A stilbene compound, a preparation method and application thereof

Abstract

The application discloses a stilbene compound, a preparation method and application thereof, and the stilbene compound is a compound as shown in formula I. The stilbene compound has a better VEGF inhibiting effect, and the effect is better than that of natural stilbene compound resveratrol. The preparation method has reasonable route design, the starting material is easy to obtain, the reagent is low in price, and post-treatment is simple, so that the production cost can be effectively reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a stilbene compound, a preparation method and application thereof. BACKGROUND

[0002] Angiogenesis refers to the development of new blood vessels from existing capillaries or post-capillary venules, which is a natural physiological phenomenon in human body and plays an important role in embryonic development and tissue repair. Under normal physiological conditions, angiogenesis-inhibiting molecules and angiogenesis-promoting molecules are in a relative balance, but under pathological conditions, angiogenesis will be out of control and grow abnormally. Pathological angiogenesis has been confirmed to be closely related to the occurrence and development of various diseases such as tumors, ocular neovascularization, arthritis, skin diseases, atherosclerosis, etc. In addition, the rapid growth and metastasis of tumors are also considered to depend on angiogenesis. Therefore, the research on anti-angiogenic drugs is of great significance for exploring the treatment methods of the above diseases.

[0003] Anti-angiogenesis is a very complex process, which includes intervention on angiogenesis regulatory factors, inhibition of degradation of vascular basement membrane and extracellular matrix, inhibition of endothelial cell division, migration and proliferation, etc. Vascular endothelial growth factor (VEGF) is considered as the most critical pro-angiogenic factor, and anti-angiogenic and anti-tumor drugs targeting VEGF and its receptor VEGFR have been the research focus in recent years.

[0004] Therefore, the development of new drugs with VEGF inhibitory activity is of great significance for improving or enriching the prevention and treatment of diseases or conditions related to VEGF. SUMMARY

[0005] The present application aims at at least solving one of the above technical problems existing in the prior art. To this end, the purpose of the present application is to provide a stilbene compound, a preparation method and application thereof.

[0006] In order to achieve the above-mentioned purpose, the technical solution adopted by the present application is as follows:

[0007] In a first aspect, the present application provides a compound of formula I, or a stereoisomer thereof, a pharmaceutically acceptable salt thereof:

[0008]

[0009] In a second aspect, the present application provides a preparation method of the compound of formula I, comprising the following steps:

[0010] The compound of formula I is prepared by substitution reaction of 4-[(E)-2-(3,5-dimethoxyphenyl)vinyl]phenol with 1,5-dihalo-3-pentanone.

[0011] In some embodiments of the present invention, the 1,5-dihalo-3-pentanone includes any one of 1,5-dichloro-3-pentanone, 1,5-dibromo-3-pentanone, and 1,5-diiodo-3-pentanone.

[0012] In some embodiments of the present invention, the substitution reaction is carried out in a strongly alkaline environment, such as any one of sodium hydride, sodium amide, metal alkoxide (e.g., sodium ethoxide, potassium tert-butoxide), lithium aluminum hydride, sodium borohydride, n-butyllithium, and lithium diisopropylamide.

[0013] In some embodiments of the present invention, the substitution reaction is carried out at 0°C to 20°C.

[0014] A third aspect of the invention provides a pharmaceutical composition comprising the compound of formula I or a stereoisomer thereof, a pharmaceutically acceptable salt thereof, and optionally, a pharmaceutically acceptable excipient and / or carrier.

[0015] In some embodiments of the present invention, the pharmaceutical composition is a vascular endothelial growth factor inhibitor.

[0016] A fourth aspect of the invention provides the use of the compound of Formula I or a stereoisomer thereof, a pharmaceutically acceptable salt thereof, and / or the pharmaceutical composition thereof in the preparation of a medicament.

[0017] In some embodiments of the present invention, the medicament is used to treat the following diseases or conditions: ocular neovascularization, psoriasis, hemangioblastoma, glomerular membranous cell proliferation, atherosclerosis, arterial restenosis, autoimmune diseases, acute inflammation, fibrotic diseases, diabetes, endometriosis, chronic asthma, arterial or post-transplant atherosclerosis, neurodegenerative diseases, lymphoma, leukemia, such as acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, and solid tumors;

[0018] The ocular neovascularization-induced disease is preferably angiogenic ophthalmopathy, which is selected from age-related macular degeneration, diabetic retinopathy, diabetic macular edema, central retinal vein occlusion, and corneal neovascularization.

[0019] The glomerular cell proliferation disease is preferably selected from diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy syndrome or transplant rejection, glomerulonephritis, hemolytic uremic syndrome, diabetic nephropathy and hypertensive nephrosclerosis;

[0020] The solid tumor is a primary or secondary solid tumor, selected from breast cancer, colon cancer, lung cancer (e.g., small cell lung cancer), prostate cancer, myeloma, and Kaposi's sarcoma.

[0021] In some embodiments of the present invention, the medicament is used to treat the following diseases or conditions: diseases caused by ocular neovascularization, such as angiogenic ophthalmopathy; said angiogenic ophthalmopathy is selected from age-related macular degeneration, diabetic retinopathy, diabetic macular edema, central retinal vein occlusion, and corneal neovascularization, such as retinopathy such as diabetic retinopathy or age-related macular degeneration; psoriasis, angioblastoma such as hemangioma (benign vascular proliferation), glomerular membranous cell proliferative diseases such as chronic or acute kidney disease, such as diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy syndrome or transplant rejection, or such as inflammatory kidney disease such as glomerulonephritis, such as membranous proliferative glomerulonephritis, hemolytic uremic syndrome. Diabetic nephropathy, hypertensive nephrosclerosis, atherosclerosis, arterial restenosis, autoimmune diseases (such as arthritis like rheumatoid arthritis), acute inflammation, fibrotic diseases (such as cirrhosis), diabetes, endometriosis, chronic asthma, arterial or post-transplant atherosclerosis, neurodegenerative diseases, lymphoma, and neoplastic diseases such as leukemia, such as acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, and other "liquid tumors," as well as solid tumors (such as cancer), such as breast cancer (such as Paget's disease), colon cancer, lung cancer (such as small cell lung cancer), prostate cancer, myeloma, or Kaposi's sarcoma, for example, the growth or metastatic spread of the above tumors and the growth of micrometastases.

[0022] In some embodiments of the present invention, the drug is used to treat the following diseases or conditions:

[0023] (1) Tumor; further, the tumor is a solid tumor;

[0024] (2) Leukemia;

[0025] (3) Psoriasis;

[0026] (4) Paget's disease;

[0027] (5) Benign angiogenesis;

[0028] (6) Arthritis;

[0029] (7) Atherosclerosis;

[0030] (8) Neovascular eye disease, further, the neovascular eye disease is a primary or secondary neovascular eye disease.

[0031] When administered as a medicine, the compounds of the present invention can be given in the form of pharmaceutical compositions. These compositions can be prepared in ways well known in the pharmaceutical field and can be administered via a variety of routes, depending on whether local or systemic treatment is required and the area to be treated. Administration can be topical (e.g., transdermal, skin, eye, and mucous membrane delivery, including intranasal, vaginal, and rectal delivery), pulmonary (e.g., by inhalation or blowing of powders or aerosols, including via nebulizers; intratracheal, intranasal), oral, or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial, such as intrathecal or intraventricular administration. Parenteral administration can be in the form of a single large dose or via, for example, a continuous infusion pump. Topically administered pharmaceutical compositions and formulations may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powder formulations, and powders. Conventional drug carriers, water, powder or oily bases, thickeners, etc., may be necessary or required.

[0032] In preparing the compositions of the present invention, the active ingredient is typically mixed with an excipient, diluted by the excipient, or contained in a carrier such as a capsule, pouch, paper, or other container. When the excipient is used as a diluent, it can be a solid, semi-solid, or liquid substance, serving as a solvent, carrier, or medium for the active ingredient. Therefore, the compositions can be in the following forms: tablets, pills, powders, lozenges, pouches, capsules, flat capsules, microcapsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols (solid or soluble in liquid solvents); ointments containing, for example, up to 10% by weight of the active compound; soft and hard gelatin capsules; suppositories; sterile injectable solutions; and sterile packaged powders.

[0033] Some examples of suitable excipients include lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, tragacanth gum, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. Formulations may also contain: lubricants such as talc, sodium stearate, magnesium stearate, sodium oleate, sodium benzoate, sodium acetate, sodium chloride, and mineral oil; humectants; emulsifiers and suspending agents; preservatives such as methyl benzoate and hydroxypropyl benzoate; sweeteners and flavoring agents. The compositions of the present invention can be formulated using methods known in the art to provide immediate, sustained, or delayed release of the active ingredient upon administration to a patient.

[0034] Compositions can be formulated in unit dosage forms, each containing approximately 5–1000 mg, more typically approximately 100–500 mg (e.g., 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg) of active ingredient. The term "unit dosage form" refers to a physically isolated single-dose unit suitable for use in human patients and other mammals, each unit containing a predetermined amount of active substance, calculated to produce the desired therapeutic effect, when mixed with suitable pharmaceutical excipients.

[0035] The effective dose range of an active compound can be quite wide, and it is usually administered at the pharmaceutically effective dose. However, it is understood that the actual amount of compound administered is usually determined by the physician based on relevant circumstances, including the condition being treated, the route of administration chosen, the actual compound administered, the patient's age, weight, and response, and the severity of the patient's symptoms.

[0036] For the preparation of solid compositions, such as tablets, the main active ingredient is mixed with a pharmaceutical excipient to form a solid preform composition containing a homogeneous mixture of the compounds of the present invention. When these preform compositions are referred to as homogeneous, it means that the active ingredient is generally uniformly distributed throughout the composition, such that the composition can be readily divided into equivalent effective unit dosage forms, such as tablets, pills, and capsules. The solid preform is then divided into unit dosage forms of the above-described type containing, for example, about 0.1 to 1000 mg (e.g., 1 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg) of the active ingredient of the present invention.

[0037] The tablets or pills of this invention can be coated or compounded to obtain dosage forms that provide the advantage of long-lasting action. For example, the tablets or pills contain an internal dose and an external dose component, the latter being a coated form of the former. The two components can be separated by an enteric coating layer, which is used to prevent disintegration in the stomach, allowing the internal component to pass through the duodenum intact or to delay release. A variety of substances can be used for such enteric coatings or coatings, including a variety of high molecular weight acids and mixtures of high molecular weight acids with such substances such as shellac, cetyl alcohol, and cellulose acetate.

[0038] The compounds and compositions of the present invention may be incorporated into liquid forms for oral or injectable administration, including aqueous solutions, suitably flavored syrups, water or oil suspensions; and emulsions flavored with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil; as well as elixirs and similar pharmaceutical solvents.

[0039] Compositions for inhalation or inhalation include solutions and suspensions, and powders, dissolved in pharmaceutically acceptable water or organic solvents or mixtures thereof. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, the composition is administered orally or via nasal inhalation to achieve local or systemic effects. The composition can be nebulized using an inert gas. The nebulized solution can be inhaled directly from a nebulizer, or the nebulizer can be connected to a face mask or intermittent positive pressure ventilation machine. Solutions, suspensions, or powder compositions can be administered orally or nasally by a device that delivers the formulation in a suitable manner.

[0040] The amount of compound or composition given to a patient is not fixed and depends on the drug being administered, the purpose of administration (e.g., prevention or treatment), the patient's condition, the method of administration, etc. In therapeutic applications, a sufficient amount of the composition may be given to a patient with an existing disease to cure or at least partially suppress the symptoms of the disease and its complications. The effective dose should depend on the disease state being treated and the judgment of the attending clinician, which depends on factors such as the severity of the disease, the patient's age, weight, and general condition.

[0041] The compositions administered to patients may be in the form of the pharmaceutical compositions described above. These compositions may be sterilized using conventional sterilization techniques or filterable sterilization. The aqueous solutions may be used as is, or lyophilized; prior to administration, the lyophilized formulation may be mixed with a sterile aqueous carrier. The pH of the compound formulation is typically 3–11, more preferably 5–9, and most preferably 7–8. It is understood that the use of certain of the aforementioned excipients, carriers, or stabilizers may result in the formation of drug salts.

[0042] The therapeutic dose of the compounds of this invention may be determined based on, for example, the specific therapeutic use, the manner of administration of the compound, the patient's health and condition, and the prescribing physician's judgment. The proportion or concentration of the compounds of this invention in the pharmaceutical composition may not be fixed and depends on a variety of factors, including dosage, chemical properties (e.g., hydrophobicity), and route of administration. For example, the compounds of this invention may be provided in a physiologically buffered aqueous solution containing about 0.1 to 10% (w / v) of the compound for parenteral administration. Some typical dosage ranges are from about 1 μg / kg to about 1 g / kg body weight / day. In some embodiments, the dosage range is from about 0.01 mg / kg to about 100 mg / kg body weight / day. The dosage is likely to depend on variables such as the type and severity of the disease or condition, the general health status of the specific patient, the relative biological potency of the selected compound, the excipient formulation, and the route of administration. The effective dose can be obtained by extrapolation from dose-response curves derived from in vitro or animal model testing systems.

[0043] According to embodiments of the present invention, the compound of Formula I or its stereoisomers, pharmaceutically acceptable salts thereof, and pharmaceutical drugs or pharmaceutical compositions thereof exert preventive or therapeutic effects on the diseases and conditions by inhibiting the activity of vascular endothelial growth factor.

[0044] The term "stereoisomer" refers to a compound composed of identical atoms bonded by the same bonds, but with different three-dimensional structures. This invention will cover various stereoisomers and mixtures thereof.

[0045] When the compounds of the present invention contain alkene double bonds, unless otherwise stated, the compounds of the present invention are intended to contain E- and Z-geometric isomers.

[0046] The term "tautomer" refers to an isomer formed when a proton is transferred from one atom of a molecule to another atom of the same molecule. All tautomer forms of the compounds of this invention are also included within the scope of this invention.

[0047] The compounds of the present invention, or pharmaceutically acceptable salts thereof, may contain one or more chiral carbon atoms, and thus may produce enantiomers, diastereomers, and other stereoisomers. Each chiral carbon atom may be defined as (R)- or (S)- based on stereochemistry. The present invention aims to include all possible isomers, as well as their racemic and optically pure forms. The preparation of the compounds of the present invention may be carried out using racemic, diastereomer, or enantiomer as a starting material or intermediate. Optically active isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as crystallization and chiral chromatography. Conventional techniques for the preparation / separation of individual isomers include chiral synthesis from suitable optically pure precursors, or resolution of racemic mixtures (or racemic mixtures of salts or derivatives) using, for example, chiral high-performance liquid chromatography.

[0048] The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0049] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the bioavailability of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobroms, sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetate, trifluoroacetate, propionates, hexanoates, octanoates, decanoates, undecenoates, glycolates, gluconates, lactates, sebates, adipates, glutarate, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamates, pyroglutamates, aspartate, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginates, ascorbic acid salts, salicylates, 4-aminosalicylic acid salts, and naphthalene disulfonates. These salts can be prepared using methods known in this field.

[0050] "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that retain the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, the following: primary amines, secondary amines, and tertiary amines; substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0051] The beneficial effects of this invention are:

[0052] The stilbene compounds in this application have superior VEGF inhibition effects, which are better than those of the natural stilbene compound resveratrol.

[0053] The preparation method of this application is reasonably designed, the starting materials are readily available, the reagents are inexpensive, and the post-processing is simple, which can effectively reduce production costs. Attached Figure Description

[0054] Figure 1 The image shows the 1H NMR spectrum of compound RE-16 obtained in Example 1 of this application.

[0055] Figure 2 The image shows the HPLC chromatogram of compound RE-16 obtained in Example 1 of this application.

[0056] Figure 3 This is a comparison of the spectra of compound RE-16, prepared in Example 1 of this application, hitting the VEGF protein and VEGFR-1 receptor binding domain in molecular docking software.

[0057] Figure 4 The binding pattern of compound RE-16, prepared in Example 1 of this application, to VEGF protein in a surface plasmon resonance (SPR) system.

[0058] Figure 5 This is a quantitative comparison diagram showing the inhibitory activity of compound RE-16, prepared in Example 1 of this application, on vascular endothelial cells in inhibiting VEGF protein-induced vascular cell growth.

[0059] Figure 6 A quantitative comparison diagram of how the compound RE-16, prepared in Example 1 of this application, inhibits HaCaT cell scratch migration.

[0060] Figure 7 This is a quantitative comparison diagram of the potential cytotoxicity of compound RE-16 from Example 1 of this application in the MTT assay of HaCaT cells. Detailed Implementation

[0061] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0062] Unless otherwise specified, the temperature parameters in this invention can be either constant temperature treatment or variations within a certain temperature range. It should be understood that the constant temperature treatment allows temperature fluctuations within the precision range controlled by the instrument. Fluctuations are permitted within ranges such as ±5℃, ±4℃, ±3℃, ±2℃, and ±1℃.

[0063] Example 1

[0064] This embodiment prepared the stilbene compound RE-16, and the specific process is as follows:

[0065]

[0066] 4-[(E)-2-(3,5-dimethoxyphenyl)vinyl]phenol (1.0 g, 3.90 mmol) and 1,5-dichloro-3-pentanone (0.30 g, 1.95 mmol) were reacted with sodium hydride (0.094 g, 3.90 mmol) at 0 °C to 20 °C for 4 hours to prepare stilbene compound RE-16 (8 mg).

[0067] The obtained product RE-16 was subjected to NMR analysis, and the resulting proton NMR spectrum is as follows: Figure 1 .

[0068] The RE-16 proton NMR data are as follows: 1 H NMR: (300MHz, CD3Cl) δ2.90(4H,t,J=4.8Hz), 3.82(12H,s), 4.24(4H,t,J=4.8Hz), 6.37(2H, t,J=1.5Hz),6.46(4H,d,J=1.5Hz),6.81-6.83(6H,m),6.91-6.92(2H,m),7.38-7.44(4H,m).

[0069] The obtained product was analyzed by HPLC, and the purity was above 97%. The test results are as follows: Figure 2 The detection method is as follows: 4 mL of 50% methanol was added to the sample, and the mixture was sonicated for 15 minutes. After centrifugation at 1000 x g for 5 minutes, the supernatant was obtained. Before injection, the supernatant was filtered through a 0.45 μm microporous membrane. The filtrate was then injected for analysis. The analytical instrument used was an Agilent liquid chromatograph equipped with an autosampler and a binary pump. The chromatographic column was an Agilent Grace VisionHT C18 (4.6 x 250 mm, 5 μm). The mobile phase was acetonitrile (solvent A) and 0.2% formic acid aqueous solution (solvent B). The flow rate was 1 mL / min, the column temperature was room temperature, and gradient elution was used. The mobile phase ratios were as follows: 0–60 min, 10–35% solvent A; 60–96 min, 35–100% solvent A. The injection volume was 20 μL, and the detection wavelength was 254 nm.

[0070] Example 2 Molecular docking experiment

[0071] 2.1. Experimental Principle

[0072] VEGF (vascular endothelial growth factor) is one of the important proteins in vascular endothelium. Under normal circumstances, VEGF binds to the VEGFR-1 receptor and provides the material basis for subsequent pathways. The binding domain of the VEGF protein to the VEGFR-1 receptor is amino acid fragment 1-165. Theoretically, if a chemical component can strike this binding domain, it can competitively bind to VEGFR, thereby effectively inhibiting the binding of VEGF to the VEGFR receptor, thus affecting subsequent pathways and ultimately inhibiting angiogenesis. This binding domain has been identified as the target of this molecular docking study.

[0073] 2.2. Experimental Procedures and Results

[0074] Using SEESAR (version 13.1) software, the binding domain (amino acid fragments 1-165) of the VEGFR-1 receptor in the VEGF protein (PDB code: 1FLT) was selected as the target, and the affinity of compound RE-16 for this target was then detected. The docking software SEESAR can analyze the target potency; the lower the potency, the more stable the binding of the chemical molecule to the target.

[0075] The results are as follows Figure 3 The results show that RE-16 can effectively bind to the VEGF target ( Figure 3 The target efficacy was predicted to be -27.4 kJ / mol. This indicates that compound RE-16 can effectively target the VEGF protein and prevent it from binding to the VEGFR-1 receptor, thus more effectively inhibiting VEGF and consequently inhibiting angiogenesis.

[0076] Example 3 Protein Binding Experiment

[0077] 3.1 Experimental Principle

[0078] Surface plasmon resonance (SPR) is an optical, label-free detection technique used to detect the bonding between two or more molecules in real time. The principle of SPR is based on total internal reflection, where light intensity decreases as it is reflected at a specific angle from the sensor's glass surface. As molecules bond, the surface refractive index changes, causing a change in the angle of minimum reflection intensity. This change in angle is proportional to the mass of the bonded material. The results can be represented by a sensing graph recording the angle as a function of time.

[0079] 3.2 Experimental Procedure

[0080] The binding of the sample to the VEGF protein was performed on a Biacore S200 instrument equipped with a GE Series dextran-coated (CM5) sensor chip. The reaction temperature was set at 25°C, and HBS-T (150 mM sodium chloride, 10 mM HCl, 0.05% polysorbate 20, 3.4 mM EDTA, pH 7.4) was selected as the run buffer. The chip sensor surface used for capture was prepared by covalently immobilizing VEGF to the chip surface according to the EDC / NHS {1-ethyl-3-[3-(dimethylamino)propyl]carboxyhydrazine hydrochloride / N-hydroxysuccinimide} coupling chemistry method. After surface activation, VEGF protein dissolved in the coupling buffer (0.1 M acetate buffer, pH 4.5) was placed on the activated surface of the chip until the detected VEGF protein RU (resonance unit) signal reached approximately 6500 RU. To remove uncoupled proteins, the chip with activated coupling surfaces was washed and reacted with 10 mM glycine hydrochloride at a pH of 1.5. Samples of different concentrations were diluted with run buffer (stepwise 2-fold dilutions from 20 μM to 0.3125 μM) and flowed through the VEGF protein-coupled chip surface.

[0081] 3.3 Experimental Results

[0082] As shown in Table 1, Figure 4 As shown, RE-16 can effectively bind to VEGF, with a maximum resonance unit (RU) of 45 and a binding constant (K). d The concentration was 2.17 μM. The results indicate that RE-16 significantly binds to VEGF, effectively inhibiting VEGF and thus more effectively suppressing VEGF-related biochemical functions such as angiogenesis.

[0083] Table 1

[0084] Detection substance Maximum resonance units RU (concentration 20 μM) Binding constant K d (μM) RE-16 45 2.17

[0085] Example 4: Vascular Endothelial Cell Viability Test

[0086] 4.1 Experimental Methods

[0087] 5×10 3One well of vascular endothelial cells (HUVECs) was seeded in 100 μL of culture medium in a sterile 96-well plate. After 24 hours of incubation, the culture medium in each well was replaced with 100 μL of fresh culture medium containing 10 ng / mL VEGF or a series of concentrations of RE-16 (1 μM, 3 μM, 10 μM). Forty-eight hours after drug treatment, 10 μL of MTT solution at a concentration of 5 mg / mL was added per well. After incubation at 37°C for 4 hours, 150 μL of 100% dimethyl sulfoxide (DMSO) was added to each well to dissolve the formalazole salt, followed by aspiration of the culture medium. The color intensity of the formalazole solution was read using a microwell spectrophotometer at a wavelength of 570 nm. The release of lactate dehydrogenase (LDH) in the mixture was detected using a Cytotoxicity Detection Kit PLUS (LDH) (Roche Diagnostics, Indianapolis, IN). The LDH content of each group was quantified according to the following formula: Cell viability (%) = (Experimental value - Low control) / (High control - Low control) × 100%.

[0088] 4.2 Experimental Results

[0089] like Figure 5 As shown, the addition of VEGF protein significantly enhanced the biological activity of vascular endothelial cells, while the addition of the positive controls Avastin and Resveratrol weakened cell activity. Notably, similar to the positive control, RE-16 significantly reduced VEGF-induced biological activity. This indicates that both can inhibit VEGF-related biochemical activities by inhibiting VEGF protein.

[0090] Example 5 Cell Scratch Assay

[0091] 5.1. Methods

[0092] HaCaT cells at 20×10 4 Cells were seeded at a density of 10 cells / well in 12-well plates and cultured overnight to allow cell adhesion. Once cells reached 80% confluence, a transverse incision was made in the center of each well using a 200 μL pipette tip. The culture medium was discarded, and the cells were washed once with PBS. Human vascular endothelial growth factor (VEGF) at 10 ng / mL was added to the culture medium, and 200 μg / mL of avastin and different concentrations of RE-16 (0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM) were administered. A blank control group (no drug administration but given an equal volume of solvent) and a control group (no drug administration) were also included. Cell coverage changes in each well were photographed at 50x magnification using a microscope equipped with a camera at 0 and 24 hours. The cell recovery rate was then quantified using TScratch software and calculated using the following formula: Recovery rate % = (At0 - At...)24 ) / At0×100%, where At0: the scratch area measured 0 hours after drug administration; At 24 The scratch area measured 24 hours after drug administration.

[0093] 5.2. Results and Conclusions

[0094] Figure 6 The graph shows the quantitative effect of positive control (A), resveratrol (RES), and RE-16 on the inhibition of HaCaT cell scratch migration. The percentage of change in cell scratch migration in the graph represents the degree of wound healing (%).

[0095] according to Figure 6 It can be seen that compound RE-16 can effectively inhibit the proliferation of HaCaT cells induced by VEGF protein and wound healing, and its effect is better than that of the positive control.

[0096] Example 6 Cytotoxicity Experiment

[0097] 6.1. Methods

[0098] The cytotoxic effect of RE-16 on human HaCaT cells was observed using the MTT assay. HaCaT cells were cultured at a concentration of 5.0 × 10⁻⁶. 3 Cells were seeded at a density of 100 μL / mL in 96-well plates. Once cells reached 80% confluence, different concentrations of RE-16 (1 μM, 10 μM, 20 μM, 50 μM, 100 μM) were added to each well. After 48 hours of incubation, 10 μL of MTT solution was added to each well, and the plates were incubated at 37°C for another 4 hours. The culture was then terminated, the supernatant was discarded, and 150 μL of LDMSO was added to each well. The plates were shaken for 10 min to dissolve the cells completely. The absorbance (OD) of each well was measured at 490 nm using a microplate reader. Wells without the drug were used as controls. Cell viability was calculated using the following formula: Cell viability (%) = (OD sample - OD control) / OD control × 100%.

[0099] 6.2. Results and Conclusions

[0100] according to Figure 7 It can be seen that RE-16 has no significant effect on cell viability, indicating that the sample has no obvious cytotoxicity.

[0101] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A compound of formula I, or a pharmaceutically acceptable salt thereof: 。 2. A method for preparing the compound of formula I according to claim 1, characterized in that: The method includes the following steps: reacting 4-[(E)-2-(3,5-dimethoxyphenyl)vinyl]phenol with 1,5-dihalo-3-pentanone to obtain the compound of formula I.

3. The method for preparing the compound of formula I according to claim 2, characterized in that: The 1,5-dihalo-3-pentanone includes any one of 1,5-dichloro-3-pentanone, 1,5-dibromo-3-pentanone, and 1,5-diiodo-3-pentanone.

4. The method for preparing the compound of formula I according to claim 2, characterized in that: The substitution reaction is carried out in a strongly alkaline environment; and / or, the substitution reaction is carried out at 0°C to 20°C.

5. A pharmaceutical composition, characterized in that: It includes the compound of formula I as claimed in claim 1 or a pharmaceutically acceptable salt thereof, and optionally, pharmaceutically acceptable excipients and / or carriers.

6. The pharmaceutical composition according to claim 5, characterized in that: The dosage forms of the pharmaceutical compositions include tablets, pills, powders, lozenges, capsules, flat capsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

7. The pharmaceutical composition according to claim 5, characterized in that: The pharmaceutical composition is a vascular endothelial growth factor inhibitor.

8. The use of a compound of formula I according to claim 1 or a pharmaceutically acceptable salt thereof, and / or the use of a pharmaceutical composition according to any one of claims 5 to 7 in the preparation of a medicament; said medicament being used to treat the following diseases or conditions: diseases caused by ocular neovascularization, psoriasis, hemangioblastoma, glomerular membranous cell proliferation disease, atherosclerosis, arterial restenosis, autoimmune diseases, acute inflammation, fibrotic diseases, diabetes, endometriosis, chronic asthma, arterial or post-transplant atherosclerosis, neurodegenerative diseases, lymphoma, and leukemia.

9. The application according to claim 8, characterized in that: The leukemias mentioned include acute lymphoblastic leukemia, acute myeloid leukemia, and chronic myeloid leukemia.

10. The use of a compound of formula I according to claim 1 or a pharmaceutically acceptable salt thereof, and / or the use of a pharmaceutical composition according to any one of claims 5 to 7 in the preparation of a medicament; said medicament being used to treat the following diseases or conditions: age-related macular degeneration, diabetic retinopathy, diabetic macular edema, central retinal vein occlusion and corneal angiogenesis; diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy syndrome or transplant rejection, glomerulonephritis, hemolytic uremic syndrome and hypertensive nephrosclerosis; breast cancer, colon cancer, lung cancer, prostate cancer, myeloma and Kaposi's sarcoma.

11. The application according to claim 10, characterized in that: The lung cancers mentioned include small cell lung cancer.

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