Anti-tumor polyketone spiro compound as well as preparation method and application thereof

Abstract

The invention relates to a preparation method for producing a polyketone compound by using a recombinant strain Streptomyces coelicola A3 (2) / p15A-KOspiH3 (the preservation number is CCTCC NO: M 20241470), and also relates to an application of the polyketone compound in tumor resistance, and the structural formula of the polyketone compound is # imgabs0 #, the structural formula of the polyketone compound is as shown in the specification, the structural formula of the polyketone compound is as shown in the specification, and the structural formula of the polyketone compound is as shown in the specification. A fermentation product containing the compound can be obtained through fermentation culture of Streptomyces coelicola A3 (2) / p15A-KOspiH3, and then the compound is obtained through separation and purification of methods such as VLC normal-phase column chromatography separation, C-18 ODS reversed-phase column chromatography separation and semi-preparative HPLC. The invention aims to obtain a novel compound with an anti-tumor effect in a gene cluster of a heterologous expression actinomycete source, and the novel compound has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of industrial microorganisms, and specifically to Streptomyces coelicolor A3(2) / p15A-KO spi H3 (Deposit number: CCTCC NO: M 20241470, deposit date: July 3, 2024, deposit unit: China Center for Type Culture Collection, deposit address: School of Life Sciences, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, 430072) A method for producing polyketide compounds; the present invention also relates to the use of such compounds in anti-tumor. Background Art

[0002] Tumors, also known as cancer, are one of the major public health problems worldwide. According to the World Health Organization (WHO), cancer is the second leading cause of death worldwide and poses a serious threat to human health. The occurrence and development of tumors is a complex multi-stage process involving abnormalities in multiple genes and signaling pathways within cells. With the rapid development of modern biomedical technology, especially the progress of molecular biology and genomics, people have a deeper understanding of the molecular mechanisms of tumors. Scientists have identified many molecular markers and signaling pathways related to the occurrence and development of tumors, providing the possibility for the development of targeted drugs. Traditional tumor treatments, such as surgery, radiotherapy and chemotherapy, although effective to a certain extent, also have limitations, such as large side effects and easy drug resistance. With the understanding of the tumor immune microenvironment and tumor metabolic mechanisms, new treatment strategies, such as immunotherapy and targeted therapy, are becoming a hot topic of research. The development of technologies such as high-throughput screening, combinatorial chemistry, genetic engineering, and bioinformatics has accelerated the discovery and development process of new drugs. Natural products play a very important role in the application of anti-tumor drugs. Not only do they provide rich chemical diversity, but many natural products have been shown to have significant anti-tumor activity. Active ingredients extracted from natural products, such as terpenes, alkaloids, polysaccharides, volatile oils, and peptides, can effectively inhibit the growth of tumor cells. In addition, natural products, as one of the important sources of anti-tumor drugs, have become a valuable resource for identifying and developing new cancer treatment options. For example, paclitaxel is a diterpenoid compound extracted from plants that has significant therapeutic effects on a variety of cancers. Triterpenoid compounds such as ginsenosides Rg3 and Rh2 also show strong anti-tumor activity. These natural products are not only widely used in clinical practice, but also an important basis for the development of new drugs. However, natural products also have some challenges in drug development, such as low content, difficult extraction, low bioavailability, and possible toxic side effects. In order to overcome these limitations, scientists are using methods such as structural modification, chemical synthesis, and biosynthesis to improve the bioavailability and therapeutic effects of natural products while reducing their side effects. Therefore, the development of more effective and lower-cost anti-tumor drugs has important socioeconomic significance. Summary of the invention

[0003] The present invention aims to provide a novel compound having strong cytotoxicity inhibitory activity. Its structural formula is

[0004]

[0005] Formula I

[0006] The compounds of formula I of the present invention can be obtained by fermentation of microorganisms to obtain fermentation products containing the compounds, and then the crude fermentation extracts are separated and purified by VLC normal phase column chromatography, C-18 ODS reverse phase column chromatography and semi-preparative HPLC. Streptomyces coelicolor A3(2) / p15A-KO spi H3 Examples of the preparation of compounds of formula I according to the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 The present invention provides Streptomyces coelicolor A3(2) / p15A-KO spi H3 gene cluster.

[0008] Figure 2 It is the key two-dimensional signal of the compound of formula I.

[0009] Figure 3 is the single crystal diffraction pattern of the compound of formula I. DETAILED DESCRIPTION

[0010] The chemical structure of the compound I in the following examples (the Arabic numerals in the structural formula are the positions of the carbon atoms in the chemical structure) is

[0011]

[0012] Compound Ⅰ

[0013] Example 1 Construction of recombinant strain, fermentation production and separation and purification of compound Ⅰ

[0014] Construction of recombinant strains

[0015] Actinomycete genome extraction method was used to extract Streptomyces sp. HDN15129 actinomycete genome; the genome was digested with MfeI / MseI restriction endonucleases to release the target gene cluster spi ; At the same time, the previously constructed primers were used to amplify the cloning vector p15A- spi; Use Red / ET recombineering technology to construct the target plasmid from the cloning vector recovered from the gel and the linear genome after enzyme cutting, and obtain the correct plasmid through transformant screening. Then re-transform the correct plasmid into GB05 colon to eliminate the influence of background; add the strong promoter kasOp* to the re-transformed correct plasmid through the linear plus circular homologous recombination (LCHR) method to start the expression of the target gene cluster. And add the site-specific recombination element pR6K-oriT-phiC31- kasOp* through this method, and transfer the plasmid into E. coli GBred-gyrA462, and pick a single colony on the plate for overnight culture and transfer to obtain OD 600 =0.4-0.6 GBred-gyrA462 carrying the knockout plasmid; with the help of the Redαβ system mediated by Red / ET homologous recombination, p15A-cm-ccdb plasmid was used as a template and knockout primers were used to amplify cm- ccdb The fragment was purified using a kit to obtain the amplified fragment. Then about 200 ng of cm-ccdB The fragments were electroporated into GBred-gyrA462 containing the knockout plasmid, and the incubated colon was finally spread on a solid LB plate containing cm / Apra resistance. After 12-18 hours of culture, a single colony was picked and enzyme digestion was performed for verification. The correct recombinant plasmid was digested with restriction endonuclease PacI at 37 °C for 2 hours to remove the inserted gene cluster. cm-ccdb After desalting with a desalting membrane at room temperature for 40 min, the fragment was electroporated. E. coli GB2005, the revived cells were plated on LB solid plates containing Apra and cultured at 37°C overnight. After obtaining a single colony, enzyme digestion verification was performed. The transformant with correct enzyme digestion was the plasmid with successful knockout. Finally, the plasmid was electroporated into ET12567 / PUZ8002; Streptomyces coelicolor ( S. coelicolor ) A3(2) in MS [medium composition (g / L): soybean powder 20 g, mannitol 20 g, agar powder 20 g, pH adjusted to 7.2] streak culture on the plate for 5-7 days, scrape the grown spores with a sterile cotton swab and place them in a 50mL centrifuge tube, heat shock at 50℃ for 10 min and naturally cool the strain as the recipient bacteria for conjugation transfer. E. coli ET12567 / pUZ8002 / p15A-KO spiH3 was grown in 100 mL of LB liquid culture medium containing 50 μg / mL kanamycin, 25 μg / mL chloramphenicol and 50 μg / mL apramycin at 37°C until the OD600 value was about 0.6. The bacteria were collected by centrifugation (9500 rpm, 1 min), washed 3 times with antibiotic-free LB, and suspended in 1 mL LB culture medium as the donor bacteria for conjugation transfer. Take 400 μL of the above-mentioned recipient bacteria and 200 μL of the donor bacteria, mix them evenly, spread them on MS solid culture medium without any antibiotics, blow dry, and culture them at 30°C for 16-20 h. Then take out the plate and cover the plate with water containing antibiotics with a final concentration of 50 μg / mL apramycin and 25 μg / mL nalidixic acid. After blowing dry, incubate in a 30°C incubator for 10-20 days and observe; extract the genomic DNA of each mutant strain, and use the heterologous expression detection primers to obtain positive clones through PCR detection, that is, obtain spi Mutant strain knocking out oxidoreductase H3 after heterologous expression of gene cluster S. coelicolor A3(2) / p15A-KO spi H3.

[0016] Example 2 Fermentation production and separation and purification of compound I

[0017] 1. Fermentation of production bacteria According to the conventional method of culturing microorganisms, Streptomyces coelicolor was taken Streptomyces coelicolor A3(2) / p15A-KO spi H3 appropriate amount, first in the antibiotic Apra 50 μ g / ml MS solid medium was cultured in a 28°C incubator for 7 days. Streptomyces coelicolor A3(2) / p15A-KO spi An appropriate amount of H3 was inoculated into a 500 mL conical flask containing 100 mL of culture medium [culture medium composition (g / L): 10 g soluble starch, 2 g peptone, 4 g yeast extract, 1 L water, pH 7.2-7.4], and cultured at 28 °C in a shaking incubator (180 rpm) for 8 days to obtain the fermentation product.

[0018] 2. The fermentation liquid was filtered through gauze to obtain the supernatant. It was extracted three times with an equal amount of ethyl acetate, all ethyl acetate phases were combined, and concentrated under reduced pressure to obtain a crude extract, a total of 50 grams.

[0019] 3. The extract (50.0 g) was dissolved in methanol, then dissolved in 90% methanol, extracted with petroleum ether to remove the oil component, evaporated to dryness, and chromatographed on a normal phase column using methanol-dichloromethane as the elution system to separate into two fractions. Component 1 was first chromatographed on a C-18 ODS reverse phase column using methanol-water as the mobile phase, and then subjected to reverse phase semi-preparative high performance liquid chromatography (methanol: water = 70:30) after gradient elution to obtain a centrosymmetric space group C 2 / c racemate. Crystal data provide the correctness of its structure. Therefore, the (+) and (-) two enantiomers can be separated by chiral high performance liquid chromatography column, the ratio is almost 1:1, and the compound I in this article is the (+) enantiomer. Compound I is a brown solid, molecular formula C 18 H 22 O3, HR-ESI-MS m / z : 321.0762 [M - H] - , (calculated value: 321.0768); IR (KBr) ν max 3449, 2928, 2855,2363, 1609,1456, 1384, 1332, 1268, 1155, 1035 cm -1 ; 1 H and 13 C NMR see Table 1.

[0020] Table 1 Compound Ⅰ 1 H and 13 C NMR data (500 and 125 MHz, in DMSO- d 6) a

[0021] position <![CDATA[ δ C ]]> <![CDATA[ δ H ( J in Hz)]]> <![CDATA[ 1 H- 1 H cosy b ]]> <![CDATA[HMBC (H→C) c ]]> 1 139.5, C 2 114.8, CH 6.64, dd (8.0, 0.8) 3 4, 6, 10 3 136.2, CH 7.48, t (8.0) 2, 4 1, 5 4 117.7, CH 6.97, dd (8.0, 0.8) 3 2, 5, 6 5 161.6, C 6 114.7, C 7 189.8, C 8 128.0, CH 6.55, s 6, 10 9 156.7, C 10 91.1, C 11 191.1, C 12 107.9, C 13 143.1, C 14 114.4, CH 6.50, s 12, 16 15 168.3, C 16 96.5, CH 6.63, s 12, 14, 15, 17 17 176.0, C 18 <![CDATA[17.6, CH3]]> 1.88, s 8, 9, 10 19 <![CDATA[17.9, CH3]]> 2.35, s 12, 13, 14 5-OH 12.24, s 4, 5, 6 15-OH 11.40, s

[0022] a) The signal assignments in this table are based on the analysis results of COSY, HSQC and HMBC spectra. Hydrogen signals are represented by s (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet).

[0023] b) The numbers and codes in this column represent 1 H- 1 The H COSY spectrum is consistent with the corresponding row 1 H gives the coupling correlation signal 1 H core.

[0024] c) The numbers and symbols in this column represent the HMBC spectrum and the corresponding rows. 1 H gives the coupling correlation signal 1 C core.

[0025] Example 3 Cytotoxicity test of compounds

[0026] 1. Experimental samples and experimental methods

[0027] Preparation of the test sample solution: The test sample is the pure compound I separated and purified in Example 1 above. Accurately weigh an appropriate amount of sample, use DMSO to prepare a 30 μM stock solution, and then use the two-fold dilution method to dilute it into 15, 7.5, 3.75, 1.875, 0.9375 μM test solutions. The positive drug doxorubicin (ADM) is prepared to 1 μM for activity testing. Test cells: L-02, NCI-H446 (SRB method). Test method: The SRB experimental method is as follows: Detection and data analysis (1) Cell treatment: adherent cells, trypsin digestion of logarithmic phase cells, prepare cell suspension. (2) Cell counting: Use a counting plate and a counter to draw 10-20 μL and count the average number of cells x in the 16 squares on the four corners (x is roughly 20-50, which is better), 40÷x=y, y is the number of milliliters of cells needed to add to a plate, and each plate requires a total of 6 mL, 6-y=z, z is the volume of culture medium to be added (counting principle, count the top but not the bottom, count the left but not the right). (The number of cells in each 96-well plate is between 380,000 and 410,000) (3) Cell plating: Add 90 μL of cell suspension to each well of a 96-well plate (4) Add sample: After the cells have grown for 24 hours, add 10 μL of compound solution of different concentrations to each well. (5) The cells were cultured in a 37°C incubator. (6) After 72 h, the culture medium was removed and 100 μL of TCA solution was added to each well. The cells were fixed at 4°C for more than 1 h, rinsed with tap water 5-6 times, and air-dried. 100 μL of SRB solution was added to each well for staining. After staining for 5-10 min, SRB was removed and the cells were rinsed with 1% glacial acetic acid solution 5-6 times. The cells were air-dried. After drying, 150 μL of Tris solution was added and the cells were shaken to mix well. The absorbance was measured at 515 nm using an ELISA reader.

[0028] 2. Experimental Results

[0029] In the cytotoxic activity test, the experimental results of different concentrations of compound I on the tested tumor cells are shown in Table 2.

[0030] Table 2 Inhibitory effect of different concentrations of compound I on NCI-H446 cells

[0031] Concentration (μM) OD value Inhibition rate% <![CDATA[IC 50 (μM)]]> Blank Group 0.70±0.05 Control group L02 0.66±0.05 ADM 25 0.08±0.01 91.92 0.80 12.5 0.08±0.01 91.83 6.25 0.12±0.01 88.37 3.125 0.26±0.01 74.92 1.5625 0.41±0.01 59.50 Compound Ⅰ 30 0.07±0.01 89.46 2.26 15 0.07±0.01 89.84 7.5 0.09±0.01 87.96 3.75 0.22±0.05 68.53 1.875 0.47±0.02 33.33 0.9375 0.53±0.03 25.34

[0032] 3. Conclusion

[0033] Compound I has strong tumor cell proliferation inhibitory activity and has good application prospects in the field of cancer treatment as an anti-tumor compound.

Claims

1. A compound as shown in the following structure 。 2. A recombinant strain Streptomyces coelicolor A3(2) / p15A-KO spi H3, characterized by being Actinomycetes from South China Sea mud Streptomyces After the whole genome of sp. HDN15129 was sequenced and analyzed by bioinformatics, the spi gene cluster was introduced into the heterologous host S. coelicolor A3(2) using the Red / ET recombination method, and the post-modification gene oxidoreductase H3 was knocked out to obtain a mutant strain. Streptomyces coelicolor A3(2) / p15A-KO spi H3.

3. The method for preparing the compound according to claim 1, characterized in that The following steps are involved: Streptomyces coelicolor Streptomyces coelicolor A3(2) / p15A-KO spi H3 (preservation number: CCTCC NO: M 20241470) fermentation culture to obtain a fermentation product containing the compound, then VLC normal phase column chromatography separation, C-18 ODS reverse phase column chromatography separation; finally, reverse phase semi-preparative high performance liquid chromatography separation and purification to obtain the compound according to claim 1, the preparation gradient is acetonitrile: water = 21:

79.

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

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