Naphthyridinomycin compound produced by streptomyces as well as preparation method and application of naphthyridinomycin compound

Abstract

The invention discloses a naphthyridine mycin compound generated by streptomyces as well as a preparation method and application thereof, and belongs to the field of medicines. The naphthyridine mycin compound and the analogue of the naphthyridine mycin compound are prepared by fermenting, extracting, separating and purifying streptomyces lageri W307 (the preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) No.32721). The invention further discloses a preparation method of the naphthyridine mycin compound and the analogue of the naphthyridine mycin compound. The invention further provides application of the eight naphthyridine mycin compounds in preparation of anti-tumor drugs and application of the eight naphthyridine mycin compounds in preparation of antibacterial drugs, and potential anti-tumor drugs and antibacterial drugs are provided clinically.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and relates to the field of screening of new anti-tumor drugs and antibacterial drugs, and in particular to eight naphthyromycin compounds produced by Streptomyces, and their preparation methods and applications. Background Art

[0002] Naphthyrimidines are the earliest discovered members of the tetrahydroisoquinoline alkaloid family, possessing a unique and complex polycyclic structure. These compounds not only exhibit antitumor activity but also demonstrate potent antibacterial activity against some clinically resistant bacteria. These antibiotics all possess a pharmacodynamic functional group, a hemiaminal, in which the hydroxyl group undergoes protonation and dehydration, with the nitrogen atom donating a lone electron pair to form an electrophilic imine species. The carbon atom of this imine is susceptible to nucleophilic attack at the N-2 position of guanine, effectively alkylating DNA. Ecteinascidin 743 (ET-743), one of the most active members in the antitumor family, is primarily used to treat soft tissue tumors. However, naphthyrimidines are extremely unstable during extraction and isolation, necessitating prior derivatization to isolate their analogs before further investigation into their biosynthesis mechanism and physiological activity. Summary of the Invention

[0003] The present invention aims to address the difficulties of separation and structural analysis of naphthyromycin compounds, and to provide eight naphthyromycin compounds produced by Streptomyces labedae W307 (culture collection number CGMCC No. 32721), as well as their preparation methods and applications.

[0004] The object of the present invention is achieved by the following technical solutions: As a first aspect, the present invention provides a naphthyromycin compound with m / z 786 (molecular formula C 41 H 43 N3O 13 , mass 785.2796Da), m / z 784 (molecular formula C 41 H 41 N3O 13 , mass 783.2639Da), m / z 720 (molecular formula C 37 H 41 N3O 12 , mass 719.2690Da), m / z 718 (molecular formula C 37 H 39 N3O 12 , mass 717.2534Da), m / z 570 (molecular formula C 29 H 35N3O9, mass 569.2373), m / z 568 (molecular formula C 29 H 33 N3O9, mass 567.2217Da), m / z 528 (molecular formula C 27 H 33 N3O8, mass 527.2268), m / z 526 (molecular formula C 27 H 31 N3O8, mass 525.2111Da), has the following structure:

[0005]

[0006] As a second aspect, a method for preparing the naphthyromycin compound is provided, which is prepared by fermentation, extraction, separation and purification of Streptomyces labedae W307; the Streptomyces labedae W307 was deposited in the General Microbiology Center of the China Culture Collection Administration on November 20, 2024, and the culture collection number is CGMCC No. 32721.

[0007] Furthermore, the extraction, separation and purification comprises the following steps: activating the Streptomyces; fermenting the actinomycetes on a large scale using an MS medium shaker; separating and purifying the compound using ODS, LH-20 and HPLC; and 1 H NMR, 13 C NMR, HSQC, COSY, and HMBC were used to analyze the planar structure of the compound.

[0008] As a third aspect, provided is the use of the naphthyromycin compound described in the first aspect in the preparation of an anti-tumor drug.

[0009] Furthermore, the diseases targeted by the anti-tumor drug include: lung cancer, gastric cancer, colon cancer, cervical cancer, chronic myeloid leukemia, renal clear cell adenocarcinoma, esophageal cancer, bladder cancer, gallbladder cancer, breast cancer, liver cancer, brain tumor, prostate cancer, thyroid cancer, pancreatic cancer, osteosarcoma, malignant melanoma, and rhabdomyosarcoma.

[0010] Furthermore, the anti-tumor drug is particularly effective against cervical cancer, chronic myeloid leukemia, esophageal cancer, bladder cancer, thyroid cancer, osteosarcoma, and rhabdomyosarcoma.

[0011] As a fourth aspect, there is provided a use of the alkaloid compound in the preparation of antibacterial drugs.

[0012] Furthermore, the pathogenic bacteria targeted by the antibacterial drug include: Enterococcus faecalis, Stenotrophomonas maltophilia, Pseudomonas aeruginosa, Klebsiella pneumoniae, Shigella flexneri, Acinetobacter baumannii, Acinetobacter pilomii, Providencia, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Enterobacter cloacae, Proteus mirabilis, methicillin-resistant Staphylococcus aureus, Streptococcus pyogenes, and Staphylococcus epidermidis.

[0013] Furthermore, the antibacterial drug is particularly effective against Acinetobacter baumannii, Bacillus subtilis, methicillin-resistant Staphylococcus aureus, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, and Stenotrophomonas maltophilia.

[0014] The beneficial effects of the present invention are that the compound obtained by separation and purification is a naphthyromycin analogue with relatively stable properties, which provides a reference for the subsequent extraction and separation of naphthyromycin and its analogues; the compound screened out good cytotoxic activity, and the inhibitory effect on some tumor cells is better than that of the positive drug doxorubicin, and the cytotoxicity of normal cells is lower than that of the positive drug doxorubicin, which is of great significance for the discovery of new anti-tumor drugs; the compound screened out good antibacterial activity, and the antibacterial activity against a variety of multidrug-resistant bacteria is better than that of the positive drug polymyxin or vancomycin, which is of great significance for the screening of new antibacterial drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the high-resolution mass spectrum of compound m / z 786;

[0016] Figure 2 This is the high-resolution mass spectrum of compound m / z 528;

[0017] Figure 3 Figure 2 is the structure diagram of the compound, where (A) is the planar structure of the compound m / z 786 and m / z 528, and (B) is a schematic diagram of the main two-dimensional nuclear magnetic resonance related signals. DETAILED DESCRIPTION

[0018] The embodiments of the present invention provide 8 naphthyromycin compounds with cytotoxic and antibacterial activities, wherein the naphthyromycin compounds are produced by Streptomyces labedae W307, which was deposited in the General Microbiology Center of the China Culture Collection Administration on November 20, 2024, with a culture collection number of CGMCC No. 32721. The fermentation method and the extraction, separation and identification method of the compound include: A) taking seed liquid from a glycerol tube to activate actinomycetes on a plate; B) using MS medium shaker to ferment actinomycetes on a large scale; C) using ODS, LH-20, HPLC and other means to separate and purify the compound; D) using 1 HNMR, 13C NMR, HSQC, COSY, HMBC, etc. were used to analyze the planar structure of the compound.

[0019] The activity test results of the isolated compounds of the above embodiments of the present invention include A) 18 classical cell lines of mainstream cancer-infecting organs in China (human lung cancer cells-A549, human gastric cancer cells-MKN-45, human colon cancer cells-HCT116, human cervical cancer cells-HeLa, human chronic myeloid leukemia cells-K-562, human renal clear cell adenocarcinoma cells-786-O, human esophageal cancer cells-TE-1, human bladder cancer cells-5637, human gallbladder cancer cells-GBC-SD, human breast cancer cells-MCF7, human Hepatocellular carcinoma cells (HepG2), human brain tumor cells (SF126), human prostate cancer cells (DU145), human thyroid cancer cells (CAL-62), human pancreatic cancer cells (PATU8988T), human osteosarcoma cells (HOS), human malignant melanoma cells (A-375), human rhabdomyosarcoma cells (A-673), and two normal cell lines (human embryonic kidney cells (293T) and human normal liver cells (L-02)) were used to screen the antitumor activity of the compounds. B) 16 pathogenic bacteria, including Enterococcus faecalis faecalis ATCC19433), Stenotrophomonas maltophilia ATCC13637, Pseudomonas aeruginosa ATCC27853, Klebsiella pneumoniae subsp. Pneumoniae ATCC13883, Shigella flexneri, Acinetobacter baumannii, Acinetobacter pylori, Providencia, Bacillus subtilis, Escherichia coli ATCC25922, Staphylococcus aureus subsp. Aureus ATCC25933, Enterobacter cloacae, Proteus mirabilis mirabilis), Methicillin-resistant Staphylococcus aureus, Streptococcus pyogenes, and Staphylococcus epidermidis for screening of the antibacterial activity of the compounds.

[0020] Example 1: Method for producing naphthyromycin compounds by fermentation with Streptomyces ragneri and identification of their structures.

[0021] 1. Fermentation method of actinomycetes

[0022] (1) Pipette the seed solution from the 30% glycerol tube and apply it to SFM solid medium to recover the strain;

[0023] (2) Scrape the spores of the strain with a pipette tip and inoculate them into LB liquid medium for seed culture;

[0024] (3) 1 mL of seed liquid was inoculated into a 1 L conical flask containing 200 mL of MS liquid medium, and 10 L of the culture medium was fermented in a shaking incubator at 28 °C and 180 rpm for 7 days.

[0025] Culture medium and ingredients used:

[0026] Solid culture medium (w / v);

[0027] SFM: 2% soybean flour, 2% mannitol, 2% agar.

[0028] Liquid culture medium (w / v);

[0029] LB: 1% tryptone, 1% sodium chloride, 0.5% yeast extract powder.

[0030] MS: 2% soybean flour, 2% mannitol, 0.3% calcium carbonate. pH 7.0.

[0031] 2. Treatment of fermentation broth

[0032] (1) Extract the fermentation broth three times with equal volumes of ethyl acetate;

[0033] (2) Evaporate the ethyl acetate extract to dryness, re-dissolve in methanol, centrifuge at 10,000 rpm, and discard the precipitate;

[0034] (3) The supernatant was added to an ODS column (C18 filler YMC*GEL ODS-A-HG, 12 nm S-10 μm) and eluted with 30%, 70%, and 100% methanol-water systems, respectively, to obtain three fractions;

[0035] (4) 30% of the fraction was washed with 100% MeOH in LH-20 (Cytiva Sephadex TM LH-20) for separation and collection in glass test tubes;

[0036] (5) The compound-enriched fraction was spin-dried and subsequently separated by HPLC (HITACHI 5430). Mobile phase A was ultrapure water containing 0.1% formic acid, and mobile phase B was methanol containing 0.1% formic acid. The liquid phase conditions were 20%-100% methanol, 35 min, C18 (YMC-Actus Triart C18 250x 20.0, S-5μm, 12nm).

[0037] 3. Structural identification of compounds

[0038] The planar structure of the compound is 1 H NMR, 13 The planar structure of the compound and the main two-dimensional NMR related signals determined by C NMR, HSQC, COSY, TOCXY, and HMBC are as follows: Figure 1 and Figure 2 Shown are naphthyromycin compounds.

[0039] Table 1 Compounds m / z 786 and m / z 528 1 H (600MHz) and 13 C (151 MHz) NMR information (DMSO-d6, δ in ppm, J in Hz).

[0040]

[0041] 4. Compound Characterization

[0042] m / z 786:brown amorphous powder; UV(MeOH)λmax:220,290nm; 1 H and 13 C NMRdata,Table 1;HR-ESI-MS:m / z 786.2910[M+H]+(calcd.for C 41 H 44 N3O 13 ,786.2874).

[0043] m / z 528:brown amorphous powder; UV(MeOH)λmax:220,290nm; 1 H and 13 C NMRdata,Table 1;HR-ESI-MS:m / z 528.2380[M+H]+(calcd.for C 27 H 34 N3O8,528.2346).

[0044] Example 2: Screening of cytotoxic activity of naphthymycin compounds.

[0045] A total of 20 cell lines, including 18 classic cell lines from mainstream cancer-causing organs in China and 2 normal cell lines, were selected to screen the cytotoxic activity of the compounds.

[0046] Experimental principle: Cell Counting Kit (CCK-8) method is used to evaluate the inhibitory activity of compounds on cell proliferation, and the half-maximal inhibitory concentration IC is determined by single-concentration activity screening and multiple-concentration determination. 50 The assay works by reducing WST-8, contained in the CCK-8 reagent, to a highly water-soluble yellow formazan product (formazan) under the action of the electron carrier 1-methoxy-5-methylphenazine methylsulfate (1-Methoxy PMS). The amount of formazan produced is proportional to the number of viable cells.

[0047] 1. Cytotoxicity assay process:

[0048] (1) Weigh the sample and dissolve it in cell-grade DMSO to a 10 mM stock solution. Dilute the sample to 10 times the concentration to be tested using cell culture medium.

[0049] (2) Cell inoculation: Prepare cells into a single cell suspension using culture medium containing 10% (v / v) fetal bovine serum, and inoculate 90 μL of 5×10 cells per well of a 96-well plate. 4 / mL of adherent cells and 9×10 4 / mL suspension cells were pre-cultured at 5% CO2, 37℃ for 24h;

[0050] (3) Add the sample solution to be tested: add 10 μL of sample solution to each well. Set one concentration for each sample in the initial activity screening, and set up 3 replicate wells; IC 50 Eight concentrations (including 0 concentration) were measured, with three replicate wells for each concentration; the cells were cultured in an incubator for 48 hours.

[0051] Experimental setup: The drug group (Drug) was prepared according to steps (1)-(3). The test sample solution in the control group (Control) was replaced with an equal volume of DMSO. The blank group (Blank) contained only basal culture medium (no cell inoculation and no test sample solution was added).

[0052] (4) Color development: Remove the old culture medium and drug solution from adherent cells (add 10 μL CCK-8 solution directly to suspended cells), add 100 μL CCK-8 solution diluted tenfold to each well, and continue culturing at 37°C, 5% CO2 for 1-4 h (keep away from light and observe in real time).

[0053] (5) Detection: Measure the absorbance at 450 nm using an enzyme-labeled instrument and record the raw data results.

[0054] (6) Perform standardization of the original data and calculate the cell proliferation inhibition rate by the OD value of each well in the initial screening. The formula is: Cell proliferation inhibition rate = (OD Control -OD Drug ) / (OD Control -OD Blank )×100%, OD Blank ,OD Control ,OD Drug Respectively represent the OD values ​​of the blank group, control group and drug group, and calculate the inhibition rate. 50 Calculated by GraphPad Prism 8.

[0055] (7) Positive control: Doxorubicin.

[0056] 2. Initial screening of compound cytotoxicity test

[0057] The initial screening concentration of the monomeric compound cytotoxic activity was 50 μM. The initial screening results are shown in Table 2.

[0058] Table 2 Results of preliminary screening of cytotoxic activity of compounds m / z 786 and m / z 528 against 20 human cell types (cell inhibition rate %)

[0059]

[0060] 3. Compound Cytotoxicity Test Rescreening

[0061] The cells with compound inhibition rates higher than 80% were subjected to activity rescreening. The activity rescreening results are shown in Table 3.

[0062] The above experimental results show that the compound has a better inhibitory effect on A-673, CAL-62, HeLa, HOS, K-562, TE-1, and 5637 cells than the positive drug doxorubicin, and is less toxic to normal human cells L-02 and 293T than the positive drug doxorubicin.

[0063] Table 3 Results of rescreening of cytotoxicity of compounds m / z 786 and m / z 528 against 14 human cell lines (IC 50 μM)

[0064]

[0065] Example 3: Antibacterial activity test of naphthyromycin compounds.

[0066] A total of 16 pathogenic bacteria were selected to conduct antibacterial experiments on the isolated monomer compounds. The information of the 16 strains and the positive controls (Positive Controls) are shown in Table 4.

[0067] 1. Antibacterial experiment operation process:

[0068] (1) The monomer compound and the positive drug were prepared at an initial concentration of 6.4 mM and diluted sequentially in a 96-well plate to 3.2 mM, 1.6 mM, 0.8 mM, 0.4 mM, 0.2 mM, 0.1 mM, 0.05 mM, 0.025 mM, 0.0125 mM, 0.00625 mM, and 0.003125 mM, for a total of 12 concentration gradients;

[0069] (2) After activating the 10 pathogenic bacteria, they were cultured in LB liquid medium at 37°C in a shaking incubator for 12 h;

[0070] (3) Pipette blank LB medium into row A of a 96-well plate, 100 μL / well;

[0071] (4) Dilute the grown bacterial solution with LB medium to an OD value between 0.01 and 0.02. Pipette the diluted bacterial solution into rows B-H of a 96-well plate, 100 μL / well.

[0072] (5) Pipette 12 monomer compound solutions with a concentration gradient of D into rows B, C, and D of a 96-well plate at 1 μL / well. Pipette 1 μL of the positive drug solution with a concentration gradient of 12 into rows E, F, and G of a 96-well plate at 1 μL / well.

[0073] (6) After 16 h of incubation in 96-well plates, the minimum inhibitory concentration of the compound was calculated.

[0074] Among them, the parameters of the microplate reader are set to use the endpoint method, the detection wavelength is 595nm, and there is no reference wavelength.

[0075] 2. Antibacterial test results

[0076] As shown in Table 4, the experimental results showed that the compound had significant inhibitory activity against multiple drug-resistant strains, such as Acinetobacter baumannii, Bacillus subtilis, methicillin-resistant Staphylococcus aureus, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, and Stenotrophomonas maltophilia.

[0077] Table 4 Antibacterial activity test results (MIC μM) of compounds m / z 786 and m / z 528 against 16 pathogenic bacteria

[0078]

[0079] It should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations are possible. Any modifications, equivalent substitutions, or improvements made without inventive effort shall fall within the scope of protection of the present invention.

Claims

1. A naphthyromycin compound, characterized in that: Any one of the following compounds:

2. A method for preparing the naphthyromycin compound according to claim 1, characterized in that: It is produced by fermentation, extraction, separation and purification of Streptomyces labedae W307.

3. The preparation method according to claim 2, characterized in that The extraction, separation and purification comprises the following steps: activating the streptomyces; fermenting the actinomycetes on a large scale using an MS medium shaker; separating and purifying the compound using ODS, LH-20 and HPLC; and using 1 H NMR, 13 C NMR, HSQC, COSY, and HMBC were used to analyze the planar structure of the compound.

4. Use of the naphthyromycin compound according to claim 1 in the preparation of anti-tumor drugs.

5. The use according to claim 4, characterized in that The anti-tumor drugs target the following diseases: lung cancer, gastric cancer, colon cancer, cervical cancer, chronic myeloid leukemia, renal clear cell adenocarcinoma, esophageal cancer, bladder cancer, gallbladder cancer, breast cancer, liver cancer, brain tumor, prostate cancer, thyroid cancer, pancreatic cancer, osteosarcoma, malignant melanoma, and rhabdomyosarcoma.

6. Use of the naphthyromycin compound according to claim 1 in the preparation of antibacterial drugs.

7. The use according to claim 6, characterized in that The pathogenic bacteria targeted by the antibacterial drugs include: Enterococcus faecalis, Stenotrophomonas maltophilia, Pseudomonas aeruginosa, Klebsiella pneumoniae, Shigella flexneri, Acinetobacter baumannii, Acinetobacter pilomii, Providencia, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Enterobacter cloacae, Proteus mirabilis, methicillin-resistant Staphylococcus aureus, Streptococcus pyogenes, and Staphylococcus epidermidis.

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