Compounds, methods, and uses

Abstract

The application discloses a novel compound, a method and application, and the method comprises the following steps: S1, mutant strain construction, interrupting a coding gene pyrK1 in a strain of Streptomyces rugosporus LL-42D005; S2, mutant strain fermentation; S3, extraction, collecting fermentation, taking supernatant after centrifugation, adjusting PH, extracting with ethyl acetate, reducing pressure and concentrating the ethyl acetate to obtain an extract; S4, separating to obtain the novel compound; the application adopts gene knockout biological technology, inactivates a biosynthesis pathway and the like to find an analogue of a natural product, and investigates biological activity and anti-tumor effects of the novel compound.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and in particular to novel compounds, methods, and applications. Background Technology

[0002] Ovarian cancer is a type of gynecological malignancy. Treatment methods include surgery and radiotherapy / chemotherapy. The prominent problems with currently used anti-tumor drugs are low efficacy, poor selectivity (high toxicity), and insensitivity to drug-resistant tumors. Furthermore, recurrence and metastasis are also challenges in cancer treatment. Therefore, there is an urgent need to find new anti-tumor drugs.

[0003] Natural products, primarily derived from the metabolites of animals, plants, and microorganisms, have always played a vital role in human health. According to a recent report in the journal *Natural Product Chemistry*, between 1981 and 2014, as many as 75% of anti-tumor drugs were directly derived from natural products or analogues based on structural modifications of natural products. Natural products possess pharmacologically exclusive structural units, which can be used as molecular probes in life science research. Therefore, effectively exploring and utilizing the pharmacologically active groups provided by nature is a significant challenge in life sciences. Furthermore, genomics and proteomics research has generated a large number of targets that can be used for drug screening. High-throughput screening based on these targets still places higher demands on the quantity and sources of new compounds. In recent years, due to the overuse of antibiotics, new drug resistance problems have emerged, making the search for new natural products even more urgent.

[0004] Organic chemistry plays an indispensable role in the synthesis and structural modification of complex natural products. However, the rapid, efficient, and simple construction of natural product libraries remains difficult to achieve through organic synthesis, and organic reagents and catalysts are highly toxic. Developing green and environmentally friendly biotechnological methods to generate natural product analogs and subsequently construct libraries of these analogs has significant academic and applied value. These methods mainly include precursor-guided biosynthesis, mutagenesis, combinatorial biosynthesis, and enzymatic transformation. Summary of the Invention

[0005] This invention mainly employs gene knockout biotechnology to inactivate biosynthetic pathways and discover analogs of natural products, and investigates the bioactivity and antitumor effects of novel compounds.

[0006] To achieve the above-mentioned objects and other advantages according to the present invention, in a first aspect, a novel compound is provided, the compound having the structural formula:

[0007] .

[0008] A second aspect of the present invention provides a method for preparing a novel compound, comprising the following steps:

[0009] S1. Construction of mutant strains: The gene encoding pyrK1 was interrupted in the strain Streptomyces rugosporus LL-42D005.

[0010] S2, fermentation of mutant strain;

[0011] S3. Extraction: Collect the fermentation broth, centrifuge, take the supernatant to adjust the pH, extract with ethyl acetate, concentrate the ethyl acetate under reduced pressure to obtain the extract;

[0012] S4. A novel compound was obtained by isolation.

[0013] Preferably, the culture of S. rugosporus LL-42D005 is first inoculated into a seed culture medium and cultured for 1-3 days. Then, a portion of the cultured culture is transferred to a fermentation culture medium and cultured for 5-15 days.

[0014] Preferably, the seed culture medium consists of: 1-2% glucose, 2-4% soluble starch, 1-2% yeast extract, 1-2% NZ Amine A, and 0.1-0.3% calcium carbonate, mixed in water and adjusted to pH 7.2.

[0015] Preferably, the fermentation medium consists of: 3-5% glucose, 1-3% peptone, 1-3% yeast extract, 0.1-0.5% ferric ammonium citrate, and calcium carbonate mixed in water, with the pH adjusted to 6.5.

[0016] Preferably, the separation method in step S4 includes: gel column chromatography (LH-20), methanol elution, and HPLC detection of the target product; reversed-phase silica column chromatography (ODS), and Semi-PHPLC to obtain the novel compound.

[0017] Preferably, the structural formula of the compound is:

[0018] .

[0019] A third aspect of the invention provides the use of the novel compounds described herein in the preparation of antitumor drugs.

[0020] Preferably, the tumor includes ovarian cancer.

[0021] Preferably, the structural formula of the compound is:

[0022] .

[0023] Compared with the prior art, the beneficial effects of this invention are: this invention provides a new compound with potential anti-tumor medicinal value and its preparation, extraction and separation method from the related gene pyrK1 of Streptomyces rugosporus LL-42D005, the cytotoxic activity of the compound against tumor cells, and the application of the compound in the preparation of anti-tumor drugs. Attached Figure Description

[0024] Figure 1 The above are the HMBC and NOESY diagrams of the compounds according to the present invention. Detailed Implementation

[0025] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0026] To better understand this invention, the technical terms used in this invention will be explained below.

[0027] Streptomyces rugosporus, a family of fungi in the order Actinomycetes. The mycelium within the substrate is unbroken, while the aerial hyphae are usually well-developed, forming long (sometimes short) sporophytes. The spores are non-motile, and the outer sheath often has wart-like, spine-like, or hair-like ornaments.

[0028] Genes are segments of DNA that have a hereditary effect (the genetic material of some viruses, such as tobacco mosaic virus and HIV, is RNA). Genes support the basic structure and function of life. They store all the information about a life's species, blood type, conception, growth, and death. The interdependence of environment and heredity drives important physiological processes such as reproduction, cell division, and protein synthesis. All life phenomena, including birth, growth, aging, disease, and death, are related to genes. They are also the intrinsic factors that determine health. Therefore, genes have a dual nature: materiality (mode of existence) and informationality (fundamental attribute).

[0029] A tumor is a new growth formed when a cell in a local tissue loses its normal regulation of growth at the gene level under the influence of various carcinogenic factors, leading to its clonal abnormal proliferation. Tumors are generally classified into two main categories: benign and malignant. Tumor development is caused by an imbalance of electrons in cells. Reactive oxygen species (ROS) are electron-deficient substances (unsaturated electron substances). After entering the body, they compete for electrons. If they steal electrons from cellular protein molecules, causing the proteins to branch and undergo alkylation, forming aberrant molecules that are carcinogenic. Because these aberrant molecules lack electrons themselves, they then steal electrons from neighboring molecules, causing those neighboring molecules to also become aberrant and carcinogenic. This vicious cycle leads to the formation of a large number of aberrant protein molecules. When these aberrant protein molecules reproduce, gene mutations occur, forming a large number of cancer cells, eventually resulting in cancer symptoms. Conversely, when free radicals or aberrant molecules steal electrons from genes, cancer develops directly. When the human body receives negative ions, these negatively charged ions, carrying excess electrons, can provide electrons to cells that are lacking, thus breaking the vicious cycle and preventing or inhibiting cancer cells. Recent research also indicates that antioxidant therapy has significant clinical value in tumor treatment. Furthermore, it has been reported that negative air ions inhibit the growth of transplanted tumors in animals, and therefore have been applied in the prevention and treatment of human tumors. Negative ions can also maintain the stability of the internal environment by regulating the acid-base imbalance and redox imbalance caused by malignant tumors, promoting normal cell metabolism, and reducing or eliminating the adverse effects of chemotherapy. They are particularly effective in aiding the recovery of cancer patients after chemotherapy.

[0030] A compound is a chemical substance composed of two or more elements bonded together by chemical bonds in a fixed molar ratio. Compounds are mainly divided into organic compounds and inorganic compounds. Organic compounds contain hydrocarbons (or hydrocarbons), such as methane (CH4), and are classified as carbohydrates, nucleic acids, lipids, and proteins. Inorganic compounds do not contain hydrocarbons, such as lead(II) sulfate (PbSO4), and are classified as acids, bases, salts, and oxides.

[0031] Example 1

[0032] 1. Construction of mutant strains

[0033] Total DNA was extracted from *Streptomyces rugosporus* LL-42D005 and whole-genome sequencing was performed, revealing a complete gene cluster related to the biosynthesis of pyrrolodoxin (PYR). This cluster covers a 105 kb region on the chromosome and contains 48 orf genes. Bioinformatics analysis identified the functions of most of these genes. Then, the coding gene pyrK1 was selected for in vivo disruption, resulting in the cessation of PYR production. LC-MS analysis confirmed the formation of a new compound.

[0034] 2. Fermentation

[0035] Fermentation employs a two-stage culture method.

[0036] Seed culture medium: 1% glucose, 2% soluble starch, 1% yeast extract, 1% NZ Amine A, 0.1% calcium carbonate, mixed in tap water, pH adjusted to 7.2, and incubated in a shaker at 28 °C and 220 rpm for 3 days.

[0037] Fermentation medium: 3% glucose, 1% peptone, 1% yeast extract, 0.1% ferric ammonium citrate, and calcium carbonate mixed in tap water, with the pH adjusted to 6.5.

[0038] First, 100 μL of the preserved Streptomyces rugosporus LL-42D005 culture was inoculated into 100 ml of seed culture medium and cultured at 28°C and 220 rpm for 48 hours. Then, 10 ml of the culture was transferred to 100 ml of fermentation medium and cultured at 28°C and 220 rpm for 10 days. Large-scale fermentation was then carried out in a volume of 30 L.

[0039] 3. Extraction

[0040] Collect the fermentation broth, centrifuge it, take the supernatant, add phosphoric acid to adjust the pH to 6.5, extract it three times with an equal volume of ethyl acetate, concentrate the ethyl acetate under reduced pressure to obtain the extract.

[0041] 4. Separation

[0042] The concentrated solutions were subjected to gel column chromatography (LH-20) with methanol elution (HPLC detection of the target product); reversed-phase silica gel column chromatography (ODS) with methanol-water, 10%-90% elution; and semi-PHPLC (methanol-water, 40%) to prepare 30 mg of novel compound 1 and 20 mg of novel compound 2.

[0043] Example 2

[0044] Compounds 1 and 2 prepared in Example 1 were isolated and identified by HRMS, MS, 1H, 13C-NMR, HMBC, HSQC, and NOESY. The structural formulas of compounds 1 (R1) and 2 (R2) are as follows:

[0045] .

[0046] The results are as follows Figure 1 As shown, Figure 1 A is the HMBC diagram of compound 1. Figure 1 B is the NOESY diagram of compound 1; Figure 1 C is the HMBC diagram of compound 2. Figure 1 D is the NOESY plot of compound 2. The NMR data of compounds 1 and 2 are shown in Table 1 below.

[0047] Table 1. NMR data of new compounds 1-2

[0048]

[0049]

[0050]

[0051] In DMSO–d6, 500MHz 1 H and 125MHz 13 C was used for nuclear magnetic resonance testing. ov = overlap. All signals were determined based on COSY, HSQC, HMBC, and NOESY correlations.

[0052] Example 3: Compounds 1-2 possess antitumor biological activity.

[0053] The antitumor activity of compounds 1-2 prepared in Example 1 and etoposide (control group) was detected by the MTT reduction method.

[0054] Logarithmic growth phase ovarian cancer cells HEY and ES-2 (purchased from Shanghai Jinyuan Biotechnology Co., Ltd.) were cultured in DMEM medium containing 10% fetal bovine serum at a concentration of 1×10⁻⁶ cells / mL. 6Cells were seeded at 100 µL / ml in 96-well plates and incubated at 37°C with 5% CO2 for 24 h. A control group and 10 experimental groups with different concentrations (50, 25, 12.5, 6.25, 3.125, 1.6, 0.8, 0.4, 0.2, 0.1 μM) were set up, with 3 replicates per group. After 24 h of incubation, the culture medium was aspirated. For the positive control (Etoposide), 100 µL of culture medium containing different concentrations of compounds 1-2 and Etoposide was added to the experimental groups, while the control group received an equal volume of culture medium. The cells were incubated at 37°C with 5% CO2 for 72 h. Then, 10 μL / well of phosphate-buffered MTT (5 mg / mL) was added to each well, and the cells were incubated for another 4 h. The supernatant was discarded, and 100 μL / well of DMSO was added. The cells were gently shaken for 10 min, and the absorbance (OD) was measured at 540 nm using a microplate reader to calculate the cell inhibition rate. Cell inhibition rate = (1 - mean value of compound A group / mean value of cell control group) × 100%. IC50 was calculated using GraphPad Prism 8. 50 value.

[0055] The results are shown in Table 2. As can be seen from Table 2, the antitumor effects of compounds 1 and 2 of this application are superior to those of the antitumor drug etoposide.

[0056] Table 2. IC50 values ​​of compounds 1 and 2 against different ovarian cancer cells HEY and ES-2.

[0057]

[0058] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A compound, characterized in that, The structural formula of the compound is: ； Where R is selected from or .

2. The method for preparing the compound according to claim 1, characterized in that, Includes the following steps: S1. Construction of mutant strains: The gene pyrK1 encoding gene was selected and interrupted in the strain Streptomyces rugosporus LL-42D005 using gene knockout technology. S2, fermentation of mutant strain; S3. Extraction: Collect the fermentation broth, centrifuge, take the supernatant to adjust the pH, extract with ethyl acetate, concentrate the ethyl acetate under reduced pressure to obtain the extract; S4. The compound is obtained by separation.

3. The method for preparing the compound according to claim 2, characterized in that, The separation method in step S4 includes: The target product was obtained by gel column chromatography (LH-20), methanol elution, and HPLC detection; ODS was obtained by reversed-phase silica column chromatography, followed by semi-PHPLC.

4. The use of the compound of claim 1 in the preparation of an antitumor drug, wherein the tumor is ovarian cancer.

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