secondary mass spectrometry MS. 2 Identify its structure.

[0125] From the HSQC spectrum combined with the carbon spectrum, the chemical shift δ assignments of H and the C connected to compound VII are shown in Table 7:

[0126] Table 7 Compound VII isolated in Example 1 1 H (600MHz) and 13 C (150 MHz) NMR data, methanol-d4 as solvent

[0127]

[0128]

[0129] HR-ESI-MS m / z of compound VII: [M+H] +The quasi-molecular ion peak is 1010.7261 (C 56 H 96 N7O9[M+H] + , calc m / z: 1010.7264) indicates that its molecular formula is C 56 H 95 N7O9, contains 13 degrees of unsaturation. According to HR-ESI-MS, 1 H-NMR, 13 C-NMR and Q-TOF-MS 2 Compared with compound nocacyclomycin III, compound nocacyclomycin VII has an additional -CH2CH2- fragment on the alkyl chain, so compound VII was named nocacyclomycin VII.

[0130] To determine the absolute configuration of amino acids, 1.0 mg of nocacyclomycin VII was weighed and acid-hydrolyzed with 6 M HCl (1 mL) at 110 °C for 24 h. The amino acid residues after acid hydrolysis were then reacted with Marfey's reagent L-FDAA. At the same time, amino acid standards (L- / D-) were reacted with L-FDAA under the same conditions. Finally, the absolute configuration of the amino acid residues in the sample was analyzed by LC-MS, which was L-Thr, L-Leu, and L-Thr. 1 , L-Phe, L-Leu 2 , L-Leu 3 , L-Leu 4 , D-Ala.

[0131] In summary, the structural formula of compound VII isolated in Example 1 can be determined to be:

[0132]

[0133] The compound VIII isolated in Example 1 was characterized by one-dimensional nuclear magnetic resonance spectroscopy (1D-NMR), two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR), high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and secondary mass spectrometry MS. 2 Identify its structure.

[0134] From the HSQC spectrum combined with the carbon spectrum, the chemical shift δ assignments of H and the C connected to compound VIII are shown in Table 8:

[0135] Table 8 Compound VIII isolated from Example 1 1 H (600MHz) and 13 C (150 MHz) NMR data, chloroform-d4 as solvent

[0136]

[0137]

[0138] HR-ESI-MS m / z of compound VIII: [M+H] + The quasi-molecular ion peak is 996.7109 (C 55 H 94 N7O9[M+H] + , calc m / z: 996.7108) indicates that its molecular formula is C 55 H 93 N7O9, contains 13 degrees of unsaturation. According to HR-ESI-MS, 1 H-NMR, 13 C-NMR and Q-TOF-MS 2 Comparison with compound nocacyclomycin VII shows that the second leucine (Leu) in compound nocacyclomycin VIII is changed to valine (Val), and compound VII is named nocacyclomycin VIII.

[0139] To determine the absolute configuration of amino acids, 1.0 mg of nocacyclomycin VIII was weighed and acid-hydrolyzed with 6 M HCl (1 mL) at 110 °C for 24 h. The amino acid residues after acid hydrolysis were then reacted with Marfey's reagent L-FDAA. At the same time, amino acid standards (L- / D-) were reacted with L-FDAA under the same conditions. Finally, the absolute configuration of the amino acid residues in the sample was analyzed by LC-MS and was L-Thr, L-Leu, and L-Thr. 1 , L-Phe, D-Val, L-Leu 2 , L-Leu 3 , D-Ala.

[0140] In summary, it can be determined that the structural formula of compound VIII isolated in Example 1 is:

[0141]

[0142] The compound IX obtained in Example 1 was separated and characterized by one-dimensional nuclear magnetic resonance spectroscopy (1D-NMR), two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR), high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and secondary mass spectrometry MS. 2 Identify its structure.

[0143] From the HSQC spectrum combined with the carbon spectrum, the chemical shift δ assignments of H and the C connected to compound IX are shown in Table 9:

[0144] Table 9 Compound IX isolated from Example 11 H (600MHz) and 13 C (150 MHz) NMR data, chloroform-d4 as solvent

[0145]

[0146]

[0147] HR-ESI-MS m / z of compound IX: [M+H] + The quasi-molecular ion peak is 1098.6637 (C 64 H 88 N7O9[M+H] + , calc m / z: 1098.6638) indicates that its molecular formula is C 64 H 87 N7O9, contains 33 degrees of unsaturation. According to HR-ESI-MS, 1 H-NMR, 13 C-NMR and Q-TOF-MS 2 It is speculated that it contains four phenylalanine residues. Preliminary NMR analysis revealed a planar structure, which differs significantly from the eight previously isolated and identified compounds. To confirm the accuracy of the structure, attempts were made to grow compound IX. Fortunately, a single crystal structure was obtained, confirming the planar structure of the compound as shown below. Compound IX was named nocacyclomycin IX.

[0148] To determine the absolute configuration of amino acids, 1.0 mg of nocacyclomycin IX was weighed and acid-hydrolyzed with 6 M HCl (1 mL) at 110 °C for 24 h. The amino acid residues after acid hydrolysis were then reacted with Marfey's reagent L-FDAA. At the same time, amino acid standards (L- / D-) were reacted with L-FDAA under the same conditions. Finally, the absolute configuration of the amino acid residues in the sample was analyzed by LC-MS and was D-Val, L-Phe 1 , L-Ile, L-Phe 2 ,LN-Me-Leu,L-Phe 3 , L-Phe 4 .

[0149] In summary, it can be determined that the structural formula of compound IX isolated in Example 1 is:

[0150]

[0151] Example 3

[0152] Screening of anti-tumor cytotoxic activity of nocacyclomycins I-IX prepared in Example 1

[0153] Principle of the MTS assay for cell viability: MTS, a new MTT analogue (3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium), is a yellow dye. Succinate dehydrogenase in the mitochondria of living cells metabolizes and reduces MTS to form a soluble formazan compound. The formazan content can be measured using a microplate reader at 490 nm. Generally, the amount of formazan produced is proportional to the number of viable cells, so the number of viable cells can be estimated based on the optical density (OD) value.

[0154] The cells used in this example include leukemia HL-60 cells, lung cancer A549 cells, liver cancer HepG2 cells, breast cancer MDA-MB-231 cells, and colon cancer SW480 cells.

[0155] The experimental method is as follows:

[0156] Cell seeding: Prepare a single cell suspension in DMEM containing 10% fetal bovine serum and seed 3,000 to 15,000 cells per well in a 96-well plate with a volume of 100 μL per well. Cells should be seeded and cultured 24 hours in advance.

[0157] Add the test compound solution: the compound is dissolved in DMSO, the compound is initially screened at a concentration of 40 μM, the final volume of each well is 200 μL, and 3 replicate wells are set for each treatment.

[0158] Color development: After 48 hours of culture at 37 degrees Celsius, discard the culture medium in the wells of adherent cells and add 20 μL of MTS solution and 100 μL of DMEM culture medium to each well; discard 100 μL of culture supernatant of suspended cells and add 20 μL of MTS solution to each well; set up 3 blank replicate wells (a mixture of 20 μL of MTS solution and 100 μL of culture medium) and continue incubation for 2-4 hours to allow the reaction to proceed fully before measuring the light absorbance value.

[0159] Colorimetry: Select 492 nm wavelength and read the absorbance of each well using a multifunctional microplate reader (MULTISKAN FC). Record the results. After data processing, plot the cell inhibition rate graph with the compound number as the horizontal axis and the cell inhibition rate as the vertical axis.

[0160] Positive control compound: Cisplatin (DDP) was set as the positive compound in each experiment. The cell growth curve was drawn with the concentration as the horizontal axis and the cell survival rate as the vertical axis. The IC value of the compound was calculated using the two-point method (Reed and Muench method). 50 The initial screening results are shown in Table 10.

[0161] Table 10 Preliminary screening results of cell inhibition rate (%) of nocacyclomycins I-IX at 40 μM concentration

[0162]

[0163] At a concentration of 40 μM, compounds nocacyclomycins Ⅰ-Ⅸ showed inhibitory activity against the in vitro tumor growth of leukemia HL-60, lung cancer A549, liver cancer SMMC-7721, breast cancer MDA-MB-231, and colon cancer SW480. Among them, compounds nocacyclomycins Ⅲ and Ⅳ showed better inhibitory activity against the in vitro tumor growth of lung cancer A549, liver cancer SMMC-7721, breast cancer MDA-MB-231, and colon cancer SW480. Next, we will continue to screen for compounds with tumor cytotoxic activity (IC 50 detection).

[0164] The same method as the initial screening was used to rescreen the compounds at concentrations of 40 μM, 8 μM, 1.6 μM, 0.32 μM, and 0.064 μM, respectively, with cisplatin as the positive control. The results of the rescreening are shown in Table 11.

[0165] Table 11 Half-maximal inhibitory concentrations of Nocacyclomycins I-IX against five tumor cell lines (μM)

[0166]

[0167] The half-maximal inhibitory concentration values ​​of nocacyclomycins I-IX on leukemia HL-60, lung cancer A549, liver cancer SMMC-7721, breast cancer MDA-MB-231 and colon cancer SW4805 tumor cell lines are shown in Table 11. Among them, compounds nocacyclomycins III and IV have good inhibitory activity on the in vitro tumor growth of lung cancer A549, liver cancer SMMC-7721, breast cancer MDA-MB-231 and colon cancer SW480, IC 50 The values ​​ranged from 3.227 ± 0.237 to 9.427 ± 0.405 μM.

Claims

1. A strain of Nocardia sungurluensis YINM00009, deposited with GDMCC No: 65367.

2. Use of Nocardia sungurluensis YINM00009 according to claim 1 in the preparation of novel cyclic lipopeptide compounds; The structural formulas of the novel cyclic lipopeptide compounds are shown in Formulas I to IX:

3. The use according to claim 2, characterized in that: The preparation method of the novel cyclolipopeptide compound is as follows: (1) Bacterial activation: Nocardia YINM00009 was inoculated into the activation medium for activation and standby use; (2) Preparation of bacterial strains: inoculating the activated bacterial cells in step (1) into a seed culture medium and culturing on a shaking table to obtain bacterial strains; (3) Fermentation process: inoculating the bacterial strain prepared in step (2) into the fermentation medium and fermenting it on a shaking table to obtain a fermentation liquid; (4) After the fermentation is completed, the fermentation broth is extracted with an equal volume of ethyl acetate and concentrated to obtain a crude extract. The crude extract is dissolved in dichloromethane-methanol and eluted by gel column chromatography using dichloromethane-methanol eluent to obtain an eluate. After TLC spotting, development and color development, the color of the spot and R f The eluates were merged according to the value; (5) The combined eluate was subjected to TLC spot plate and HPLC analysis again, and the eluate with more TLC spot plate reaction points and more HPLC analysis peaks was selected for further separation. The selected eluate was evenly mixed with silica gel, and gradient elution was performed on a silica gel column using petroleum ether-ethyl acetate as eluent. Finally, semi-preparative high performance liquid chromatography was used to purify the compounds represented by Formula I to Formula IX.

4. The use according to claim 3, characterized in that: The components of the activation culture medium in step (1) are: yeast extract 4.0±0.5 g / L, glucose 4.0±0.5 g / L, malt extract 10.0±0.5 g / L, agar 15.0±0.5 g / L, pH 7.0±0.2, culture temperature 28±0.5°C, and culture time 5 to 7 days.

5. The use according to claim 3, characterized in that: The components of the seed culture medium in step (2) are: yeast extract 4.0±0.5 g / L, glucose 4.0±0.5 g / L, malt extract 10.0±0.5 g / L, pH 7.0±0.2; the culture temperature is 28±0.2° C., and the culture time is 3 to 5 days.

6. The use according to claim 3, characterized in that: The fermentation medium in step (3) comprises the following components: sucrose 100±5 g / L, glucose 10±0.5 g / L, acid hydrolyzed casein 0.12±0.1 g / L, yeast extract 5 g±0.5 / L, 3-(N-morpholino)propanesulfonic acid 21±0.5 g / L, trace elements 1.0 mL, K2SO4 0.25±0.1 g / L, MgCl2·6H2O 10 g±0.5 / L, and pH 7.0±0.2; the inoculum size of the bacterial strain is 10% of the volume of the fermentation medium; the culture temperature is 28±0.2°C, and the culture time is 10 to 20 days.

7. The use according to claim 3, characterized in that: In step (4), extraction with ethyl acetate is performed three times; in the dichloromethane-methanol solution used to dissolve the crude extract, the volume ratio of dichloromethane to methanol is 1:2; the gel column chromatography filler used is hydroxypropyl dextran gel; and in the dichloromethane-methanol eluent in the gel column chromatography elution, the volume ratio of dichloromethane to methanol is 1:

1.

8. The use according to claim 3, characterized in that: In step (5), the developing solvent of the TLC spot plate is a mixed solution of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 15:1; the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate eluent used in the forward silica gel column chromatography gradient elution is 30:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:5, and 1:10, respectively.

9. Use of the novel cyclic lipopeptide compound of claim 2 in the preparation of a drug for treating lung cancer, liver cancer, breast cancer or colon cancer, characterized in that: Use of the compound represented by formula I in the preparation of drugs for treating lung cancer and liver cancer; use of the compound represented by formula II in the preparation of drugs for treating leukemia, lung cancer, liver cancer and colon cancer; use of the compound represented by formula III in the preparation of drugs for treating leukemia, lung cancer, liver cancer, breast cancer and colon cancer; use of the compound represented by formula IV in the preparation of drugs for treating leukemia, lung cancer, liver cancer and colon cancer; use of the compound represented by formula V in the preparation of drugs for treating lung cancer, liver cancer and colon cancer; use of the compound represented by formula VI in the preparation of drugs for treating lung cancer; use of the compound represented by formula VII in the preparation of drugs for treating leukemia, lung cancer, liver cancer, breast cancer and colon cancer; use of the compound represented by formula VIII in the preparation of drugs for treating leukemia, lung cancer and breast cancer; use of the compound represented by formula IX in the preparation of drugs for treating leukemia, lung cancer, liver cancer, breast cancer and colon cancer.

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