TW201514151A - Inhibitor of VEGF-2/3 receptor and protein kinase and pharmaceutical use thereof - Google Patents

Abstract

The present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein X is NH or O; Y is NH or O; Z is aryl group or heteroaryl group including N, O, P, or S; and R1 is H, halogen, C1-C6 alkoxyl. The present disclosure also provides a pharmaceutical composition including the compound of formula (I) or the pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable vehicle. The present disclosure also provides use of the compound for preparing a medicament for treating a cell proliferative disorder.

Description

VEGF-2/3 receptor and protein tyrosine kinase inhibitor and their medical use

The present invention relates to protein tyrosine kinase inhibitors and their medical uses, and in particular to a VEGF-2/3 receptor and protein tyrosine kinase inhibitors and their medical use.

Vascular endothelial growth factor (VEGF) is a major factor regulating vascular endothelial cell division and regulating vascular permeability, which affects two very important physiological functions of angiogenesis and vasculogenesis. There are three major types of vascular endothelial growth factor receptors on vascular endothelial cells, namely VEGFR-1 (or flt-1), VEGFR-2 (or KDR/flk-1) and VEGFR-3 (or flt-4). ). In the early stages of studying vascular endothelial growth factor receptors, these vascular endothelial growth factor receptors were thought to be expressed only on endothelial cells. However, subsequent studies have shown that these receptors are also expressed in vascular smooth muscle cells, osteoblasts, and cardiomyocytes. , myofibroblasts, neurogenic cells and various tumor cells. VEGFR-2 plays a key role in mitosis, angiogenesis and vascular invasion of endothelial cells. VEGFR-3 binds to VEGFR-C or VEGFR-D and affects the formation of embryonic lymphatics (Lymphangiogenesis).

In addition, most human cancers are characterized by over-expression of tumor cells. VEGF and tumor-associated blood vessels are now overexpressing VEGF receptors. In cutaneous squamous cell carcinoma, VEGF also appears to affect very early tumor development. VEGF-C also acts on VEGFR-2 and VEGFR-3, and its performance is considered to be critical for Kaposi's sarcoma.

Tumor cells require oxygen for their growth and metastasis. Oxygen has a very limited diffusion range. Therefore, for tumors that grow beyond a very limited size, they cannot rely on passive oxygen transport, so they must establish active oxygen transport, ie Must attract the blood vessels of the host. The nutrients required for the tumor are also supplied by blood vessels. No tumors occur in the blood vessel region or in the final expanded avascular zone, resulting in low pO ₂ and the pH, and these factors trigger the concentration of VEGF in the tumor cells increases. In the absence of adequate oxygen and nutrient supply, tumor cells are necrotic or apoptotic, and the tumor will therefore stop growing and may even subside. Angiogenesis is considered absolutely necessary for tumors that grow more than about 1 to 2 mm in diameter; before reaching this limit, oxygen and nutrients can be supplied to tumor cells by diffusion. Therefore, each tumor, regardless of its origin and its cause, depends on angiogenesis after it reaches a certain size.

High volume of human tumors (especially gliomas and carcinomas) VEGF. This has led to the hypothesis that VEGF released by tumor cells stimulates the growth of capillaries and the proliferation of tumor endothelium in a paracrine manner and accelerates tumor growth via a modified blood supply. Enhanced VEGF performance may explain the presence of cerebral edema in patients with gliomas. In addition, in studies that inhibit VEGF expression or VEGF activity, there is evidence directly indicating the role of VEGF as a tumor angiogenic factor in vivo. This was achieved using an anti-VEGF antibody, a dominant negative VEGFR-2 mutant that inhibits signal transduction, and antisense VEGF RNA technology. All methods have slowed the growth of glioma cell lines or other tumor cell lines in vivo due to inhibition of tumor angiogenesis. In addition, studies have shown that the degree of angiogenesis of cancer tumors and the malignancy of their tumors and clinical Prognosis is related.

In addition, there is clear evidence that angiogenic factors (specifically The performance of VEGF) is an important cause of proliferative diabetic retinopathy (PDR). Pre-retinal vascularization is a major cause of blindness in this condition and other conditions such as retinopathy of prematurity, sickle cell retinopathy, age-related macular degeneration, retinal vein occlusion, and Eales disease. New blood vessels grow from the inner retinal vascular structure into the vitreous fluid. This can result in loss of vision due to vitreous hemorrhage and/or traction retinal detachment due to contraction of fibrous tissue associated with new blood vessels.

Therefore, inhibitors of vascular endothelial growth factor receptors can be used to treat diseases such as cancer, macular degeneration and retinopathy. In view of this, there is a need in the industry for an inhibitor that is effective in inhibiting vascular endothelial growth factor receptors.

The present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof: Wherein X is NH or O; Y is NH or O; Z is an aryl group or a heteroaryl group containing nitrogen, oxygen, phosphorus or sulfur; and R ₁ is H, a halogen group or a C ₁ to C ₆ alkoxy group. .

The invention further provides a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

The invention further provides the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, for the manufacture of a pharmaceutical composition for treating a cell proliferative disorder in a subject in need thereof.

The above and other objects, features, and advantages of the invention will be apparent from

The compound of the formula (I) of the present invention or a pharmaceutically acceptable salt thereof will be described in detail below. The invention will be followed by a number of different embodiments to implement different features of the invention. It is to be understood that the various embodiments of the invention are set forth in the various embodiments of the invention, and are not intended to limit the scope of the invention.

Unless otherwise defined, "substitution" as used herein means that one or more hydrogen atoms on a group are independently substituted with a substituent including, but not limited to, -F, -Cl, -Br. , -I, -OH, protecting hydroxy, -NO ₂ , -SH, -CN, C ₁ -C ₁₂ alkyl optionally substituted by halogen, C ₂ -C ₁₂ aliphatic alkenyl optionally substituted by halogen, A halogen-substituted C ₂ -C ₁₂ alkynyl group, an amine group, a protected amine group.

The invention provides a compound of formula (I):

Wherein X is NH or O; Y is NH or O. Z may be a substituted or unsubstituted aryl group or a substituted or unsubstituted heteroaryl group containing nitrogen, oxygen, phosphorus or sulfur. For example, in an embodiment of the invention, the substituted or unsubstituted aryl group can be phenyl, naphthyl, anthracenyl, fluorenyl or indanyl. Further, the substituted or unsubstituted heteroaryl group containing nitrogen, oxygen, phosphorus or sulfur may be pyridyl, quinolyl, isoquinolyl, indolyl, tetrazolyl, furyl, thiazolyl, imidazolyl, Imidazo[1,2-a]pyrimidinyl, pyrazolyl, oxazolyl, oxadiazolyl, thiophenyl, 1,2,4-triazolyl, isoxazolyl, thienyl , pyrazinyl, pyrimidinyl, [1,2,3]triazolyl, isothiazolyl, imidazo[2,1-b]thiazolyl, benzimidazolyl, benzofuranyl or benzofuranyl . Further, R ₁ may be H, a halogen group, or a linear or branched alkoxy group of C ₁ to C ₆ . For example, in an embodiment of the present invention, the halogen group includes fluorine, chlorine, bromine or iodine, and the linear or branched alkoxy group of C ₁ to C ₆ may be, for example, a methoxy group, an ethoxy group or a propoxy group. Base, butoxy and tert-butoxy.

In some embodiments, X is NH and Y is NH. In other embodiments, X is O and Y is NH. In still other embodiments, X is NH and Y is O. In still other embodiments, X is O and Y is O.

Additionally, in some embodiments, the compound is:

Wherein Z may be a substituted or unsubstituted aryl group or a substituted or unsubstituted heteroaryl group containing nitrogen, oxygen, phosphorus or sulfur. And R ₂ may be H, a halogen group, or a linear or branched alkoxy group of C ₁ to C ₆ .

Some specific examples of the compounds of the present invention are listed below, but are not limited thereto:

In addition, the invention also provides pharmaceutically acceptable compounds of formula (I) The salt, which is water or oil soluble or dispersible, is suitable for treating diseases without undue toxicity, irritation and allergic response. For example, such pharmaceutically acceptable salts include, but are not limited to, non-toxic acid addition salts, salts of amine groups, and inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or organic acids, For example, acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid is added or prepared by other methods in the art, such as ion exchange. Other pharmaceutically acceptable salts, including but not limited to: hexanoate, alginate, ascorbate, aspartate, besylate, benzoate, hydrogen sulfate, borate, butyrate , camphorate, camphor Acid salts, citrates, cyclopentane propionates, digluconates, lauryl sulfates, ethanesulfonates, formates, fumarates, glucohesates, glycerol phosphates, Oxalate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurate, malate, Maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinic acid salt, nitrate, oleate, oxalate, palmitate, pamoate, ALT, persulfate, 3-phenylpropionate, phosphate, picrate, trimethylacetate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate Acid salt, p-toluenesulfonate, eleven carbonate, pentane salt and the like. Representative alkali or alkaline earth metal salts include: sodium, lithium, potassium, calcium, magnesium, and the like. Other pharmaceutically acceptable salts, including the appropriate use of counterions such as chlorides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyl groups having from 1 to 6 carbon atoms, sulfonates and aryl groups Sulfonic acid, non-toxic ammonium, quaternary ammonium and amine cations formed.

The compound of the formula (I) or a pharmaceutically acceptable salt thereof has a molecular weight of from 200 to 2,000. For example, it is 300 to 1,000.

Further, according to an embodiment of the present invention, the compound of the formula (I) can be obtained by an addition reaction of the precursors of the formula (II) and the formula (III):

Wherein A ₁ is a halogen group; A ₂ and A ₃ each independently comprise H, or a linear or branched alkoxy group of C ₁ to C ₆ , B is boron, and X, Y, Z have the same formula ( I). This reaction can be carried out by using a catalyst such as tetrakis(triphenylphosphine)palladium (Pd(PPh ₃ ) ₄ ) at room temperature or under heating.

The present invention also uses a fluorescence assay to determine the inhibitory effect of a compound of formula (I). The kinase activity inhibition assay mainly uses DELFIA (dissociation enhanced lanthanide fluoroimmunoassay) system time-resolved fluorometry (TRF) to determine the inhibitory effect of the compound of formula (I). First, the kinase, the acceptor peptide, and the ATP were reacted, and the amount of the peptide that was phosphorylated by the kinase was measured using a spectrometer. Next, in another set of experiments, the compound of formula (I) was added to the kinase, the acceptor peptide and the ATP reaction, and then the amount of the peptide that was phosphorylated by the kinase was also measured using a spectrometer. Comparing the above two tests, the inhibitory effect of the compound of the formula (I) on the kinase activity can be known. In an embodiment of the invention, the compound of formula (I) may have an inhibitory effect on VEGFR2 of from about 60% to about 90%, and the compound of formula (I) may have an inhibitory effect on VEGFR3 of from about 30% to about 95%. .

Unless otherwise specified, the "effective amount" as used in this specification is defined to be necessary to bring the above effects to the individual when administered to an individual in need thereof. The amount of the compound of formula (I).

For therapeutic use, when a therapeutically effective amount of a compound of formula (I) And when the therapeutically acceptable salt can be administered as the original chemical, the active ingredient can be presented as a pharmaceutical composition. Accordingly, the present invention further provides a pharmaceutical composition for inhibiting vascular endothelial growth factor receptor (VEGF receptor) and protein tyrosine kinase, which comprises the compound of formula (I) or a pharmaceutically acceptable salt thereof as effective Ingredients; and a pharmaceutically acceptable carrier. The compound of the formula (I) or a pharmaceutically acceptable salt thereof constitutes from 0.1% by weight to 99.9% by weight of the pharmaceutical composition. Further, an effective amount of the compound of the formula (I) or a pharmaceutically acceptable salt thereof ranges from about 0.001 mg/kg to about 1000 mg/kg per day, for example, in one embodiment, an effective amount is about 0.01 mg per day. /Kg to about 0.09 mg/Kg per day, and in another embodiment, an effective amount is from about 200 mg/kg to about 900 mg/kg per day. However, as will be appreciated by those skilled in the art, the effective amount will vary depending on the type of effect, the route of administration, the application of the excipient, and the likelihood of being combined with other therapeutic means.

This pharmaceutically acceptable carrier includes any and all solvents, dispersions Medium, film, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. are compatible with pharmaceutical administration. For example, in some embodiments, the pharmaceutically acceptable carrier can be, for example, a saccharide such as lactose, glucose, and sucrose, a cyclodextrin such as alpha, beta, gamma-cyclodextrin; a starch such as corn starch and potato starch; Cellulose and its derivatives, for example, sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered gum tragacanth; malt; gelatin or talc: additionally, for liquid formulations, pharmaceutically acceptable The acceptable carrier can be an aqueous or non-aqueous solution, suspension, emulsion or oil. The non-aqueous solvent can be, for example, propylene glycol, polyethylene glycol, and an injectable organic ester such as ethyl oleate. The aqueous carrier can be, for example, water, an alcoholic/aqueous solution, an emulsion or a suspension Floating fluid, including brine and buffered media. The oil which can be used as a carrier can be petroleum, animal oil, vegetable oil or synthetic oil such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil and cod liver oil.

The pharmaceutical composition formulation can be presented in unit dosage form per unit dose Contains a predetermined amount of active ingredient. Such a unit may contain, for example, 0.5 mg to 1 g, preferably 1 mg to 700 mg, more preferably 5 mg to 100 mg of the compound of formula (I), depending on the condition being treated, the severity of the symptoms, the time of administration, The route of administration, the rate of excretion of the compound employed, the duration of treatment and the age, sex, weight and symptoms of the patient, or the pharmaceutical formulation may be presented in unit dosage form, each containing a predetermined amount of active ingredient. Further, such pharmaceutical formulations can be made by any of the methods conventional in the art of pharmacy.

The pharmaceutical composition can be administered by any suitable route, for example by the oral cavity (including cheek or sublingual), rectal, nasal, topical (including cheek, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) routes. The pharmaceutical composition can be made by any method known in the art of pharmacy, for example, by bringing the active ingredient into association with a carrier or excipient. Furthermore, the compounds of the invention may be administered using conventional drug delivery techniques, such as intra-arterial stents.

Suitable for oral administration of pharmaceutical compositions, which can be presented as discrete units. For example, capsules, tablets, powders, granules, solutions or suspensions in aqueous or non-aqueous liquids, edible foams or blistering creams, or oil-based liquid emulsions or water-in-oil emulsions.

For example, for oral administration in the form of tablets or capsules, The active pharmaceutical ingredient is combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. The powder is pulverized to a suitable fine size by It is prepared by mixing with a comminuted pharmaceutical carrier which may be a carbohydrate for consumption, such as starch or mannitol. The pharmaceutical composition may also contain a flavoring, preservative, dispersing or coloring agent.

The capsule is filled with the above-mentioned powder mixture to the gelatin which has been formed. Made of outer casing. Glidants and lubricants, such as colloidal cerium oxide, talc, magnesium stearate, calcium stearate or solid polyethylene glycol, can be added to the powder mixture prior to filling operations. A disintegrant or a solubilizing agent such as agar, calcium carbonate or sodium carbonate may also be added to improve the availability of the agent when the capsule is ingested.

In addition, the present invention also provides a compound of formula (I) or a pharmaceutically acceptable compound thereof For the use of the salt, it can be used to prepare a pharmaceutical composition for treating a cell proliferative disorder in a subject in need thereof. Such cell proliferative disorders include: papilloma, blastoglioma, Kaposi's sarcoma, melanoma, non-small cell lung cancer, ovarian cancer, prostate cancer, squamous cell tumor, astrocytoma, Head, neck, bladder, breast, lung, colorectal, thyroid, pancreatic, stomach, liver, leukemia, lymphoma, Hodgkin's disease, Burkitt's Disease), or macular degeneration of the eye.

In summary, the present invention provides a compound of formula (I) or a pharmaceutical thereof An acceptable salt and use thereof, and a pharmaceutical composition comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof. The compound of the formula (I) or a pharmaceutically acceptable salt thereof can be used as a VEGF-2/3 receptor and a protein tyrosine kinase inhibitor, and the pharmaceutical composition can be used to treat a cell proliferative disorder.

[Example 1]

The precursor of formula (a1) (600 mg, 4 mmol) and hydrazine (hydrazine, 869.7 mg, 4 mmol) were dissolved in dichloromethane (12 ml), and nitrogen was passed through the reaction flask, followed by reaction at room temperature. After an hour, the solvent was drained and the solid was washed with n-hexane for 1 hour, and filtered to give 1.27 g of intermediate product (white solid, yield 86.3%).

Next, the above intermediate product (white solid, 2.27 g, 9.2 mmol) was mixed with sodium hydrogen carbonate (1.14 g, 13.57 mmol) and ethanol (20 ml) at room temperature, and the mixture was refluxed for 24 hours. After cooling to room temperature, the reaction was quenched by the addition of water, followed by stirring in an ice bath for 2 hr and then filtered to afford 1.96 g of the compound of formula (a2) (yellow solid, yield 81%).

After dissolving the compound of the formula (a3) (1 g, 4.56 mmol) in 20 ml of dichloromethane (dichloromethane, DCM), nitrogen was added to the reaction flask, and the compound of the formula (a4) (1.1 ml, 5.15 mmol) was added. Then, 3 ml of triethylamine (TEA, 21.5 mmol) was further added and reacted at room temperature for 20 hours, followed by the addition of water to terminate the reaction. The solution from which the reaction was terminated was poured into a separatory funnel to remove the aqueous layer in the solution, and the organic layer was left. The organic layer was extracted with a saturated aqueous solution of sodium hydrogencarbonate, water and brine, and then the organic layer was evaporated over anhydrous magnesium sulfate. The product obtained by extracting the organic solvent is purified by column chromatography, which is washed with n-hexane and ethyl acetate (percentage of n-hexane: ethyl acetate = 5:1). 1 g of the compound of the formula (a5) was obtained. (Yellow solid, yield 65%).

The compound of the formula (a5) (900 mg, 2.67 mmol) and the compound of the formula (a2) (741.4 mg, 2.86 mmol) and sodium carbonate (848.6 mg, 8 mmol) were placed in a round bottom flask, and toluene (15 ml) and ethanol were added. (15 ml) and water (15 ml) were added to a round bottom flask to remove oxygen, and then tetrakis(triphenylphosphine)palladium (Pd(PPh ₃ ) ₄ , 154.2 mg, 0.13 mmol) was added and refluxed for 16 hours. Then, it was diluted with ethyl acetate. This diluted solution of ethyl acetate was poured into a separatory funnel to remove the aqueous layer from the solution, leaving an organic layer. The organic layer was extracted with a saturated aqueous solution of sodium hydrogencarbonate, water and brine, and then the organic layer was evaporated over anhydrous magnesium sulfate. The product obtained by extracting the organic solvent is purified by column chromatography, and the purification is carried out by using methanol and dichloromethane (volume percentage: methanol: dichloromethane = 1:40) to obtain the formula (a6). The compound was 700 mg (brown solid, yield 72%).

The compound of the formula (a6) was subjected to mass spectrometry, and the measured mass to mass ratio was 342 (m/z). ¹ H NMR analysis of the compound of the formula (a6), ¹ H NMR (DMSO-d ₆ ) 11.73 (1H, bs), 10.83 (1H, s), 7.75 (2H, d), 7.42-7.31 (6H) , m), 7.29-7.23 (3H, m), 6.80 (1H, m), 4.31 (2H, bs), 3.68 (2H, s).

[Example 2]

After dissolving the compound of the formula (b3) (2.49 g, 10 mmol) in dichloromethane (dichloromethane, DCM, 30 ml), nitrogen was added to the reaction flask, and the compound of the formula (b4) (1.65 ml, 12.5 mmol) was added. Further, triethylamine (TEA, 3.5 ml, 25 mmol) was added and reacted at room temperature for 20 hours, followed by the addition of water to terminate the reaction. Pour the solution that has terminated the reaction into a separatory funnel and remove The aqueous layer in the solution leaves an organic layer. The organic layer was extracted with a saturated aqueous solution of sodium hydrogencarbonate, water and brine, and then the organic layer was evaporated over anhydrous magnesium sulfate. The product obtained by extracting the organic solvent is purified by column chromatography, which is washed with n-hexane and ethyl acetate (percentage of n-hexane: ethyl acetate = 5:1). 2.1 g of the compound of the formula (b5) (yellow solid, yield 57.2%) was obtained.

The compound of formula (b5) (1.84 g, 5 mmol) and the compound of formula (a2) (1.42 g, 5.5 mmol, as described in Example 1) and sodium carbonate (1.7 g, 16 mmol) were placed in a round bottom bottle. Toluene (30 ml), ethanol (30 ml) and water (30 ml) were added, and after nitrogen was purged into a round bottom bottle to remove oxygen, tetrakis(triphenylphosphine)palladium (Pd(PPh ₃ ) ₄ , 290 mg, 0.25 was added. After mmol), it was refluxed for 16 hours. Then, it was diluted with ethyl acetate. This diluted solution of ethyl acetate was poured into a separatory funnel to remove the aqueous layer from the solution, leaving an organic layer. The organic layer was extracted with a saturated aqueous solution of sodium hydrogencarbonate, water and brine, and then the organic layer was evaporated over anhydrous magnesium sulfate. The product obtained by extracting the organic solvent is purified by column chromatography, and the purification is carried out by using methanol and dichloromethane (volume percentage: methanol: dichloromethane = 1:40) to obtain the formula (b6). The compound was 980 mg (brown solid, yield 52.6%).

The compound of the formula (b6) was subjected to mass spectrometry, and the measured mass-to-mass ratio was 372 (m/z). ¹ H NMR analysis of the compound of the formula (b6), ¹ H NMR (DMSO-d ₆ ) 11.74 (1H, bs), 10.31 (1H, s), 7.58 (2H, d), 7.41-7.29 (5H) , m), 7.25-7.16 (3H, m), 6.80-6.76 (1H, m), 4.31 (2H, bs), 3.81 (3H, s), 3.68 (2H, s).

[Example 3]

After dissolving the compound of the formula (c3) (2.53 g, 10 mmol) in dichloromethane (dichloromethane, DCM, 30 ml), nitrogen was added to the reaction flask, and the compound of the formula (c4) (1.8 ml, 12.5 mmol) was added. Further, triethylamine (TEA, 3.5 ml, 25 mmol) was added and reacted at room temperature for 20 hours, followed by the addition of water to terminate the reaction. The solution from which the reaction was terminated was poured into a separatory funnel to remove the aqueous layer in the solution, leaving an organic layer. The organic layer was extracted with a saturated aqueous solution of sodium hydrogencarbonate, water and brine, and then the organic layer was evaporated over anhydrous magnesium sulfate. The product obtained by extracting the organic solvent is purified by column chromatography, which is washed with n-hexane and ethyl acetate (percentage of n-hexane: ethyl acetate = 5:1). 1.7 g of the compound of the formula (c5) (yellow solid, yield: 43.6%) were obtained.

The compound of formula (c5) (1.5 g, 3.89 mmol) and the compound of formula (a2) (1.04 g, 4 mmol, as described in Example 1) and sodium carbonate (1.33 g, 12.5 mmol) were placed in a round bottom bottle. Toluene (25 ml), ethanol (25 ml) and water (25 ml) were added, and nitrogen gas was introduced into a round bottom flask to remove oxygen, and then tetrakis(triphenylphosphine)palladium (Pd(PPh ₃ ) ₄ , 290 mg, was added. After 0.25 mmol), it was refluxed for 16 hours. Then, it was diluted with ethyl acetate. The solution diluted with ethyl acetate was poured into a separatory funnel to remove the aqueous layer from the solution, and the organic layer was left. The organic layer was extracted with a saturated aqueous solution of sodium hydrogencarbonate, water and brine, and then the organic layer was evaporated over anhydrous magnesium sulfate. The product obtained by extracting the organic solvent is purified by column chromatography, and the purification is carried out by using methanol and dichloromethane (volume percentage: methanol: dichloromethane = 1:40) to obtain the formula (c6). Compound 920 mg (brown solid, yield 60.5%).

The compound of the formula (c6) was subjected to mass spectrometry, and the measured mass-to-mass ratio was 390.1 (m/z). ¹ H NMR analysis of the compound of formula (c6), ¹ H NMR (DMSO-d ₆ ) 11.66 (1H, bs), 10.81 (1H, s), 7.73 (2H, d), 7.51-7.24 (5H) , m), 7.19-7.13 (2H, m), 6.70 (1H, m), 4.31 (2H, bs), 3.62 (2H, s), 2.28 (3H, s).

[Kinase Activity Inhibition Experiment 1 - Determination of the inhibitory effect of compounds of formula (a6, b6 and c6) on VEGFR2]

The following experiment can determine the inhibitory effect of the compounds of the formulae (a6, b6 and c6) in Examples 1-3 on VEGFR2. First, 10 μL of ATP (10 mM, purchased from cell signaling, product number 9804) was added to 0.625 mL of biotinylated receptor peptide (12 μM, available from ANASPEC, product number 60329-1) to form a mixture. This receiving quality biotinylated gastrin peptide is (Gastrin) precursor of biotinylated peptides (biotinylated peptide, sequence: Biotin-EGPWLEEEEEAYGWMDF-NH _2, having Tyr87 as phosphorylation site). Next, this mixture was diluted to 2.5 mL with deionized water to prepare a 2X ATP/biotinylated receptor peptide mixture ([ATP] = 40 μM, [Biotinylated receptor peptide] = 3 μM).

KDR (VEGFR2) kinase was immediately transferred from -80 °C to ice. KDR (VEGFR2) kinase was thawed on ice. A slight microcentrifugation at 4 ° C allowed the liquid to settle in the bottom of the small tube and the KDR (VEGFR2) kinase was immediately returned to the ice.

10 μL of DTT (1.25 M, purchased from cell signaling) was added to 2.5 mL of 4X HTScanR tyrosine kinase buffer (240 mM HEPES pH 7.5, 20 mM MgCl ₂ , 20 mM MnCl ₂ , 12 μM Na ₃ VO ₄ , purchased from cell signaling, Product No. 9805) to formulate DTT/kinase buffer. Next, 100 μL of KDR (VEGFR2) kinase (100 ng/μL, purchased from Uptate, product number 14-630M) was added to 1.25 mL of DTT/kinase buffer to prepare a 4X reaction mixture (this 4X reaction mixture [ KDR kinase] = 8 ng / μL). Next, 12.5 μL of the 4X reaction mixture was incubated with 12.5 μL/well of the pre-diluted compound of formula (a6, b6 and c6) (usually about 10 μM) for 5 minutes at room temperature.

Next, 25 μL of 2X ATP/biotinylated receptor peptide mixture was added to 25 μL/well of the pre-incubation reaction mixture/compound (the inhibition conditions of the compounds of formula (a6, b6 and c6) at this time were determined to be 60 mM HEPES ( pH 7.5), 5 mM MgCl ₂ , 5 mM MnCl ₂ , 20 μM ATP, 1.25 mM DTT, 3 μM Na ₃ VO ₄ , 1.5 μM biotinylated receptor peptide and 100 ng KDR (VEGFR2) kinase). The reaction plates were incubated for 30 minutes at room temperature. Next, 50 μL/well of stop buffer (50 mM EDTA, pH 8) was added to stop the reaction.

25 μL of each reaction solution that has stopped reacting and 75 μL of separation The sub-water/well was transferred to a 96 well coated streptavidin plate (purchased from PerkinElmer Life Sciences) and incubated for 60 minutes at room temperature. It was then washed 3 times with 200 μL/well of PBS/T.

Dilute primary anti-stain with PBS/T containing 1% bovine serum albumin (BSA) The primary antibody was phosphorylated tyrosine mAb (P-Tyr-100) (purchased from cell signaling, product number 9411), and the diluted concentration was primary antibody: PBS/T = 1 volume: 1000 volumes. 100 μL/well of primary antibody was then added and incubated for 60 minutes at room temperature. It was then washed 3 times with 200 μL/well PBS/T.

Labeled with PBS/T diluted with 1% bovine serum albumin (BSA) Secondary antibody (Europium labeled secondary antibody), this secondary antibody is anti-mouse IgG (purchased from PerkinElmer Life Sciences) or anti-rabbit IgG (purchased from PerkinElmer Life Sciences). The diluted concentration was anti-mouse IgG: PBS/T = 1 volume: 500 volumes, or anti-rabbit IgG: PBS / T = 1 volume: 1000 volumes. 100 μL/well of diluted secondary antibody was then added and incubated for 30 minutes at room temperature. It was then washed 5 times with 200 mL/well PBS/T.

Next, add 100 μL / well DELFIA enhancement solution (purchased from PerkinElmer Life Sciences, product number 1244-105), and incubated for 5 minutes at room temperature. Next, the 615 nm fluorescence emitted by the secondary antibody was detected by a time-resolved fluorescent plate reader (SpectraMax M5 Microplate Reader) (excitation light was 340 nm, and retardation was 400 ms). The measured fluorescence intensity represents the amount of biotinylated receptor peptide phosphorylated by KDR (VEGFR2) kinase. Comparing the amount of fluorescence measured by the control group to which the compound of the formula (a6, b6, and c6) was not added, and the amount of fluorescence measured by the experimental group of the above-mentioned compound of the formula (a6, b6, and c6), that is, The inhibitory effect of the compounds of the formula (a6, b6 and c6) on the activity of KDR (VEGFR2) kinase, that is, [(the amount of fluorescence measured in the control group) - (the amount of fluorescence measured by the experimental group)] / ( The amount of fluorescence measured in the control group = the amount of biotinylated receptor peptide that was not phosphorylated by the inhibitory effect of the compound of formula (a6, b6, and c6) / the total amount of biotinylated receptor peptide. Table 1 shows the inhibitory effects on the KDR (VEGFR2) kinase activity measured by the above experiments on the compounds of the formulas (a6, b6 and c6) of 1 μM and 0.1 μM.

[Kinase Activity Inhibition Experiment 2 - Determination of the inhibitory effect of compounds of formula (a6, b6 and c6) on VEGFR3]

The manner of measuring the inhibitory effect of the compound of the formula (a6, b6 and c6) in Example 1-3 on VEGFR3 was similar to the experiment of the above kinase activity inhibition experiment 1. However, this experiment used 100 ng/μL of VEGFR3 kinase (purchased from Uptate, product number 14-681M), and the concentration of this VEGFR3 kinase in the 4X reaction mixture was 20 ng/μL. In addition, the biotinylated receptor peptide used in this experiment was Bioein-MH10 (purchased from ANASPEC), the sequence of which was Biotin-MYDKEYYSVH-NH ₂ , and its initial use amount was 1.042 μL (12 μM). In the assay conditions the inhibitory effect of the compounds of this experiment of formula (a6, b6 and c6) is of 60mM HEPES (pH7.5), 5mM MgCl 2, 5mM MnCl 2, 20μM ATP, 1.25mM DTT, 3μM Na 3 VO 4, 2.5 μM biotinylated receptor peptide and 250 ng VEGFR3 kinase. Table 2 shows the inhibitory effect on the VEGFR3 (flt-4) kinase activity measured by the above experiments on the compounds of the formulas (a6, b6 and c6) of 1 μM and 0.1 μM:

Although the embodiments of the present invention and its advantages are disclosed above, it should be understood that those skilled in the art can make modifications, substitutions, and refinements without departing from the spirit and scope of the invention. In addition, the scope of the present invention is not limited to the processes, machines, manufacture, compositions, devices, methods, and steps in the specific embodiments described in the specification. Any one of ordinary skill in the art can. The processes, machines, fabrications, compositions, devices, methods, and procedures that are presently or in the future are understood to be used in accordance with the present invention as long as they can perform substantially the same function or achieve substantially the same results in the embodiments described herein. Accordingly, the scope of the invention includes the above-described processes, machines, manufactures, compositions, devices, methods, and steps. In addition, the scope of each of the claims constitutes an individual embodiment, and the scope of the invention also includes the combination of the scope of the application and the embodiments.

Claims (11)

a compound of formula (I) or a pharmaceutically acceptable salt thereof: Wherein X is NH or O; Y is NH or O; Z is an aryl group or a heteroaryl group containing nitrogen, oxygen, phosphorus or sulfur; and R ₁ is H, a halogen group or a C ₁ to C ₆ alkoxy group. . The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound or a pharmaceutically acceptable salt thereof has a molecular weight of from 300 to 1,000. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein X is NH and Y is NH. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein X is O and Y is NH. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein X is NH and Y is O. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein X is O and Y is O. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is: The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is: Wherein Z is an aryl group or a heteroaryl group containing nitrogen, oxygen, phosphorus or sulfur; and R ₂ is H, a halogen group or a C ₁ to C ₆ alkoxy group. A pharmaceutical composition for inhibiting vascular endothelial growth factor receptor (VEGF receptor) and protein tyrosine kinase, comprising: a compound of any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof as an active ingredient; and a pharmaceutically acceptable carrier. A use of a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical composition for treating a cell proliferative disorder in a subject in need thereof. The use according to claim 10, wherein the cell proliferative disorder comprises: papilloma, blastoglioma, Kaposi's sarcoma, melanoma, non-small cell lung cancer, ovarian cancer, Prostate cancer, squamous cell tumor, astrocytoma, head cancer, neck cancer, bladder cancer, breast cancer, lung cancer, colorectal cancer, thyroid cancer, pancreatic cancer, stomach cancer, liver cancer, leukemia, lymphoma, Hodgkin Hodgkin's disease, Burkitt's disease, or ocular macular degeneration.