Nocardia YINM00009 and application thereof in preparation of cyclic lipopeptide compounds

Abstract

The invention discloses nocardia YINM00009 and application of the nocardia YINM00009 in preparation of cyclic lipopeptide compounds, and belongs to the technical field of microorganisms. The nocardia YINM00009 is an endophyte of polygonatum kingianum in Kunming of Yunnan province, and a large number of novel cyclic lipopeptide compounds can be metabolized after fermentation; the fermentation step mainly comprises the following steps: fermenting the strain under proper culture conditions to obtain fermentation liquor, extracting and concentrating through a solvent, and combining with column chromatography to prepare nine cyclic lipopeptide compounds, the total concentration of the nine cyclic lipopeptide compounds in the fermentation liquor can reach 10.2-16.5 mg / L, the purity can reach 99% or above after extraction and separation, and the cyclic lipopeptide compounds can be used for preparing the cyclic lipopeptide compounds. Wherein the two cyclic lipopeptide compounds have very strong anti-tumor cytotoxic activity. The method is relatively low in cost, simple in process and environment-friendly, can be used for mass production and preparation of the cyclic lipopeptide compounds, not only meets the requirements of low-carbon economy, but also lays a foundation for later production of the cyclic lipopeptide compounds and development of related products.

Description

Technical Field

[0001] The present invention relates to a strain of Nocardia YINM00009 and its application in the preparation of cyclic lipopeptide compounds, belonging to the field of microbial technology. Background Art

[0002] In recent years, due to the characteristics of microorganisms such as short metabolic cycle, mild reaction conditions, few by-products, and strong stereoselectivity, the method of preparing target active compounds through microbial fermentation has attracted more and more attention. In actual production, there have been a large number of application examples of producing some compounds with medicinal value or economic value through microbial fermentation.

[0003] Cyclic lipopeptide refers to a class of amphiphilic cyclic structure molecules formed by fatty acids and peptide fragments through ester bonds or amide bonds. The components of cyclic lipopeptides mainly come from the secondary metabolites of microorganisms, and have environmentally friendly characteristics such as low toxicity, small irritation, and biodegradability. In addition, most cyclic lipopeptides also have special biological activities, including antibacterial and anti-tumor, etc., which have attracted extensive attention of researchers in recent years. Daptomycin is a cyclic lipopeptide antibiotic with a completely new structure extracted from the fermentation broth of Streptomyces reseosporus. It was discovered by Eli Lilly and Company in the United States in the 1980s and developed successfully by Cubist Pharmaceuticals in 1997. It not only has a novel chemical structure, but also its mode of action is different from any approved antibiotic: it disrupts the transport of amino acids across the cell membrane, thereby hindering the biosynthesis of peptidoglycan in the bacterial cell wall, changing the properties of the cytoplasmic membrane, and can damage the functions of the bacterial cell membrane in many aspects and quickly kill Gram-positive bacteria. In addition to acting on most clinically relevant Gram-positive bacteria, daptomycin is more importantly highly active against isolated strains with drug-resistant properties such as methicillin and vancomycin in vitro, and this property has very important clinical significance for critically ill patients. At present, cyclic lipopeptide compounds are mainly obtained through the isolation and purification of microbial fermentation metabolites, and the strains capable of metabolizing cyclic lipopeptide compounds are mostly actinomycetes from plants or soil sources, and their yields of metabolizing cyclic lipopeptide compounds are low, and the too low yields restrict the development and utilization of cyclic lipopeptide compounds.

[0004] Nocardia Nocardia sungurluensis YINM00009 is an endophytic actinomycete obtained from Polygonatum kingianum in Kunming, Yunnan. Through the analysis of its secondary metabolites, it is found that the yield of novel cyclic lipopeptide compounds is large and easy to isolate and purify. Therefore, this strain has the development potential to be used as an engineering bacterium to produce novel cyclic lipopeptide compounds. Summary of the Invention

[0005] One of the objectives of the present invention is to provide a strain of Nocardia YINM00009, which was deposited at the Guangdong Provincial Culture Collection of Microorganisms (abbreviated as GDMCC) on October 28, 2024. The deposit address is: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 65367. The taxonomic naming is: Nocardia sungurluensis .

[0006] Another objective of the present invention is to provide the application of Nocardia YINM00009 in the preparation of novel cyclic lipopeptide compounds.

[0007] There are 9 novel cyclic lipopeptide compounds prepared by the present invention. 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 a novel cyclic lipopeptide compound by Nocardia YINM00009, which mainly includes the following steps:

[0026] (1) Bacterial activation: Inoculate Nocardia YINM00009 into a strain activation medium for activation and standby.

[0027] (2) Strain preparation: Inoculate the activated bacteria in step (1) into a seed medium for cultivation to obtain the strain.

[0028] (3) Fermentation process: Inoculate the strain prepared in step (2) into a fermentation medium for fermentation culture to obtain a fermentation broth.

[0029] (4) After the fermentation is completed, the fermentation broth is extracted with an equal volume of ethyl acetate, concentrated to obtain a crude extract. After the crude extract is dissolved in dichloromethane-methanol, gel column chromatography elution is carried out with a dichloromethane-methanol eluent to obtain 200 tubes of eluent. After TLC plate development and coloration, tubes 1-27, 28-32, 33-47, 48-54, 55-70, 71-86, 87-100, 101-115, 116-135, and 136-200 are combined to obtain 10 portions of eluent.

[0030] (5) Select the eluent of tubes 28-32, 33-47, 48-54, and 55-70 obtained in step (4) for further separation. Positive silica gel column chromatography gradient elution is carried out with a petroleum ether-ethyl acetate eluent, and finally purification is carried out by semi-preparative high performance liquid chromatography to obtain the compounds shown in 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 d.

[0032] Preferably, the components of the seed 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 3-5 d.

[0033] Preferably, the components of the fermentation medium in step (3) are as follows: 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 inoculation amount 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 d.

[0034] Preferably, in step (4), ethyl acetate extraction is performed 3 times; in the dichloromethane-methanol solution used for dissolving the crude extract, the volume ratio of dichloromethane to methanol is 1:2; the gel column chromatography packing used is hydroxypropyl dextran gel (Sephadex LH-20).

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

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

[0037] Preferably, in the normal-phase silica column chromatography gradient elution in step (5), the volume ratios of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate eluent are successively 30:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:5, 1:10. Among them, the compounds shown in Formula I and Formula II are contained in the petroleum ether-ethyl acetate eluent with a volume ratio of 1:1 in tubes 28 - 32; the compounds shown in Formula IV and Formula VIII are contained in the petroleum ether-ethyl acetate eluent with a volume ratio of 1:1 in tubes 33 - 47; the compounds shown in Formula III and Formula VII are contained in the petroleum ether-ethyl acetate eluent with a volume ratio of 2:1 in tubes 48 - 54; the compounds shown in Formula V and Formula VI are contained in the petroleum ether-ethyl acetate eluent with a volume ratio of 1:2 in tubes 55 - 70; the compound shown in Formula IX is contained in the petroleum ether-ethyl acetate eluent with a volume ratio of 5:1 in tubes 33 - 47.

[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] Advantages of the present invention

[0040] (1) The present invention provides a Nocardia strain capable of highly producing cyclic lipopeptides Nocardia sungurluensis YINM00009, which can produce 9 novel cyclic lipopeptides with high yields.

[0041] (2) Nine novel cyclic lipopeptides were obtained for the first time in the present invention, enriching the variety of cyclic lipopeptides.

[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, there is no pollution to the environment, and it is suitable for large-scale production of cyclic lipopeptide compounds. Brief Description of the Drawings

[0043] Figure 1 It is the crystal structure of Compound V provided by the present invention.

[0044] Figure 2 It is the crystal structure of Compound VI provided by the present invention.

[0045] Figure 3 It is the crystal structure of Compound IX provided by the present invention. Detailed Embodiments

[0046] The technical solutions of the present invention will be further described below in conjunction with the drawings and through specific embodiments. However, the following embodiments are only simple examples of the present invention and do not represent or limit the scope of the protection of the present invention. The scope of protection of the present invention is subject to the claims, and the technical operations without special description are well-known operations to those skilled in the art.

[0047] Polygonatum kingianum collected from Kunming, Yunnan was surface sterilized by soaking in sodium hypochlorite solution (effective chlorine concentration 5.0%), 75% ethanol, and washing with sterile water. Then, the processed small pieces of plant tissue were implanted into the activated medium sterilized at 121°C, and after culturing at 28°C for 7 days, the endophyte was continuously purified using the activated medium sterilized at 121°C Nocardia sungurluensis YINM00009, with the preservation number of GDMCC No. 65367.

[0048] The Nocardia YINM00009 described in the present invention is a rare actinomycete. Its surface is smooth on the ISP2 medium, without spore production and aerial hyphae. The 16s RNA sequence of Nocardia YINM00009 is shown in SEQ ID No. 1:

[0049]

[0050] The formulations of the culture media used in the examples are as follows. All culture media need to be autoclaved at 121 °C for 20 min before use.

[0051] (1) The components of the activation medium are: 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 components of the seed medium are: yeast extract 4.0 g / L, glucose 4.0 g / L, malt extract 10.0 g / L, pH 7.0.

[0053] (3) The components of the fermentation medium are: 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] Using Nocardia sp. YINM00009 to prepare novel cyclic lipopeptide compounds, which specifically include the following steps:

[0056] (1) Bacterial activation: Inoculate Nocardia sp. YINM00009 into the activation medium, culture it in a constant temperature incubator at 28 °C for 5 d, and then place it in a refrigerator at 4 °C for standby.

[0057] (2) Strain preparation: Inoculate the activated bacteria into the seed medium and ferment at 28 °C and 200 rpm for 3 d.

[0058] (3) Fermentation process: Add the strain prepared in step (2) to 20 L of the 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) according to a volume fraction of 10%, and ferment at 28 °C and 200 rpm for 15 d to obtain the 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 a 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 fillers using a dropper, and the gel column was eluted with an eluent having 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 combined to obtain 10 eluates, which were represented by Fr.1-Fr.10.

[0060] (5) Take 10 aliquots of the eluent for TLC spotting and HPLC analysis. Tubes 28-32, 33-47, 48-54, and 55-70 were subjected to TLC spotting (developing solvent: dichloromethane / methanol volume ratio of 15 / 1) and showed that there were many spots. 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 eluents of fractions Fr.2, Fr.3, Fr.4 and Fr.5 were selected for further separation, and the eluents 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 eluents were sequentially used with petroleum ether:ethyl acetate volume ratios of 30:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:5, and 1:10 for forward silica gel column (300-400 mesh) chromatography gradient elution to obtain the eluent.

[0061] According to the results of TLC spot plate and HPLC analysis, the fraction Fr.2 was selected with an eluent of 1:1 volume ratio of petroleum ether to ethyl acetate, and semi-preparative HPLC purification (semi-preparative gradient elution method: 0 min, 95% methanol; 5 min, 100% methanol; 15 min, 100% methanol) was performed 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 RThe purity (t = 32.5 min) reached over 99% (analysis gradient elution method: 0 min, 30% methanol; 30 min, 100% methanol).

[0062] Based on the TLC plate spotting and HPLC analysis results, the eluent with a petroleum ether - ethyl acetate volume ratio of 1:1 in fraction Fr.3 was selected for purification by semi - preparative high - performance liquid chromatography (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). By HPLC analysis, the purity of compound Ⅳ (retention time t R = 32.7 min) and compound Ⅷ (retention time t R = 33.7 min) reached over 99% (analysis gradient elution method: 0 min, 30% methanol; 30 min, 100% methanol).

[0063] Based on the TLC plate spotting and HPLC analysis results, the eluent with a petroleum ether - ethyl acetate volume ratio of 2:1 in fraction Fr.4 was selected for purification by semi - preparative high - performance liquid chromatography (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). By HPLC analysis, the purity of compound Ⅲ (retention time t R = 33.0 min) and compound Ⅶ (retention time t R = 34.1 min) reached over 99% (analysis gradient elution method: 0 min, 30% methanol; 30 min, 100% methanol).

[0064] Based on the TLC plate spotting and HPLC analysis results, the eluent with a petroleum ether - ethyl acetate volume ratio of 1:2 in fraction Fr.5 was selected for purification by semi - preparative high - performance liquid chromatography (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). By HPLC analysis, the purity of compound Ⅴ (retention time t R = 32.2 min) and compound Ⅵ (retention time t R = 33.2 min) reached over 99% (analysis gradient elution method: 0 min, 30% methanol; 30 min, 100% methanol).

[0065] Based on the results of TLC plate spotting and HPLC analysis, the eluent with a petroleum ether - ethyl acetate volume ratio of 5:1 in fraction Fr.2 was selected and purified by semi-preparative high performance liquid chromatography (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). By HPLC analysis, the purity of compound IX (retention time t R = 34.5 min) reached over 99% (Analysis gradient elution method: 0 min, 30% methanol; 30 min, 100% methanol).

[0066] Finally, by 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; 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] Take compound I separated in Example 1 and identify its structure 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 tandem mass spectrometry (Q-TOF-MS 2 ).

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

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

[0072]

[0073]

[0074] The 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, containing 15 degrees of unsaturation. Through the 1 H-NMR, 13 C-NMR and HSQC spectral analysis, it can be known that compound I contains 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, β-C / β-H and saturated fatty alkyl chains was found. Preliminary analysis indicated that the substance was a macrocyclic lipopeptide compound. To determine the types and connection order of amino acid residues in the macrocyclic lipopeptide compound, Q-TOF-MS 2 secondary mass spectrometry was tested, the fragment ions were analyzed, and the planar structure of the macrocyclic lipopeptide was preliminarily determined. By querying relevant databases and relevant literature, it was found that this molecule was a new unreported cyclic lipopeptide compound, named nocacyclomycin Ⅰ.

[0076] To determine the absolute configuration of the amino acids, 1.0 mg of nocacyclomycin Ⅰ was weighed and hydrolyzed with 6M HCl (1 mL) at 110 °C for 24 h. Then, the amino acid residues after hydrolysis were 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 as L-Thr, L-Leu 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] Take the compound Ⅱ isolated from Example 1 and identify its structure 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 tandem mass spectrometry (Q-TOF-MS 2 )

[0080] From the HSQC spectrum combined with the carbon spectrum, the chemical shift δ assignments of the H of compound Ⅱ and the C to which it is attached are shown in Table 2:

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

[0082]

[0083]

[0084] The 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, containing 15 degrees of unsaturation. According to HR-ESI-MS, 1 1H-NMR and 13 13C-NMR and comparison with compound nocacyclomycin Ⅱ, compound Ⅱ has one less -CH2CH2- fragment than Ⅰ, and this compound is named nocacyclomycin Ⅱ.

[0085] To determine the absolute configuration of the amino acids, weigh 1.0 mg of nocacyclomycin Ⅱ and perform acid hydrolysis with 6M HCl (1 mL) at 110 °C for 24 h. Then, react the amino acid residues after acid hydrolysis with Marfey's reagent L-FDAA. At the same time, react the amino acid standards (L- / D-) with L-FDAA under the same conditions. Finally, analyze the absolute configuration of the amino acid residues in the sample by LC-MS as 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 as follows:

[0087]

[0088] Take Compound III isolated in Example 1, and identify its structure 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 tandem mass spectrometry (Q-TOF-MS 2 ).

[0089] From the HSQC spectrum combined with the carbon spectrum, the chemical shift δ assignments of the H and the connected C of Compound III are shown in Table 3:

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

[0091]

[0092]

[0093] The 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, with 13 degrees of unsaturation. According to HR-ESI-MS, Q-TOF-MS 2 , 1 1H-NMR and 13 13C-NMR comparison with Compound nocacyclomycin II shows that the phenylalanine (Phe) residue of Compound III replaces the tryptophan (Trp) residue of Compound II, and Compound III is named nocacyclomycin III.

[0094] To determine the absolute configuration of the amino acids, weigh 1.0 mg of nocacyclomycin III and perform acid hydrolysis with 6M HCl (1 mL) at 110 °C for 24 h. Then, react the amino acid residues after acid hydrolysis with Marfey's reagent L-FDAA. At the same time, react the amino acid standards (L- / D-) with L-FDAA under the same conditions. Finally, analyze the absolute configuration of the amino acid residues in the sample by LC-MS as L-Thr, L-Leu1 , L-Phe, L-Leu 2 , L-Leu 3 , L-Leu 4 , D-Ala.

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

[0096]

[0097] Take the compound Ⅳ isolated in Example 1, and identify its structure 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 tandem mass spectrometry MS 2 to identify its structure.

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

[0099] Table 4 H (600 MHz) and 1 C (150 MHz) NMR data of the compound D isolated in Example 1, with chloroform-d as the solvent 13

[0100]

[0101]

[0102] The 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, containing 13 degrees of unsaturation. According to HR-ESI-MS, 1 1H-NMR, 13 13C-NMR and Q-TOF-MS 2 and comparison with compound nocacyclomycin Ⅲ, it can be seen that the second leucine (Leu 2 ) in compound nocacyclomycin Ⅳ has become valine (Val), and compound Ⅳ is named nocacyclomycin Ⅳ.

[0103] ​To determine the absolute configuration of the amino acids, 1.0 mg of nocacyclomycin Ⅳ was weighed and hydrolyzed with 6 M HCl (1 mL) at 110 °C for 24 h. Then, the amino acid residues after hydrolysis were 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 configurations of the amino acid residues in the sample were analyzed by LC-MS as 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 as follows:

[0105]

[0106] Compound Ⅴ isolated in Example 1 was taken and its structure was identified 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 tandem mass spectrometry MS 2 to identify its structure.

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

[0108] Table 5 H (600 MHz) and 1 C (150 MHz) NMR data of compound D isolated in Example 1 13 with DMSO-d6 as the solvent

[0109]

[0110]

[0111] The HR-ESI-MS m / z of compound Ⅴ: [M+H] + The quasi-molecular ion peak was 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, containing 15 degrees of unsaturation. According to HR-ESI-MS, 1 1H-NMR, 13 13C-NMR and Q-TOF-MS 2Comparison with the compound nocacyclomycin Ⅱ shows that the second leucine (Leu 2 ) in the compound nocacyclomycin Ⅴ is changed to valine (Val), and the compound Ⅴ is named nocacyclomycin Ⅴ.

[0112] To determine the absolute configuration of the amino acids, 1.0 mg of nocacyclomycin Ⅴ was weighed and hydrolyzed with 6 M HCl (1 mL) at 110 °C for 24 h. Then, the amino acid residues after hydrolysis were 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 configurations of the amino acid residues in the sample were analyzed by LC-MS as 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 the compound Ⅴ isolated in Example 1 is:

[0114]

[0115] The compound Ⅵ isolated in Example 1 was taken, and its structure was identified 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 tandem mass spectrometry MS 2 identification.

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

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

[0118]

[0119]

[0120] The HR-ESI-MS m / z of the 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, which has 15 degrees of unsaturation. According to HR-ESI-MS, 1 H-NMR, 13 C-NMR and Q-TOF-MS 2 and comparison with compound nocacyclomycin Ⅴ, it can be seen that there is one more -CH2CH2- fragment on the alkyl chain in compound nocacyclomycin Ⅵ. Compound Ⅵ was named nocacyclomycin Ⅵ.

[0121] To determine the absolute configuration of the amino acids, 1.0 mg of nocacyclomycin Ⅵ was weighed and hydrolyzed with 6 M HCl (1 mL) at 110 °C for 24 h. Then the amino acid residues after hydrolysis were reacted with Marfey's reagent L-FDAA. At the same time, the 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 as L-Thr, L-Leu 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] Compound Ⅶ isolated in Example 1 was taken and its structure was identified 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 tandem mass spectrometry MS 2 Identify its structure.

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

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

[0127]

[0128]

[0129] The 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, with 13 degrees of unsaturation. According to HR-ESI-MS, 1 H-NMR, 13 C-NMR and Q-TOF-MS 2 and comparison with compound nocacyclomycin Ⅲ, it can be seen that there is an additional -CH2CH2- fragment in the alkyl chain of compound nocacyclomycinⅦ. Compound Ⅶ was named nocacyclomycin Ⅶ.

[0130] To determine the absolute configuration of the 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. Then, the amino acid residues after acid hydrolysis were 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 as L-Thr, L-Leu 1 , L-Phe, L-Leu 2 , L-Leu 3 , L-Leu 4 , D-Ala.

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

[0132]

[0133] Compound Ⅷ isolated in Example 1 was taken, and its structure was identified 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 tandem mass spectrometry MS 2 .

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

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

[0136]

[0137]

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

[0139] To determine the absolute configuration of the amino acids, 1.0 mg of nocacyclomycin VIII was weighed and hydrolyzed with 6 M HCl (1 mL) at 110 °C for 24 h. Then the amino acid residues after hydrolysis were reacted with Marfey's reagent L-FDAA. At the same time, the 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 as L-Thr, L-Leu 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 the compound VIII isolated in Example 1 is:

[0141]

[0142] Take the compound IX isolated in Example 1 and identify its structure 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 tandem mass spectrometry MS 2 to identify its structure.

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

[0144] Table 9 of the compound IX isolated in Example 11 H (600 MHz) and 13 C (150 MHz) NMR data, with chloroform-d4 as the solvent

[0145]

[0146]

[0147] HR-ESI-MS m / z of Compound IX: [M+H] + The quasi-molecular ion peak was 1098.6637 (C 64 H 88 N7O9 [M+H] + , calc m / z: 1098.6638) indicating a molecular formula of C 64 H 87 N7O9, with 33 degrees of unsaturation. Based on HR-ESI-MS, 1 H-NMR, 13 C-NMR and Q-TOF-MS 2 It was speculated that it contains four phenylalanine residues. Based on the NMR data, the planar structure was initially resolved, which is very different from the first eight isolated and identified compounds. To confirm the accuracy of the structure, attempts were made to grow crystals of Compound IX, and fortunately, a single crystal structure was obtained, confirming the planar structure of the compound as shown in the following figure. Compound IX was named nocacyclomycin IX.

[0148] To determine the absolute configuration of the 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. Then, the amino acid residues after acid hydrolysis were 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 as D-Val, L-Phe 1 , L-Ile, L-Phe 2 , L-N-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] Antitumor cytotoxic activity screening of nocacyclomycins Ⅰ-Ⅸ prepared in Example 1

[0153] Principle of detecting cell viability by MTS method: MTS is a brand-new MTT analogue, with the full name of 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium, which is a yellow dye. Succinate dehydrogenase in the mitochondria of living cells can metabolically reduce MTS to generate soluble formazan compounds, and the content of formazan can be measured by an enzyme-labeled instrument at 490 nm. Under normal circumstances, the amount of formazan generated is proportional to the number of living cells. Therefore, the number of living cells can be inferred based on the optical density OD value.

[0154] The cells used in this example are 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] Inoculating cells: Prepare a single-cell suspension with a culture medium (DMEM) containing 10% fetal bovine serum, and inoculate 3000 - 15000 cells per well into a 96-well plate, with a volume of 100 μL per well. The cells are inoculated and cultured 24 hours in advance.

[0157] Adding the compound solution to be tested: The compound is dissolved in DMSO, and the compound is initially screened at a concentration of 40 μM, with a final volume of 200 μL per well. Each treatment is set with 3 replicate wells.

[0158] Color development: After culturing at 37 °C for 48 hours, for adherent cells, discard the culture medium in the wells, add 20 μL of MTS solution and 100 μL of DMEM culture medium to each well; for suspension cells, discard 100 μL of the culture supernatant, and add 20 μL of MTS solution to each well; set 3 blank replicate wells (a mixture of 20 μL of MTS solution and 100 μL of culture medium), and continue to incubate for 2 - 4 hours. After the reaction proceeds sufficiently, measure the light absorption value.

[0159] Colorimetry: Select a wavelength of 492 nm, and use a multi-functional enzyme-labeled instrument (MULTISKAN FC) to read the light absorption values of each well, record the results, and after data processing, plot the cell inhibition rate graph with the compound number as the abscissa and the cell inhibition rate as the ordinate.

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

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

[0162]

[0163] At a concentration of 40 μM, compounds nocacyclomycins Ⅰ-Ⅸ had inhibitory activity on 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 had better 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. Next, continue to perform the screening of tumor cell cytotoxic activity compounds (IC 50 detection).

[0164] Using the same method as the primary screening, the compounds were rescreened at concentrations of 40 μM, 8 μM, 1.6 μM, 0.32 μM, and 0.064 μM, with cisplatin as the positive control. The rescreening results are shown in Table 11.

[0165] Table 11 Half-maximal inhibitory concentration values (μM) of compounds Nocacyclomycins Ⅰ-Ⅸ against 5 tumor cell lines

[0166]

[0167] The half-maximal inhibitory concentration values of Nocacyclomycins Ⅰ-Ⅸ against leukemia HL-60, lung cancer A549, liver cancer SMMC-7721, breast cancer MDA-MB-231, and colon cancer SW480 5 tumor cell lines are shown in Table 11. Among them, compounds nocacyclomycins Ⅲ and Ⅳ had better 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, and the IC 50 values were between 3.227 ± 0.237 and 9.427 ± 0.405 μM.

Claims

1. A strain of Nocardia ( Nocardia sungurluensis ), YINM00009, with the deposit number GDMCC No: 65367.

2. Use of the Nocardia sp. YINM00009 according to claim 1 in the preparation of novel cyclic lipopeptide compounds.

3. The application according to claim 2, characterized in that: The structural formulas of the novel cyclic lipopeptide compounds are shown in Formulas I to IX: Formula Ⅰ; Formula II; Formula III; Formula IV; Formula V; Formula VI; Formula VII; Formula VIII; Formula IX.

4. The application according to claim 2, characterized in that: The preparation method of the novel cyclic lipopeptide compounds is as follows: (1) Bacterial activation: Inoculate Nocardia sp. YINM00009 into an activation medium for activation and standby; (2) Strain preparation: Inoculate the activated bacteria in step (1) into a seed medium and culture them in a shaker to obtain a strain; (3) Fermentation process: Inoculate the strain prepared in step (2) into a fermentation medium and culture it by shaking fermentation to obtain a fermentation broth; (4)After fermentation, the fermentation broth was extracted with an equal volume of ethyl acetate, and the concentrate was obtained. The crude extract was dissolved in dichloromethane-methanol and eluted by a dichloromethane-methanol eluent on a gel column chromatography to obtain an eluate. After TLC spotting, developing and coloring, the eluate was combined according to the color of the spots and the size of the R f value. (5) Perform TLC spotting plate and HPLC analysis on the combined eluate again. Select the eluate with more reaction spots on the TLC spotting plate and more peaks in the HPLC analysis to continue the separation of the eluate. Mix the selected eluate evenly with silica gel, and perform gradient elution on a normal-phase silica gel column using a petroleum ether-ethyl acetate eluent. Finally, purify it using semi-preparative high-performance liquid chromatography to obtain the compounds shown in Formulas I to IX.

5. The application according to claim 4, characterized in that: 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, culture temperature is 28 ± 0.5 °C, and culture time is 5 - 7 d.

6. The application according to claim 4, wherein: The components of the seed 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; culture temperature is 28 ± 0.2 °C, and culture time is 3 - 5 d.

7. The application according to claim 4, characterized in that: The components of the fermentation medium in step (3) are: 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 inoculation amount of the strain is 10% of the volume of the fermentation medium; culture temperature is 28 ± 0.2 °C, and culture time is 10 - 20 d.

8. The application according to claim 4, characterized in that: In step (4), perform ethyl acetate extraction 3 times; in the dichloromethane-methanol solution used to dissolve the crude extract, the volume ratio of dichloromethane to methanol is 1:2; the filler used for gel column chromatography is hydroxypropyl dextran gel; in the dichloromethane-methanol eluent for gel column chromatography, the volume ratio of dichloromethane to methanol is 1:

1.

9. The application according to claim 4, wherein: In step (5), the developing agent for TLC spotting plate is a mixed solution of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 15:1; in the gradient elution of normal-phase silica gel column chromatography, the volume ratios of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate eluent are 30:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:5, 1:10 in sequence.

10. Use of the novel cyclic lipopeptide compound according to claim 2 in the preparation of a medicament for treating lung cancer, liver cancer, breast cancer or colon cancer.

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