Enantiomeric eudesmane sesquiterpenoids and applications

Abstract

This application relates to the technical field of sesquiterpene compounds, specifically to enantiomeric-eudesmane sesquiterpene compounds and their applications. The enantiomeric-eudesmane sesquiterpene compounds are as shown, and they have the effect of inhibiting angiogenesis.

Description

Technical Field

[0001] The present application relates to the technical field of sesquiterpenoid compounds, and particularly relates to enantiomeric-eudesmane sesquiterpenoid compounds and their applications. Background Art

[0002] Terpenoid compounds (terpenes) are hydrocarbons and their oxygen-containing derivatives with molecular formulas that are multiples of isoprene units. They are a large class of natural products with high diversity, including monoterpenes, sesquiterpenes, diterpenes, sesterterpenes, triterpenes, and so on. Many terpene derivatives have been developed into important drugs for treating cancer, bacterial infections, malaria, and various other human diseases. Therefore, the synthesis of terpenoids is very important. However, due to factors such as the still incompletely elucidated biosynthetic pathways, the non-modular structures of terpenoid compounds, and the lack of a universal and unified synthetic strategy, the source of terpenoid compounds depends on the extraction of natural products.

[0003] In addition to terrestrial plants, sesquiterpenoid compounds (sesterterpenes) are also one of the main secondary metabolites of various sponges, algae, and their endophytic fungi in the ocean. Existing technologies show that very few microorganisms from marine environments can produce such compounds, especially enantiomeric sesquiterpenoid compounds. Summary of the Invention

[0004] The present application provides enantiomeric-eudesmane sesquiterpenoid compounds and their applications. These enantiomeric-eudesmane sesquiterpenoid compounds have the effect of inhibiting angiogenesis.

[0005] To this end, the embodiments of the present application at least disclose the following technical solutions:

[0006] In the first aspect, the embodiments disclose the following compounds and their salts:

[0007]

[0008] In the second aspect, the embodiments disclose a pharmaceutical composition comprising the compound or its salt described in the first aspect and a pharmaceutically acceptable excipient, especially a pharmaceutically acceptable salt, especially in unit dosage.

[0009] In the third aspect, the embodiments disclose the use of the compound or its salt described in the first aspect in the preparation of a drug for treating cancer. The treatment comprises the step of administering the compound or its salt described in the first aspect to a person determined to be in need thereof.

[0010] In the fourth aspect, the embodiments disclose a pharmaceutical composition comprising the compound or its salt described in the first aspect and different anti-cancer agents. These components can be co-packaged or co-formulated, and / or in unit dosage.

[0011] In embodiments, different anti-cancer agents are used for cancers (including tumors, neoplasms, malignancies, etc.) and are preferably labeled or approved by a government (such as the FDA) for such use; examples include fluorouracil, bevacizumab, irinotecan hydrochloride, capecitabine, cetuximab, leucovorin calcium, oxaliplatin, panitumumab, regorafenib, aflibercept.

[0012] The term "pharmaceutically acceptable salts" means salts of the active compounds prepared with relatively non-toxic acids or bases, depending on the specific substituents found on the compounds described herein. When the compounds of the present application contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the required base (either pure or in a suitable inert solvent). Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts, or the like. When the compounds of the present application contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the required acid (either pure or in a suitable inert solvent). Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate, phosphoric acid, hydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, or phosphorous acid, and the like, as well as from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, oxalic acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like. Also included are salts of amino acids such as arginine, etc., and salts of organic acids such as glucuronic acid or galacturonic acid. Certain specific compounds of the present application contain both basic and acidic functional groups, which enable the compounds to be converted into base addition salts or acid addition salts.

[0013] The neutral form of the compounds can be regenerated by contacting the salts with an acid or a base and isolating the parent compound in the usual manner. The parent form of the compounds differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but for the purposes of the present application, these salts are equivalent to the parent form of the compounds.

[0014] In addition to the desalted form, the invention provides prodrug forms of the compounds. As used herein, a prodrug of a compound refers to those compounds that undergo chemical change under physiological conditions to provide the compounds of the present application. In addition, prodrugs can be converted into the compounds of the present application by chemical or biochemical methods in an in vitro environment. For example, when placed in a transdermal patch reservoir together with a suitable enzyme or chemical reagent, the prodrug can be slowly converted into the compound of the present application. Prodrugs are often effective because they may be easier to administer than the parent drug, may have better oral bioavailability than the parent drug, and may have increased solubility in a pharmaceutical composition compared to the parent drug. A variety of prodrug derivatives are known in the art, such as those that rely on their hydrolytic cleavage or oxidative activation. An unrestricted example of a prodrug is a compound of the present application administered as an ester ("prodrug"), but then hydrolytically metabolized to the carboxylic acid active form.

[0015] Certain compounds of the present application may exist in unsolvated as well as solvated forms, including hydrate forms. In general, the solvated forms are equivalent to the unsolvated forms and are included within the scope of the present application. Certain compounds of the present application may exist in polymorphic or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present application and are included within the scope of the present application.

[0016] In addition, the compounds have asymmetric carbon atoms (optical centers) or double bonds, and racemates, diastereomers, geometric isomers, and specifically designed or described chiral forms are preferred and, in many cases, decisive for optimal activity; however, all such isomers are intended to be included within the scope of the present application.

[0017] The term "therapeutically effective amount" refers to the amount of the target compound that elicits a biological or medical response in a tissue, system, animal, or human to a certain significant degree, which responses are sought by a researcher, veterinarian, physician, or other clinician, e.g., when administered, is sufficient to arrest its development, or to alleviate to some extent one or more symptoms of the disease or disorder being treated. A therapeutically effective amount will vary depending on the compound, the disease and its severity, and the age, weight, etc. of the mammal being treated.

[0018] The administered composition can be made into a bulk solution, suspension, or bulk powder. However, more commonly, the composition is presented in unit dosage forms for easy and accurate dosing. The term "unit dosage form" refers to physically discrete units suitable for unit doses for human subjects and other mammals, each unit containing a predetermined amount of the active material calculated to produce the desired therapeutic effect, as well as suitable pharmaceutical excipients. Typical unit dosage forms include ampoules or syringes prefilled with a predosed liquid composition, or in the case of solid compositions, pills, tablets, capsules, etc. In these compositions, the compound is usually a minor component (about 0.1 to 50% by weight or preferably about 1 to about 40% by weight), and the remainder consists of various excipients and processing aids to help form the desired dosage form.

[0019] Suitable excipients or carriers and methods for preparing the administrable components are known to or within the grasp of those skilled in the art and are described in detail in some publications, such as Remington′s Pharmaceutical Science, Mack Publishing Co, NJ (2013). In addition, such compounds can be advantageously used in combination with other therapeutic agents described herein or known in the art, particularly other anti-necrosis agents. Thus, these drugs can be administered alone, jointly, or in combination in a single dosage unit.

[0020] The dosage depends on the formulation of the compound, the mode of administration, etc., and is usually determined empirically in routine tests and will have to vary according to the target, host, mode of administration, etc. Generally, depending on the specific application, the amount of the active compound in a unit dosage formulation can vary or be adjusted from about 1, 3, 10, or 30 to about 30, 100, 300, or 1000 mg. In a specific embodiment, the unit dosage form can be packaged in a multi-piece pack suitable for first use, such as a blister pack, containing at least 6, 9, or 12 unit dosage form tablets. The actual dose used can vary according to the patient's needs and the severity of the condition being treated. Determining the correct dose in a particular case is within the skill of the art. Generally, treatment starts with a smaller dose, which is less than the optimal dose of the compound. Thereafter, the dose can be increased in small increments until the optimal effect in that case is achieved. If desired, for convenience, the total daily dose can be divided and administered in fractions during the day.

[0021] The compound can be administered in a variety of ways, including but not limited to parenterally, topically, orally, or locally, such as by aerosol or transdermally, for prevention and / or treatment. And according to the knowledge of experienced clinicians, the treatment regimen (such as the dosage and frequency of administration) will vary depending on the observed effect of the administered therapeutic agent on the patient and the observed response of the disease to the administered therapeutic agent.

[0022] The treatment in the present application can administer a therapeutically effective dose and total amount during the process of implementing an effective treatment plan for a patient. For more effective compounds, an amount of micrograms (μg) per kilogram of the patient may be sufficient. For example, from about 1, 10, or 100 μg / kg to about 0.01, 0.1, 1, 10, or 100 mg / kg based on the patient's body weight, although the optimal dose is specific to the compound and is usually determined empirically.

[0023] Generally, conventional tests in clinical trials will determine the specific range to achieve the desired therapeutic effect. Each treatment, each dosing regimen, and the dosing for a specific patient will also be adjusted within the effective and safe range according to the patient's condition and response to the first dose. However, the final dosing regimen will be adjusted according to the judgment of the participating clinicians, considering factors such as the patient's age, condition, and weight, as well as the potency of the compound and the severity of the disease being treated. For example, the dosing regimen of the compound can be an oral dose from 10 mg to 2000 mg / day, preferably from 10 to 1000 mg / day, more preferably from 50 to 600 mg / day, administered in 2 - 4 (preferably 2) divided doses. Intermittent therapy (such as 1 week out of 3 weeks or 3 weeks out of 4 weeks) can also be used for treatment.

[0024] In a fifth aspect, the embodiments disclose a preparation method of the compound described in the first aspect. The preparation method includes:

[0025] Activating the Eutypella sp. F0219 strain;

[0026] Obtaining a seed solution by liquid culturing the Eutypella sp. F0219 colonies;

[0027] Obtaining a fermentation broth by scale-up culturing the seed solution;

[0028] Collecting the mycelium of Eutypella sp. F0219 from the fermentation broth;

[0029] Extracting the compound described in the first aspect from the mycelium.

[0030] In some embodiments, the Eutypella sp. F0219 strain is activated using a PDA plate.

[0031] In some embodiments, the seed solution is obtained by culturing with a potato liquid medium. The potato liquid medium contains 200 g / L potatoes and 20 g / L glucose.

[0032] In some embodiments, the fermentation broth is obtained by scale-up culturing with a rice solid medium. The rice solid medium contains 80 g / L rice and 0.3 wt% sea salt.

[0033] In some embodiments, the steps of extracting the compound described in the first aspect from the mycelium include: successively soaking and extracting the fermented rice ferment with petroleum ether and ethyl acetate, combining the ethyl acetate extracts, and concentrating under reduced pressure to obtain a crude ethyl acetate extract.

[0034] In some embodiments, the steps of extracting the compound described in the first aspect from the crude ethyl acetate extract include: subjecting the crude extract to ethyl acetate extraction to obtain an ethyl acetate extract; performing silica gel column chromatography on the extract, with the eluent being a chloroform-methanol solvent (100:0 - 1:1, v / v), performing gradient elution successively, collecting fractions at about 1 L each time, combining similar fractions by TLC detection, and dividing them into 5 components, namely Fr.1 - 5. Component Fr.2 is identified for its structure by comprehensively applying separation methods such as ODS column chromatography, Sephadex LH-20 column chromatography, and HPLC preparation, and by comprehensively applying physical and chemical analysis, modern spectroscopic methods such as NMR and MS, and single crystal X-ray diffraction to obtain a new enantiomeric eudesmane sesquiterpenoid compound I.

[0035] It should be understood that the embodiments and implementation schemes described herein are only for illustrative purposes, and various modifications and variations thereof will be foreseeable to those skilled in the art and are included in the spirit and scope of this application, as well as the scope of protection of the appended claims. All publications, patents, and patent applications cited herein, including those cited therein, are hereby incorporated by reference in their entirety for all purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 1H NMR spectrum of compound I provided for the example 1 1H NMR spectrum.

[0037] Figure 2 13C NMR spectrum of compound I provided for the example 13 13C NMR spectrum.

[0038] Figure 3 DEPT135 spectrum of compound I provided for the example

[0039] Figure 4 1H- 1 H COSY spectrum of compound I provided for the example 1 1H-1H COSY spectrum.

[0040] Figure 5 HSQC spectrum of compound I provided for the example

[0041] Figure 6 HMBC spectrum of compound I provided for the example

[0042] Figure 7NOESY spectrum of Compound I provided for the example.

[0043] Figure 8 HR-ESIMS spectrum of Compound I provided for the example.

[0044] Figure 9 UV spectrum of Compound I provided for the example.

[0045] Figure 10 Crystal structure diagram of Compound I provided for the example.

[0046] Figure 11 Result diagram showing that Compound I provided for the example has no toxic effect on HEMC-1 cells.

[0047] Figure 12 Result diagram of the cell scratch healing test of Compound I provided for the example on HEMC-1 cells. Scale bar: 50 μm. Data are presented as mean ± SEM, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the blank group. Figure 12 A is a representative image of the cell scratch healing experiment of HEMC-1 cells (0 is the blank group, 20 μM, 40 μM, 80 μM are the drug-treated groups respectively); Figure 12 B is the corresponding quantitative data analysis diagram (0 is the blank group, 20, 40, 80 are the drug-treated groups respectively).

[0048] Figure 13 Result diagram of the Transwell chamber migration and invasion experiments of Compound I provided for the example on HEMC-1 cells. Scale bar: 50 μm. Data are presented as mean ± SEM, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the blank group. Figure 13 A is a representative image of Transwell chamber migration (0 is the blank group, 20 μM, 40 μM, 80 μM are the drug-treated groups respectively). Figure 13 B is a representative image of Transwell chamber invasion (0 is the blank group, 20 μM, 40 μM, 80 μM are the drug-treated groups respectively). Figure 13 C is the quantitative data analysis diagram of Transwell chamber migration (0 is the blank group, 20 μM, 40 μM, 80 μM are the drug-treated groups respectively). Figure 13 D is the quantitative data analysis diagram of Transwell chamber invasion (0 is the blank group, 20 μM, 40 μM, 80 μM are the drug-treated groups respectively).

[0049] Figure 14Graph showing the results of the lumen formation assay of Compound I provided in the example on HEMC-1 cells. Scale bar: 50 μm. Data are presented as mean ± SEM, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the blank group. Figure 14 A is a representative image of lumen formation in HEMC-1 cells (0 is the blank group, 20 μM, 40 μM, 80 μM are the drug-treated groups respectively). Figure 14 B is the corresponding quantitative data analysis graph (0 is the blank group, 20 μM, 40 μM, 80 μM are the drug-treated groups respectively).

[0050] Figure 15 Graph showing the results of the sprouting assay of Compound I provided in the example on rat arterial rings. Scale bar: 50 μm. Data are presented as mean ± SEM, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the blank group. Figure 15 A is a representative image of sprouting in rat arterial rings (0 is the blank group, 20 μM, 40 μM, 80 μM are the drug-treated groups respectively). Figure 15 B is the corresponding quantitative data analysis graph (0 is the blank group, 20 μM, 40 μM, 80 μM are the drug-treated groups respectively). Detailed implementation manners

[0051] In order to make the objectives, technical solutions and advantages of this application more clear and understandable, the following further details this application in conjunction with examples. It should be understood that the specific examples described herein are only used to explain this application and are not used to limit this application. The reagents not specifically described in detail in this application are all conventional reagents and can be obtained commercially; the methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.

[0052] This application discloses a sesquiterpenoid compound from marine fungi. This compound is an enantiomeric-eudesmane-type sesquiterpenoid. This sesquiterpenoid is isolated from the secondary metabolites of the marine fungus Eutypella sp. F0219. The marine fungus Eutypella sp. F0219 was deposited on July 3, 2023 at the China Center for Type Culture Collection, Wuhan University, Wuhan, Hubei, and the deposit number given by the deposit center to this strain is CCTCC NO: M20231180.

[0053] Strain source

[0054] The strain (Eutypella sp.) F0219 provided in this application was isolated from the seabed sediment at a depth of 75 m in the northern South China Sea (114.6609°E, 21.5942°N). Through morphological and molecular biological identification, this strain is a fungus of the genus Eutypella. The strain was sent to a sequencing company for identification (testing company: Shanghai Sangon Biotech Co., Ltd.). The obtained base sequence was compared for similarity in the GenBank database, and then the BLAST program was used to search for homologous sequences for comparison. It was determined that the F0219 strain is of the genus Eutypella sp., with a sequence similarity of 99%. It was deposited in the China Center for Type Culture Collection at Wuhan University, Wuhan, Hubei on July 3, 2023, and the deposit number given by the deposit center for this strain is CCTCC NO: M20231180.

[0055] Enantiocaryophyllane sesquiterpenoids

[0056] It was also found in the embodiment of this application that the Eutypella sp. F0219 strain can ferment to produce an enantiomeric eudesmane sesquiterpenoid compound. The specific steps are as follows:

[0057] 1. Fermentation culture of Eutypella sp. F0219

[0058] (1) Strain preservation: The Eutypella sp. F0219 strain was preserved at -80 °C in a PDB-glycerol medium. Among them, the PDB-glycerol medium contains 200 g / L potatoes, 20 g / L glucose, and 30 wt% glycerol.

[0059] (2) Strain activation: The Eutypella sp. F0219 strain preserved at -80 °C was taken out, pre-cultured in an incubator at 28 °C for 24 h, and then inoculated onto a PDA plate and activated at 28 °C for 3 days.

[0060] (3) Fermentation culture: The activated Eutypella sp. F0219 cells the size of broad beans were inoculated into a potato liquid medium and cultured in a constant temperature shaker at 28 °C for 3 days to obtain a seed solution. The seed solution was transferred to a rice solid medium and fermented statically at 28 °C for 40 days to obtain a rice fermented product. Among them, the rice solid medium contains 80 g / L rice and 0.3 wt% sea salt.

[0061] 2. Isolation and purification of the compound

[0062] (1) Extraction: The fermented mycelia were successively soaked and extracted 3 times with petroleum ether and ethyl acetate, and the ethyl acetate extracts were combined and concentrated under reduced pressure to obtain a total of 78 g of ethyl acetate crude extract.

[0063] (2) Column chromatography: The crude extract (78 g) was subjected to normal-phase silica gel column chromatography (100 - 200 mesh), and the eluent was chloroform-methanol solvent (100:01:1, v / v) for gradient elution in sequence, and the fractions were collected at about 1.0 L each time. Similar fractions were combined by TLC detection and divided into 5 sub-components, namely Fr.1 - Fr.5. Component Fr.3 was subjected to gradient elution by using reversed-phase C18 column chromatography (MeOH:H2O:30:70 - 100:0) to obtain 73 sub-components Fr.3.1 - Fr.3.73. Among them, component Fr.3.19 was subjected to Sephadex LH-20 column chromatography to obtain 15 sub-fractions Fr.3.19.1 - Fr.3.19.15. Among them, sub-fraction Fr.3.19.7 was further separated and purified by semi-preparative high performance liquid chromatography (CH3CN:H2O = 45:55) to obtain the compound I (13.6 mg), and the structural formula is as shown.

[0064] In addition, the compound I obtained by the above semi-preparative high performance liquid chromatography was analyzed by using an MCP5100 polarimeter (Anton Paar GmbH, Austria, the measuring temperature was 25 °C, and the length of the polarimeter tube was 100 mm), and its was -352.9 (c0.14, MeOH). The academic name of the compound I is (4aS,6S,8aS)-4a-hydroxy-6-(2-hydroxypropan-2-yl)-8a-methyl-4-methylideneoctahydronaphthalen-1(2H)-one. This indicates that the compound I obtained by fermenting Eutypellasp. F0219 provided in this application is an optically pure chiral compound, which has an important decisive role in inhibiting angiogenesis.

[0065] Structure identification of Compound I

[0066] Modern structure identification techniques such as one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy, high-resolution mass spectrometry and single crystal X-ray diffraction were used to determine the chemical structure of the compound I. 1 1H-NMR, 13 13C-NMR, DEPT135, 1 1H- 1The H COSY, HSQC, HMBC and NOE NMR spectra were measured on a Bruker Advance-400 NMR spectrometer, and the ESI-HRMS data were measured on a Bruker maXis ESI-QTOF mass spectrometer (Bruker Daltonics, Bremen, Germany); the single-crystal X-ray diffraction data were collected on a Rigaku Oxford Diffraction Supernova X-ray source diffractometer equipped with an Atlas S2 CCD using Cu Kα radiation (λ = 1.54184 Å) at 150 K (Rigaku Americas Corporation, The Woodlands, Texas, USA).

[0067] Compound I was a colorless crystal (its NMR data are shown in Table 1);

[0068] Table 1 NMR data of Compound I in deuterated methanol (400 MHz / 125 MHz, δ in ppm, J in Hz)

[0069]

[0070] a Overlapped signals assigned by HSQC and HMBC spectra without designating multiplicity.

[0071] According to HRESIMS [M + H - H2O] + m / z 235.1696, C 15 H 23 O2, the calculated value was 235.1698, and the molecular formula of the compound was determined to be C 15 H 24 O3, and the degree of unsaturation was 4. 1 The 1H-NMR spectrum and HSQC spectrum showed that the compound had two exocyclic double-bond hydrogen signals [δ H 5.00 (m, H-15), 5.07 (m, H-15)]; three methyl signals [δ H 1.19 (s, H3-12), 1.19 (s, H3-13), 1.05 (s, H3-14)]; one methine hydrogen signal [δ H 1.80 (m, H-7)]; ten methylene hydrogen signals [δ H2.70(m,H-2α),2.28(ddd,J=15.2,7.2,1.2Hz,H-2β); 2.47(ddd,J=13.6,8.4,1.2Hz H-3α),3.00(m,H-3β);1.77(m,H-6α),1.77(m,H-6β);1.72(m,H-8α),1.26( m, H-8β); 1.47 (dt, J = 13.2, 3.6 Hz, H-9 α), 2.05 (td, J = 14.0, 4.0 Hz, H-9 β)]. 13 C-NMR and HSQC spectra show that there are 15 carbon signals in compound I, including 5 quaternary carbons, 1 methine, 6 methylene, and 3 methyl groups. The planar structure of compound I was determined by further analyzing its 1H-1H COSY, HSQC, and HMBC two-dimensional spectra. As shown in the figure above, the absolute configuration of compound I was determined by X-ray single crystal diffraction.

[0072] Inhibitory angiogenesis activity test of Compound I

[0073] 1. CCK-8 test

[0074] Test Example The CCK-8 test was used to test the non-toxicity of compound I to cells. The test process included: inoculating 5×10 3 Human dermal microvascular endothelial cells (HMEC-1, catalog number CP-H098, Pronocell) were cultured at 37°C and 5% CO2 overnight until adhered. The culture medium was removed and the cells were divided into a drug-added group and a blank group. In the drug-added group, culture medium with different concentrations of compound I was added to each well at 12.5μM, 25μM, 50μM, 100μM, 200μM, and 400μM, respectively. The blank group was added with the same volume of drug-free culture medium, and then 10μL CCK-8 reagent was added after culturing for 48 hours. After incubation for another 2 hours, the absorbance was detected at 450nm using an enzyme reader (Bio-Rad, Hercules, CA, USA). The results were processed and analyzed using Graphpad Prism, and the cell proliferation curve was drawn to calculate the drug concentration at 50% inhibition rate, i.e., IC 50 The experiment was repeated 3 times.

[0075] like Figure 11 As shown, compound I has no toxicity to HMEC-1 cells.

[0076] 2. Cell scratch healing test

[0077] After washing, digestion, centrifugation, and resuspending, HMEC-1 cells were seeded in each well of a 6-well plate at 3 × 10 4HEMC-1 cells were cultured at 37°C and 5% CO2 until the adhesion reached 90% to 100% and a monolayer of cell membrane was formed at the bottom of each well. The cell solution was discarded, washed twice with PBS buffer, serum-free medium was added, and incubation continued for 12 hours; a 200μL pipette head shoulder was used to take a direction perpendicular to the long axis of the plate along the ruler, and a scratch was formed on the bottom of the well with gentle force. After washing the dead cells, fresh medium containing 20μM, 40μM, 80μM compound I or no compound I was added to the cells and placed in a cell culture incubator for 8 hours. The same field of view was taken at 0 and 8h using an inverted microscope, and the migrating cells were counted using IPP6 software.

[0078] like Figure 12 A and Figure 12 As shown in B, the wound of the blank group without drug addition was almost healed, and compound I reduced cell migration in a concentration-dependent manner.

[0079] 3. Transwell chamber migration and invasion assay

[0080] 100 μL of 2×10 4 The serum-free culture medium of HEMC-1 cells was added to the drug-addition group at the same time, and 20μM, 40μM and 80μM of compound I were added to the blank group. 600μL of complete culture medium was added to the lower chamber of Transwell and cultured for another 24h. After rinsing the lower chamber with PBS, the cells on the surface of the lower chamber were fixed with 4% paraformaldehyde and identified by crystal violet staining. Images of migrating cells were taken with an inverted microscope. IPP6 software was used to count the number of cell migrations in 5 randomly selected pictures. The experimental process of the Transwell chamber invasion assay was the same as that of the Transwell chamber migration assay, except that the upper chamber was coated with matrigel matrix gel.

[0081] like Figure 13 As shown in AD, the Transwell migration and invasion assays showed that compound I inhibited the migration and invasion ability of HMEC-1 cells in a concentration-dependent manner, indicating that compound I has a strong inhibitory effect on the movement of endothelial cells required for the angiogenesis process.

[0082] 4. Lumen formation test

[0083] Add 50 μL of thawed matrigel to each well of a 96-well plate, and place the plate in a 37°C incubator for more than 1 hour to allow the matrigel to solidify. Treat HEMC-1 cells in the logarithmic growth phase, culture and resuspend in EGM-2 complete medium or 0.5% low-serum EGM-2 medium to obtain a cell suspension with a cell concentration of 4×105 ~6×10 5 , a volume of 200 μL was placed in an EP tube, and 20 μM, 40 μM and 80 μM compound I or a blank group was added and placed in an incubator for 1 hour. The cell suspension was then inoculated in a 96-well plate covered with matrigel matrix gel, with three replicates in each group. After 6 hours of incubation at 37°C and 5% CO2, the formation of tubular structures of vascular endothelial cells was observed under a microscope and photographed and recorded, and the images were analyzed using Image-Pro Plus 6 (IPP6).

[0084] like Figure 14 A and Figure 14 As shown in B, endothelial cells were observed to differentiate and form tubular structures on the matrix gel in the blank group. In the drug-added group, compound I obviously destroyed the formation of segments in a dose-dependent manner, where only about 10% of the tubular structure formation was observed in the 80 μM compound I relative to the blank group.

[0085] 5. Animal Testing

[0086] SD rats (Animal Experiment Center, Wuhan University) were killed with carbon dioxide, and the aorta was isolated from the thoracic region of the SD rats. After removing the peripheral fat, the aorta was cut into 1-1.5 mm rings, and the rings were inoculated on a 96-well plate coated with matrigel matrix gel, and then covered with 80 μL of matrigel matrix gel to the sandwich structure. After incubation at 37 ° C for 1 hour, fresh culture medium containing 20 μM, 40 μM, 80 μM compound I or blank group was added to the culture dish and cultured for another 6 days. The microvessels around the rings were observed and photographed using an inverted microscope. The density of microvessels was quantitatively determined using IPP6 software.

[0087] like Figure 15 A and Figure 15 As shown in B, compound I significantly reduced the sprouting of microvessels in a concentration-dependent manner, and the segment formation of the ring and the total length of microvessels were significantly reduced, which again showed that compound I has the effect of inhibiting angiogenesis.

[0088] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. The compound and its salt as shown below:

2. A pharmaceutical composition comprising the compound or salt thereof according to claim 1 and a pharmaceutically acceptable excipient.

3. Use of the compound or salt thereof according to claim 1 in the preparation of a medicament for treating cancer, wherein the treatment comprises the step of administering the compound or salt thereof according to claim 1 to a person determined to be in need thereof.

4. A pharmaceutical composition comprising the compound or a salt thereof according to claim 1, and different anticancer agents.

5. The pharmaceutical composition according to claim 4, wherein the different anticancer agents are selected from fluorouracil, bevacizumab, irinotecan hydrochloride, capecitabine, cetuximab, leucovorin calcium, oxaliplatin, panitumumab, regorafenib, and aflibercept.

6. A method for preparing the compound according to claim 1, comprising: The Eutypella sp.F0219 strain was activated using PDA plates; The Eutypella sp. F0219 colony is cultured in a potato liquid medium to obtain a seed solution; the potato liquid medium comprises 200 g / L potato and 20 g / L glucose; The seed liquid is cultured with a rice solid culture medium to obtain a fermentation liquid; the rice solid culture medium comprises 80 g / L rice and 0.23 wt % sea salt; Collecting mycelium of Eutypella sp.F0219 from the fermentation broth; The mycelium obtained by fermentation is extracted by soaking in petroleum ether and ethyl acetate respectively, the ethyl acetate extract is combined, and concentrated under reduced pressure to obtain a crude ethyl acetate extract; Extracting the crude extract with ethyl acetate to obtain an ethyl acetate extract; The extract was subjected to silica gel column chromatography, wherein the eluent A was chloroform and the eluent B was methanol, and a gradient elution was performed from 100% of the eluent A and the eluent B to 50% of the eluent A and the eluent B, and the component Fr.2 was collected by TLC detection; The component Fr.2 was separated by comprehensive use of ODS column chromatography, Sephadex LH-20 column chromatography and HPLC preparation, and its structure was identified to obtain the target compound.

7. According to the preparation method according to claim 6, the Eutypella sp.F0219 strain was deposited in the China Type Culture Collection at Wuhan University, Wuhan, Hubei on July 3, 2023, and the collection center gave the strain a collection number of CCTCC NO:M20231180.

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