Benzamide compound as well as preparation method, pharmaceutical composition and application thereof

Abstract

The invention belongs to the technical field of pharmacy, and relates to a benzamide compound as well as a preparation method, a pharmaceutical composition and application thereof. The benzamide compound is shown as a formula (I), and is an EPHB4 inhibitor. An in-vitro phosphorylation reaction verifies that the compound has an inhibitory effect on the activity of EPHB4 kinase. Meanwhile, an insulin-resistant cell model system verifies that the compound has the effect of increasing the insulin sensitivity of model cells.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a benzamide compound, its preparation method, uses, and pharmaceutical composition. Background Technology

[0002] In recent years, the incidence of metabolic diseases such as obesity and diabetes has shown a rapid upward trend. Related research data indicates that in 2021 alone, the number of people diagnosed with diabetes in my country exceeded 4 million, of which more than 98% were type 2 diabetes patients. The total number of people with diabetes in China has exceeded 117 million, making it the country with the largest number of diabetes patients in the world. If obesity and diabetes are not treated and controlled effectively in a timely manner, they can also induce related metabolic syndromes affecting the cardiovascular system, kidneys, eyes, and other organs, posing a serious threat to the physical and mental health of the population while also imposing a heavy economic burden on society.

[0003] Insulin signaling plays a crucial role in regulating glucose homeostasis in the body, and insulin resistance is a key pathological feature of type 2 diabetes and a recognized major contributing factor to many metabolic diseases. Studies have shown that insulin resistance can develop 10-20 years before the diagnosis of type 2 diabetes, possibly due to chronic tissue inflammation, endoplasmic reticulum stress, and gut microbiota factors. In cases of insulin resistance, the body compensates by increasing insulin secretion, leading to hyperinsulinemia. Our research indicates that high insulin levels further promote the interaction between the insulin receptor and the EphB4 protein. EphB4, through binding to the adaptor protein Ap2, promotes the cloathrin-dependent endocytosis of the insulin receptor and its degradation via the lysosomal pathway, thereby inhibiting insulin signaling and exacerbating insulin resistance.

[0004] Currently, there is still a lack of effective insulin sensitizers in clinical practice. Thiazolidinediones (TZDs), which have been proven to improve insulin resistance and increase insulin sensitivity, target PPARγ, belonging to the mammalian nuclear receptor superfamily. However, TZDs have been withdrawn from the market or have their use restricted due to side effects such as increased weight and cardiovascular risk. Therefore, there are currently no effective insulin sensitizers in practical use. Our research demonstrates that EphB4 can serve as a target for insulin sensitizers. Therefore, this invention aims to develop novel inhibitors of EphB4 as a target, and to present them as new insulin sensitizers to address the current problem. Summary of the Invention

[0005] The purpose of this invention is to provide a novel EPHB4 inhibitor that has strong inhibitory activity against EPHB4 kinase, thereby having preventive and / or therapeutic effects on EPHB4-mediated diseases, especially insulin resistance and related metabolic diseases.

[0006] To solve the technical problem of this invention, the present invention provides the following technical solution:

[0007] The first aspect of the present invention is to provide a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0008]

[0009] Wherein: R1 is selected from hydrogen, C 1-3 alkyl.

[0010] Specifically, the preferred compounds according to the present invention are as follows:

[0011]

[0012] A second aspect of the present invention is to provide a method for preparing the compound, which includes the following steps:

[0013]

[0014] (1) Compound B was prepared by reacting compound A with 4-pyridineboronic acid via a Suzuki reaction.

[0015] (2) Compound B was hydrolyzed with concentrated sulfuric acid to prepare compound C;

[0016] (3) Compound C was brominated to prepare compound D;

[0017] (4) Compound D is prepared by the Suzuki reaction to obtain the compound shown in formula (I) or a pharmaceutically acceptable salt thereof;

[0018] A third aspect of the present invention is to provide a pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier and / or excipient; preferably, the pharmaceutical composition further comprises one or more pharmaceutically active ingredients for the prevention and / or treatment of tumors, in addition to the compound or a pharmaceutically acceptable salt thereof.

[0019] In another aspect, the present invention also provides a pharmaceutical formulation comprising at least one of the compounds described herein or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier, diluent, or excipient; preferably, the pharmaceutical formulation is selected from the following dosage forms: parenteral formulations, such as injectable solutions or suspensions; enteral formulations, such as oral formulations, like tablets or capsules; topical formulations, such as lotions, gels, ointments, emulsions, nasal formulations, suppositories, transdermal formulations, or ophthalmic formulations.

[0020] In another aspect, the present invention also provides the use of the said compound or a pharmaceutically acceptable salt thereof, or the said pharmaceutical composition, in the preparation of a medicament for the prevention and / or treatment of tumors. In other words, the present invention provides a method for the prevention and / or treatment of tumors, comprising administering to a subject in need a preventive and / or therapeutically effective amount of the said compound or a pharmaceutically acceptable salt thereof, or the said pharmaceutical composition.

[0021] The following are definitions of some of the terms used in this invention; other undefined terms have meanings known to those skilled in the art.

[0022] "alkyl" refers to a compound with the general formula C n H 2n+1 The alkyl group is a hydrocarbon group. This alkyl group can be straight-chain or branched. For example, the term "C1-C3 alkyl" should be understood to refer to a straight-chain or branched saturated monovalent hydrocarbon group having 1, 2, or 3 carbon atoms. Examples of such groups include, but are not limited to, methyl, ethyl, propyl, and isopropyl.

[0023] “C 1-3 "Alkyl" refers to an alkyl group containing 1 to 3 carbon atoms. Examples of such groups include, but are not limited to, methyl, ethyl, propyl, and isopropyl.

[0024] The term "inhibitor" refers to a compound or agent that can inhibit the biological function of a target protein or polypeptide, such as inhibiting the activity or expression of the target protein or polypeptide.

[0025] The term "effective amount" refers to the amount of a compound or pharmaceutical composition described herein sufficient to achieve the intended application as described below, including but not limited to the treatment of a disease. Effective amounts may vary depending on: the intended application (in vivo or in vitro); or the individual being treated and the disease condition, such as the individual's weight and age, the severity of the disease; the route of administration, etc. Effective amounts can be readily determined by those skilled in the art.

[0026] The compounds described in this invention also include their isotope-labeled compounds. The term "isotope-labeled compound" refers to a compound in which one or more atoms are replaced by atoms having the same atomic number but a different atomic mass number than those normally found in nature. Examples of isotopes suitable for this invention include, but are not limited to, isotopes of hydrogen. 2 H and 3 H; carbon isotopes 11 C 13 C and 14 C; isotopes of chlorine 36 Cl; isotopes of fluorine 18 F; Isotopes of iodine 123 I and 125 I; Nitrogen isotopes 13 N and 15 N; isotopes of oxygen 15 O、 17 O and 18 O; isotopes of phosphorus 32 Isotopes of P and sulfur 35 S.

[0027] Various solvates and hydrates of the compounds or their salts described in this invention, as well as their polymorphs, are also included within the scope of this invention.

[0028] The term "solvent" refers to a compound that also includes stoichiometric or non-stoichiometric solvents bound by non-covalent intermolecular forces. A solvate can be the disclosed compound or a pharmaceutically acceptable salt thereof. When the solvent is water, the solvate is a "hydrate". Pharmaceutically acceptable solvates and hydrates are, for example, complexes that may include, for example, one to about 100, one to about 10, one to about 2, about 3, or about 4 solvent or water molecules. The term "polymorph" refers to a compound that exists in two or more different crystalline forms.

[0029] Prodrugs of the compounds described in this invention are also included within the scope of this invention. Some derivatives of the compounds described in this invention possess weak or no pharmacological activity, but when these derivatives are administered into the body, they can be converted into pharmacologically active compounds of this invention through processes such as hydrolysis and cleavage. These derivatives are called "prodrugs". Further information on the uses of prodrugs can be found in *Pro-drugs as Novel Delivery Systems*, Vol. 14, ACS Symposium Series (T. Higuchi and W. Stella) and *Bioreversible Carriers in Drug Design*, Pergamon Press, 1987 (ed. E.B. Roche, American Pharmaceutical Association).

[0030] The compounds described in this invention include pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" refers to a salt that is pharmaceutically acceptable and possesses the pharmacological activity required of the parent compound. Pharmaceutically acceptable salts are described in detail by Berge et al. in J. Pharma. Sci., 1977, 66, 1-19, which is incorporated herein by reference. The compounds described in this invention may contain sufficient acidic groups, sufficient basic groups, or both types of functional groups, and accordingly react with some inorganic or organic base, or inorganic and organic acid, to form pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, phosphates, monohydrophosphates, dihydrophosphates, metaphosphates, pyrophosphates, hydrochlorides, hydrobromates, hydroiodates, acetates, propionates, decanoates, octanoates, acrylates, formates, isobutyrates, hexanoates, heptanates, propynates, oxalates, malonates, succinates, octanoates, sebates, fumarates, maleates, butyn-1,4-dicitates, hexyn-1,6-dicitates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, gamma-hydroxybutyrate, glycolate, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, and mandelates.

[0031] When used as a pharmaceutical, the compounds described herein are typically administered in the form of a pharmaceutical composition. Therefore, pharmaceutical compositions comprising the compounds described herein and pharmaceutically acceptable carriers, diluents, or excipients are also included within the scope of this invention. Carriers, excipients, and additives as used herein include any and all solvents, diluents or other liquid excipients, dispersants or suspending agents, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc., suitable for the desired particular dosage form. Various carriers for formulating pharmaceutically acceptable compositions and known techniques for their preparation are disclosed in Remington: The Science and Practice of Pharmacy, 21st edition, 2005, ed. D.B. Troy, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J.C. Boylan, 1988–1999, Marcel Dekker, New York, the contents of which are incorporated herein by reference.

[0032] The compositions of this invention can be administered via any route suitable for the condition to be treated. In particular, administration is possible via: parenteral administration, for example, as an injectable solution or suspension; enteral administration, for example, orally, in tablet or capsule form; topical administration, for example, as a lotion, gel, ointment, or emulsion, or via nasal or suppository form. Topical application is, for example, to the skin. Another form of topical administration is administration to the eye.

[0033] Pharmaceutical compositions can be administered in solid, semi-solid, liquid, or gaseous form, or may be in the form of dry powders, such as lyophilized forms. Pharmaceutical compositions can be packaged in easily deliverable forms, including, for example, solid dosage forms such as capsules, pouches, sachets, gelatin, paper, tablets, suppositories, granules, pills, lozenges, and tablets. The type of packaging will generally depend on the route of administration. Implantable, sustained-release formulations and transdermal formulations are also covered.

[0034] Examples of materials that can serve as pharmaceutically acceptable carriers include, but are not limited to: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffers (e.g., phosphates), glycine, sorbic acid or potassium sorbate, mixtures of metaglycerides of saturated vegetable fatty acids, water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene block copolymers, lanolin, sugars (e.g., lactose, glucose, and sucrose), and starches (e.g., corn starch). The composition may contain: potato starch, cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth gum powder; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols, such as propylene glycol or polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; and phosphate buffer, as well as other non-toxic and compatible lubricants, such as sodium lauryl sulfate and magnesium stearate. Colorants, releasing agents, coating agents, sweeteners, flavorings and aromas, preservatives and antioxidants may also be present in the composition, at the discretion of the formulation personnel.

[0035] The compounds described in this invention can be used alone or in combination with other therapeutic agents for treating the diseases or conditions described in this invention (e.g., cancer). In some embodiments, the compounds described in this invention are combined in a pharmaceutical combination formulation with a second compound having anti-proliferative properties or for treating highly proliferative diseases (e.g., cancer), or in a dosing regimen as a combination therapy. The second compound in the pharmaceutical combination formulation or dosing regimen preferably has an activity complementary to that of the compounds described in this invention so that they do not adversely affect each other. Such compounds are suitably present in the combination in an amount effective for the intended purpose. In one embodiment, the compounds of this invention are combined with other antitumor drugs.The antitumor drugs include: alkylating agents, including but not limited to cyclophosphamide, nitrogen mustard, melphalan, cyclophosphamide, and carmustine; platinum-based drugs, including but not limited to carboplatin, cisplatin, and oxaliplatin; topoisomerase inhibitors, including but not limited to topotecan, camptothecin, topotecan, and irinotecan; antibiotics, including but not limited to cyclophosphamide, actinomycin D, daunorubicin, doxorubicin, mitoxantrone, bleomycin, and procainoxantrone; antimicrotubule or antimitotic agents, including but not limited to paclitaxel, vinorelbine, docetaxel, and doxorubicin; and antimetabolites, including but not limited to fluorouracil, methotrexate, cytarabine, and mecaptopurine. Thioguanine and gemcitabine; antibodies, including but not limited to Herceptin and bevacizumab; hormones, including but not limited to Letrazole, vorazole, tamoxifen, toremifene, fulvestrant, flutamide, nilumethoxazole, and triptorelin; kinase inhibitors, including but not limited to EGFR kinase inhibitors such as gefitinib, erlotinib, lapatinib, and afatinib; and VEGFR inhibitors such as sorafenib and regoraflini. Enib, sunitinib, cabozantinib, pazopanib, vandetanib, axitinib; ALK inhibitors, including but not limited to crizotinib, ceritinib, alectinib; Bcr-Abl inhibitors, including but not limited to imatinib, ponatinib, nilotinib, dasatinib; BTK inhibitors Drugs, including but not limited to ibrutinib; B-RAF inhibitors, including but not limited to vemurafenib; cyclin-dependent kinase CDK4 / 6 inhibitors, such as palbociclib; mTOR inhibitors, including but not limited to rapamycin and everolimus; deacetylase inhibitors, including but not limited to vorinostat; PD1 / PDL1 antibodies, such as Keytruda (pembrolizumab) and Opdivo (nivolumab).

[0036] Beneficial technical effects:

[0037] The compounds of this invention have a unique skeletal structure and exhibit high inhibitory activity against EPHB4; they also have a strong binding affinity to the EPHB4 protein; and in vivo pharmacodynamic studies have shown that the compounds of this invention can activate insulin signaling in a dose-dependent manner. Attached Figure Description

[0038] Figure 1 The activity of the EphB4 kinase inhibitor was tested, which showed that Example 1 could effectively inhibit EPHB4 kinase activity.

[0039] Figure 2 For cytotoxicity testing, the IC50 of Example 1 was 26.87 μM.

[0040] Figure 3 It has an agonistic effect on insulin signaling; Example 1 shows that it can activate insulin signaling in a dose-dependent manner. Detailed Implementation

[0041] The following are specific embodiments of the present invention, which further describe the technical solution of the present invention. However, the scope of protection of the present invention is not limited to these embodiments. Any changes or equivalent substitutions that do not depart from the concept of the present invention are included within the scope of protection of the present invention.

[0042] Preparation method

[0043] The compounds described in this invention can be synthesized according to the synthetic schemes described herein and / or techniques well known in the art. For example, the compounds provided by this invention can be prepared according to the following general synthetic methods.

[0044] In a general synthetic method, the compound shown in formula (Ⅰ) is prepared according to method-1.

[0045] Method-1

[0046]

[0047] Specifically, in Method-1, the benzamide compounds of the present invention can be prepared by a four-step reaction. Starting with compound A, compound B is prepared by a Suzuki reaction with 4-pyridineboronic acid; compound B is hydrolyzed with concentrated sulfuric acid to prepare compound C; compound C is brominated to prepare compound D; and compound D is then reacted with a Suzuki reaction to obtain the compound shown in formula (Ⅰ).

[0048] The compounds described in this invention can be synthesized according to one or more synthetic schemes described herein and / or techniques well known in the art. Those skilled in the art will recognize that the synthetic methods of some embodiments described in detail herein can be readily applied to the synthesis of other embodiments. In some embodiments, the compounds described herein can be prepared by appropriate combinations of synthetic methods well known in the art. Many starting materials and other reagents are available from commercial suppliers, such as Alfaisa (China) Chemical Co., Ltd., or can be readily prepared using synthetic methods commonly used in the art.

[0049] 1 H NMR spectra are recorded on instruments operating at 400 MHz or 500 MHz. 1 ¹H NMR spectra were obtained in solution form (reported in ppm), using CDCl₃ (7.26 ppm), DMSO-d₆ (2.50 ppm), or the internal standard tetramethylsilane (0.00 ppm) as reference standards. When reporting peak multiplicity, the following abbreviations were used: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad peak), dd (doublet), dt (doubletuplet). Coupling constants are given in Hertz (Hz).

[0050] In the following preparation methods and examples, "PE" refers to petroleum ether, "EA" refers to ethyl acetate, "MeOH" refers to methanol, "DMSO-d6" refers to deuterated dimethyl sulfoxide, "DCM" refers to dichloromethane, "NBS" refers to N-bromosuccinimide, "Pd(dppf)Cl2" refers to [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride, "M" refers to volumetric molar concentration, "rt" refers to room temperature, "h" refers to hours, and "mL" refers to milliliters.

[0051] “μL” refers to microliters, “mmol” refers to millimoles, “μM” refers to micromoles, “nM” refers to nanomoles, and “℃” refers to degrees Celsius.

[0052] Preparation of intermediate 2-amino-3-bromo-5-(pyridin-4-yl)benzamide

[0053] Step 1: Synthesis of 2-amino-5-(pyridin-4-yl)benzonitrile

[0054]

[0055] 2-Amino-5-bromobenzonitrile (9.85 g, 50 mmol), 4-pyridineboronic acid (8.60 g, 70 mmol), 200 mL dioxane, 75 mL 2M potassium carbonate aqueous solution, and Pd(dppf)Cl2 (1.83 g, 2.5 mmol) were added sequentially to the reaction flask. The mixture was purged with argon and reacted at 100 °C for 10 h. The mixture was filtered through diatomaceous earth, extracted with EA (300 mL × 3), washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and subjected to silica gel column chromatography (DCM / MeOH = 10 ...

[0056] (25:1, v / v) yielded 8.08 g of brown solid, with a yield of 82.9%.

[0057] 1 H NMR (400MHz, DMSO-d6) δ8.57–8.51(m,2H),7.95(d,J=2.3Hz,1H),7.82(dd,J=8.9,2.3Hz,1H),7.68–7.62(m,2H),6.90(d,J=8.8Hz,1H),6.45(s,2H).

[0058] Step 2: Synthesis of 2-amino-5-(pyridin-4-yl)benzamide

[0059]

[0060] 2-Amino-5-(pyridin-4-yl)benzonitrile (2.02 g, 10.4 mmol) was added to 25 mL of concentrated sulfuric acid at 0 °C, and the reaction was carried out overnight at rt. The reaction solution was poured into ice water, the pH was adjusted to 7 with saturated sodium bicarbonate solution, and the mixture was extracted with EA (150 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give 1.79 g of a yellow solid, yield 81.3%.

[0061] 1 H NMR(500MHz,DMSO-d6)δ8.55–8.50(m,2H),8.05(d,J=

[0062] 2.2Hz,2H),7.72–7.64(m,3H),7.23(s,1H),6.98(s,2H),6.82(d,J=8.6Hz,1H).

[0063] Step 3: Synthesis of 2-amino-3-bromo-5-(pyridin-4-yl)benzamide

[0064]

[0065] 2-Amino-5-(pyridin-4-yl)benzamide (535 mg, 2.5 mmol) was dissolved in 10 mL of glacial acetic acid, and NBS (463 mg, 2.6 mmol) was added in portions. The reaction mixture was reacted overnight at rt. The reaction solution was diluted with methanol, concentrated, and subjected to silica gel column chromatography (DCM / MeOH = 10:1, v / v) to give 700 mg of a pale yellow solid, with a yield of 95.6%.

[0066] 1 H NMR (400MHz, DMSO-d6) δ8.59–8.53(m,2H),8.20(s,1H),8.11–8.04(m,2H),7.78–7.72(m,2H),7.48(s,1H),7.03(s,2H).

[0067] Example 1: Synthesis of 2-amino-3-(1H-indazol-4-yl)-5-(pyridin-4-yl)benzamide

[0068]

[0069] 2-Amino-3-bromo-5-(pyridin-4-yl)benzamide (50 mg, 0.17 mmol), 1H-indazole-4-boric acid (39 mg, 0.24 mmol), 3 mL dioxane, 250 μL of 2M potassium carbonate aqueous solution, and Pd(dppf)Cl2 (14 mg, 0.02 mmol) were added sequentially to a reaction flask. The mixture was purged with argon and reacted at 100 °C for 10 h. After concentration, preparative thin-layer chromatography (DCM / MeOH = 10:1, v / v) yielded 41 mg of a yellow solid, with a yield of 73.2%.

[0070] 1 H NMR (400MHz, DMSO-d6) δ13.22(s,1H),8.53(d,J=6.1Hz,2H),8.19(s,1H),8.14(d,J=2.2Hz,1H),7.81(s,1H),7.75(d,J=6. 2Hz, 2H), 7.68 (d, J = 2.2Hz, 1H), 7.60 (d, J = 8.5, 1H), 7.48 (t, J = 7.1Hz, 1H), 7.40 (s, 1H), 7.17 (d, J = 7.0Hz, 1H), 6.52 (s, 2H).

[0071] MS(ESI+) m / z: 330.1 [M+H] + .

[0072] Example 2: 2-Amino-3-(5-methyl-1H-indazol-4-yl)-5-(pyridin-4-yl)benzamide

[0073]

[0074] The title compound was prepared from 5-methyl-1H-indazole-4-boronic acid according to the method in Example 1.

[0075] 1 H NMR (400MHz, DMSO-d6) δ13.07(s,1H),8.52(d,J=5.3Hz,2H),8.19(s,1H),8.17(d,J=2.2Hz,1H),7.75(d,J=5.5Hz ,2H),7.59(d,J=2.1Hz,1H),7.51(d,J=8.0Hz,2H),7.39(s,1H),7.35(d,J=8.5Hz,1H),6.29(s,2H),2.20(s,3H).

[0076] MS(ESI+) m / z: 344.2 [M+H] + .

[0077] Pharmacological activity evaluation

[0078] Experimental Example 1: Detection of EphB4 kinase inhibitor activity in Example 1

[0079] An in vitro phosphorylation reaction was used to establish a validation system for an inhibitor of EphB4 kinase activity. A universal kinase substrate peptide (Poly(4:1Glu,Tyr) Peptide, Yiqiao Shenzhou, P61-58) was used as the substrate at a final concentration of 0.4 μg / μl. This was incubated with purified human EPHB4 (0.1 μg / ml), ATP (100 μM), and solvent / positive control (NVP-BHG712, 20 nM) / Example 1 (1 μM) in kinase reaction buffer (20 mM MgCl2, 2 mM MnCl2, 40 mM Tris pH 7.5, 0.1 mg / ml BSA, 2 mM DTT, 100 μM sodium vanadate) at 37 °C for 30 min. The reaction was then terminated, and the sample was spotted onto a nitrocellulose membrane for dot blot analysis. The degree of substrate phosphorylation was detected using an anti-pan-tyrosine phosphorylation kinase antibody (PY-1000, CST, #8954), and the basal protein level was detected using an anti-GST antibody. Results are as follows: Figure 1 As shown, Example 1 can effectively inhibit EPHB4 kinase activity.

[0080] Experimental Example 2: Cytotoxicity Detection in Example 1

[0081] HepG2 cells were seeded into 96-well plates, with 100 μL of culture medium added to each well, resulting in a cell density of 102. 4 Cells / well. Under conditions of 37°C and 5%... The cells were cultured in an incubator for 24 hours. Then, the original culture medium was discarded, and culture medium containing different concentrations of the medium from Example 1 was added: 1.5625 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 400 μM. Each concentration had eight independent replicate wells, and a solvent control group and a blank control group were also included. After 24 hours of treatment with Example 1, 10 μL of CCK-8 working solution was added to each well, and incubation continued for 2 hours. The absorbance (OD value) was then measured using a microplate reader at a wavelength of 450 nm. Cell viability (%) was calculated as follows: [OD value = ...] 450 Value (dosage) - OD 450 (blank)] / [OD 450 (solvent)-OD 450 The cell viability was calculated using the formula [(blank)]×100, and the half-maximal inhibitory concentration (IC50) of Example 1 was also calculated. The results are attached. Figure 2 As shown, the IC50 of Example 1 is 26.87 μM.

[0082] Experimental Example 3: The agonistic effect of Example 1 on insulin signaling

[0083] HepG2 cells were seeded in culture plates. When the cell confluence reached 50%-60%, the culture medium was replaced with complete medium containing 30 mM glucose and treated for 24 hours. Then, cells were treated overnight (12 hours) with the positive control drug NVP-BHG712 (abbreviated as "NVP") as described in Example 1. The treatment concentrations in Example 1 were 0.2 μM, 0.5 μM, 2.0 μM, and 5.0 μM, respectively. The NVP-BHG712 treatment concentration was 10 nM. Before harvesting the cells, they were stimulated with 10 nM insulin for 20 min, and then Western blotting was performed to analyze the relative levels of phosphorylated insulin receptor (pIR) and phosphorylated Akt (pAkt), markers of insulin signal activation. The results are attached. Figure 3 As shown, Example 1 can activate insulin signaling in a dose-dependent manner.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof: in: R1 is selected from hydrogen, C 1-3 alkyl.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

3. A pharmaceutical composition comprising at least one compound according to any one of claims 1-2 or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier and / or excipient.

4. The pharmaceutical composition according to claim 3, further comprising a pharmaceutically active ingredient other than the compound or a pharmaceutically acceptable salt thereof.

5. The use of the compound according to any one of claims 1-2 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to any one of claims 3 or 4, in the preparation of a medicament for the prevention and / or treatment of diseases associated with EPHB4.

6. Use of the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-2, or the pharmaceutical composition according to any one of claims 3 or 4, in the preparation of a medicament for the prevention and / or treatment of insulin resistance and related metabolic diseases.

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