Vascular endothelial growth factor inhibitor as well as preparation method and application thereof

Abstract

The invention discloses a vascular endothelial growth factor inhibitor as well as a preparation method and application thereof. The vascular endothelial growth factor inhibitor comprises a compound # imgabs0 # as shown in a formula I. A stilbene compound disclosed by the invention has a better VEGF (vascular endothelial growth factor) inhibition effect, and the effect of the stilbene compound is superior to that of a natural stilbene compound resveratrol. The preparation method of the stilbene compound is reasonable in route design, the starting raw materials are easy to obtain, the reagent price is low, aftertreatment is simple, and the production cost can be effectively reduced.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a vascular endothelial growth factor inhibitor and a preparation method and application thereof. Background Art

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

[0003] Anti-angiogenesis is an extremely complex process, including intervention in angiogenesis regulatory factors, inhibition of vascular basement membrane and extracellular matrix degradation, inhibition of endothelial cell division, migration and proliferation, etc. Vascular endothelial growth factor (VEGF) is regarded as the most critical angiogenesis factor, and anti-angiogenesis and anti-tumor drugs targeting VEGF and its receptor VEGFR have been a hot topic in recent years.

[0004] Bevacizumab (avilimus) is the first monoclonal antibody that targets the vascular endothelial growth factor (VEGF) protein and prevents the binding of kinases to its receptors. However, it has been reported that bevacizumab may cause cardiotoxic reactions, and patients are advised to stop treatment when this side effect occurs. As for small molecule inhibitors, pazopanib and sunitinib are antagonists against multiple VEGF receptors (such as VEGFR-1 and VEGFR-2), while sorafenib has been confirmed as an inhibitor of multiple kinases such as CRAF, BRAF, VEGF, VEGFR-2, etc. However, after the use of these three drugs, patients are at risk of arrhythmias, heart failure, and myocardial ischemia. In short, the adverse reactions of the treatment options currently on the market indicate that it is still urgent to develop more VEGF-targeted therapies with higher efficacy and lower toxicity. Summary of the invention

[0005] The present invention aims to solve at least one of the above technical problems existing in the prior art. To this end, the present invention aims to provide a vascular endothelial growth factor inhibitor and a preparation method and application thereof.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

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

[0008]

[0009] The second aspect of the present invention provides a method for preparing the compound of formula I, comprising the following steps:

[0010] 4,4'-(1,5-pentanediyl)dioxybenzaldehyde and a compound of formula II Reaction is carried out, followed by deprotection to obtain a compound of formula I;

[0011] Wherein, R1 and R2 are independently selected from hydroxyl protecting groups.

[0012] In some embodiments of the present invention, the reaction is a Horner-Wadsworth-Emmons reaction.

[0013] In some embodiments of the present invention, the Horner-Wadsworth-Emmons reaction is carried out at -78°C to 10°C (such as -20°C to 5°C, -10°C to 3°C, 0°C, etc.) for 3 to 8 hours (such as 4 to 6 hours).

[0014] In some embodiments of the present invention, the hydroxyl protecting group includes any one of methyl ether (MOM), benzyl ether (Bn), p-methoxybenzyl ether (PMB), TBS (tert-butyldimethylsilyl), tert-butyldiphenylsilyl (TBDPS), trimethylsilyl (TMS), acetyl (Ac), and benzoyl (Bz). This type of protecting group is a common hydroxyl protecting group in the art and can be introduced and deprotected by conventional means in the art.

[0015] In some embodiments of the present invention, the deprotection is carried out in the presence of a nucleophilic reagent, and the nucleophilic reagent includes any one of tetra-n-butylammonium fluoride (TBAF), hydrofluoric acid (HF), potassium fluoride (KF), cesium fluoride (CsF), and boron tribromide (BBr3).

[0016] In some embodiments of the present invention, the deprotection is carried out at -78°C to 10°C (such as -20°C to 5°C, -10°C to 3°C, 0°C, etc.) for 10 to 50 min (such as 20 to 40 min).

[0017] The third aspect of the present invention provides a pharmaceutical composition comprising the compound of formula I or its stereoisomer, a pharmaceutically acceptable salt thereof, and optionally, a pharmaceutically acceptable excipient and / or carrier.

[0018] In some embodiments of the present invention, the pharmaceutical composition is a vascular endothelial growth factor inhibitor.

[0019] The fourth aspect of the present invention provides a use of the compound of formula I or its stereoisomer, a pharmaceutically acceptable salt thereof, and / or a pharmaceutical composition thereof in the preparation of a drug.

[0020] In some embodiments of the invention, the medicament is used to treat the following diseases or conditions: diseases caused by ocular neovascularization, psoriasis, hemangioblastoma, mesangial cell proliferation diseases, 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 lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia and solid tumors;

[0021] The disease caused by ocular neovascularization is preferably an angiogenic eye disease, and the angiogenic eye disease is selected from age-related macular degeneration, diabetic retinopathy, diabetic macular edema, central retinal vein occlusion and corneal neovascularization;

[0022] The mesangial cell proliferative disease is preferably selected from diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy syndrome or transplant rejection, glomerulonephritis, hemolytic uremic syndrome, diabetic nephropathy and hypertensive nephrosclerosis;

[0023] The solid tumor is a primary or secondary solid tumor selected from breast cancer, colon cancer, lung cancer (eg, small cell lung cancer), prostate cancer, myeloma, and Kaposi's sarcoma.

[0024] In some embodiments of the invention, the medicament is used to treat the following diseases or conditions: diseases caused by ocular neovascularization, such as angiogenic eye diseases; the angiogenic eye diseases are 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, hemangioblastoma such as hemangioma (benign vascular proliferation), mesangial cell proliferation 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 diseases such as glomerulonephritis, such as mesangial proliferative glomerulonephritis, hemolytic uremic syndrome , diabetic nephropathy, hypertensive nephrosclerosis, atherosclerosis, arterial restenosis, autoimmune diseases (e.g. arthritis such as rheumatoid arthritis), acute inflammation, fibrotic diseases (e.g. cirrhosis), diabetes, endometriosis, chronic asthma, arterial or post-transplant atherosclerosis, neurodegenerative diseases, lymphomas and, for example, neoplastic diseases such as leukemias, such as acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia and other "liquid tumors", as well as solid tumors (e.g. cancers), such as breast cancer (e.g. Paget's disease), colon cancer, lung cancer (e.g. small cell lung cancer), prostate cancer, myeloma or Kaposi's sarcoma, such as the growth of the above tumors or metastatic spread and the growth of micrometastases.

[0025] In some embodiments of the present invention, the medicament is used to treat the following diseases or conditions:

[0026] (1) Tumor; further, the tumor is a solid tumor;

[0027] (2) Leukemia;

[0028] (3) Psoriasis;

[0029] (4) Paget's disease;

[0030] (5) benign vascular proliferation;

[0031] (6) Arthritis;

[0032] (7) Atherosclerosis;

[0033] (8) Neovascular eye disease, further, the neovascular eye disease is primary or secondary neovascular eye disease.

[0034] As a drug, the compounds of the present invention can be administered in the form of a pharmaceutical composition. These compositions can be prepared in a manner well known in the pharmaceutical field, and they can be administered by a variety of routes, depending on whether local or systemic treatment is required and the area to be treated. It can be administered topically (e.g., transdermal, skin, eye and mucous membranes including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizers; intratracheal, intranasal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, such as intrathecal or intraventricular administration. It can be administered parenterally in a single large dose form, or it can be administered by, for example, a continuous infusion pump. Topically administered pharmaceutical compositions and preparations can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powder preparations and powders. Conventional pharmaceutical carriers, water, powder or oily bases, thickeners, etc. may be necessary or required.

[0035] In preparing the compositions of the present invention, the active ingredient is usually mixed with an excipient, diluted by an excipient or enclosed in such a carrier in the form of a capsule, sachet, paper or other container. When the excipient serves as a diluent, it can be a solid, semisolid or liquid substance, which acts as a solvent, carrier or medium for the active ingredient. Thus, the composition can be in the form of tablets, pills, powders, lozenges, sachets, cachets, microcapsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols (solid or dissolved in a liquid medium); 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.

[0036] Some examples of suitable excipients include lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methylcellulose. The formulation may also contain lubricants such as talc, sodium stearate, magnesium stearate, sodium oleate, sodium benzoate, sodium acetate, sodium chloride and mineral oil; wetting agents; emulsifiers and suspending agents; preservatives such as methyl benzoate and hydroxypropyl benzoate; sweeteners and flavoring agents. The composition of the present invention may be formulated using methods known in the art to provide an immediate release, sustained release or delayed release of the active ingredient after administration to the patient.

[0037] The composition can be formulated in unit dosage form, each dose containing about 5 to 1000 mg, more usually about 100 to 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 physically discrete single dosage units suitable for use in human patients and other mammals, each unit containing a predetermined amount of active material calculated to produce the desired therapeutic effect in admixture with a suitable pharmaceutical excipient.

[0038] The effective dosage range of the active compound can be very large, and is generally administered in a pharmaceutically effective amount. 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 selected, the actual compound being administered; the age, weight and response of the individual patient; the severity of the patient's symptoms, etc.

[0039] For preparing solid compositions such as tablets, the main active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of the compound of the invention. When these preformulation compositions are referred to as homogeneous, it is meant that the active ingredient is generally evenly distributed throughout the composition so that the composition can be easily divided into equally effective unit dosage forms such as tablets, pills and capsules. The solid preformulation is then divided into unit dosage forms of the above 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 invention.

[0040] The tablets or pills of the present invention may be coated or compounded to obtain dosage forms that provide the advantage of prolonged action. For example, the tablet or pill contains an inner dose and an outer dose component, the latter being a film-coated form of the former. The two components may be isolated by an enteric layer that is used to prevent disintegration in the stomach so that the inner component passes intact through the duodenum or is delayed in release. A variety of materials may be used for such enteric layers or coatings, including a variety of polymeric acids and mixtures of polymeric acids and such materials as shellac, cetyl alcohol, and cellulose acetate.

[0041] Liquid forms in which the compounds and compositions of the present invention may be incorporated for oral or injectable administration include aqueous solutions, appropriately flavored syrups, aqueous 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 pharmaceutically acceptable vehicles.

[0042] Compositions for inhalation or insufflation include solutions and suspensions, 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 certain embodiments, the composition is administered by oral or nasal respiratory route to achieve local or systemic effect. The composition may be atomized by using an inert gas. The atomized solution may be inhaled directly from an atomizing device, or the atomizing device may be connected to a mask curtain or an intermittent positive pressure breathing machine. Solutions, suspensions or powder compositions may be administered orally or by a device that delivers the formulation in an appropriate manner through the nose.

[0043] The amount of compound or composition administered to a patient is not fixed and depends on the drug being administered, the purpose of administration, such as prevention or treatment; the patient's condition, the mode of administration, etc. In therapeutic applications, a patient already suffering from a disease may be administered a composition in an amount sufficient to cure or at least partially suppress the symptoms of the disease and its complications. The effective dose will 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.

[0044] The composition administered to the patient may be in the form of a pharmaceutical composition as described above. These compositions may be sterilized by conventional sterilization techniques or by filtration sterilization. The aqueous solution may be packaged for use as is, or lyophilized, and the lyophilized preparation may be mixed with a sterile aqueous carrier before administration. The pH of the compound preparation is generally 3 to 11, more preferably 5 to 9, and most preferably 7 to 8. It will be appreciated that the use of some of the aforementioned excipients, carriers or stabilizers may result in the formation of a pharmaceutical salt.

[0045] The therapeutic dose of the compounds of the present invention may be determined, for example, based on the specific use of the treatment, the mode of administering the compound, the health and condition of the patient, and the judgment of the prescribing physician. The ratio or concentration of the compounds of the present invention in the pharmaceutical composition may not be fixed, depending on a variety of factors, including dosage, chemical properties (e.g., hydrophobicity), and route of administration. For example, the compounds of the present 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 about 1 μg / kg to about 1 g / kg body weight / day. In certain embodiments, the dosage range is about 0.01 mg / kg to about 100 mg / kg body weight / day. The dosage is likely to depend on such variables as the type and extent of the disease or condition, the general health status of the specific patient, the relative biological efficacy of the selected compound, the excipient formulation, and its route of administration. The effective dose can be obtained by extrapolation of a dose-response curve derived from an in vitro or animal model test system.

[0046] According to an embodiment of the present invention, the compound represented by Formula I or its stereoisomer, its pharmaceutically acceptable salt, and its medicine or pharmaceutical composition have a preventive or therapeutic effect on the diseases and conditions by inhibiting the activity of vascular endothelial growth factor.

[0047] The term "stereoisomer" refers to compounds composed of the same atoms, bonded by the same bonds, but having different three-dimensional structures. The present invention is intended to encompass various stereoisomers and mixtures thereof.

[0048] When the compounds of the present invention contain olefinic double bonds, and unless specified otherwise, it is intended that the compounds of the present invention include both E- and Z- geometric isomers.

[0049] The term "tautomer" refers to an isomer formed when a proton is shifted from one atom of a molecule to another atom of the same molecule. All tautomeric forms of the compounds of the present invention are intended to be encompassed within the scope of the present invention.

[0050] The compounds of the present invention or their pharmaceutically acceptable salts may contain one or more chiral carbon atoms and may therefore produce enantiomers, diastereomers and other stereoisomeric forms. Each chiral carbon atom may be defined as (R)- or (S)- based on stereochemistry. The present invention is intended to include all possible isomers, as well as racemates and optically pure forms thereof. The compounds of the present invention may be prepared using racemates, diastereomers or enantiomers as starting materials or intermediates. 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 preparing / separating individual isomers include chiral synthesis from suitable optically pure precursors, or resolving racemates (or racemates of salts or derivatives) using, for example, chiral high performance liquid chromatography.

[0051] The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0052] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, nitrates, phosphates, and the like; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetates, trifluoroacetates, propionates, caproates, caprylates, decanoates, undecylenates, glycolates, gluconates, lactates, sebacates, adipates, glutarates, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamate, pyroglutamate, aspartate, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginate, ascorbate, salicylates, 4-aminosalicylates, naphthalene disulfonates, and the like. These salts may be prepared by methods known in the art.

[0053] "Pharmaceutically acceptable base addition salt" refers to a salt formed with an inorganic base or an organic base that can maintain the biological effectiveness of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts. The salt derived from organic base includes but is not limited to the following salt: primary amines, secondary amines and tertiary amines, substituted amines, including natural 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 profession.

[0054] The beneficial effects of the present invention are:

[0055] The diphenylethylene compound of the present invention has a better VEGF inhibitory effect, and the effect is better than that of the natural diphenylethylene compound resveratrol.

[0056] The preparation method of the stilbene compound of the present invention has a reasonable route design, the starting raw materials are easily available, the reagent price is relatively low, the post-treatment is simple, and the production cost can be effectively reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is the H NMR spectrum of compound RE-15 prepared in Example 1 of the present application.

[0058] Figure 2 HPLC charts of compound RE-15 and prepared in Example 1 of the present application.

[0059] Figure 3 This is a comparison chart of the compound RE-15 prepared in Example 1 of the present application hitting the VEGF protein and the VEGFR-1 receptor binding domain in the molecular docking software.

[0060] Figure 4 This is the binding spectrum of the compound RE-15 prepared in Example 1 of the present application with VEGF protein in a surface plasmon resonance (SPR) system.

[0061] Figure 5 This is a quantitative comparison chart of the activity of the compound RE-15 prepared in Example 1 of the present application in inhibiting the vascular cell growth induced by VEGF protein in vascular endothelial cells.

[0062] Figure 6 A quantitative comparison chart of the inhibition of HaCaT cell scratch migration by the compound RE-15 prepared in Example 1 of the present application.

[0063] Figure 7 This is a quantitative comparison diagram of the potential cytotoxicity of the compound RE-15 in Example 1 of the present application in the MTT test of HaCaT cells. DETAILED DESCRIPTION

[0064] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0065] The temperature parameters in the present invention, if not specifically limited, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0066] Example 1

[0067] In this example, a stilbene compound RE-15 was prepared, and the specific process was as follows:

[0068]

[0069] Triethylamine (2.78 g) and tert-butyldimethylsilyl compound (TBSOTf, 14.1 mL) were added to 3,5-dihydroxybenzoic acid methyl ester (compound 1, 5 g) in dichloromethane (100 mL) at room temperature, and the mixture was reacted for 30 minutes to obtain 3,5-di-(tert-butyl-dimethyl-silyloxy)-benzoic acid methyl ester (compound 2, 11.8 g);

[0070] Compound 2 (11.8 g) was slowly added with lithium aluminum hydride (LiAlH4, 2.26 g) at 0°C in tetrahydrofuran (THF, 50 mL), and reacted for 2 hours to obtain [3,5-di-(tert-butyl-dimethyl-silanyloxy)-phenyl]-methanol (compound 3, 10.9 g);

[0071] Compound 3 (10.9 g) was added with I2 (11.3 g) and PPh3 (11.8 g) at room temperature in dichloromethane (50 mL), and the mixture was reacted for 5 hours to obtain [3,5-di-(tert-butyl-dimethyl-silanyloxy)-phenyl]-iodomethyl (compound 4, 5.6 g);

[0072] Compound 4 (4.3 g) was placed in P(OEt)3 (10 mL) and heated to 140°C for 50 minutes to generate diethyl 3,5-di(tert-butyldimethylsilyloxy)benzylphosphonate (compound 5, 4.75 g);

[0073] 4-Hydroxybenzaldehyde (Compound 6, 4.75 g) and 1,5-dibromopentane (Compound 7, 5.0 g) were added with potassium carbonate (11.5 g) at room temperature in acetonitrile (100 mL), and reacted for 48 hours to obtain 4,4'-(1,5-pentanediyl)dioxybenzaldehyde (Compound 8, 4.71 g);

[0074] Compound 8 (0.77 g) and compound 5 (3.0 g) were placed in a THF solution (20 mL), and t-BuOK (2.15 g) was added at 0°C for 5 hours to obtain compound 9 (2.41 g);

[0075] Compound 9 (2.41 g) was added with tetra-n-butylammonium fluoride (TBAF, 10 mL) in THF (8 mL) solution, and the mixture was reacted at 0° C. for 30 minutes to generate the final product RE-15 (0.30 g).

[0076] The obtained product RE-15 was subjected to nuclear magnetic resonance analysis, and the obtained hydrogen spectrum NMR was as follows Figure 1 .

[0077] The obtained product was tested by HPLC, and the purity was above 97%. Figure 2. The detection method is as follows: 4 mL and 50% methanol are added to the sample respectively, and after ultrasonic treatment for 15 minutes, the sample is centrifuged at 1000 x g for 5 minutes to obtain the supernatant. Before the supernatant is injected, it is filtered with a 0.45 μm microporous filter membrane, and the filtrate is taken for injection analysis. The analytical instrument used is an Agilent liquid phase with an automatic sampler and a binary pump, the chromatographic column is Agilent, Grace VisionHT C18 (4.6x 250mm, 5μm), the mobile phase is acetonitrile (solvent A) and 0.2% formic acid aqueous solution (solvent B), the flow rate is 1 mL / min, the column temperature is room temperature, the gradient elution, and the mobile phase is as follows: 0-60 minutes, 10-35% solvent A; 60-96 minutes, 35-100% solvent A. The injection volume is 20 μL, and the detection wavelength is 254 nm.

[0078] Example 2 Molecular docking experiment

[0079] 2.1 Experimental Principle

[0080] VEGF (vascular endothelial growth factor) is one of the important proteins of vascular endothelium. Under normal circumstances, VEGF binds to the VEGFR-1 receptor and provides a material basis for subsequent pathways. Among them, the binding domain of the VEGF protein with the receptor VEGFR-1 is an amino acid fragment 1-165. Theoretically, if the chemical component can hit this binding domain, it will competitively bind to VEGFR, thereby effectively inhibiting the binding of VEGF to the receptor VEGFR, thereby affecting the subsequent pathways and ultimately inhibiting the growth of new blood vessels. This binding domain has also been identified as the target of this molecular docking.

[0081] 2.2. Experimental procedures and results

[0082] Using the software SEESAR (version 13.1), the binding domain (amino acid fragment 1-165) of the VEGF protein (PDB code: 1FLT) with the VEGFR-1 receptor was selected as the target, and the affinity of the compound RE-15 with the target was then tested. The docking software - SEESAR can analyze the target efficacy. The lower the efficacy, the more stable the binding of the chemical molecule to the target.

[0083] The results are as follows Figure 3 It was shown that RE-15 can effectively bind to the VEGF target ( Figure 3 ), where the target potency was predicted to be -33.6KJ / mol. This indicates that compound RE-15 can effectively hit VEGF protein and prevent the protein from binding to VEGFR-1 receptor, which can more effectively inhibit VEGF and thus more effectively inhibit angiogenesis.

[0084] Example 3 Protein Binding Experiment

[0085] 3.1 Experimental Principle

[0086] Surface Plasmon Resonance (SPR) is an optical, label-free detection technique that can be used to detect binding interactions between two or more molecules in real time. The principle of SPR is based on total reflection, which causes a decrease in light intensity when light is reflected at a specific angle on the sensor glass surface. As molecules bind, the surface refractive index changes, causing a change in the angle of minimum reflection intensity. The change in angle is proportional to the mass of the bound substance. The results can be represented by a sensor plot that records the angle change over time.

[0087] 3.2 Experimental operation

[0088] The binding between the sample and the VEGF protein was performed on a Biacore S200 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 Hepes, 0.05% polysorbate 20, 3.4 mM EDTA, pH 7.4) was selected as the running buffer. The chip sensor surface 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. After surface activation, the VEGF protein dissolved in the coupling buffer (0.1 M acetic acid buffer, pH 4.5) was placed on the activated surface of the chip until the RU (resonance unit) signal of the detected VEGF protein reached about 6500 RU. To remove uncoupled proteins, the chip with activated coupled surface was washed and reacted with 10 mM glycine HCl, pH set to 1.5. Samples of different concentrations were diluted with running buffer (stepwise 2-fold dilution from 20 μM to 0.3125 μM) and flowed over the chip surface coupled with VEGF protein.

[0089] 3.3 Experimental Results

[0090] like Figure 4 As shown in Table 1, RE-15 can effectively bind to VEGF, with a maximum resonance unit (RU) of 473 and a binding constant (K d ) is 1.67 μM. The results show that RE-15 has a significant binding effect with VEGF and can effectively inhibit VEGF and thus more effectively inhibit angiogenesis and other VEGF-related biochemical functions.

[0091] Table 1

[0092] Detection object Maximum resonance unit RU (concentration 20μM) <![CDATA[Binding constant K d (μM)]]> RE-15 473 1.67

[0093] Example 4 Vascular endothelial cell activity test

[0094] 4.1 Experimental methods

[0095] 5×10 3 Human vascular endothelial cells (HUVECs) were seeded in sterile 96-well plates in 100 μL of culture medium. After 24 h of incubation, the culture medium in each well was replaced with fresh culture medium (100 μL) containing 10 ng / mL VEGF or a series of concentrations of RE-15. 48 h after drug treatment, MTT solution (10 μL per well) at a concentration of 5 mg / mL was added. After incubation at 37°C for 4 h, 150 μL of 100% dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazol salt, and the culture medium was then removed. The color intensity of the formazol solution was read in a microwell spectrophotometer with a wavelength set to 570 nm. The release of lactate dehydrogenase (LDH) in the mixture was detected by Cytotoxicity Detection kit PLUS (LDH) (Roche Diagnostics, Indianapolis, IN). The LDH content in each group was quantified according to the following formula: Cell activity (%) = (experimental value - low control) / (high control - low control) × 100%.

[0096] 4.2 Experimental Results

[0097] like Figure 5 As shown in the figure, after adding VEGF protein, the biological activity of vascular endothelial cells was significantly enhanced, while after adding positive control avastin and resveratrol, the cell activity was weakened. It is worth noting that, like the positive control, RE-15 can significantly reduce the biological activity induced by VEGF. This shows that both can inhibit VEGF-related biochemical activity by inhibiting VEGF protein.

[0098] Example 5 Cell scratch test

[0099] 5.1. Methods

[0100] HaCaT cells were grown at 20×10 4Cells were inoculated at a density of 100 μg / well in a 12-well plate and cultured overnight to allow the cells to adhere to the wall. After the cells reached 80% fusion, a 200 μL pipette tip was used to scratch a horizontal wound in the middle of each well, the culture medium was discarded, and the cells were washed once with PBS. 10 ng / mL of human vascular endothelial growth factor VEGF was added to the culture medium separately, and 200 μg / mL of avastin and different concentrations of RE-15 samples (0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM) were administered respectively. In addition, a blank group (no drug but an equal volume of solvent) and a control group (no drug) were set up. A microscope equipped with a camera was used to take pictures of the changes in the cell layer coverage in each well at 50 times magnification after 0 and 24 hours. The recovery growth rate was then digitized using TScratch software, and the cell recovery growth rate was calculated according to the following formula: Recovery rate % = (At0-At 24 ) / At0×100%, where At0 is the scratch area measured 0 hours after administration; At 24 : The scratch area measured 24 hours after administration.

[0101] 5.2. Results and conclusions

[0102] Figure 6 Figure 1 is a quantitative graph showing the inhibition of HaCaT cell scratch migration by positive control (A), resveratrol (RES), and RE-15. Cell scratch migration (% of change) in the figure represents the degree of wound recovery (%).

[0103] according to Figure 6 It can be seen that compound RE-15 can effectively inhibit the proliferation of HaCaT cells and wound healing induced by VEGF protein, and its effect is better than that of the positive control.

[0104] Example 6 Cytotoxicity Experiment

[0105] Methods

[0106] The toxicity of RE-15 to human HaCaT cells was observed by MTT colorimetry. 3The cells were inoculated at a density of 100 μL / mL in a 96-well culture plate, and 100 μL was added to each well. After the cells reached 80% fusion, different concentrations of RE-15 (1 μM, 10 μM, 20 μM, 50 μM, 100 μM) were added to the wells. After 48 hours, 10 μL of MTT solution was added to each well. After 4 hours of continuous culture at 37°C, the culture was terminated, the culture supernatant was discarded, 150 μL of DMSO was added to each well, and the cells were fully dissolved by shaking for 10 minutes. The wavelength of 490 nm was selected, and the absorbance (OD) value of each well was measured on an ELISA reader. The wells without drug addition were used as controls, and the cell viability was calculated according to the following formula: Cell viability (%) = (OD sample-OD control substance) / OD control substance × 100%.

[0107] Results and conclusions

[0108] according to Figure 7 It can be seen that RE-15 has no obvious effect on cell activity, indicating that the sample has no obvious cytotoxicity.

[0109] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A compound of formula I, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

2. A method for preparing the compound of formula I according to claim 1, characterized in that: The following steps are involved: 4,4'-(1,5-pentanediyl)dioxybenzaldehyde and a compound of formula II Reaction is carried out, followed by deprotection to obtain a compound of formula I; Wherein, R1 and R2 are independently selected from hydroxyl protecting groups.

3. The method for preparing the compound of formula (I) according to claim 2, characterized in that: The reaction is a Horner-Wadsworth-Emmons reaction; and / or, the reaction is carried out at -78°C to 10°C for 3 to 8 hours.

4. The method for preparing the compound of formula (I) according to claim 2, characterized in that: The hydroxyl protecting group includes any one of methyl ether, benzyl ether, p-methoxybenzyl ether, TBS, tert-butyldiphenylsilyl, trimethylsilyl, acetyl, and benzoyl.

5. A pharmaceutical composition comprising the compound of formula I according to claim 1, and optionally, a pharmaceutically acceptable excipient and / or carrier.

6. The pharmaceutical composition according to claim 5, characterized in that: Dosage forms of the pharmaceutical composition include tablets, pills, powders, lozenges, sachets, cachets, 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 or 6, characterized in that: The pharmaceutical composition is a vascular endothelial growth factor inhibitor.

8. Use of the compound of formula I according to claim 1 or its stereoisomer, its pharmaceutically acceptable salt, and / or the pharmaceutical composition according to claim 5 in the preparation of a medicament.

9. The use according to claim 7, characterized in that: The medicament is used to treat the following diseases or conditions: diseases caused by ocular neovascularization, psoriasis, hemangioblastoma, mesangial cell proliferative diseases, 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 lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia and solid tumors.

10. The use according to claim 7, characterized in that: The medicament is used to treat the following diseases or conditions: age-related macular degeneration, diabetic retinopathy, diabetic macular edema, central retinal vein occlusion and corneal neovascularization; diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy syndrome or transplant rejection, glomerulonephritis, hemolytic uremic syndrome, diabetic nephropathy and hypertensive nephrosclerosis; breast cancer, colon cancer, lung cancer (e.g., small cell lung cancer), prostate cancer, myeloma and Kaposi's sarcoma.

Images