A vascular endothelial growth factor inhibitor and its preparation method and application

Abstract

The application discloses a vascular endothelial growth factor inhibitor and a preparation method and application thereof. The vascular endothelial growth factor inhibitor comprises a compound of formula I. The stilbene compound of the application has a better VEGF inhibiting effect and is better than natural stilbene resveratrol. The preparation method of the stilbene compound of the application has a reasonable route design, starting materials are easy to obtain, reagents are 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 field of medicine, in particular to a vascular endothelial growth factor inhibitor and 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 the 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 believed 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 vascular basement membrane and extracellular matrix degradation, inhibition of endothelial cell division, migration and proliferation, etc. Vascular endothelial growth factor (VEGF) is considered to be the most critical pro-angiogenic factor, and anti-angiogenic and anti-tumor drugs targeting VEGF and its receptor VEGFR have been the focus of research in recent years.

[0004] Bevacizumab (Avastin) is the first monoclonal antibody targeting vascular endothelial growth factor (VEGF) protein and preventing the binding of kinases to its receptors. However, it has been reported that bevacizumab can cause cardiotoxicity, and when this side effect occurs, patients are advised to stop treatment. As for small molecule inhibitors, pazopanib and sunitinib are antagonists against multiple VEGF receptors (such as VEGFR-1 and VEGFR-2), while sorafenib is identified 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 arrhythmia, heart failure and myocardial ischemia. In short, the current treatment options on the market have adverse reactions, which indicates that there is still an urgency to develop more targeted VEGF treatment methods with higher efficacy and lower toxicity. SUMMARY

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

[0006] To achieve the above object, the present application adopts the technical scheme of:

[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 method for preparing the compound of Formula I, comprising the steps of:

[0010] reacting 4,4'-(1,5-pentanediyl) dioxybenzaldehyde with a compound of Formula II to obtain a compound of Formula I.

[0011] wherein R1 and R2 are independently selected from a hydroxyl protecting group.

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

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

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

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

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

[0017] In a third aspect, the present application 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.

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

[0019] In a fourth aspect of the present application, there is provided a use of a compound of formula I, or a stereoisomer thereof, a pharmaceutically acceptable salt thereof, and / or a pharmaceutical composition as described, in the manufacture of a medicament.

[0020] In some embodiments of the application, the medicament is for the treatment of a disease or condition selected from the group consisting of ocular neovascularization, psoriasis, hemangio blastoma, glomerular 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 ocular neovascularization is preferably an ocular angiogenic disease selected from the group consisting of age-related macular degeneration, diabetic retinopathy, diabetic macular edema, central retinal vein occlusion, and corneal neovascularization.

[0022] The glomerular cell proliferation disease is preferably selected from the group consisting of diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy syndromes or transplant rejection, glomerulonephritis, hemolytic-uremic syndrome, diabetic nephropathy, and hypertensive renal sclerosis.

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

[0024] In some embodiments of the application, the medicament is for use in the treatment of a disease or condition selected from the group consisting of diseases caused by ocular neovascularization, such as angiogenic eye diseases; the angiogenic eye diseases are selected from the group consisting of age-related macular degeneration, diabetic retinopathy, diabetic macular edema, central retinal vein occlusion and corneal neovascularization, for example retinal diseases such as diabetic retinopathy or age-related macular degeneration; psoriasis, hemangio-blastoma such as hemangioma (benign vascular hyperplasia), glomerular membrane cell proliferation diseases such as chronic or acute nephropathy, for example diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy syndrome or transplant rejection, or for example inflammatory kidney diseases such as glomerulonephritis, for example glomerular membrane proliferative glomerulonephritis, hemolytic uremic syndrome, diabetic nephropathy, hypertensive renal sclerosis, atherosclerosis, arterial restenosis, autoimmune diseases (for example arthritis such as rheumatoid arthritis), acute inflammation, fibrotic diseases (for example cirrhosis), diabetes, endometriosis, chronic asthma, arterial or post-transplant atherosclerosis, neurodegenerative diseases, lymphoma and for example neoplastic diseases such as leukemia, for example acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia and other "liquid tumors", as well as solid tumors (such as carcinomas), for example breast cancer (for example Paget's disease), colon cancer, lung cancer (for example small cell lung cancer), prostate cancer, myeloma or Kaposi's sarcoma, for example growth of the above-mentioned tumors or metastatic spread and growth of micrometastases.

[0025] In some embodiments of the application, the medicament is for use in the treatment of a disease or condition selected from the group consisting of:

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

[0027] (2) leukemia;

[0028] (3) psoriasis;

[0029] (4) Paget's disease;

[0030] (5) benign vascular hyperplasia;

[0031] (6) arthritis;

[0032] (7) atherosclerosis;

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

[0034] As pharmaceuticals, the compounds of the present application can be given as pharmaceutical compositions. These compositions can be prepared in a manner widely known in the pharmaceutical art, and administered by a variety of routes depending upon whether local or systemic treatment is desired and upon the area to be treated. They can be administered topically (e.g., transdermal, transcutaneous, opthalmic, and mucosal 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 infusions include intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injections or infusions; or intracranial, e.g., intrathecal or intracerebroventricular administration. Parenteral infusions can be given as a single bolus, or can be given by, e.g., continuous infusion pump. Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders, and aerosols, as is well known to the pharmaceutical art. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable.

[0035] In making the compositions of the application, the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, e.g., a capsule, sachet, paper or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, caplets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (solid or liquid), ointments containing, e.g., up to 10% by weight of active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders.

[0036] Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methyl cellulose. The formulations can additionally include: lubricating agents such as talc, sodium stearate, magnesium stearate, glycine, sodium chloride, and waxes; wetting agents such as lecithin and phosphatidylcholine; emulsifying and suspending agents; preservatives such as methyl and propylhydroxybenzoates; sweetening agents; and flavoring agents. The compositions of the application can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art.

[0037] The compositions can be formulated in unit dosage form, each dosage containing from 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 units suitable for single administration of a predetermined dosage of active ingredient which, as is known in the art, can be packaged in a hermetically sealed container, or in a non-retardable dosage unit form, with an appropriate pharmaceutical excipient.

[0038] The effective dosage of active compound will vary widely, and will be determined by such factors as the particular condition being treated, the severity of the patient's symptoms, the particular active compound being administered, the mode of administration, and the age and condition of the patient. However, it is expected that the dosage will be in the range of 0.01 to 100 mg / kg, preferably 0.1 to 50 mg / kg, more preferably 0.5 to 10 mg / kg, most preferably 1 to 5 mg / kg, of body weight per day, given in one or more dosages.

[0039] For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present application. When referring to these preformulation compositions as "homogeneous", it is meant that the active ingredient is dispersed essentially evenly throughout the composition so that the composition can be readily subdivided into equally effective dosage forms such as tablets, pills and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described above containing, for example, from about 0.1 to about 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 application.

[0040] The tablets or pills of the present application can be coated or otherwise compounded to provide a dosage form affording the release of the active ingredient in a particular phase, as in galenical dosage form coating, enteric coating, and the like. For example, the tablets or pills can be coated with an enteric coating, to provide a dosage form having a delayed release of the active ingredient. A wide variety of materials can be used for this purpose, such as a number of film-coatings and coatings known in the art. Thus, the compositions can be formulated to provide slow or delayed release of the active ingredient.

[0041] Liquid forms in which the compounds and compositions of the present application can be incorporated for administration orally or by injection include aqueous solutions, suitably flavored suspensions, aqueous or oil suspensions, and flavored, emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

[0042] Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable volatile carrier(s) such as halogenated hydrocarbon, volatile alcohols, or other materials. Preferably, the compositions are administered by inhalation or insufflation. Liquid or solid compositions can contain suitable quantities of pharmaceutically-acceptable buffers, diluents, or carriers. In certain embodiments, the compositions are administered in an acceptable diluent or carrier via oral or nasal respiratory routes, to provide local or systemic effects. The compositions can be nebulized or aerosolized for delivery via inhalation. The aerosol can be delivered from a pressurized container or by atomizing the composition. The atomizing can be accomplished by use of a suitable propellant. The compositions can be administered directly to the lungs by inhalation through the mouth or nose from a device that delivers the formulation in a form appropriate for the patient. Alternatively, the compositions can be administered in the form of a solution, suspension, or powder either by oral or nasal inhalation from devices that deliver the formulation in an appropriate manner.

[0043] The amount of a compound or composition administered to a patient will vary depending upon what is being administered, the purpose of the administration, e.g., prophylactic or therapeutic; the state of the patient, the mode of administration; and the like. In therapeutic applications, an amount sufficient to cure or at least partially arrest symptoms of the disease and its complications is administered to a patient already suffering from the disease. Effective dosages should be determined by considering various factors, e.g., the severity of the disease, the age, weight and general health condition of the patient, and the like.

[0044] The compositions administered to a patient can be in the form of a pharmaceutical composition described above. These compositions can be sterilized by conventional sterilization techniques or can be sterile filtered. Aqueous solutions can be packaged in sterile vials or pre-filled syringes and stored in lyophilized condition. The pH and exact concentration of the compound within the composition can vary according to the particular use. It will be understood that use of certain of the foregoing excipients, carriers, or diluents can result in the formation of pharmaceutical salts of the compounds.

[0045] Therapeutic dosages of the compounds of the present application can vary according to, e.g., the particular use for which the therapy is made, the mode of administration, the health and condition of the patient, and the judgment of the prescribing physician. The proportion or concentration of a compound of the present application in a pharmaceutical composition can not be fixed, and will vary with factors such as the dosage of the compound, chemical properties of the compound (e.g., hydrophobicity), and the route of administration. For example, a typical dose of a compound of the present application for intravenous administration to an adult can range from about 0.1 to 10% (w / v). Some typical doses are in the range from about 1 μg / kg to about 1 g / kg body weight per day. In certain embodiments, the dose is in the range from about 0.01 mg / kg to about 100 mg / kg body weight per day. The dosage, of course, will depend on such variables as the type and extent of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the compound selected, formulation of the excipient, and its route of administration. Effective doses will be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0046] According to embodiments of the present application, the compounds of Formula I or stereoisomers thereof, pharmaceutically acceptable salts thereof, and pharmaceutical or pharmaceutical compositions thereof produce a prophylactic or therapeutic effect on the diseases and conditions by inhibiting the activity of vascular endothelial growth factor.

[0047] The term "stereoisomers" refers to compounds which have the same atomic constitution but differ in the arrangement of the atoms in space. The present application will encompass all possible stereoisomers and mixtures thereof.

[0048] When the compounds of the present application contain an olefinic double bond, the compounds of the present application are intended to include both E- and Z- geometric isomers, unless otherwise noted.

[0049] The term "tautomer" refers to structural isomers that exist in equilibrium with each other. All tautomeric forms of the compounds of the present application are also intended to be included within the scope of the present application.

[0050] The compounds of the present application, or pharmaceutically acceptable salts thereof, can contain one or more chiral carbon atoms and can therefore give rise to enantiomers, diastereomers, and other stereoisomeric forms. Each chiral carbon atom can be defined as either an (R)- or (S)- based on the stereochemistry. The present application is intended to include all possible isomers, as well as their racemic and optically pure forms. The preparation of the compounds of the present application can select either the racemate, diastereomer, or enantiomer as starting material or intermediate. The optically active isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as crystallization and chromatography on chiral supports, etc. Conventional techniques for the preparation / isolation of individual isomers include chiral synthesis from appropriate optically pure precursors, or resolution of the racemate (or salt or derivative of the racemate) using, for example, chiral high pressure 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 salt" refers to salts of the free base which retain the biological effectiveness and non-toxicity of the free base and which are formed with inorganic acids or organic acids. Inorganic acid salts include, but are not limited to, hydrochloride, hydrobromide, sulfate, nitrate, phosphate, and the like. Organic acid salts include, but are not limited to, formate, acetate, 2,2-dichloroacetate, trifluoroacetate, propionate, hexanoate, octanoate, decanoate, undecylenate, glycolate, gluconate, lactate, sebacate, adipate, pimelate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, 4-aminosalicylate, naphthalene-2, 6-disulfonate, and the like. These salts can be prepared by methods known in the art.

[0053] "Pharmaceutically acceptable base addition salt" refers to salts of the free acid which retain the biological effectiveness and non-toxicity of the free acid and which are formed with inorganic or organic bases. 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, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, 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, dimethyl ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0054] The present application has the following advantages:

[0055] The stilbene compound of the present application has a better VEGF inhibiting effect, which is better than that of natural stilbene compound resveratrol.

[0056] The preparation method of the stilbene compound of the present application has reasonable route design, starting material is easy to obtain, reagent price is relatively low, and post-treatment is simple, which can effectively reduce production cost. BRIEF DESCRIPTION OF DRAWINGS

[0057] Figure 1 NMR spectrum of compound RE-15 prepared for Example 1 of the present application.

[0058] Figure 2 HPLC chart of compound RE-15 prepared for Example 1 of the present application.

[0059] Figure 3 Graphical comparison of compound RE-15 prepared for Example 1 of the present application in docking the VEGF protein to the VEGFR-1 receptor binding domain in molecular docking software.

[0060] Figure 4 Binding profile of compound RE-15 prepared for Example 1 of the present application to the VEGF protein in surface plasmon resonance (SPR) system.

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

[0062] Figure 6 Quantitative comparison of compound RE-15 prepared for Example 1 of the present application in inhibiting HaCaT cell scratch migration.

[0063] Figure 7 Quantitative comparison of potential cytotoxicity of compound RE-15 prepared for Example 1 of the present application in MTT test of HaCaT cells. DETAILED DESCRIPTION

[0064] The present application will be further described in details by specific examples. The raw materials, reagents or apparatus used in the examples and comparative examples are commercially available or can be obtained by prior art methods unless otherwise specified. The test or test method is the conventional method in the art unless otherwise specified.

[0065] The temperature parameter in the present application, unless otherwise specified, allows for constant temperature treatment and also allows for variation within a certain temperature range. It should be understood that the constant temperature treatment allows for fluctuation within the accuracy range controlled by the instrument. Fluctuation within the range of, for example, ±5°C, ±4°C, ±3°C, ±2°C, ±1°C is allowed.

[0066] Example 1

[0067] In this example, stilbene compound RE-15 was prepared by the following process:

[0068]

[0069] Methyl 3,5-dihydroxybenzoate (Compound 1, 5 g) was dissolved in dichloromethane (100 mL) and triethylamine (2.78 g) and tert-butyldimethylsilyl compound (TBSOTf, 14.1 mL) were added at room temperature. After 30 minutes of reaction, methyl 3,5-di-(tert-butyl-dimethyl-silanyloxy)-benzoate (Compound 2, 11.8 g) was obtained.

[0070] Compound 2 (11.8 g) was dissolved in tetrahydrofuran (THF, 50 mL) and lithium aluminum hydride (LiAlH4, 2.26 g) was added at 0°C. After 2 hours of reaction, [3,5-di-(tert-butyl-dimethyl-silanyloxy)-phenyl]-methanol (Compound 3, 10.9 g) was obtained.

[0071] Compound 3 (10.9 g) was dissolved in dichloromethane (50 mL) and I2 (11.3 g) and PPh3 (11.8 g) were added at room temperature. After 5 hours of reaction, [3,5-di-(tert-butyl-dimethyl-silanyloxy)-phenyl]-iodomethyl (Compound 4, 5.6 g) was obtained.

[0072] Compound 4 (4.3 g) was dissolved in P(OEt)3 (10 mL) and heated to 140°C for 50 minutes to obtain diethyl 3,5-di(tert-butyldimethylsiloxy)benzylphosphonate (Compound 5, 4.75 g).

[0073] 4-Hydroxybenzaldehyde (Compound 6, 4.75 g) and 1,5-dibromopentane (Compound 7, 5.0 g) were dissolved in acetonitrile (100 mL) and potassium carbonate (11.5 g) was added at room temperature. After 48 hours of reaction, 4,4'-(1,5-pentanediyl)dobenzaldehyde (Compound 8, 4.71 g) was obtained.

[0074] Compound 8 (0.77 g) and Compound 5 (3.0 g) were dissolved in THF (20 mL) and t-BuOK (2.15 g) was added at 0°C. After 5 hours of reaction, Compound 9 (2.41 g) was obtained.

[0075] Compound 9 (2.41 g) was dissolved in THF (8 mL) and tetra-n-butylammonium fluoride (TBAF, 10 mL) was added at 0°C. After 30 minutes of reaction, final product RE-15 (0.30 g) was obtained.

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

[0077] The obtained product was subjected to HPLC detection and the purity was more than 97% and the detection results were as follows: Figure 2The detection method is as follows: 4 mL of 50% methanol is added to the sample, and after ultrasonic treatment for 15 minutes, centrifugation is performed at 1000x g for 5 minutes to obtain the supernatant. Before sample injection, the supernatant is filtered through a 0.45 μm microporous filter, and the filtrate is taken for injection and analysis. The analytical instrument used is an Agilent liquid phase with an automatic sampler and a binary pump, and the chromatographic column is an Agilent, Grace VisionHT C18 (4.6x 250mm, 5 μm). 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, and the gradient elution 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 VEGFR-1 receptor and provides a material basis for the subsequent pathway. Among them, the binding domain of VEGF protein to VEGFR-1 receptor is amino acid fragment 1-165. In theory, if the chemical composition can hit this binding domain, it can compete with VEGFR to effectively inhibit the binding of VEGF to the receptor VEGFR, thereby affecting the subsequent pathway and ultimately inhibiting the growth of new blood vessels. And this binding domain is also determined as the target of this molecular docking.

[0081] 2.2. Experimental operation and results

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

[0083] The results are shown in Figure 3 , which shows that RE-15 can effectively bind to the VEGF target Figure 3 , and the target efficiency is predicted to be -33.6 KJ / mol. This indicates that compound RE-15 can effectively hit the VEGF protein and prevent the protein from binding to the VEGFR-1 receptor, and can more effectively inhibit VEGF and 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 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.

[0087] 3.2 Experimental Procedure

[0088] 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.

[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 The concentration was 1.67 μM. The results indicate that RE-15 significantly binds to VEGF, effectively inhibiting VEGF and thus more effectively suppressing VEGF-related biochemical functions such as angiogenesis.

[0091] Table 1

[0092] Detection substance Maximum resonance units RU (concentration 20 μM) Binding constant K d (μM) RE-15 473 1.67

[0093] Example 4: Vascular Endothelial Cell Viability Test

[0094] 4.1 Experimental Methods

[0095] 5×10 3 One 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 fresh medium (100 μL) containing 10 ng / mL VEGF or a series of concentrations of RE-15. 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 formalazole salt, and then the culture medium was aspirated. The color intensity of the formalazole solution was read using a microwell spectrophotometer at a wavelength of 570 nm. The amount of lactate dehydrogenase (LDH) released in the mixture was detected using the Cytotoxicity Detection Kit PLUS (LDH) (Roche Diagnostics, Indianapolis, IN). The LDH content in each group was quantified using the following formula: Cell viability (%) = (Experimental value - Low control) / (High control - Low control) × 100%.

[0096] 4.2 Experimental Results

[0097] 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-15 significantly reduced VEGF-induced biological activity. This indicates that both can inhibit VEGF-related biochemical activities by inhibiting VEGF protein.

[0098] Example 5 Cell Scratch Assay

[0099] 5.1. Methods

[0100] HaCaT cells at 20 × 10 4Cells 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-15 (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 in each well was 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.

[0101] 5.2. Results and Conclusions

[0102] Figure 6 The graph shows the quantitative effect of scratch migration of HaCaT cells on positive control (A), resveratrol (RES), and RE-15. The percentage of change in cell scratch migration in the graph represents the degree of wound healing (%).

[0103] according to Figure 6 It can be seen that compound RE-15 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.

[0104] Example 6 Cytotoxicity Experiment

[0105] 6.1. Methods

[0106] The cytotoxic effect of RE-15 on human HaCaT cells was observed using the MTT assay. HaCaT cells were inoculated at a concentration of 5.0 × 10⁻⁶ cells / cells. 3The cells were cultured in 96-well culture plates at a density of 1 x 104 / mL, 100 μL per well. After the cells grew to 80% confluence, 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, and the culture was continued at 37°C for 4 hours. Then the culture was terminated, the supernatant was discarded, 150 μL of DMSO was added to each well, and the cells were fully dissolved by shaking for 10 minutes. The absorbance (OD) value of each well was measured on an enzyme marker at a wavelength of 490 nm. The well without the drug was used as a control. The cell viability was calculated according to the following formula: cell viability (%) = (OD sample - OD control) / OD control x 100%.

[0107] 6.2. Results and Conclusion

[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 examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples. Any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods, and shall be included in the protection scope of the present application.

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: Includes the following steps: 4,4'-(1,5-pentanediyl)dioxydibenzaldehyde was reacted with compound of formula II The reaction proceeds, followed by deprotection, to obtain compound I. R1 and R2 are each 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 a compound of formula I as claimed in claim 1, and optionally, a pharmaceutically acceptable excipient and / or carrier.

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 or 6, 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 claim 5 in the preparation of a medicament; said medicament being used to treat the following diseases or conditions: ocular neovascularization, psoriasis, hemangioblastoma, glomerular 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 claim 5 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.

Images