WO2022237843A1 - Axl inhibitor - Google Patents

Abstract

A class of Axl inhibitors. Specifically, disclosed are novel compounds as represented by formulae (1), (2), (3), and (4) and/or pharmaceutically acceptable salts thereof, and a composition containing the compounds as represented by formulae (1), (2), (3), and (4) and/or a pharmaceutically acceptable salt thereof, a preparation method therefor, and a use thereof as an Axl inhibitor in preparation of antitumor drugs.

Description

Axl inhibitor

This application claims the priority of Chinese application CN202110518860.4 with a filing date of May 12, 2021. This application cites the full text of the aforementioned Chinese application.

technical field

The present invention relates to the field of medicinal chemistry, and more specifically relates to a new compound with Axl kinase inhibitory effect, a preparation method thereof, and an application of the compound in the preparation of antitumor drugs.

Background technique

Receptor tyrosine kinases (RTKs) sense extracellular cell signals, activate cytoplasmic and intranuclear signal transduction pathways, and regulate cell survival, growth, differentiation, adhesion, and migration. Abnormal expression or activation of RTK is involved in cancer cell transformation, tumor formation and metastasis. Tyrosine kinase inhibitors have been used clinically to treat tumors.

Axl is a member of the TAM subfamily of RTK kinases. Other members of this family include MER and Tyro-3. TAM family kinases contain an extracellular domain that binds to ligands, a transmembrane domain that mediates signal transmission, and a conserved intracellular kinase domain. Axl and MER are overexpressed in various types of leukemia and a variety of solid tumor cells, promoting the survival, metastasis and drug resistance of cancer cells. In addition to cancer cells, AXL in the tumor microenvironment of vascular epithelial cells and smooth cells (Korshunov et al., Circ.Res.2006,98,1446), as well as many cells including macrophages, dendritic cells, natural killer cells Expressed in various immune cells, it promotes angiogenesis, immunosuppression and immune escape of tumor cells (Sharif et al., J.Exp.Med.2006, 203, 1891; Rothlin et al., Cell.2007, 131, 1124) (Lu et al., Nature . 1999, 398, 723; Lu & Lemke, Science. 2001, 293, 306; Prasad et al., Mol. Cell Neurosci. 2006, 3, 96; Shankar et al., J. Neurosci. 2003, 23, 4208). It can be seen that AXL participates in the occurrence and development of tumors through multiple mechanisms.

In addition to tumors, overactivation of the Axl pathway is involved in vascular diseases induced by dysfunction of immunity, platelet function, vascular calcification, thrombin-induced vascular smooth muscle cells, and acute and chronic glomerulonephritis, diabetic nephropathy, and chronic allogenic Allograft rejection and other pathological processes. Axl inhibitors are expected to provide therapeutic benefit in a variety of diseases including cancer (including solid tumors such as carcinomas and sarcomas, leukemia and lymphoid malignancies), vascular diseases (including but not limited to thrombosis, atherosclerosis and restenosis) , renal disease (including but not limited to acute and chronic glomerulonephritis, diabetic nephropathy, and transplant rejection) and disorders of angiogenesis induced diseases with serious consequences (including but not limited to diabetic retinopathy, retinopathy, psoriasis , rheumatoid arthritis, atheroma, Kaposi's sarcoma, and hemangioma).

Therefore, the development of small molecule inhibitors of highly active Axl kinase has important clinical value.

WO2015012298A1 reported compound A with Axl inhibitory activity (Example 5 in WO2015012298A1). The clinical phase I trial of the compound for the treatment of osimertinib-resistant non-small-cell lung cancer (Osimertinib-resistant non-small-cell lung cancer) is underway;

Contents of the invention

The present invention provides the compound represented by formula (1)-formula (4) or its various isomers, various crystal forms, pharmaceutically acceptable salts, hydrates or solvates:

Another object of the present invention is to provide a pharmaceutical composition, which contains a pharmaceutically acceptable carrier, diluent and/or excipient, and the compound shown in the present invention, or its various isomers, and various crystal forms , a pharmaceutically acceptable salt, hydrate or solvate as the active ingredient.

Another object of the present invention is to provide the compound shown in the present invention, or its various isomers, various crystal forms, pharmaceutically acceptable salts, hydrates or solvates or the above-mentioned pharmaceutical composition for the preparation of treatment, regulation Or the application in medicines for preventing diseases related to Axl protein.

Another object of the present invention is also to provide a method for treating, regulating or preventing diseases related to Axl protein, comprising administering to the subject a therapeutically effective amount of the compound shown in the present invention, or its isomers, crystal forms, A pharmaceutically acceptable salt, hydrate or solvate or the above pharmaceutical composition.

By synthesizing and carefully studying many kinds of new compounds related to Axl inhibition, the inventors found that among the compounds shown in formulas (1), (2), (3), and (4), compound (1) and compound A In comparison, it has unexpected improved PK properties and enhanced Axl inhibitory activity.

It is to be understood that both the foregoing general description and the following detailed description of the present invention are exemplary and explanatory and are intended to provide further explanation of the invention as claimed.

Further forms of compounds

"Pharmaceutically acceptable" here refers to a substance, such as a carrier or diluent, that does not abolish the biological activity or properties of the compound, and that is relatively nontoxic, e.g., does not cause unwanted biological effects or Interact in a harmful manner with any of its components.

The term "pharmaceutically acceptable salt" refers to a form of a compound which does not cause significant irritation to the organism to which it is administered and which does not abolish the biological activity and properties of the compound. In some specific aspects, pharmaceutically acceptable salts are obtained by reacting the compounds shown in the present invention with acids, such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid and other inorganic acids, formic acid, acetic acid, Propionic acid, oxalic acid, trifluoroacetic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. Organic acids and acidic amino acids such as aspartic acid and glutamic acid.

References to pharmaceutically acceptable salts are understood to include solvent added forms or crystalline forms, especially solvates or polymorphs. Solvates contain stoichiometric or non-stoichiometric solvents and are selectively formed during crystallization with pharmaceutically acceptable solvents such as water, ethanol, and the like. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is ethanol. Solvates of the compounds shown in this invention are conveniently prepared or formed according to the methods described herein. For example, the hydrates of the compounds shown in the present invention are conveniently prepared by recrystallization from a mixed solvent of water/organic solvent, and the organic solvent used includes but not limited to tetrahydrofuran, acetone, ethanol or methanol. Furthermore, the compounds mentioned herein can exist in unsolvated as well as solvated forms. In general, the solvated forms are considered equivalent to the unsolvated forms for purposes of the compounds and methods provided herein.

In other embodiments, the compounds shown herein are prepared in different forms including, but not limited to, amorphous, pulverized and nano-particle sized forms. Furthermore, the compounds shown in the present invention include crystalline forms and may also exist as polymorphs. Polymorphs include different lattice arrangements of the same elemental composition of a compound. Polymorphs usually have different X-ray diffraction spectra, infrared spectra, melting points, densities, hardness, crystal forms, optical and electrical properties, stability and solubility. Different factors such as recrystallization solvent, crystallization rate and storage temperature may cause a single crystal form to predominate.

In another aspect, the compounds shown in the present invention may exhibit chiral centers and/or axial chirality and thus exist as racemates, racemic mixtures, single enantiomers, diastereoisomeric compounds and single diastereomers. enantiomers, and cis-trans isomers. Each chiral center or axial chirality will independently give rise to two optical isomers, and all possible optical isomers and diastereomeric mixtures as well as pure or partially pure compounds are included within the scope of the invention. The present invention is meant to include all such isomeric forms of these compounds.

The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compounds. For example, compounds can be labeled with radioactive isotopes such as tritium ( ³ H), iodine-125 ( ¹²⁵ I), and C-14 ( ¹⁴ C). For another example, heavy hydrogen can be used to replace hydrogen atoms to form deuterated compounds. The bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Stability, enhanced curative effect, extended drug half-life in vivo and other advantages. All changes in isotopic composition of the compounds of the invention, whether radioactive or not, are encompassed within the scope of the invention.

the term

Unless otherwise stated, the terms used in the present application, including the specification and claims, are defined as follows. It must be noted that in the specification and appended claims, the singular form "a" and "an" includes plural references unless the context clearly dictates otherwise. If not stated otherwise, conventional methods of mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology are used. In this application, the use of "or" or "and" means "and/or" if not stated otherwise.

Certain pharmaceutical and medical terms

The term "acceptable", as used herein, means that a formulation ingredient or active ingredient does not have an undue adverse effect on health for the general purpose of treatment.

The term "treatment", "course of treatment" or "therapy" as used herein includes alleviating, suppressing or improving the symptoms or conditions of a disease; inhibiting the development of complications; improving or preventing the underlying metabolic syndrome; inhibiting the development of diseases or symptoms, Such as controlling the development of a disease or condition; alleviating a disease or a symptom; causing a disease or a symptom to regress; alleviating a complication caused by a disease or a symptom, or preventing or treating a symptom caused by a disease or a symptom. As used herein, a certain compound or pharmaceutical composition, after administration, can improve a certain disease, symptom or condition, especially improve its severity, delay the onset, slow down the progression of the disease, or reduce the duration of the disease. Circumstances that may be attributable to or related to the administration, whether fixed or episodic, continuous or intermittent.

"Active ingredient" refers to the compounds shown in the present invention, and the pharmaceutically acceptable inorganic or organic salts of the compounds shown in the present invention. The compounds of the present invention may contain one or more asymmetric centers (chiral centers or axial chirality) and thus exist as racemates, racemic mixtures, single enantiomers, diastereomeric compounds and single non- Enantiomers occur in the form of enantiomers. The asymmetric centers that can exist depend on the nature of the various substituents on the molecule. Each such asymmetric center will independently give rise to two optical isomers and all possible optical isomers and diastereomeric mixtures as well as pure or partially pure compounds are included within the scope of the invention. The present invention is meant to include all such isomeric forms of these compounds.

The terms "compound", "composition", "agent" or "medicine or medicament" are used interchangeably herein and refer to In animals), a compound or composition capable of inducing a desired pharmaceutical and/or physiological response through local and/or systemic action.

The term "administered, administering, or administration" as used herein means direct administration of the compound or composition, or administration of a prodrug, derivative, or analog of the active compound Wait.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the relative numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently inherently contain standard deviations resulting from their individual testing methodology. As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Alternatively, the term "about" means that the actual value falls within an acceptable standard error of the mean, as considered by those skilled in the art. Except for the experimental examples, or unless otherwise expressly stated, all ranges, quantities, numerical values and percentages used herein should be understood (for example, to describe the amount of material used, the length of time, temperature, operating conditions, quantitative ratios and other similar Those) are modified by "about". Therefore, unless otherwise stated to the contrary, the numerical parameters disclosed in the specification and the appended claims are approximate values and may be changed as required. At a minimum, these numerical parameters should be understood as the number of significant digits indicated plus the usual rounding method.

Unless otherwise defined in this specification, the meanings of scientific and technical terms used herein are the same as the usual meanings understood by those skilled in the art. In addition, the singular nouns used in this specification include the plural forms of the nouns, and the plural nouns used also include the singular forms of the nouns, unless the context conflicts with the context.

therapeutic use

The present invention provides that the compounds or pharmaceutical compositions shown herein are generally useful for inhibiting Axl kinase and thus for treating one or more conditions associated with Axl kinase activity. Accordingly, in certain embodiments, the present invention provides a method for treating an Axl kinase-mediated disorder comprising the step of administering a compound of the present invention, or a pharmaceutically acceptable composition thereof, to a patient in need thereof .

Cancers treatable with compounds of the invention include, but are not limited to, hematological malignancies (leukemias, lymphomas, myelomas including multiple myeloma, myelodysplastic syndromes, and myeloproliferative syndromes) and solid tumors (carcinomas such as prostate, breast, , lung, colon, pancreas, kidney, ovary and soft tissue cancer and osteosarcoma, and stromal tumors) etc.

Route of administration

The compounds of the present invention and their pharmaceutically acceptable salts can be made into various preparations, which contain the compounds of the present invention or their pharmaceutically acceptable salts and pharmaceutically acceptable excipients or carriers within the range of safe and effective amounts . Wherein, "safe and effective amount" means: the amount of the compound is sufficient to obviously improve the condition without causing severe side effects. The safe and effective dose of the compound is determined according to the specific conditions such as the age, condition, and course of treatment of the subject to be treated.

"Pharmaceutically acceptable excipient or carrier" means: one or more compatible solid or liquid filler or gel substances, which are suitable for human use and must be of sufficient purity and low enough toxicity . "Compatibility" herein means that the components of the composition can be blended with the compound of the present invention and with each other without significantly reducing the efficacy of the compound. Examples of pharmaceutically acceptable excipients or carrier parts include cellulose and derivatives thereof (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants ( Such as stearic acid, magnesium stearate), calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as

), wetting agent (such as sodium lauryl sulfate), coloring agent, flavoring agent, stabilizer, antioxidant, preservative, pyrogen-free water, etc.

When the compounds of the present invention are administered, they can be administered orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), topically.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is admixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with (a) fillers or extenders, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, For example, glycerol; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow agents, such as paraffin; (f) Absorption accelerators such as quaternary ammonium compounds; (g) wetting agents such as cetyl alcohol and glyceryl monostearate; (h) adsorbents such as kaolin; and (i) lubricants such as talc, hard Calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shell materials, such as enteric coatings and others well known in the art. They may contain opacifying agents and, in such compositions, the release of the active compound or compounds may be in a certain part of the alimentary canal in a delayed manner. Examples of usable embedding components are polymeric substances and waxy substances. The active compounds can also be in microencapsulated form, if desired, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, etc.

Besides such inert diluents, the compositions can also contain adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols, and suitable mixtures thereof.

Dosage forms for topical administration of a compound of this invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required, if necessary.

The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds. When using a pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage is a pharmaceutically effective dosage when administered, for a person with a body weight of 60kg, the daily The dosage is usually 1-2000 mg, preferably 50-1000 mg. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

The above-mentioned features mentioned in the present invention, or the features mentioned in the embodiments can be combined arbitrarily. All the features disclosed in the specification of this case can be used in combination with any combination, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equivalent or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equivalent or similar features.

Detailed ways

In the following description, various specific aspects, characteristics and advantages of the above-mentioned compounds, methods, and pharmaceutical compositions will be described in detail, so that the content of the present invention will become very clear. It is to be understood that the following detailed description and examples describe specific embodiments and are provided for informational purposes only. After reading the description of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent situations also fall within the scope defined in the present application.

In all the examples, ¹ H-NMR was recorded by a Varian Mercury 400 nuclear magnetic resonance apparatus, and the chemical shifts were expressed in δ (ppm); the silica gel used for separation was 200-300 mesh, and the ratio of the eluent was volume ratio.

The present invention adopts the following abbreviations: _CDCl represents deuterated chloroform; EtOAc represents ethyl acetate; Hexane represents n-hexane; HPLC represents high performance liquid chromatography; MeCN represents acetonitrile; DCM represents dichloromethane; DIPEA represents diisopropyl Ethylamine; DMF stands for N,N-dimethylformamide; DMAP stands for 4-(dimethylamino)pyridine; DMSO stands for dimethylsulfoxide; HATU stands for O-(7-azabenzotriazole-1- base)-NNN'N'-tetramethyluronium hexafluorophosphate; hr stands for hour; min stands for minute; K ₂ CO ₃ stands for potassium carbonate; min stands for minute; MeOH stands for methanol; MS stands for mass spectrum; NMR stands for nuclear magnetic resonance; Pd/C stands for palladium on carbon; POCl ₃ stands for phosphorus oxychloride; TEA stands for triethylamine; TLC stands for thin layer chromatography.

The synthesis of embodiment 1 compound 1

Step 1: Synthesis of compound int_1-2:

Add int_1-1 (200mg, 0.951mmol), DMF (5mL), anhydrous potassium carbonate (5.92g, 42.9mmol) and fluoroiodomethane (229mg, 1.43mmol) into a 100mL single-necked bottle, and the mixture was replaced with argon and stirred at room temperature Reacted for 16 hours, after LC-MS detected that the reaction was complete, ethyl acetate (10mL) and water (10mL) were added to the mixed solution, stirred, separated, the aqueous phase was extracted with ethyl acetate (10mL), and the combined organic phases were washed with saturated Sodium chloride solution (10mL*2) was washed twice, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a light brown solid product (211mg, yield: 90.5%).

ESI-MS m/z:242[M+H] ⁺

Step 2: Synthesis of compound int_1-3:

Add int_1-2 (211mg, 0.86mmol), DMF (5mL), DIPEA (1.5mL) and 6-nitropyridin-3-alcohol (121mg, 0.86mmol) in the 50mL one-port bottle, and the mixture is heated to Stir and react at 100°C for 72 hours. After the reaction was detected by LC-MS, ethyl acetate (10mL) and water (10mL) were added to the mixture, stirred, separated, and the aqueous phase was extracted with ethyl acetate (10mL) and combined. The organic phase was washed successively with saturated sodium bicarbonate solution (10 mL), saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give a brown oil (228 mg, yield: 76%). ESI-MS m/z:346[M+H] ⁺

Step 3: Synthesis of compound int_1-4:

Add int_1-3 (228mg, crude product, 0.66mmol), MeOH (10mL), DCM (1mL), 10% palladium on carbon (50mg, wet=55%) into a 100mL single-necked bottle, replace hydrogen three times, and hydrogenate with stirring at room temperature and normal pressure. After 72 hours, LC-MS detected that the reaction was complete, the mixture was filtered through diatomaceous earth, and the filtrate was concentrated to obtain the product (220 mg, yield: 100%).

ESI-MS m/z:316[M+H] ⁺

Step 4: Synthesis of compound 1:

Add int_1-4 (93mg, 0.295mmol), DMF (5mL), DIPEA (114mg, 0.885mmol), HATU (170mg, 0.442mmol) and int_1-5 (84mg, 0.295mmol) in a 50mL one-port bottle, and mix liquid argon After replacement, the temperature was raised to 60°C and the reaction was stirred for 4 hours. After the reaction was detected by LC-MS, the mixture was purified by preparative LC-MS to obtain the product (16 mg, yield: 9.3%).

¹ H NMR (400MHz, Chloroform-d) δ11.94(s, 1H), 9.33(s, 1H), 8.55(d, J=5.3Hz, 1H), 8.50(d, J=9.0Hz, 1H), 8.21(d, J=2.8Hz, 1H), 7.92(s, 1H), 7.66–7.54(m, 4H), 7.48(s, 1H), 7.27(d, J=1.7Hz, 1H), 7.25(s ,1H),6.44(d,J＝5.3Hz,1H),5.98(s,1H),5.84(s,1H),4.05(s,3H),2.65–2.54(m,4H),2.12(q, J＝6.3Hz, 2H).

ESI-MS m/z:581[M+H] ⁺

The synthesis of embodiment 2 compound 2

Step 1: the synthesis of compound int_2-2:

Add int_2-1 (200mg, 0.951mmol), DMF (5mL), anhydrous potassium carbonate (5.92g, 42.9mmol) and fluoroiodomethane (229mg, 1.43mmol) into a 100mL single-necked bottle, and the mixture was replaced with argon and stirred at room temperature Reacted for 16 hours, after LC-MS detected that the reaction was complete, ethyl acetate (10mL) and water (10mL) were added to the mixture, stirred, separated, the aqueous phase was extracted with ethyl acetate (10mL), and the combined organic phases were washed with saturated Sodium chloride solution (10mL*2) was washed twice, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a light brown solid product (208mg, yield: 90%).

ESI-MS m/z:242[M+H] ⁺

Step 2: the synthesis of compound int_2-3:

Add int_2-2 (208mg, 0.86mmol), DMF (5mL), DIPEA (1.5mL) and 6-nitropyridin-3-alcohol (121mg, 0.86mmol) in the 50mL one-port bottle, and the mixture is heated to Stir and react at 100°C for 72 hours. After the reaction was detected by LC-MS, ethyl acetate (10mL) and water (10mL) were added to the mixture, stirred, separated, and the aqueous phase was extracted with ethyl acetate (10mL) and combined. The organic phase was washed successively with saturated sodium bicarbonate solution (10 mL), saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give a brown oil (220 mg, yield: 76%). ESI-MS m/z:346[M+H] ⁺

Step 3: Synthesis of compound int_2-4:

Add int_2-3 (220mg, crude product, 0.66mmol), MeOH (10mL), DCM (1mL), 10% palladium on carbon (50mg, wet=55%) into a 100mL single-necked bottle, replace hydrogen three times, and hydrogenate with stirring at room temperature and normal pressure. After 72 hours, LC-MS detected that the reaction was complete, the mixture was filtered through diatomaceous earth, and the filtrate was concentrated to obtain the product (220 mg, yield: 100%).

ESI-MS m/z:316[M+H] ⁺

Step 4: Synthesis of compound 2:

Add int_2-4 (100mg, 0.317mmol), DMF (5mL), DIPEA (114mg, 0.885mmol), HATU (170mg, 0.442mmol) and int_1-5 (84mg, 0.295mmol) in a 50mL one-port bottle, and mix liquid argon After replacement, the temperature was raised to 60°C and the reaction was stirred for 4 hours. After LC-MS detection, the reaction was almost complete, and the mixture was purified by preparative LC-MS to obtain the product (19 mg, yield: 10.3%).

¹ H NMR (400MHz, Chloroform-d) δ11.95(s, 1H), 9.32(s, 1H), 8.54(d, J=5.3Hz, 1H), 8.51(d, J=9.0Hz, 1H), 8.22(d, J=2.8Hz, 1H), 7.80(s, 1H), 7.66–7.54(m, 5H), 7.27(d, J=1.5Hz, 1H), 6.50(d, J=5.3Hz, 1H ),6.00(s,1H),5.87(s,1H),4.06(s,3H),2.59(dt,J=13.3,6.3Hz,4H),2.11(q,J=6.3Hz,2H).

ESI-MS m/z:581[M+H] ⁺ .

The synthesis of embodiment 3 compound 3

Step 1: the synthesis of compound int_3-2:

Add 40 ml of nitric acid (64%) to a 100 ml single-necked bottle, add 1-fluoro-2,3-dimethoxybenzene (8 g, 51.23 mmol) dropwise under ice bath, stir for 15 minutes under ice bath, then naturally rise to room temperature Stirring was continued for 15 minutes. After the reaction was monitored by TLC, the reaction solution was poured into 200 ml of cold water and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was subjected to column chromatography to obtain the target product (6 g, yield: 58%, white solid).

¹ H NMR (400MHz, Chloroform-d) δ7.68 (ddd, J = 10.7, 2.7, 0.8Hz, 1H), 7.61 (t, J = 2.2Hz, 1H), 4.07 (dd, J = 2.2, 0.8Hz ,3H),3.96(d,J=0.8Hz,3H).

Step 2: the synthesis of compound int_3-3:

Int_3-2 (6 g, 29.83 mmol), 10% palladium on carbon (600 mg, wet=55%) were suspended in methanol (150 mL) at room temperature. After hydrogen replacement three times, the mixture was stirred at room temperature and pressure overnight. A large amount of product was detected by LC-MS, the mixed solution was filtered through diatomaceous earth, and the filtrate was concentrated to obtain the target product (5 g, yield 97%, gray solid).

ESI-MS m/z:172[M+H] ⁺

Step 3: the synthesis of compound int_3-5:

Int_3-3 (5.11 g, 29.85 mmol) and int_3-4 (6.46 g, 29.85 mmol) were suspended in ethanol (150 mL) at room temperature. Stir at reflux for 1 hour under the protection of argon. The completion of the reaction was monitored by LC-MS. After cooling to room temperature, it was concentrated under reduced pressure, and the residue was subjected to column chromatography to obtain the target product (10 g, yield 98%, brown liquid).

Step 4: the synthesis of compound int_3-6:

Suspend int_3-5 (10.2 g, 29.88 mmol) in diphenyl ether (100 mL) at room temperature, heat up to 250° C. and reflux for 1 hour. The completion of the reaction was monitored by LC-MS. After cooling to room temperature, the insoluble matter was collected by filtration to obtain the target product (2.6 g, yield 30%, brown solid). The crude product was directly used in the next reaction.

ESI-MS m/z:296[M+H] ⁺

Step 5: Synthesis of compound int_3-7:

Suspend int_3-6 (3g, 10.16mmol) in ethanol (20mL) at room temperature, add aqueous sodium hydroxide solution (4M, 80mL), and stir the reaction solution at 90°C for 2 hours. LC-MS monitors the completion of the reaction and cools to room temperature Afterwards, the pH was adjusted to 1 with concentrated hydrochloric acid, and the solid was collected, washed with cold dilute hydrochloric acid, and dried to obtain the target product (2.6 g, yield: 95%, gray-brown solid). ESI-MS m/z:268[M+H] ⁺

Step 6: Synthesis of compound int_3-8:

Suspend int_3-6 (2.6 g, 9.73 mmol) in diphenyl ether (120 mL) at room temperature, heat up to 250° C. and reflux for 1 hour. The completion of the reaction was monitored by LC-MS. After cooling to room temperature, the insoluble matter was collected by filtration to obtain the target product (2 g, yield 92%, gray solid).

ESI-MS m/z:224[M+H] ⁺

Step 7: Synthesis of compound int_3-9:

Int_3-8 (1.7 g, 7.62 mmol) was dissolved in phosphine oxychloride (15 mL) at room temperature. Stir at 100°C for 1 hour, LC-MS monitors the completion of the reaction, concentrate under reduced pressure to remove excess phosphine oxychloride, add the residue into a large amount of water and stir for 10 minutes. DCM was extracted, the organic phase was dried over anhydrous sodium sulfate, the filtrate was concentrated under reduced pressure, and the residue was subjected to column chromatography to obtain the target product (1.3 g, yield: 61%, gray solid).

¹ H NMR (400MHz, DMSO-d6) δ8.68 (d, J = 4.8Hz, 1H), 7.56 (d, J = 4.8Hz, 1H), 7.41 (d, J = 1.9Hz, 1H), 3.99 ( s,3H),3.92(s,3H).

ESI-MS m/z:242[M+H] ⁺

Step 8: Synthesis of compound int_3-10:

Add int_3-9 (300mg, 1.24mmol), int_3-10 (260.7mg, 1.24mmol), chlorobenzene (12mL), DMAP (227mg, 1.86mmol) into a 50mL single-necked bottle, and heat up the mixture to 130°C after argon replacement The reaction was stirred for 12 hours. After the reaction was detected by LC-MS, ethyl acetate (10mL) and water (10mL) were added to the mixture, stirred, separated, and the aqueous phase was extracted with ethyl acetate (10mL), and the organic phase was combined. It was washed with saturated sodium bicarbonate solution (10 mL) and saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give a brown oil (68 mg, yield: 17.5%).

ESI-MS m/z:316[M+H] ⁺

Step 9: Synthesis of compound 3:

Add int_3-11 (5 7mg, 0.17mmol), DMF (5mL), DIPEA (66mg, 0.5mmol), HATU (100mg, 0.34mmol) and int_1-5 (97mg, 0.34mmol) in a 50mL one-port bottle, and mix liquid argon After gas replacement, the temperature was raised to 60°C and the reaction was stirred for 4 hours. After the reaction was almost complete as detected by LC-MS, the mixture was purified by preparative LC-MS to obtain the product (20 mg, yield: 41.5%, white solid).

¹ H NMR (400MHz, DMSO-d6) δ11.97(s, 1H), 8.97(s, 1H), 8.63(d, J=5.5Hz, 1H), 8.42(d, J=9.1Hz, 1H), 8.34(d, J=2.9Hz, 1H), 7.86(dd, J=9.0, 2.9Hz, 1H), 7.69–7.57(m, 3H), 7.46(dd, J=7.0, 1.8Hz, 2H), 7.37 (d,J=1.7Hz,1H),6.69(d,J=5.5Hz,1H),4.00(s,3H),3.91(s,3H),2.53(d,J=5.9Hz,4H),1.98 (p,J=6.1Hz,2H).

ESI-MS m/z:581[M+H] ⁺

The synthesis of embodiment 4 compound 4

Step 1: the synthesis of compound int_4-2:

Add int_4-1 (210mg, 0.86mmol), DMF (5mL), DIPEA (1.5mL) and 6-nitropyridin-3-alcohol (121mg, 0.86mmol) in the 50mL one-port bottle, and the mixture is heated to Stir and react at 100°C for 72h. After the reaction was detected by LC-MS, ethyl acetate (10mL) and water (10mL) were added to the mixture, stirred, separated, and the aqueous phase was extracted with ethyl acetate (10mL). The phase was washed successively with saturated sodium bicarbonate solution (10 mL) and saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give an oil (198 mg, yield: 68%).

ESI-MS m/z:346[M+H] ⁺

Step 2: the synthesis of compound int_4-3:

Add int_4-2 (198mg, 0.57mmol), MeOH (10mL), DCM (1mL), 10% palladium carbon (50mg, wet=55%) into a 100mL single-necked bottle, replace hydrogen three times, and hydrogenate with stirring at room temperature and pressure. After 72 hours, LC-MS detected that the reaction was complete, the mixture was filtered through diatomaceous earth, and the filtrate was concentrated to obtain the product (180 mg, yield: 100%).

ESI-MS m/z:316[M+H] ⁺

Step 4: Synthesis of compound 4:

Add int_4-3 (100mg, 0.317mmol), DMF (5mL), DIPEA (114mg, 0.885mmol), HATU (170mg, 0.442mmol) and int_1-5 (84mg, 0.295mmol) into a 50mL single-necked bottle, mixed liquid argon After replacement, the temperature was raised to 60° C. and the reaction was stirred for 4 h. After the reaction was detected by LC-MS, the mixture was purified by preparative LC-MS to obtain the product (29 mg, yield: 15.8%).

ESI-MS m/z:581[M+H] ⁺

Embodiment 5 Compounds of the present invention inhibit Axl enzyme activity test in vitro

Compounds diluted in DMSO gradients and Axl or c-Met recombinant protein were mixed, and after standing at room temperature for 10 minutes, biotin-labeled TK substrate (TK) and ATP were added. After reacting at room temperature for 40 minutes, Sa-XL 665 and Crytate-labeled TK antibody were added, and after incubation at room temperature for 1 hour, the fluorescence intensity at 615 and 665 nm was detected. Calculate the ratio of the 665 nm and 615 nm fluorescence intensities. Compound _IC50 was calculated compared to DMSO control. The results are shown in Table 1 below.

Table 1. The inhibitory activity of the compounds of the present invention to recombinant protein Axl

compound	Axl enzyme activity inhibition (IC 50 ,nM)	c-Met enzyme activity inhibition (IC 50 ,nM)
1	0.8	5.49
2	+++	-
3	+++	-
4	+++	-
Compound A	1.1	13.35

+++ means IC ₅₀ less than or equal to 50nM

++ indicates an _IC50 of 50nM to 100nM

+ indicates _IC50 greater than 100 nM.

- Indicates untested activity

It can be seen from the data in Table 1 that the compounds of the present invention have better inhibitory activity on the enzyme activity of recombinant protein Axl. And compared with compound A, compound 1 has an unexpected improvement in the inhibitory activity of c-Met enzyme.

Example 6 The compounds of the present invention have antiproliferative activity in vitro on BaF3-Axl cells

10000/well of Axl-overexpressed BaF3 (BaF3-Axl) cells were planted in a 96-well plate, and then the compound diluted with DMSO was added. After 3 days of treatment, the intracellular ATP level was measured by CTL-PLUS. Compared with the DMSO group, the percentage of inhibition of cell growth and the IC ₅₀ of the compounds were calculated, and the results are shown in Table 2 below.

Table 2 The compound of the present invention is to the antiproliferative activity of BaF3-Axl cell

compound	IC 50 (nM) of anti-proliferation activity of BaF3-Axl cells
1	0.5
2	3.5
3	5
4	>5
Compound A	1.2

From the data in Table 2, it can be seen that the compound of the present invention has stronger anti-proliferation activity on BaF3/Axl cells, but the anti-proliferation activity of compound 2 and compound 3 on BaF3/Axl cells is weaker than that of compound 1. This result indicates that in compounds 1, 2, and 3, the position of fluorine atom substitution has an unexpected effect on the activity of the compounds. And compared with compound A, compound 1 has unexpectedly improved anti-BaF3/Axl proliferation activity.

Embodiment 7 In vivo pharmacokinetic experiment of the compound of the present invention

CD-1 female mice aged 7 to 10 weeks were selected, and the doses of intravenous and oral administration were 2 mg/Kg and 10 mg/Kg, respectively. Mice were fasted for at least 12 hours before administration, fed again 4 hours after administration, and had free access to water throughout the experiment. On the day of the experiment, the animals in the intravenous group were administered with the corresponding compound through a single injection of the tail vein, and the administration volume was 10 ml/Kg. Animals in the oral group were given the corresponding compound by intragastric single injection, and the administration volume was 10 ml/Kg. The animals were weighed before administration, and the administration volume was calculated according to the body weight. Sample collection time: 0.083 (injection group), 0.167, 0.5, 1, 2, 4, 8, 24h. At each time point, about 200 uL of whole blood was collected through the submandibular venous plexus to prepare plasma for concentration determination by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). All animals were euthanized under CO ₂ anesthesia after collecting the PK samples at the last time point. The non-compartmental model of Phoenix WinNonlin ^TM version8.3 (Certara) pharmacokinetic software was used to process the plasma concentration, and the linear logarithmic trapezoidal method was used to calculate the pharmacokinetic parameters. The in vivo pharmacokinetic results are shown in Table 3 below.

Table 3 In vivo pharmacokinetic evaluation results of the compounds of the present invention

It can be seen from the data in Table 3 that in the mouse pharmacokinetic experiment, compared with compound A, the Vdss of compound 1 of the present invention increased, the clearance rate in the body decreased, the half-life was prolonged, and the AUC increased. Compound 1 has unexpectedly improved pharmacokinetic properties compared to Compound A.

Although the specific implementations of the present invention have been described above, those skilled in the art should understand that these are only examples, and various changes or changes can be made to these implementations without departing from the principle and essence of the present invention. Revise. Accordingly, the protection scope of the present invention is defined by the appended claims.

Claims (3)

1. A compound represented by formula (1)-formula (4) or its isomers, crystal forms, pharmaceutically acceptable salts, hydrates or solvates:

   
2. A pharmaceutical composition, characterized in that it contains a pharmaceutically acceptable excipient or carrier, and the compound as claimed in claim 1, or its isomers, crystal forms, and pharmaceutically acceptable salts , hydrate or solvate as the active ingredient.
3. A compound as claimed in claim 1, or each isomer thereof, each crystal form, a pharmaceutically acceptable salt, hydrate or solvate, or a pharmaceutical composition as claimed in claim 2 in the preparation of a treatment and Application in drugs for Axl protein-related diseases.