Enantio-cineole type sesquiterpenoids and application thereof

Abstract

The invention relates to the technical field of sesquiterpenoids, in particular to an enantiomer-cineole type sesquiterpenoids and application thereof. The enantiomer-cineole type sesquiterpenoids compound is shown as # imgabs0 #, and has the effect of inhibiting angiogenesis.

Description

Technical Field

[0001] The present application relates to the technical field of sesquiterpenoid compounds, and in particular to ent-eudesane-type sesquiterpenoid compounds and applications thereof. Background Art

[0002] Terpenes are hydrocarbons and their oxygen-containing derivatives with a molecular formula of multiples of isoprene units. They are a large class of natural products with high diversity, including monoterpenes, sesquiterpenes, diterpenes, dipterpenes, triterpenes, etc. Many terpenoid derivatives have been developed as important drugs for the treatment of cancer, bacterial infections, malaria and various other human diseases, so the synthesis of terpenes is very important. However, due to the fact that the biosynthetic pathway has not been fully elucidated, terpenoids have a non-modular structure, and there is no universal unified synthetic strategy, the source of terpenoids depends on the extraction of natural products.

[0003] In addition to terrestrial plants, sesterterpenes are also one of the main secondary metabolites of various marine sponges and algae and their endophytic fungi. Existing technology shows that very few microorganisms from the marine environment can produce such compounds, especially enantio-sesquiterpenes. Summary of the invention

[0004] The present application provides ent-eudesane type sesquiterpenoid compounds and applications thereof. These ent-eudesane type 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 salts thereof:

[0007]

[0008] In a second aspect, the embodiments disclose a pharmaceutical composition comprising the compound or a salt thereof according to the first aspect, and a pharmaceutically acceptable excipient, especially a pharmaceutically acceptable salt, especially in a unit dose.

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

[0010] In a fourth aspect, an embodiment discloses a pharmaceutical composition comprising the compound or a salt thereof according to the first aspect, and different anticancer agents, wherein these components can be co-packaged or co-formulated, and / or in a unit dose.

[0011] In an embodiment, the various anticancer agents are used for cancer (including tumors, neoplasia, malignancies, etc.) and are preferably labeled or approved by the government (such as the FDA) for this use; examples include fluorouracil, bevacizumab, irinotecan hydrochloride, capecitabine, cetuximab, leucovorin, oxaliplatin, panitumumab, regorafenib, aflibercept.

[0012] The term "pharmaceutically acceptable salt" is meant to include salts of the active compounds prepared with relatively nontoxic 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 desired base (either neat 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 desired acid (either neat or in a suitable inert solvent). Examples of pharmaceutically acceptable acid addition salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid or phosphorous acid and the like, and from relatively nontoxic 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, 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 allow the compounds to be converted into base addition salts or acid addition salts.

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

[0014] In addition to the salt form, the invention provides compounds in the form of prodrugs. Compound prodrugs referred to herein refer to those compounds that undergo chemical changes under physiological conditions to provide compounds of the present application. In addition, prodrugs can be converted into 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 slowly be converted into a 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 an increased solubility that exceeds the parent drug in a pharmaceutical composition. Various prodrug derivatives are known in the art, such as those prodrugs that rely on their hydrolytic cleavage or oxidative activation. An unrestricted example of a prodrug is that the compound of the present application is administered as an ester ("prodrug"), but then is hydrolyzed and metabolized into a carboxylic acid active body.

[0015] Some compounds of the present application may exist in non-solvated and solvated forms, including hydrate forms. In general, solvated forms are equivalent to non-solvated forms and are included in the scope of the present application. Some compounds of the present application may exist in polymorphic or amorphous forms. In general, all physical forms are equivalent for the purposes considered in the present application and are included in the scope of the present application.

[0016] In addition, where the compounds have asymmetric carbon atoms (optical centers) or double bonds, racemates, diastereomers, geometric isomers and specifically designed or described chirality are preferred and in many cases are critical 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 causes a biological or medical response of a tissue, system, animal or human to a significant degree, which is sought by the researcher, veterinarian, doctor or other clinician, for example, when administered, it is sufficient to prevent its development, or to alleviate to some extent one or more symptoms of the condition or disorder being treated. The 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 composition for administration can be prepared as a bulk solution, suspension or bulk powder. However, it is more common for the composition to be presented in unit dosage form to facilitate accurate measurement. The term "unit dosage form" refers to physically separate units that are suitable for unit doses of human subjects and other mammals, each unit including a predetermined amount of active material, which is calculated to produce the desired therapeutic effect, and a suitable pharmaceutical excipient. Typical unit dosage forms include ampoules or syringes pre-filled with predicted liquid compositions, or pills, tablets, capsules, etc. in the case of solid compositions. In these compositions, the compound is usually a small component (about 0.1 to 50% by weight or preferably about 1 to about 40% by weight), and the rest are various excipients and processing aids to help form the desired dosage form.

[0019] Suitable excipients or carriers and methods for preparing administrable compositions are known or mastered by 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, this compound can be advantageously used in combination with other therapeutic agents described herein or otherwise known in the art, particularly other anti-necrosis agents. Therefore, these drugs can be administered alone, in combination, 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 experiments, and will necessarily change according to the target, host and mode of administration, etc. Generally, according to the specific application, the amount of active compound in the unit dose of the preparation can be changed or 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 package suitable for use in advance, such as a blister package, containing at least 6, 9 or 12 tablets of unit dosage forms. The actual dosage used can vary according to the patient's needs and the severity of the condition being treated. Determining the correct dosage in a particular case belongs to the technical scope 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 continuously increased in small amounts to achieve the best effect in the case. If necessary, for convenience, the total daily dose can be divided and administered in divided doses within the day.

[0021] The compounds can be administered in a variety of ways, including but not limited to parenteral, topical, oral, or local administration, such as by aerosol or transdermal, for prevention and / or treatment. And according to the knowledge of experienced clinicians, the treatment regimen (such as 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] Treatment in the present application can be administered in a therapeutically effective dose and total amount during the course of implementing a therapeutically effective regimen for a patient. For more effective compounds, micrograms (ug) per kilogram of a patient may be sufficient, for example, from about 1, 10 or 100 ug / kg to about 0.01, 0.1, 1, 10 or 100 mg / kg according to the patient's body weight, although the optimal dose is compound-specific and is usually determined empirically.

[0023] In general, the conventional test of clinical trial will determine the specific range of desired therapeutic effect, and each treatment, each dosage regimen and the administration of specific patients will also be adjusted within the effective and safe range according to the patient's condition and the reaction to the first administration. However, the final dosage regimen will be adjusted according to the judgment of the participating clinicians, taking into account factors such as the patient's age, condition and weight, as well as the efficacy of the compound and the severity of the disease being treated. For example, the dosage regimen of the compound can be oral administration of a dose from 10 mg to 2000 mg / day, preferably from 10 to 1000 mg / day, more preferably from 50 to 600 mg / day, divided into 2-4 (preferably 2) divided doses for administration. Intermittent therapy (such as 1 week in 3 weeks or 3 weeks in 4 weeks) can also be used for treatment.

[0024] In the fifth aspect, the embodiment discloses a method for preparing the compound described in the first aspect. The preparation method comprises:

[0025] Activate the Eutypella sp.F0219 strain;

[0026] The Eutypella sp. F0219 colony was cultured in liquid to obtain seed solution;

[0027] The seed liquid is expanded and cultured to obtain a fermentation liquid;

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

[0029] The compound described in the first aspect is extracted 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 culture medium, wherein the potato liquid culture medium comprises 200 g / L potato and 20 g / L glucose.

[0032] In some embodiments, the fermentation broth is obtained by expanding the culture using a rice solid culture medium, wherein the rice solid culture medium comprises 80 g / L rice and 0.3 wt % sea salt.

[0033] In some embodiments, the step of extracting the compound described in the first aspect from the mycelium includes: soaking and extracting the fermented rice product obtained by fermentation with petroleum ether and ethyl acetate respectively, combining the ethyl acetate extracts, and concentrating under reduced pressure to obtain a crude ethyl acetate extract.

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

[0035] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications and variations thereof will be foreseeable by those skilled in the art, and are included in the spirit and scope of the present application, as well as the protection scope 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 Compound I provided in the embodiment 1 H NMR spectrum.

[0037] Figure 2 Compound I provided in the embodiment 13 C NMR spectrum.

[0038] Figure 3 DEPT135 spectrum of Compound I provided in the examples.

[0039] Figure 4 Compound I provided in the embodiment 1 H- 1 H COSY spectrum.

[0040] Figure 5 This is the HSQC spectrum of Compound I provided in the Examples.

[0041] Figure 6 This is the HMBC spectrum of Compound I provided in the Examples.

[0042] Figure 7This is the NOESY spectrum of Compound I provided in the Examples.

[0043] Figure 8 This is the HR-ESIMS spectrum of Compound I provided in the Examples.

[0044] Fig. 9 This is the UV spectrum of Compound I provided in the examples.

[0045] Fig.10 The crystal structure diagram of Compound I provided in the examples.

[0046] Fig.11 This is a graph showing the results of compound I provided in the example having no toxic effect on HEMC-1 cells.

[0047] Fig.12 The results of the cell scratch healing test of compound I provided in the example on HEMC-1 cells are shown in Figure 1. Scale bar: 50 μm. Data are expressed as mean ± SEM, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the blank group. Fig.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, and 80 μM are the drug-added groups respectively); Fig.12 B is the corresponding quantitative data analysis chart (0 is the blank group, 20, 40, and 80 are the drug-added groups).

[0048] Fig.13 The results of the Transwell chamber migration and invasion assay of HEMC-1 cells by Compound I provided in the Example. Scale bar: 50 μm. Data are expressed as mean ± SEM, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the blank group. Fig.13 A is a representative image of Transwell chamber migration (0 is the blank group, 20 μM, 40 μM, and 80 μM are the drug-added groups). Fig.13 B is a representative image of Transwell chamber invasion (0 is the blank group, 20 μM, 40 μM, and 80 μM are the drug-added groups). Fig.13 C is the quantitative data analysis diagram of Transwell chamber migration (0 is the blank group, 20 μM, 40 μM, and 80 μM are the drug-added groups). Fig.13 D is the quantitative data analysis diagram of Transwell chamber invasion (0 is the blank group, 20 μM, 40 μM, and 80 μM are the drug-added groups).

[0049] Fig.14The results of the test of compound I provided in the example on the tube formation of HEMC-1 cells. Scale bar: 50 μm. Data are expressed as mean ± SEM, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the blank group. Fig.14 A is a representative image of lumen formation in HEMC-1 cells (0 is the blank group, 20 μM, 40 μM, and 80 μM are the drug-added groups). Fig.14 B is the corresponding quantitative data analysis diagram (0 is the blank group, 20 μM, 40 μM, and 80 μM are the drug-added groups respectively).

[0050] Fig.15 The results of the test of compound I provided in the example on rat arterial ring sprouting. Scale bar: 50 μm. Data are expressed as mean ± SEM, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the blank group. Fig.15 A is a representative image of rat arterial ring sprouting (0 is the blank group, 20 μM, 40 μM, and 80 μM are the drug-treated groups). Fig.15 B is the corresponding quantitative data analysis diagram (0 is the blank group, 20 μM, 40 μM, and 80 μM are the drug-added groups respectively). DETAILED DESCRIPTION

[0051] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0052] The present application discloses a sesquiterpenoid compound from marine fungi. The compound is an enantio-eudesane-type sesquiterpenoid compound. The sesquiterpenoid compound is isolated from the secondary metabolites of the marine fungus Eutypella sp. F0219. The marine fungus Eutypella sp. F0219 was deposited in the China Type Culture Collection at Wuhan University in Wuhan, Hubei Province on July 3, 2023, and the collection center gave the strain a collection number of CCTCC NO: M20231180.

[0053] Source of strains

[0054] The strain (Eutypella sp.) F0219 provided in this application was isolated from seabed sediments at a depth of 75m in the northern part of the South China Sea (114.6609°E, 21.5942°N). The strain was identified as a strain of the genus Eutypella by morphology and molecular biology. The strain was sent to a sequencing company for identification (testing company: Shanghai Sangon Biotechnology Co., Ltd.), and 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 was Eutypella sp., with a sequence similarity of 99%. It was deposited in the China Type Culture Collection at Wuhan University in Wuhan, Hubei Province on July 3, 2023. The collection center gave the strain a collection number of CCTCC NO: M20231180.

[0055] Enantio-eucalyptane-type sesquiterpenoids

[0056] The present application example also found that the Eutypella sp. F0219 strain can ferment and produce an enantioeuline-type sesquiterpenoid compound. The specific steps are as follows:

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

[0058] (1) Preservation of strains: Eutypella sp. F0219 strains were preserved in PDB-glycerol medium at -80°C, wherein the PDB-glycerol medium contained 200 g / L potato, 20 g / L glucose and 30 wt% glycerol.

[0059] (2) Activation of strains: The strain Eutypella sp. F0219 stored at -80°C was taken out, placed in a 28°C incubator for pre-culture for 24 h, and inoculated onto a PDA plate for activation culture at 28°C for 3 days.

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

[0061] 2. Separation and purification of compounds

[0062] (1) Extraction: The mycelium obtained by fermentation was soaked and extracted three times with petroleum ether and ethyl acetate respectively, 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, and about 1.0 L of fractions were collected each time. Similar fractions were combined by TLC detection and separated into 5 sub-fractions, namely Fr.1-Fr.5. Fraction Fr.3 was purified by reverse phase C18 column chromatography (MeOH:H 2 0:30:70-100:0) was used for gradient elution to obtain 73 subfractions Fr.3.1-Fr.3.73. Among them, component Fr.3.19 was subjected to gel 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 subjected to semi-preparative high performance liquid chromatography (CH 3 CN:H 2 O=45:55) was further separated and purified to obtain the compound I (13.6 mg), the structural formula of which is as follows shown.

[0064] In addition, the compound I obtained by the semi-preparative HPLC was analyzed using an MCP5100 polarimeter (Anton Paar GmbH, Austria, measuring temperature was 25 degrees Celsius, and the length of the polarimeter tube was 100 mm). The compound I is -352.9 (c0.14, MeOH). The academic name of the compound I is (4aS, 6S, 8aS)-4a-hydroxy-6-(2-hydroxypropane-2-yl)-8a-methyl-4-methyleneoctahydroxynaphthalene-1(2H)-one. This indicates that the compound I obtained by fermentation of Eutypellasp.F0219 provided in the present application is an optically pure chiral compound, which plays an important role in inhibiting angiogenesis.

[0065] Structural identification of compound I

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

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

[0068] Table 1 NMR data of compound I in deuterated methanol (400MHz / 125MHz, δinppm, JinHz)

[0069]

[0070] a Overlapped signals assigned by HSQC andHMBC spectra without designing multiplicity.

[0071] According to HRESIMS [M+HH 2 O] + m / z 235.1696, C 15 H 23 O 2 The calculated value is 235.1698, and the molecular formula of the compound is determined to be C 15 H 24 O 3 , the unsaturation is 4. 1 H-NMR and HSQC spectra showed that the compound had two exocyclic double bond hydrogen signals [δ H 5.00 (m, H-15), 5.07 (m, H-15)]; 3 methyl signals [δ H 1.19(s,H 3 -12),1.19(s,H 3 -13),1.05(s,H 3 -14)]; 1 methine hydrogen signal [δH 1.80 (m, H-7)]; 10 methylene hydrogen signals [δ H 2.70(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] Test of the angiogenesis inhibition activity 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 placed at 37°C and 5% CO 2 Culture overnight until adherence. The culture medium was removed and the cells were divided into a drug-added group and a blank group. In the drug-added group, 12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM, and 400 μM of culture medium containing compound I were added to each well, and the blank group was added with the same volume of culture medium without drugs. After culturing for another 48 hours, 10 μL of CCK-8 reagent was added. After incubation for another 2 hours, the absorbance was detected at 450 nm using an ELISA reader (Bio-Rad, Hercules, CA, USA). Graphpad Prism was used to process and analyze the results, draw the cell proliferation curve, and calculate the drug concentration at 50% inhibition rate, i.e., IC 50 The experiment was repeated 3 times.

[0075] like Fig.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 4 HEMC-1 cells, 37°C, 5% CO 2 Culture until the adhesion reaches 90% to 100% and a monolayer of cell membrane is formed at the bottom of each well. Discard the cell solution, wash twice with PBS buffer, add serum-free medium, and continue incubation for 12 hours; use the shoulder of a 200μL pipette to take a direction perpendicular to the long axis of the plate along the ruler, and gently slide the line to form a scratch at the bottom of the well. After washing the dead cells, add fresh medium containing 20μM, 40μM, 80μM compound I or no compound I to the cells, and place them in a cell culture incubator for 8 hours. Use an inverted microscope to take the same field of view at 0 and 8h, and use IPP6 software to count the migrating cells.

[0078] like Fig.12 A and Fig.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 Fig.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 4×10 5 ~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 per group, at 37°C and 5% CO 2 After 6 h of culture, the formation of tubular structures of endothelial cells was observed under a microscope and photographed, and the images were analyzed using Image-Pro Plus 6 (IPP6).

[0084] like Fig.14 A and Fig.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 Fig.15 A and Fig.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 a compound according to claim 1 or a salt thereof, and a pharmaceutically acceptable excipient, especially a pharmaceutically acceptable salt, especially in a unit dose.

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: Activate the Eutypella sp.F0219 strain; The Eutypella sp. F0219 colony was cultured in liquid to obtain seed solution; The seed liquid is expanded and cultured to obtain a fermentation liquid; Collecting mycelium of Eutypella sp.F0219 from the fermentation broth; The compound according to claim 1 is extracted from the mycelium.

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.

8. The preparation method according to claim 6, wherein the Eutypella sp. F0219 strain is activated using a PDA plate; The seed solution is obtained by culturing with a potato liquid culture medium, wherein the potato liquid culture medium contains 200 g / L potato and 20 g / L glucose. The fermentation liquid is obtained by expanding the culture with rice solid culture medium, wherein the rice solid culture medium contains 80 g / L rice and 0.23 wt % sea salt.

9. The preparation method according to claim 6, wherein the step of extracting the compound according to claim 1 from the mycelium comprises: The mycelium obtained by fermentation was soaked and extracted with petroleum ether and ethyl acetate respectively, and the ethyl acetate extracts were combined and concentrated under reduced pressure to obtain a crude ethyl acetate extract.

10. The preparation method according to claim 6, wherein the step of extracting the compound according to claim 1 from the mycelium comprises: 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 is separated by comprehensive use of ODS column chromatography, Sephadex LH-20 column chromatography and HPLC preparation, and its structure is identified to obtain the compound as claimed in claim 1.

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