An antitumor polyketide spiro compound, its preparation method and uses

Abstract

The present invention relates to a preparation method of polyketide compounds using a recombinant strain Streptomyces coelicolor A3(2) / p15A-KO spi H3 (Deposit No.: CCTCC NO: M 20241470), and also relates to the use of such compounds in anti-tumor applications. The structural formula thereof is:; It can be obtained by fermenting and culturing Streptomyces coelicolor Streptomyces coelicolor A3(2) / p15A-KO spi H3 to obtain a fermentation product containing such compounds, and then separating and purifying it by methods such as VLC normal-phase column chromatography, C-18 ODS reverse-phase column chromatography, and semi-preparative HPLC. The purpose of the present invention is to obtain new compounds with anti-tumor effects by heterologously expressing gene clusters from actinomycetes, and it has good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of industrial microorganisms, and specifically relates 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, Hubei 430072) method for producing polyketide compounds; the present invention also relates to the application of such compounds in anti-tumor Background Art

[0002] Tumors, also known as cancers, are one of the major public health problems globally. According to the data of the World Health Organization (WHO), cancer is the second leading cause of death worldwide, posing 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 technologies, 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 tumor occurrence and development, providing the possibility for the development of targeted drugs. Traditional tumor treatment methods, such as surgery, radiotherapy, and chemotherapy, although effective to a certain extent, also have limitations, such as large side effects and easy development of 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 the focus of research. The development of technologies such as high-throughput screening, combinatorial chemistry, genetic engineering, and bioinformatics has accelerated the process of new drug discovery and development. Natural products play a very important role in the application of anti-tumor drugs. They not only provide rich chemical diversity, but many natural products have been proven to have significant anti-tumor activities. Active ingredients extracted from natural products, such as terpenoids, alkaloids, polysaccharides, volatile oils, and polypeptides, 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 regimens. For example, Paclitaxel is a diterpenoid compound extracted from plants and has significant therapeutic effects on various cancers. Triterpenoid compounds such as ginsenoside Rg3 and Rh2 also show strong anti-tumor activities. These natural products are not only widely used clinically but also an important basis for new drug research and development. However, there are also some challenges in the development of natural products as drugs, such as low content, difficult extraction, low bioavailability, and possible toxic side effects. 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 cost-effective anti-tumor drugs has important social and economic significance. Summary of the Invention

[0003] The present invention aims to provide a new compound with strong cytotoxic inhibitory activity. Its structural formula is

[0004]

[0005] Formula I

[0006] The compound of formula I of the present invention can be obtained by microbial fermentation culture to obtain a fermented product containing such compounds, and then the crude extract of the fermentation is separated and purified by methods such as VLC normal phase column chromatography, C-18 ODS reverse phase column chromatography, and semi-preparative HPLC. The following examples of the present invention list the use of Streptomyces coelicolor A3(2) / p15A-KO spi H3 to prepare an example of the compound of formula I of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 is provided by the present invention Streptomyces coelicolor A3(2) / p15A-KO spi H3 gene cluster.

[0008] Figure 2 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 OF THE INVENTION

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

[0011]

[0012] Compound I

[0013] Example 1 Construction, Fermentation Production and Separation and Purification of Recombinant Strain of Compound I

[0014] Construction of Recombinant Strain

[0015] The actinomycete genome extraction method is used to extract Streptomyces the actinomycete genome of sp. HDN15129; the genome is digested with MfeI / MseI restriction endonucleases to release the target gene cluster spi ; at the same time, the previously constructed primers are used to amplify and clone the vector p15A- spi; Use Red / ET recombination engineering technology to construct the target plasmid from the cloned vector after gel recovery and the digested linear genome, and obtain the correct plasmid through transformant screening. Then re-transform the correct plasmid into Escherichia coli GB05 to eliminate background effects; add the strong promoter kasOp* to the correct plasmid by linear plus circular homologous recombination (LCHR) to initiate the expression of the target gene cluster. And add the site-specific recombination element pR6K-oriT-phiC31-kasOp* by this method, and transfer this plasmid into E. coli GBred-gyrA462 by electroporation, pick single colonies on the plate for overnight culture and subculture to obtain GBred-gyrA462 carrying the knocked-out plasmid with OD 600 = 0.4 - 0.6; Mediated by the Redαβ system in Red / ET homologous recombination, use the p15A-cm-ccdb plasmid as a template and amplify the cm- ccdb fragment with knockout primers, and purify the amplified fragment using a kit. Then electrotransfer about 200 ng of the cm-ccdB fragment into GBred-gyrA462 containing the knocked-out plasmid. Finally, spread the incubated Escherichia coli on an LB solid plate containing cm / Apra resistance. After culturing for 12 - 18 h and picking single colonies, perform restriction enzyme digestion verification. After digesting the correct recombinant plasmid with the restriction enzyme PacI at 37 °C for 2 h, remove the cm-ccdb fragment inserted into the gene cluster. Then desalt at room temperature using a desalting membrane for 40 min, and electrotransfer it into E. coli GB2005. After recovery, spread the cells on an LB solid plate containing Apra and culture overnight at 37 °C. After obtaining single colonies, perform restriction enzyme digestion verification. The transformant with correct restriction enzyme digestion is the plasmid with successful knockout. Finally, electrotransfer this plasmid into ET12567 / PUZ8002; Streptomyces coelicolor( S. coelicolor ) A3(2) is streaked on an MS [medium composition (g / L): soybean powder 20 g, mannitol 20 g, agar powder 20 g, pH adjusted to 7.2] plate and cultured for 5 - 7 days. The grown spores are scraped off with a sterile cotton swab and placed in a 50 mL centrifuge tube. The strain heat-shocked at 50 °C for 10 min and then naturally cooled is used as the recipient bacterium for conjugation transfer. The donor bacterium E.coli ET12567 / pUZ8002 / p15A-KO spiWhen H3 grew to an OD600 value of approximately 0.6 at 37 °C in 100 mL of LB liquid medium containing 50 μg / mL kanamycin, 25 μg / mL chloramphenicol, and 50 μg / mL apramycin, the cells were collected by centrifugation (9500 rpm, 1 min), washed three times with antibiotic-free LB, and suspended in 1 mL of LB medium to serve as the donor strain for conjugation. Mix 400 μL of the above recipient strain and 200 μL of the donor strain evenly, spread them on MS solid medium without any antibiotics, air-dry, and then culture at 30 °C for 16 - 20 h. Then take out the plate, cover the plate with water containing antibiotics, with a final concentration of 50 μg / mL apramycin and 25 μg / mL nalidixic acid, air-dry, and observe after culturing in a 30 °C incubator for 10 - 20 days; Extract the genomic DNA of each mutant strain, and use the detection primers for heterologous expression to obtain positive clones by PCR, that is, obtain spi The mutant strain with the oxidoreductase H3 knocked out after heterologous expression of the gene cluster S. coelicolor A3(2) / p15A-KO spi H3.

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

[0017] 1. Fermentation culture of the production strain According to the conventional method for culturing microorganisms, take an appropriate amount of Streptomyces coelicolor Streptomyces coelicolor A3(2) / p15A-KO spi H3, first culture it on MS solid medium containing 50 μ μg / ml of antibiotic Apra in a 28 °C incubator for 7 days. Take an appropriate amount of Streptomyces coelicolor Streptomyces coelicolor A3(2) / p15A-KO spi H3 that has been slant-cultured for 7 days, inoculate it into a 500 mL conical flask containing 100 mL of medium [medium composition (g / L): soluble starch 10 g, peptone 2 g, yeast extract 4 g, water 1 L, pH 7.2 - 7.4], and culture it on a shaker at 28 °C (180 rmp) for 8 days to obtain the fermentation product.

[0018] 2. Filter the fermentation broth with gauze to obtain the supernatant. Extract it three times with an equal volume of ethyl acetate, combine all the ethyl acetate phases, and concentrate under reduced pressure to obtain a crude extract, a total of 50 grams.

[0019] 3. The extract (50.0 g) was dissolved in methanol, and then dissolved in 90% methanol. It was extracted with petroleum ether to remove the fat components. After evaporation to dryness, normal-phase column chromatography was carried out using methanol-dichloromethane as the elution system, and it was divided into 2 fractions. Fraction 1 was first subjected to C-18 ODS reversed-phase column chromatography with methanol-water as the mobile phase. After gradient elution, it was further separated by reversed-phase semi-preparative high-performance liquid chromatography (methanol: water = 70:30) to obtain the racemate belonging to the centrosymmetric space group C 2 / c. The crystal data provided the correctness of its structure. Therefore, the two enantiomers (+) and (-) with a ratio of almost 1:1 could be separated by a chiral high-performance liquid chromatography column. Compound I in this article is the (+) enantiomer. Compound I is a brown solid, with the 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 are shown in Table 1.

[0020] Table 1 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 results of COSY, HSQC and HMBC spectra analysis. The hydrogen signals are represented by s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet).

[0023] b) The numbers and codes in this column represent the 1 H- 1 H COSY spectrum 1 H 1 H nucleus that gives coupling-related signals with the

[0024] c) The numbers and codes in this column represent the 1 H 1 C nucleus that gives coupling-related signals with the

[0025] Example 3 Cytotoxic Activity Test of Compounds

[0026] 1. Experimental Samples and Methods

[0027] Preparation of the sample solution to be tested: The test sample was the pure compound I separated and purified in Example 1 above. An appropriate amount of the sample was accurately weighed and dissolved in DMSO to prepare a stock solution of 30 μM, and then diluted by the two-fold dilution method to prepare test solutions of 15, 7.5, 3.75, 1.875, and 0.9375 μM. The positive drug doxorubicin (ADM) was prepared at 1 μM for testing the activity. Test cells: L-02, NCI-H446 (SRB method). Test method: The SRB experimental method was as follows for detection and data analysis. (1) Cell treatment: For adherent cells, trypsinize the cells in the logarithmic growth phase to prepare a cell suspension. (2) Cell counting: Using a counting chamber and a counter, pipette 10 - 20 μL and count the average number x of cells in 16 squares at the four corners (it is better if x is approximately 20 - 50). 40 ÷ x = y, where y is the volume in milliliters of cells to be added to one plate. A total of 6 mL is required for each plate, and 6 - y = z, where z is the volume of the culture medium to be added (counting principle: count the cells above and to the left, not below and to the right). (The number of cells in each 96-well plate is 380,000 - 410,000). (3) Cell seeding: Add 90 μL of the cell suspension to each well of a 96-well plate. (4) Add the sample: After the cells adhere and grow for 24 h, add 10 μL of the compound solution at different concentrations to each well. (5) Incubate the cells in an incubator at 37 °C. (6) After 72 h, remove the culture medium, add 100 μL of TCA solution to each well, fix at 4 °C for more than 1 h, rinse 5 - 6 times with tap water, air dry naturally, add 100 μL of SRB solution to each well for staining. After staining for 5 - 10 min, remove the SRB, rinse 5 - 6 times with 1% glacial acetic acid solution, air dry naturally. After drying, add 150 μL of Tris solution, shake well, and measure the absorbance at 515 nm using an enzyme-linked immunosorbent assay reader.

[0028] 2. Experimental Results

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

[0030] Table 2 Inhibition Rates of Compound I at Different Concentrations 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 inhibitory activity on tumor cell proliferation and has good application prospects as an anti-tumor compound in the field of cancer treatment.

Claims

1. A compound having the following structural formula 。 2. A recombinant strain Streptomyces coelicolor A3(2) / p15A-KO spi H3, characterized in that it is Using actinomycetes sourced from the South China Sea mud Streptomyces After whole-genome sequencing of Streptomyces sp. HDN15129 and bioinformatics analysis, the spi gene cluster was introduced into the heterologous host Streptomyces coelicolor A3(2) using the Red / ET recombination method. Subsequently, the post-modification gene oxidoreductase H3 was knocked out to obtain the mutant strain Streptomyces coelicolor A3(2) / p15A-KO spi H3.

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

79.

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

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