A quinoline compound, preparation method thereof, pharmaceutical composition and medical use thereof

Abstract

The present invention belongs to the field of medicine, and specifically relates to a compound represented by formula (I), a pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer, solvate or polymorph thereof; and also relates to a method for preparing a compound, a pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer, solvate or polymorph thereof, a pharmaceutical composition comprising the above compound, a pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer, solvate or polymorph thereof, and their medical uses. The compounds of the present invention can block the VEGF / VEGFR pathway, and can prevent or treat diseases related to the VEGF / VEGFR pathway, especially tumor diseases.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to quinoline compounds, pharmaceutically acceptable salts, prodrugs, stable isotope derivatives, isomers, solvates or polymorphs thereof, and also relates to preparation methods, pharmaceutical compositions and medical uses of the above compounds. Background Art

[0002] New blood vessel formation is divided into angiogenesis and angiogenesis. The former refers to the process of angioblasts differentiating into blood cells and mature endothelial cells to form new blood vessels during embryonic development, and the latter refers to the process of existing capillaries sprouting to form new blood vessels. New blood vessel formation mainly occurs in physiological and pathological processes such as embryonic development, wound healing, ovarian corpus luteum formation, and tumors. In 1971, Judah Folkman first proposed that angiogenesis is an important process for the growth and proliferation of solid tumors. When the tumor diameter reaches about 0.2-2.0mm, it will be hypoxic. In the absence of angiogenesis, the tumor volume will be limited. Therefore, targeting tumor angiogenesis is an effective treatment for various cancers.

[0003] Angiogenesis is controlled and regulated by endogenous active factors and a variety of proteins. There are about 30 known endogenous pro-angiogenic factors and about 30 known endogenous anti-angiogenic factors. Among them, the endothelial vascular growth factor and its receptor (VEGF / VEGFR) family are the most studied pro-angiogenic regulatory factors, which have the function of inducing endothelial cell differentiation and angiogenesis. Vascular endothelial growth factor (VEGF) belongs to the homodimeric glycoprotein family and is essential for angiogenesis, lymphangiogenesis and pathological angiogenesis during embryonic development. VEGF is involved in regulating the occurrence and development of many diseases, such as tumor growth and metastasis, macular degeneration, diabetic retinopathy, rheumatoid arthritis, myocardial ischemia, preeclampsia, etc. Studies have found that there are five main receptors that VEGF ligands bind to: VEGFR-1, VEGFR-2, VEGFR-3, neuropilin-1 (NP-1) and neuropilin-2 (NP-2). Among them, three vascular endothelial growth factor receptors (VEGFR-1, VEGFR-2, and VEGFR-3) belong to receptor tyrosine kinases, which are mainly expressed in vascular and lymphatic endothelial cells and highly expressed in most tumor cells. After VEGF binds to VEGFR, it activates the tyrosine kinase in the intracellular domain of VEGFR and phosphorylates intracellular tyrosine residues, thereby activating the intracellular signal transduction pathway and playing a regulatory role. Therefore, inhibiting VEGFR activity and blocking its signal transduction can effectively inhibit angiogenesis inside the tumor, thereby achieving the effect of inhibiting tumor growth and metastasis and controlling tumor growth. As an important anti-tumor target, the FDA has approved 12 VEGFR small molecule inhibitors, and the NMPA has approved 3 VEGFR small molecule inhibitors independently developed in China.

[0004] VEGFR inhibitors are not effective in treating tumors due to the development of drug resistance. For example, patients with renal cell carcinoma often develop primary or acquired drug resistance within one year of treatment. Secondly, the VEGFR inhibitors on the market still have certain adverse reactions. Therefore, it is still necessary to develop the next generation of drugs to overcome drug resistance and reduce adverse reactions. Summary of the invention

[0005] The purpose of the present invention is to provide a new type of small molecule compound that acts on VEGFR, which can block the VEGF / VEGFR pathway and can prevent or treat diseases related to the VEGF / VEGFR pathway, such as having a potent angiogenesis inhibitory effect and anti-tumor activity, and has an improving, preventing and treating effect on various other diseases accompanied by abnormal proliferation of new blood vessel formation.

[0006] The present inventors have found through research that the compound having the following general formula (I) can act on the VEGF / VEGFR pathway, significantly inhibit the activity of protein kinases such as VEGF, and has a significant anti-tumor effect.

[0007] The first aspect of the present invention relates to a compound represented by formula (I), a pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer, solvate or polymorph thereof;

[0008]

[0009] in,

[0010] R 1 and R 2 Each is independently selected from hydroxy, aminoalkanoyl, alkoxy, alkylaminocarbonyl;

[0011] R 3 Selected from -(NR 5 ) n1 -C(O)-(NR 6 ) n2 -, R 5 and R 6 Each is independently selected from H, alkyl; n1 and n2 are each independently selected from 0, 1;

[0012] R 4 is selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl, cycloalkenyl, alkenyl, alkynyl, alkylaminocarbonyl, aminocarbonyl, cyano; the substitution is substituted by one or more of the following substituents: halogen, alkyl, alkoxy, haloalkyl, nitro, cycloalkyl, aryl. In any embodiment of the first aspect of the present invention, R 1 and R 2 Each independently selected from hydroxyl, amino C 1 -C 6 Alkanoyl, C 1 -C 6 Alkoxy, C 1 -C 6 Alkylaminocarbonyl.

[0013] In any embodiment of the first aspect of the present invention, R 1 and R 2 Each independently selected from amino C 1 -C 6 Alkanoyl, C 1 -C 6 Alkoxy.

[0014] In any embodiment of the first aspect of the present invention, R1 and R 2 Each is independently selected from carbamoyl and methoxy.

[0015] In any embodiment of the first aspect of the present invention, R 3 Selected from -(NR 5 ) n1 -C(O)-(NR 6 ) n2 -, R 5 and R 6 Each independently selected from H, C 1 -C 6 Alkyl; n1 and n2 are each independently selected from 0, 1.

[0016] In any embodiment of the first aspect of the present invention, R 3 Selected from -C(O)NH-, -NH-C(O)-NH-, -C(O)-, -C(O)N(CH 3 )-,-N(CH 3 )-C(O)-N(CH 3 )-.

[0017] In any embodiment of the first aspect of the present invention, R 3 Selected from -(NR 5 ) n1 -C(O)-(NR 6 ) n2 -, R 5 and R 6 is H; n1 and n2 are each independently selected from 0 and 1.

[0018] In any embodiment of the first aspect of the present invention, R 3 Selected from -C(O)NH-, -NH-C(O)-NH-,

[0019] In any embodiment of the first aspect of the present invention, R 4 Selected from substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted 5-10 membered aryl, substituted or unsubstituted 3-8 membered cycloalkyl, substituted or unsubstituted 5-10 membered heteroaryl, substituted or unsubstituted 5-10 membered heterocyclyl; the substitution is substituted by one or more of the following substituents: halogen, C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, halogenated C 1 -C 6Alkyl, nitro, 3-8 membered cycloalkyl, 5-10 membered aryl.

[0020] In any embodiment of the first aspect of the present invention, R 4 is selected from substituted or unsubstituted 5-10 membered aryl, substituted or unsubstituted 3-8 membered cycloalkyl; the substitution is substituted by one or more of the following substituents: halogen, C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy.

[0021] In any embodiment of the first aspect of the present invention, R 4 Selected from substituted or unsubstituted phenyl, substituted or unsubstituted cyclopropyl; the substitution is substituted by one or more of the following substituents: fluorine, methyl, methoxy.

[0022] In any embodiment of the first aspect of the present invention, R 4 Selected from Cyclopropyl,

[0023] In any embodiment of the first aspect of the present invention, the compound includes (but is not limited to):

[0024]

[0025] The second aspect of the present invention provides a method for preparing the compound of the first aspect of the present invention, its pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer, solvate or polymorph, which is the following method A or method B;

[0026] The method A comprises:

[0027] 4-nitronaphthylamine is subjected to diazotization reaction and substitution reaction to obtain intermediate A2;

[0028] Substitution reaction of intermediate A2 with 4-hydroxyquinoline to obtain intermediate A3;

[0029] Reducing the nitro group of intermediate A3 to obtain intermediate A4;

[0030] The intermediate A4 is subjected to different condensation reactions to obtain target products A5-A7 respectively;

[0031]

[0032] The method B comprises:

[0033] Substitution reaction of 4-nitronaphthol and 4-chloroquinoline was performed to obtain intermediate B2;

[0034] Reducing the nitro group of intermediate B2 to obtain intermediate B3;

[0035] The intermediate B3 is subjected to different condensation reactions to obtain the target products B4-B6 respectively;

[0036]

[0037] The above R 1 , R 2 and R 4 is as defined in the first aspect of the present invention.

[0038] The preparation method of the compound of the present invention includes (but is not limited to) the above method. In the specific operation process, the steps in the method can be expanded or combined as needed.

[0039] The third aspect of the present invention relates to a pharmaceutical composition comprising the compound of the first aspect of the present invention, its pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer, solvate or polymorph;

[0040] Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0041] In any embodiment of the third aspect of the present invention, the pharmaceutical composition further comprises other drugs and / or immunomodulators for treating tumors (such as immune checkpoint inhibitors, antibiotics, alkylating agents, antimetabolites, hormone drugs, immunoactive agents, interferon-type active agents and mixed active agents).

[0042] In any embodiment of the third aspect of the present invention, the other drugs for treating tumors are drugs for treating tumors other than the compounds of the first aspect of the present invention, their pharmaceutically acceptable salts, prodrugs, stable isotope derivatives, isomers, solvates and polymorphs.

[0043] The fourth aspect of the present invention relates to the use of the compound of the first aspect of the present invention, its pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer, solvate or polymorph, or the pharmaceutical composition of the third aspect of the present invention in the preparation of a medicament for preventing and / or treating diseases related to the VEGF / VEGFR pathway.

[0044] The fifth aspect of the present invention relates to a method for preventing and / or treating diseases related to the VEGF / VEGFR pathway, comprising administering an effective amount of the compound of the first aspect of the present invention, its pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer, solvate or polymorph, or an effective amount of the pharmaceutical composition of the third aspect of the present invention to a subject in need.

[0045] The compound of the first aspect of the present invention, its pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer, solvate or polymorph or the pharmaceutical composition of the third aspect of the present invention is used for preventing and / or treating diseases related to the VEGF / VEGFR pathway.

[0046] In any embodiment, the disease associated with the VEGF / VEGFR pathway is selected from one or more of atherosclerosis, pulmonary fibrosis, retinopathy, endometriosis, arthritis and cancer.

[0047] In any embodiment, the cancer is selected from one or more of lung cancer, kidney cancer, gastric cancer, hepatocellular carcinoma, thyroid cancer, melanoma, breast cancer, pancreatic cancer, colon cancer, rectal cancer, prostate cancer, bladder cancer, ovarian cancer, cervical cancer and glioma.

[0048] The foregoing compounds are listed below:

[0049]

[0050]

[0051]

[0052] The products, methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant personnel can modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention, and the resulting technical solutions are all included in the scope of protection required by the present invention.

[0053] The term "pharmaceutically acceptable salt" in the present invention includes a salt formed by a compound of the present invention with a pharmaceutically acceptable inorganic or organic acid or a basic salt formed with a pharmaceutically acceptable base. The acid salt includes, but is not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, hydrogen phosphate, acetate, propionate, butyrate, oxalate, trimethylacetate, adipate, alginate, lactate, citrate, tartrate, succinate, maleate, fumarate, picrate, aspartate, gluconate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate; the basic salt includes, but is not limited to, ammonium salt, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, organic base salts such as dicyclohexylamine and N-methyl-D-glucosamine salts, and amino acid salts such as arginine and lysine salts.

[0054] The term "base" in the present invention is generally any substance known as a base in organic synthesis, and is not particularly limited, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, sodium hydride, potassium hydride, sodium tert-butoxide, potassium tert-butoxide, cesium carbonate, triethylamine, trimethylamine, N-methylmorpholine, N,N-dimethylaniline, pyridine, isoquinoline, potassium hydroxide, sodium hydroxide, sodium methoxide, potassium methoxide, etc.

[0055] The term "alkyl" in the present invention refers to a hydrocarbon group formed by removing a hydrogen atom from an alkane molecule. 1-6 Alkyl, C 1-4 Alkyl, C 1-2 Alkyl; specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, and the like.

[0056] The term "alkoxy" in the present invention refers to a group provided in the form of alkyl-O-, wherein "alkyl" is as defined above. 1-6 Alkoxy, C 1-4 Alkoxy, C 1-2 Alkoxy; specific examples include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, and the like.

[0057] The term "alkoxycarbonyl" in the present invention refers to a group provided in the form of alkoxy-C(O)-, wherein "alkoxy" is as defined above. 1-6 Alkoxycarbonyl, C 1-4 Alkoxycarbonyl, C 1-2 Alkoxycarbonyl; specific examples include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl, n-butoxycarbonyl and the like.

[0058] The term "alkanoyl" in the present invention refers to a group formed by removing the hydroxyl group from a saturated fatty acid containing a single carboxyl group. 1-6 Alkanoyl, C 1-4 Alkanoyl, C 1-2 Alkanoyl; specific examples include, but are not limited to, formyl, acetyl, n-propionyl, isopropionyl, and the like.

[0059] The term "aminoalkanoyl" in the present invention is NH 2 -alkanoyl-, wherein "alkanoyl" is as defined above. 1-6 Alkanoyl, amino C 1-4 Alkanoyl, amino C 1-2 Alkanoyl; specific examples include, but are not limited to, carbamoyl, aminoacetyl, amino-n-propionyl, amino-isopropionyl, amino-n-butyryl, and the like.

[0060] The term "alkylaminoacyl" in the present invention refers to a group provided in the form of alkyl-NH-C(O)-, wherein "alkyl" is as defined above. 1-6 Alkyl, C 1-4 Alkyl, C 2-6 Alkyl, C 1-2 Alkyl; specific examples include, but are not limited to, methylaminoacyl, ethylaminoacyl, n-propylaminoacyl, isopropylaminoacyl, n-butylaminoacyl and the like.

[0061] The term "aryl" in the present invention refers to a monocyclic or condensed ring group having aromaticity, such as a 5-10-membered aryl, a 5-8-membered aryl, a 5-6-membered aryl; specific examples include but are not limited to phenyl, naphthyl, anthracenyl, phenanthrenyl, etc.

[0062] The term "heteroaryl" in the present invention refers to a monocyclic or condensed ring group with aromaticity containing at least one N, O or S heteroatom, such as 5-10 membered heteroaryl, 5-8 membered heteroaryl, 5-7 membered heteroaryl, 5-6 membered heteroaryl; specific examples include but are not limited to porphyrinyl, pyrazolyl, pyrrolyl, thiazolyl, pyridyl, imidazolyl, quinolyl, etc.

[0063] The term "heterocyclic group" in the present invention refers to a monocyclic or polycyclic saturated hydrocarbon group containing at least one heteroatom of N, O or S, such as a 5-10-membered heterocyclic group, a 5-8-membered heterocyclic group, a 5-7-membered heterocyclic group, a 5-6-membered heterocyclic group; specific examples include but are not limited to piperazinyl, morpholinyl, etc.

[0064] The term "cycloalkyl" in the present invention refers to a group formed by removing a hydrogen atom from a monocyclic saturated alkane ring, such as a 3-6-membered cycloalkyl group, a 5-6-membered cycloalkyl group; specific examples include but are not limited to cyclobutane, cyclopentane, cyclohexane, etc.

[0065] The term "halogen" in the present invention refers to fluorine, chlorine, bromine, iodine, astatine and the like.

[0066] The term "haloalkyl" in the present invention refers to a group formed by replacing at least one hydrogen atom in an alkyl group with a halogen, wherein "alkyl" is as defined above.

[0067] The "pharmaceutically acceptable carrier" in the present invention includes, but is not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum proteins, buffer substances such as phosphates, glycerol, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, beeswax, polyethylene-polyoxypropylene block polymers and lanolin.

[0068] The compound of the present invention, its pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer, solvate or polymorph or the pharmaceutical composition of the present invention has systemic and / or local effects, therefore, it can be administered by a suitable route, such as oral, parenteral, pulmonary, nasal, sublingual, lingual, buccal, rectal, transdermal, conjunctival, topical administration or administration in the form of an implant. It can be administered in an administration form suitable for these administration routes.

[0069] Suitable for oral administration are known administration forms which deliver the active ingredient rapidly and / or in a modified manner, such as tablets (uncoated or coated, such as tablets with enteric coating or film coating), capsules, dragees, granules, pellets, powders, emulsions, suspensions and aerosols.

[0070] With parenteral administration it is possible to avoid the absorption step (intravenous, intraarterial, intracardiac, intraspinal or intralumbar administration) or to include the absorption (intramuscular, subcutaneous, intradermal, transdermal or intraperitoneal administration). Suitable administration forms for parenteral administration are, in particular, preparations in the form of solutions, suspensions, emulsions, lyophilizates and sterile powders for injection and infusion.

[0071] Suitable other routes of administration include, for example, drugs for inhalation (particularly powder inhalation, spray), nasal drops / solutions, sprays; tablets or capsules for tongue, sublingual or buccal administration, suppositories, preparations for ears and eyes, vaginal capsules, aqueous suspensions (lotions, shaken mixtures), lipophilic suspensions, ointments, creams, emulsions, pastes, dusting powders or implants, such as stents.

[0072] The active ingredient can be converted into the administration form by methods known per se. It can be achieved with inert, non-toxic, suitable pharmaceutical excipients. It particularly includes carriers (e.g. microcrystalline cellulose), solvents (e.g. liquid polyethylene glycol), emulsifiers (e.g. sodium lauryl sulfate), dispersants (e.g. polyvinyl pyrrolidone), synthetic and natural biopolymers (e.g. proteins), stabilizers (e.g. antioxidants and ascorbic acid), colorants (e.g. inorganic pigments such as iron oxide) or flavoring agents and / or taste masking agents. In suitable cases, the active ingredient can be present in one or more of the above-mentioned carriers in the form of microencapsulation.

[0073] In addition to the compound of the present invention, its pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer, solvate or polymorph, the above-mentioned pharmaceutical preparation may further contain other pharmaceutically active ingredients.

[0074] The present invention has achieved the following beneficial effects:

[0075] The compounds of the present invention, their pharmaceutically acceptable salts, prodrugs, stable isotope derivatives, isomers, solvates or polymorphs can block the VEGF / VEGFR pathway, thereby preventing or treating diseases related to the VEGF / VEGFR pathway, such as improving, preventing and treating tumor diseases and various other diseases accompanied by abnormal proliferation of neovascularization. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 The curves of tumor volume changes over time in mice in the model control group, test group and positive control group are shown.

[0077] Figure 2 The photos show the changes of tumor appearance over time in mice of model control group, test group and positive control group.

[0078] Figure 3 It is the tumor weight of mice in the model control group, test group and positive control group after the experiment. DETAILED DESCRIPTION

[0079] The following examples are provided to provide those skilled in the art with a complete disclosure of how to perform and evaluate the methods and compounds claimed herein. This example merely illustrates the present invention and does not limit the scope of the present invention.

[0080] The preparation method of the compound of formula (I) of the present invention is described in more detail below, but these specific methods do not constitute any limitation to the present invention. The compounds of the present invention can also be prepared more conveniently by combining the various synthetic methods described in the optional description or known in the art, and such combination can be more easily performed by a technician in the field to which the present invention belongs. The following examples are preferred illustrative preferred embodiments of the present invention and do not constitute any limitation to the present invention.

[0081] For all the following examples, standard operations and purification methods known to those skilled in the art can be used. Unless otherwise indicated, all temperatures are expressed in ° C (Celsius). The structures of the compounds are confirmed by nuclear magnetic resonance (NMR) or mass spectrometry (MS). The melting points of the compounds are determined by RY-1 type melting point apparatus, and the thermometer is uncalibrated and is given in ° C. 1 HNMR was measured by a JNM-ECA-400 nuclear magnetic resonance instrument from Japan Electronics. Mass spectra were measured by an API3000 (ESI) mass spectrometer. All reaction solvents were not subjected to standardization pretreatment unless otherwise specified. In the following examples, % refers to mass percentage unless otherwise specified. Silica gel for column chromatography was produced by Qingdao Ocean Chemical Plant (200-300 mesh); silica gel plates for thin layer chromatography were prefabricated thin layer chromatography silica gel plates produced by Yantai Institute of Chemical Industry. The compounds of the present invention can be prepared by referring to conventional methods in the art and using appropriate reagents, raw materials and purification methods known to those skilled in the art.

[0082] Example 1 Synthesis of 7-methoxy-4-((4-(3-methoxybenzamide)naphthalen-1-yl)oxy)quinoline-6-carboxamide (1)

[0083]

[0084] Step (a) Preparation of 7-methoxy-4-((4-nitronaphthalen-1-yl)oxy)quinoline-6-carboxamide

[0085] Dissolve 4-hydroxy-7-methoxyquinoline-6-carboxamide (3.00 g, 13.75 mmol) in dimethyl sulfoxide (DMSO, 30 mL), add sodium bicarbonate (3.46 g, 41.25 mmol). Stir at room temperature for 30 min, add 1-chloro-4-nitronaphthalene (3.13 g, 15.12 mmol). Heat to 90 ° C for 24 h, cool to room temperature, then add 80 mL of water, precipitate a large amount of solid, filter to obtain a yellow solid. The yellow solid is purified by silica gel column chromatography (dichloromethane / methanol=80 / 1) to obtain 2.41 g of the target product, a yellow solid, with a yield of 45.0%.

[0086] Step (b) Preparation of 4-((4-aminonaphthalen-1-yl)oxy)-7-methoxyquinoline-6-carboxamide

[0087] Dissolve ammonium chloride (1.43 g, 26.71 mmol) in water (15 mL) to prepare an ammonium chloride solution. Dissolve 7-methoxy-4-((4-nitronaphthalene-1-yl)oxy)quinoline-6-carboxamide (2.60 g, 6.68 mmol) in 95% ethanol (100 mL), and then add the prepared ammonium chloride solution. Heat to 85°C, and after an obvious reflux state is observed, add iron powder (2.24 g, 40.07 mmol). Continue to reflux for 2 hours, and filter through diatomaceous earth. Remove the solvent from the filtrate under reduced pressure, and purify the residue by silica gel column chromatography (dichloromethane / methanol=40 / 1) to obtain 1.86 g of the target compound as an orange solid with a yield of 77.5%.

[0088] Step (c) Synthesis of 7-methoxy-4-((4-(3-methoxybenzamide)naphthalen-1-yl)oxy)quinoline-6-carboxamide

[0089] Dissolve 3-methoxybenzoic acid (0.09 g, 0.61 mmol) in N,N-dimethylformamide (5 mL), then add 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 0.42 g, 1.11 mmol), N,N-diisopropylethylamine (DIPEA, 0.18 g, 1.39 mmol). After stirring at room temperature for 30 min, add 4-((4-aminonaphthalen-1-yl)oxy)-7-methoxyquinoline-6-carboxamide (0.2 g, 0.57 mmol). After heating to 90°C for 6 h, cool to room temperature, add 50 mL of water, precipitate a large amount of solid, filter to obtain a reddish brown solid. The reddish brown solid is purified by silica gel column chromatography (dichloromethane / methanol=20 / 1) to obtain 0.06 g of the target compound as a white solid with a yield of 22.2%. Mp.157-158℃. ESI-MS M / Z:[M+H] + 494.16. 1 HNMR (400 MHz, DMSO-d 6 )δ(ppm): 10.54(s,1H),8.91(s,1H),8.66(s,1H),8.13(d,J=8.3Hz,1H),7.93(d,J=8.3H z, 2H), 7.85–7.45 (m, 9H), 7.23 (d, J = 7.2Hz, 1H), 6.41 (s, 1H), 4.09 (s, 3H), 3.90 (s, 3H). 13 C NMR (151 MHz, DMSO-d 6 )δ(ppm): 166.54,166.38,162.37,159.79,158.71,153.90,152.23,147.69,136.21,132.51,131.16,130.15,127.70,127.5 3,127.02,125.86,125.14,124.76,124.65,121.67,120.57,118.14,117.74,114.80,113.44,108.53,103.47,56.72,55.86.

[0090] Example 2 4-((4-(Cyclopropanecarboxamide)naphthalen-1-yl)oxy)-7-methoxyquinoline-6-carboxamide (2)

[0091]

[0092] Prepared according to the method of Example 1, 0.07 g of white solid was obtained, with a yield of 29.6%, mp. 244-245°C. ESI-MS m / z: [M+H] + 428.15.1 H NMR (600 MHz, DMSO-d 6 )δ(ppm): 10.28(s,1H),8.87(s,1H),8.61(d,J=5.3Hz,1H),8.24(d,J=8.6Hz,1H),7.92–7.89(m,1H),7.84(d,J=8.3Hz,2H),7.79–7.76(m,1H), 7.67(ddd,J=8.4,6.8,1.3Hz,1H), 7.56(d,J=6.9Hz,2H), 7.46(d,J=8.2Hz,1H), 6.35(d,J=5.3Hz,1H), 4.06(s,3H), 0.88(dd,J=6.7,3.8Hz,4H). 13 C NMR (151 MHz, DMSO-d 6 )δ(ppm): 173.09,166.16,163.92,159.42,152.43,149.90,146.02,132.81,129.36,127.69,127.36,1 26.78,126.60,125.28,124.10,121.80,121.56,117.97,114.70,106.61,103.47,56.88,14.64,7.83.

[0093] Example 3 Synthesis of 7-methoxy-4-((4-(3-(3-methoxyphenyl)ureido)naphthalen-1-yl)oxy)quinoline-6-carboxamide (3)

[0094]

[0095] Dissolve 4-((4-aminonaphthalen-1-yl)oxy)-7-methoxyquinoline-6-carboxamide (0.20 g, 0.57 mmol) in tetrahydrofuran / N,N-dimethylformamide (3 / 2 mL), and then add 3-methoxyphenyl isocyanate (0.10 g, 0.67 mmol). React at room temperature for 24 hours, and solid is generated. Filter to obtain a white solid, wash twice with water, dry, and then wash twice with dichloromethane, dry to obtain the target compound, 0.12 g of white solid, yield 42.5%. Mp.240-241℃. ESI-MS m / z: [M+H] + 509.17. 1 H NMR (400 MHz, DMSO-d 6)δ(ppm): 10.00(s,1H),9.53(s,1H),8.95(s,1H),8.81(d,J=6.3Hz,1H),8.58(d,J=8.6 Hz,1H),8.24(d,J=8.4Hz,1H),8.02(s,1H),7.91(s,1H),7.84(d,J=8.4Hz,1H),7.73(d ,J=9.4Hz,2H),7.59(t,J=8.3Hz,2H),7.32(s,1H),7.23(t,J=8.1Hz,1H),7.05(d,J=8. 1Hz, 1H), 6.65 (d, J = 6.2Hz, 1H), 6.59 (dd, J = 8.4, 2.5Hz, 1H), 4.12 (s, 3H), 3.77 (s, 3H). 13 C NMR (151 MHz, DMSO-d 6 )δ(ppm): 166.39,162.70,160.25,158.67,153.86,153.40,152.15,144.85,141.44,133.19,130.15,127.97,127.54,127.2 4,127.00,125.74,125.16,122.78,121.88,118.35,118.14,114.73,110.97,108.48,107.87,104.41,103.20,56.70,55.41.

[0096] The following compounds were prepared in a similar manner to the procedure described in Example 1

[0097] Example 4 4-((4-(3-(3-fluorophenyl)ureido)naphthalen-1-yl)oxy)-7-methoxyquinoline-6-carboxamide (4)

[0098]

[0099] The method was similar to that of Example 3, and 0.08 g of white solid was obtained, with a yield of 28.9%, mp. 266-267°C. ESI-MS m / z: [M+H] + 497.15. 1 H NMR (600 MHz, DMSO-d 6)δ(ppm): 9.31(s,1H),8.95(s,1H),8.89(s,1H),8.60(d,J=5.3Hz,1H),8.24(d,J=8.6H z,1H),8.08(d,J=8.3Hz,1H),7.91(s,1H),7.84(d,J=8.5Hz,1H),7.77(s,1H),7.71(t, J=7.7Hz,1H),7.58(d,J=8.9Hz,3H),7.49(d,J=8.3Hz,1H),7.35(q,J=7.8Hz,1H),7.19 (dd, J=8.2, 2.0Hz, 1H), 6.82 (td, J=8.5, 2.6Hz, 1H), 6.34 (d, J=5.3Hz, 1H), 4.07 (s, 3H). 13 C NMR (151 MHz, DMSO-d 6 )δ (ppm): 166.39, 162.96 (d, J = 239.9Hz), 162.62, 158.65, 153.89, 153.38, 15 2.21,145.13,142.10(d,J=11.0Hz),132.93,130.91(d,J=9.7Hz),128.17,127 .58,127.32,127.01,125.73,125.15,122.85,121.90,118.56,118.29,114.74 ,114.41,108.74(d,J=21.6Hz),108.53,105.36(d,J=26.6Hz),103.23,56.70.

[0100] Example 5 7-methoxy-4-((4-(3-(4-methoxyphenyl)ureido)naphthalen-1-yl)oxy)quinoline-6-carboxamide (5)

[0101]

[0102] The method was similar to that of Example 3, and 0.09 g of a white solid was obtained, with a yield of 31.3%, mp. 248-249°C. ESI-MS m / z: [M+H] + 509.17. 1 H NMR (400 MHz, DMSO-d 6)δ (ppm): 8.93 (s, 1H), 8.91 (s, 1H), 8.84 (s, 1H), 8.60 (d, J = 5.2Hz, 1H), 8.27 ( d,J=8.6Hz,1H),8.13(d,J=8.4Hz,1H),7.93(s,1H),7.84(dd,J=8.5,1.2Hz,1H ),7.79(s,1H),7.71(ddd,J=8.4,6.8,1.3Hz,1H),7.61–7.53(m,2H),7.52–7. 42(m,3H),6.96–6.89(m,2H),6.35(d,J=5.3Hz,1H),4.08(s,3H),3.75(s,3H). 13 CNMR (151MHz, DMSO-d 6 )δ(ppm): 166.39,162.82,158.71,155.03,153.78,153.61,152.01,144.56,133.52,133.24,127.79,127.51,127.16,12 6.99,125.77,125.17,122.79,121.85,120.44(2C),118.39,117.68,114.72,114.58(2C),108.36,103.18,56.71,55.66.

[0103] Example 6 4-((4-(3-cyclopropylureido)naphthalen-1-yl)oxy)-7-methoxyquinoline-6-carboxamide (6)

[0104]

[0105] The method was similar to that of Example 3, and 0.06 g of white solid was obtained, with a yield of 25.2%, mp. 237-238°C. ESI-MS m / z: [M+H] + 443.16. 1 H NMR (400 MHz, DMSO-d 6)δ(ppm): 8.90(s,1H),8.69(s,1H),8.65(d,J=5.5Hz,1H),8.26(d,J=8.6Hz,1H),8.11(d,J=8 .4Hz,1H),7.95(s,1H),7.84–7.76(m,2H),7.66(ddd,J=8.4,6.8,1.3Hz,1H),7.60(s,1H),7.5 4(ddd,J=8.1,6.8,1.0Hz,1H),7.47(d,J=8.4Hz,1H),7.01(d,J=3.0Hz,1H),6.39(d,J=5.5Hz, 1H), 4.09 (s, 3H), 2.66 (tp, J=6.9, 3.5Hz, 1H), 0.71 (td, J=6.9, 4.7Hz, 2H), 0.54–0.46 (m, 2H). 13 C NMR (151 MHz, DMSO-d 6 )δ(ppm): 166.39,162.84,158.69,156.87,153.77,152.01,144.18,134.01,127.38,126.93(2C),1 25.73,125.17,122.85,121.74,118.39,114.70,108.36(2C),103.11(2C),56.70,23.01,6.92(2C).

[0106] Example 7 7-Methoxy-4-((4-(3-(p-tolyl)ureido)naphthalen-1-yl)oxy)quinoline-6-carboxamide (7)

[0107]

[0108] The method was similar to that of Example 3, and 0.11 g of a white solid was obtained, with a yield of 39.0%, mp: 251-252°C. ESI-MS m / z: [M+H] + 493.17. 1 H NMR (400 MHz, DMSO-d 6)δ(ppm): 9.01(s,1H),8.89(d,J=12.2Hz,2H),8.61(d,J=5.3Hz,1H),8.27 (d,J=8.3Hz,1H),8.14(d,J=8.0Hz,1H),7.93(s,1H),7.87–7.78(m,2H),7. 72(t,J=7.6Hz,1H),7.59(s,2H),7.49(d,J=8.3Hz,1H),7.44(d,J=8.0Hz,2 H), 7.15 (d, J = 6.8Hz, 2H), 6.35 (d, J = 5.3Hz, 1H), 4.08 (s, 3H), 2.29 (s, 3H). 13 C NMR (151 MHz, DMSO-d 6 )δ(ppm): 166.40,162.70,158.65,153.88,153.46,152.19,144.67,137.62,133.37,131.26,129.76(2C),127.83,127.5 1,127.18,127.00,125.70,125.17,122.76,121.87,118.77(2C),118.37,117.80,114.73,108.50,103.18,56.70,20.83.

[0109] Example 8 Synthesis of N-(4-((6-carbamoyl-7-methoxyquinolin-4-yl)oxy)naphthalen-1-yl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (8)

[0110]

[0111] Dissolve 1-(4-fluorophenylcarbamoyl)cyclopropanecarboxylic acid (0.11 g, 0.50 mmol) in N,N-dimethylformamide (5 mL), add 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 0.42 g, 1.11 mmol), and add N,N-diisopropylethylamine (DIPEA, 0.18 g, 1.39 mmol) under ice bath. After reacting in ice bath for 30 min, add 4-((4-aminonaphthalen-1-yl)oxy)-7-methoxyquinoline-6-carboxamide (0.20 g, 0.57 mmol). After heating to 60°C for 24 h, the reaction solution was diluted with ethyl acetate and extracted with water. The organic layer was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol=40 / 1) to obtain the target product as a white solid (0.11 g). Yield: 33.6%, mp. 239-240°C. ESI-MS m / z: [M+H]+ 565.17. 1 H NMR (400 MHz, DMSO-d 6 )δ (ppm): 10.69 (s, 1H), 10.23 (s, 1H), 8.87 (s, 1H), 8.61 (d, J = 5.2Hz, 1H), 8 .13(d,J=8.5Hz,1H),7.87(dd,J=16.5,9.2Hz,3H),7.77(s,1H),7.66(ddd, J=10.1,7.3,3.1Hz,3H),7.56(d,J=6.4Hz,2H),7.49(d,J=8.2Hz,1H),7.18 (t, J = 8.6 Hz, 2H), 6.32 (d, J = 5.2 Hz, 1H), 4.07 (s, 3H), 1.66 (t, J = 4.4 Hz, 4H). 13 C NMR (151 MHz, DMSO-d 6 )δ (ppm): 168.73, 168.57, 165.28, 161.34, 157.84 (d, J = 240.7Hz), 157 .61,152.77,151.12,145.89,134.32,131.04,128.90,126.58,126.50, 125.84,124.72,124.05,122.77,122.14,122.08,121.64,120.63,116. 72,114.62,114.47,113.68,107.43,102.28,55.62,29.69,15.88(2C).

[0112] Example 9 Synthesis of N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)naphthalen-1-yl)-3-methoxybenzamide (9)

[0113]

[0114] Step (a) Preparation of 6,7-dimethoxy-4-((4-nitronaphthalene-1-yl)oxy)quinoline

[0115] Dissolve 6,7-dimethoxyquinolin-4-ol (2.00 g, 9.75 mmol) in N,N-dimethylformamide (15 mL), add cesium carbonate (3.18 g, 9.75 mmol). After reacting at room temperature for 30 min, add 1-chloro-4-nitronaphthalene (2.02 g, 9.75 mmol). After heating to 90 ° C for 24 h, cool to room temperature, add 100 mL of water, and a large amount of solid precipitates. Filter to obtain a yellow solid, and dry it. The yellow solid is purified by silica gel column chromatography (petroleum ether / ethyl acetate = 40 / 1) to obtain the target product, a yellow solid, 2.50 g, with a yield of 68.3%.

[0116] Step (b) Preparation of 4-((6,7-dimethoxyquinolin-4-yl)oxy)naphthalene-1-amine

[0117] Dissolve ammonium chloride (1.05 g, 19.68 mmol) in water (10 mL) to prepare an ammonium chloride solution. Dissolve 6,7-dimethoxy-4-((4-nitronaphthalene-1-yl)oxy)quinoline (1.85 g, 4.92 mmol) in 95% ethanol (70 mL), and add the prepared ammonium chloride solution. Heat to 85°C, and after an obvious reflux state is observed, add iron powder (1.65 g, 29.52 mmol). Continue to reflux for 2 hours, and filter through diatomaceous earth. Remove the solvent from the filtrate under reduced pressure, and purify the residue by silica gel column chromatography (dichloromethane / methanol=80 / 1) to obtain 1.40 g of the target compound as an orange solid with a yield of 82.4%.

[0118] Step (c) Synthesis of N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)naphthalen-1-yl)-3-methoxybenzamide

[0119] Dissolve 3-methoxybenzoic acid (0.09 g, 0.58 mmol) in N,N-dimethylformamide (5 mL), add 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 0.44 g, 1.16 mmol), N,N-diisopropylethylamine (DIPEA, 0.19 g, 1.45 mmol). After stirring at room temperature for 30 min, add 4-((6,7-dimethoxyquinolin-4-yl)oxy)naphthalene-1-amine (0.20 g, 0.58 mmol). After heating to 60°C for 10 h, cool to room temperature, add 50 mL of water, a large amount of solid precipitates, and filter to obtain a reddish brown solid. The reddish brown solid is purified by silica gel column chromatography (dichloromethane / methanol=80 / 1) to obtain 0.05 g of the target compound as a pinkish white solid with a yield of 18.1% and mp.247-248°C. ESI-MS m / z: [M+H] + 481.17. 1 H NMR (600 MHz, DMSO-d6 )δ(ppm): 10.60(s,1H),8.77(d,J=6.6Hz,1H),8.17(d,J=8.6Hz,1H),7.99(s,1H),7 .89(d,J=8.4Hz,1H),7.83(d,J=8.1Hz,1H),7.71(t,J=8.2Hz,3H),7.67(t,J=2.1Hz ,1H),7.62(ddd,J=8.2,6.7,1.1Hz,1H),7.59(s,1H),7.52(t,J=7.9Hz,1H),7.23(d d,J=8.6,2.6Hz,1H),6.74(d,J=6.7Hz,1H),4.11(s,3H),4.09(s,3H),3.88(s,3H). 13 CNMR (151MHz, DMSO-d 6 )δ(ppm): 166.02,165.51,159.22,156.14,151.21,145.89,143.44,137.42,135.59,133.08,130.47,129.62,127.55,127.2 4,125.96,124.36,123.80,120.95,120.04,117.80,117.58,115.22,112.96,103.17,100.40,100.10,56.57,56.46,55.31.

[0120] Example 10 Synthesis of 1-(4-((6,7-dimethoxyquinolin-4-yl)oxy)naphthalen-1-yl)-3-(3-methoxyphenyl)urea (10)

[0121]

[0122] Dissolve 4-((6,7-dimethoxyquinolin-4-yl)oxy)naphthalene-1-amine (0.20 g, 0.58 mmol) in tetrahydrofuran / N,N-dimethylformamide (3 / 2 mL), and then add 3-methoxyphenyl isocyanate (0.10 g, 0.69 mmol). Heat to 80°C for 24 h, cool to room temperature, add 50 mL of water, and solid will be generated. Filter to obtain a gray-brown solid, and dry it. The gray-brown solid is purified by silica gel column chromatography (dichloromethane / methanol=40 / 1) to obtain the target compound, 0.065 g of white solid, yield 22.7%, mp.180-181°C. ESI-MS m / z: [M+H] + 496.17. 1 H NMR (600 MHz, DMSO-d 6)δ(ppm): 9.14(s,1H),8.89(s,1H),8.40(d,J=5.3Hz,1H),8.25(dt,J=8.7,0.9Hz,1H),8.11(d,J=8.3H z,1H),7.88–7.83(m,1H),7.73(s,1H),7.70(ddd,J=8.4,6.8,1.3Hz,1H),7.56(ddd,J=8.1,6.8,1.1Hz ,1H),7.46(d,J=8.6Hz,2H),7.28(t,J=2.2Hz,1H),7.22(t,J=8.1Hz,1H),7.01(ddd,J=8.1,2.0,0.9Hz ,1H),6.59(ddd,J=8.3,2.5,0.9Hz,1H),6.30(d,J=5.3Hz,1H),3.99(s,3H),3.98(s,3H),3.76(s,3H). 13 C NMR (151 MHz, DMSO-d 6 )δ(ppm): 160.37,159.67,152.84,152.62,149.42,148.75,146.30,144.64,140.87,132.36,129.57,127.38,126.82,126 .60(2C),122.14,121.52,117.79,117.56,114.74,110.39,107.78,107.29,103.83,102.65,99.02,55.69,54.84,54.82.

[0123] Example 11: 1-(4-((6,7-dimethoxyquinolin-4-yl)oxy)naphthalen-1-yl)-3-(3-fluorophenyl)urea (11) was prepared in a similar manner to the procedure described in Example 10.

[0124]

[0125] The method was similar to that of Example 10, and 0.11 g of a white solid was obtained, with a yield of 39.4%, mp. 234-235°C. ESI-MS m / z: [M+H] + 484.16. 1 H NMR (600 MHz, DMSO-d 6)δ(ppm): 9.25(s,1H),8.96(s,1H),8.43(d,J=5.4Hz,1H),8.29(d,J=8.6Hz,1H),8.10(d,J=8.2Hz,1H),7.89–7.81(m,1H),7.75(s,1H),7.69( ddd,J=8.4,6.7,1.3Hz,1H),7.59–7.53(m,3H),7.46(d,J=8.9Hz,2H),7.17(t,J=8.8Hz,2H),6.34(d,J=5.3Hz,1H),4.00(s,3H),3.99(s,3H). 13 C NMR (151 MHz, DMSO-d 6 )δ (ppm): 160.81, 157.31 (d, J = 238.6Hz), 153.03, 152.90, 149.57, 148.33, 145.59, 144.51, 136.03, 132.55, 127.39, 126.87, 1 26.62,126.55,122.27,121.47,119.81,119.76,117.82,117.47,115.38,115.23,114.78,107.17,102.71,99.13,55.75(2C).

[0126] Example 12 Synthesis of N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)naphthalen-1-yl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (12)

[0127]

[0128] Dissolve 1-(4-fluorophenylcarbamoyl)cyclopropanecarboxylic acid (0.13 g, 0.58 mmol) in N,N-dimethylformamide (5 mL), add 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 0.44 g, 1.16 mmol), and add N,N-diisopropylethylamine (DIPEA, 0.19 g, 1.45 mmol) under ice bath. After reacting at 0°C for 30 min, add 4-((6,7-dimethoxyquinolin-4-yl)oxy)naphthalene-1-amine (0.20 g, 0.58 mmol). After reacting at room temperature for 4 h, add 50 mL of water to produce a large amount of solid. Filter to obtain a reddish-brown solid and dry it. The solid was purified by silica gel column chromatography (dichloromethane / methanol=70 / 1) to obtain the target product, yellow solid 0.057 g, yield 17.9%, mp: 233-234°C. ESI-MS m / z: [M+H] + :552.19.1 H NMR (400 MHz, DMSO-d 6 )δ(ppm): 10.93(s,1H),10.12(s,1H),8.74(d,J=6.6Hz,1H),8.19(d,J=8.5Hz,1H),7.99(d,J=8.2Hz,1H),7.96(s,1H),7.85(d,J=8.4H z,1H),7.65(tt,J=19.1,7.5Hz,5H),7.56(s,1H),7.19(t,J=8.7Hz,2H),6.68(d,J=6.6Hz,1H),4.09(s,3H),4.08(s,3H),1.67(s,3H). 13 C NMR (151 MHz, DMSO-d 6 )δ (ppm): 169.66, 169.45, 164.27, 158.74 (d, J = 240.7Hz), 155.42, 151 .01,145.93,145.33,140.39,135.06,132.59,129.49,127.67,127.56, 126.39,123.54,123.14,123.09,121.96,121.46,117.99,115.47,115 .39,115.33,103.36,102.71,100.28,56.63,56.54,30.50,16.83(2C).

[0129] Example 13 Test of the inhibitory activity of compounds on VEGFR-2 kinase

[0130] The inhibitory activity of the 12 compounds of the present invention on VEGFR-2 kinase was evaluated using an in vitro kinase assay. The experimental method is summarized as follows:

[0131] 1. Experimental materials and equipment

[0132]

[0133] 2. Experimental steps:

[0134] 2.1 Preparation of compound stock solution:

[0135] All compounds were dissolved in DMSO and prepared as 10 mM stock solutions. Compounds used within three months were stored in a desiccator at room temperature, and others could be stored long-term at -20°C.

[0136] 2.2 Preparation of working solution:

[0137] The compound stock solution and the positive reference compound Lenvatinib were diluted with DMSO, with an initial concentration of 1000 nM, and a 3-fold gradient dilution was performed, and 10 concentration points were obtained by dilution 10 times.

[0138] A 1000x positive control (1 mM, Lenvatinib) and a 1000x blank control (100% DMSO) were prepared.

[0139] Oscillate on a shaker for 5 min.

[0140] Prepare kinase buffer (1X): 1 volume of 5X enzyme buffer (the reagent in the HTRF KinEASE-TK kit) and 4 volumes of distilled water; the prepared kinase buffer (1X) contains: 5mM MgCl 2 ; 1mM DTT; 1mM MnCl 2 .

[0141] 2.3 Titration of kinase VEGFR-2 (100 μM ATP, 1 μM TK-substrate-biotin, both reagents in HTRFKinEASE-TK kit):

[0142] a) Use Echo 550 to transfer each compound dilution into each well of a test plate (784075, Greiner).

[0143] b) Seal the assay plate and centrifuge the composite plate at 1000 g for 1 min.

[0144] c) Prepare 2X VEGFR-2 using 1X kinase buffer.

[0145] d) Add 5 μL of 2X VEGFR-2 into each well of the above 384-well assay plate (784075, Greiner).

[0146] e) Centrifuge the test plate at 1000 g for 30 seconds and leave at room temperature for 10 minutes.

[0147] f) 5 μM 5X TK-substrate-biotin and 500 μM 5X ATP were prepared using 1X kinase buffer, and then mixed at a volume ratio of 1:1 to obtain a mixture.

[0148] g) Add 5 μL of TK-substrate-biotin and ATP mixture to each well of the above 384-well assay plate to start the reaction.

[0149] h) Centrifuge at 1000 g for 30 seconds. Seal the test plate and leave it at room temperature for 40 minutes.

[0150] i) Prepare 250 nM 4X Sa-XL 665 using HTRF detection buffer (the reagent in the HTRF KinEASE-TK kit).

[0151] j) Add 5 μL of Sa-XL665 and 5 μL of TK-antibody-Cryptate (reagents in HTRF KinEASE-TK kit) prepared in each well of the above 384 experimental plates.

[0152] k) Centrifuge at 1000 g for 30 seconds and leave at room temperature for 1 hour.

[0153] l) Fluorescence values ​​were read at 620 nm (Cryptate) and 665 nm (XL665) on an Envision 2104 plate reader.

[0154] 3. Data Analysis:

[0155] a) Calculate the ratio of 665 / 620 for each well;

[0156] b) Calculate the inhibition percentage:

[0157] Inhibition percentage = 1-100% × (Signal cmpd -Signal Ave_PC ) / (Signal Ave_VC -Signal Ave_PC ).

[0158] Among them, Signal cmpd Indicates the 665 / 620 ratio of the experimental well; Signal Ave_PC Indicates the 665 / 620 ratio of the blank well (without cells and compounds); Signal Ave_VC represents the 665 / 620 ratio of the control well (without compound);

[0159] c) Draw curves and calculate IC of compounds 50 ;

[0160] The relationship between the percent inhibition and the logarithm of the compound concentration was fitted using Graphpad 5.0 by the method of nonlinear regression (dose response - variable slope).

[0161] Y=Bottom+(Top-Bottom) / (1+10^((LogIC 50 -X)*Hill Slope))

[0162] Y is the inhibition percentage, X is the logarithm of the compound concentration, Bottom is the maximum inhibition percentage, Top is the minimum inhibition percentage, and HillSlope is the current slope coefficient.

[0163] 4. Experimental Results

[0164] As shown in Table 1, the experimental results show that the compounds of the present invention have a significant inhibitory effect on the enzyme activity of VEGFR-2, and compounds 3, 6, and 7 are better than the positive drug Lenvatinib in inhibiting VEGFR-2 kinase.

[0165] Table 1. IC of the tested compounds on VEGFR-2 kinase inhibition 50 Value (nM)

[0166]

[0167] Note: The compound number is the example number.

[0168] Example 14 Test of the inhibitory activity of the compound on relevant tumor cells

[0169] The in vitro cell proliferation inhibition assay was used to evaluate the inhibitory activity of the 12 compounds of the present invention on A549, HepG2, and HUVEC cells. The experimental method is summarized as follows:

[0170] 1. Experimental Materials and Instruments

[0171] Materials and reagents Manufacturer Cat# A549 ATCC CCL-185 HepG2 ATCC HB-8065 HUVEC ATCC PCS-100-010 CCK-8 Dojindo CK04 DMEM GIBCO C11995500BT RPMI-1640 GIBCO C11875500BT Fetal Bovine Serum(FBS) Hyclone SH30406.05 DMSO Sigma D8418 Consumables and instruments Manufacturer Cat# 96-well polypropylene plate Corning 3599 Plate shaker Thermo 4625-1CECN / THZ Q Centrifuge Eppendorf 5810R Envision 2104multi-label Reader PerkinElmer 74785 Echo Labcyte 550

[0172] 2. Experimental Procedure

[0173] 2.1 Preparation of compound stock solutions

[0174] Selected compounds were dissolved in DMSO to prepare a 10 mM stock solution. Compounds used within three months were stored in a desiccator at room temperature, and others could be stored long-term at -20°C.

[0175] Preparation of working solution:

[0176] The above compound stock solutions and positive reference compound Lenvatinib were diluted with DMSO, with a starting concentration of 1000 nM, and 2-fold gradient dilutions were performed in A549, HepG2, and HUVEC cell experiments, with 8 concentration points. Oscillating on an oscillator for 5 minutes.

[0177] 2.2 Cell Dosing

[0178] 2.2.1 A549, HepG2, and HUVEC cells in the logarithmic growth phase were seeded in 96-well plates, with 3000-4000 cells per well. The plates were pre-cultured in an incubator for 24 hours (at 37°C, 5% CO). 2 under the conditions of ).

[0179] 2.2.2 Replace the culture medium in the well plate and add 100 μL of the corresponding concentration of compound and positive reference compound to the culture plate.

[0180] 2.2.3 Incubate the culture plate in an incubator for 72 hours, add 10 μL of CCK-8 solution to each well, and then incubate the culture plate in an incubator for 1-4 hours.

[0181] 2.2.4 Measure the absorbance at 450 nm using an enzyme-labeled instrument.

[0182] 3. Data Analysis

[0183] Calculate the inhibition percentage:

[0184] Inhibition percentage = 1-100% × (Signal cmpd -Signal Ave_PC ) / (Signal Ave_VC -Signal Ave_PC ).

[0185] Among them, Signal cmpd Indicates the absorbance of the experimental well; Signal Ave_PC Indicates the absorbance of blank wells (without cells and compounds); Signal Ave_VC represents the absorbance of the control well (without compound);

[0186] Calculate the IC of a compound 50 And Plot effect dose curve:

[0187] IC was calculated by fitting the logarithm of percent inhibition and compound concentration to a nonlinear regression (dose response - variable slope) using GraphPad 6.0. 50 value.

[0188] Y=Bottom+(Top-Bottom) / (1+10^((LogIC 50 -X)*HillSlope))

[0189] Y is the inhibition percentage, X is the logarithm of the compound concentration, Bottom is the maximum inhibition percentage, Top is the minimum inhibition percentage, and HillSlope is the current slope coefficient.

[0190] 4. Experimental Results

[0191] As shown in Table 2, the experimental results show that the compounds of the present invention, compounds 1-12, have excellent proliferation inhibition activity on A549, HepG2, and HUVEC.

[0192] Table 2. IC values ​​of the tested compounds for inhibition of proliferation of A549, HepG2 and HUVEC cells 50 Value (μM)

[0193]

[0194]

[0195] Example 15: Evaluation of the anti-proliferation activity of compounds against various tumor cells

[0196] Referring to the method of Example 14, the starting concentration was 27 μM, and 2-fold gradient dilution was performed, with a total of 8 concentration points. The inhibitory activity of the compound on various tumor cells was determined, including human lung cancer (EBC-1, NCI-H1975), human liver cancer (SMMC-7721, PLCPRF5), human breast cancer (MCF7, ZR75-1, Hs578T, MDA-MB-231, HCC-38), human ovarian cancer (A2780, SKOV3), human renal cancer 786-O, human colorectal cancer (HT-29, NCI-H23, HCT-116), human gastric cancer Hs746T and human cervical cancer Hela cells.

[0197] 1. Experimental materials and instruments

[0198]

[0199]

[0200] 2. The test results are shown in Table 3: The four compounds have good anti-proliferative activity against the eight types of tumor cells tested, among which compounds 1, 8, and 11 have better anti-proliferative activity than Lenvatinib against 17 cells.

[0201] Table 3 Antiproliferative activity of compounds against various tumor cells (IC 50 value)

[0202]

[0203] ND: Not detected. The IC values ​​of compounds with inhibition rates less than 50% at the highest concentration of 27 μM were not fitted. 50 value.

[0204] Example 16 In vivo activity of compound 8 against HepG2 nude mouse transplanted tumors

[0205] 1. Experimental materials and instruments

[0206]

[0207]

[0208] 2. Establishment of tumor model

[0209] Human hepatoma cell line HepG2 (from ATCC) was cultured in high-glucose DMEM containing 10% fetal bovine serum at 37°C and 5% CO 2 After routine culture in an incubator, after three generations in vitro, when the cells grew to more than 80% and the fusion rate reached the required amount, the cells were digested and collected, and the cells were counted after washing with PBS, and the cell concentration was adjusted to about 5x10 7 Place the 500 μg / mL in a 4 mL centrifuge tube and place on ice for later use.

[0210] Female 4-5 week old Balb / C nude mice were selected and inoculated subcutaneously with human hepatoma cell line HepG2 tumor cells. The mice were grasped and placed on their side. The axilla of the forelimbs was disinfected with 75% alcohol. 100 μL of cell suspension was drawn with a 1 mL syringe and injected into the axilla subcutaneously, i.e., 5×10 6 cells / mouse / 100μL.

[0211] 3. Experimental Animal Grouping and Drug Administration

[0212] When the tumor grows to 90-150 mm 3 Afterwards, the animals were randomly divided into groups, with 6 animals in each group, and fed according to different dosing forms, namely:

[0213] Model control group: gavage with the same volume of blank solvent (DMSO: 0.5% sodium carboxymethyl cellulose: distilled water = 1:1:8) every day;

[0214] Test group: 50 mg / kg (mouse body weight) of Compound 8 solution was intragastrically administered every day.

[0215] Positive control group: 50 mg / kg (mouse body weight) of Lenvatinib solution was intragastrically administered daily.

[0216] The administration route was oral gavage, and the administration frequency was once a day for 13 consecutive days. The first day of administration was defined as the first day of the experiment. The changes in the tumor volume of the mice were measured and recorded every two days. Tumor volume was measured using a vernier caliper to measure the long diameter (a) and short diameter (b) of the tumor and calculate the tumor volume. The tumor volume V (mm 3 )=a×b 2 / 2. After the experiment, the mice were dissected and the tumor weight was weighed. The data were entered and statistically analyzed using GraphPad Prism 6 software. The data were expressed as mean±SEM (Standard Error of Mean, standard error), and one-way ANOVA was used. P≤0.05 was considered statistically significant.

[0217] 4. Experimental results

[0218] Figure 1 The curves of tumor volume changes over time in mice of the model control group, test group and positive control group; Figure 2 The photos are the changes of tumor appearance over time in mice of model control group, test group and positive control group; Figure 3 It is the tumor weight of mice in the model control group, test group and positive control group after the experiment.

[0219] like Figure 1-3 As shown in Table 4, the experimental results show that compound 8 significantly inhibits the growth of human liver cancer HepG2 cell transplanted tumors. At the same dose, the effect is comparable to that of the positive drug Lenvatinib.

[0220] Table 4. Inhibitory effect of compound 8 on HepG2 xenograft tumors in nude mice

[0221] Compound <![CDATA[Average volume (mm 3 )(Day 13)]]> Tumor inhibition rate TGI (%) Control 1908.55 — Lenvatinib 654.10 80.78％ 8 605.00 78.50％

[0222] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the technical principles of the present invention, and these improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A compound represented by formula (I) or a pharmaceutically acceptable salt thereof; in, R1 and R2 are each independently selected from amino C1-C6 alkanoyl, C1-C6 alkoxy; R3 is selected from -(NR5) n1 -C(O)-(NR6) n2 -, ; R5 and R6 are H; n1 is 1; n2 is selected from 0, 1; R4 is selected from substituted or unsubstituted 5-10 membered aryl, substituted or unsubstituted 3-8 membered cycloalkyl; the substitution is substituted by one or more of the following substituents: halogen, C1-C6 alkyl, C1-C6 alkoxy.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R1 and R2 are each independently selected from carbamoyl and methoxy.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R4 is selected from substituted or unsubstituted phenyl, substituted or unsubstituted cyclopropyl; the substitution is substituted by one or more of the following substituents: fluorine, methyl, methoxy.

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R4 is selected from , cyclopropyl, , , , .

5. The compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, wherein The compound is selected from: 。 6. A method for preparing the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, which is the following method A or method B; The method A comprises: 4-nitronaphthylamine is subjected to diazotization reaction and substitution reaction to obtain intermediate A2; Substitution reaction of intermediate A2 with 4-hydroxyquinoline to obtain intermediate A3; Reducing the nitro group of intermediate A3 to obtain intermediate A4; The intermediate A4 is subjected to different condensation reactions to obtain target products A5-A7 respectively; ； The method B comprises: Substitution reaction of 4-nitronaphthol and 4-chloroquinoline was performed to obtain intermediate B2; Reducing the nitro group of intermediate B2 to obtain intermediate B3; The intermediate B3 is subjected to different condensation reactions to obtain the target products B4-B6 respectively; ； The above R1, R2 and R4 are defined as in any one of claims 1 to 5.

7. A pharmaceutical composition comprising the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof.

8. The pharmaceutical composition according to claim 7, wherein The pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

9. The pharmaceutical composition according to claim 7 or 8, further comprising other drugs and / or immunomodulators for treating tumors.

10. The pharmaceutical composition according to claim 7 or 8, further comprising one or more selected from immune checkpoint inhibitors, antibiotics, alkylating agents, antimetabolites, hormones, immunoactive agents, interferon-type active agents and mixed active agents.

11. Use of the compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 or the pharmaceutical composition according to any one of claims 7 to 10 in the preparation of a medicament for preventing and / or treating diseases related to the VEGF / VEGFR pathway.

12. The use according to claim 11, wherein The diseases related to the VEGF / VEGFR pathway are selected from one or more of atherosclerosis, pulmonary fibrosis, retinopathy, endometriosis, arthritis and cancer.

13. The use according to claim 12, wherein The cancer is selected from one or more of lung cancer, kidney cancer, gastric cancer, hepatocellular carcinoma, thyroid cancer, melanoma, breast cancer, pancreatic cancer, colon cancer, rectal cancer, prostate cancer, bladder cancer, ovarian cancer, cervical cancer and glioma.

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