CN117412953A

CN117412953A - Axl inhibitors

Info

Publication number: CN117412953A
Application number: CN202280034595.5A
Authority: CN
Inventors: 谢雨礼; 吴应鸣; 钱立晖; 樊后兴
Original assignee: Wigen Biomedicine Technology Shanghai Co Ltd
Current assignee: Wigen Biomedicine Technology Shanghai Co Ltd
Priority date: 2021-05-12
Filing date: 2022-05-11
Publication date: 2024-01-16
Also published as: WO2022237843A1

Abstract

A class of Axl inhibitors. In particular to a novel compound shown as a formula (1), (2), (3) and (4) and/or pharmaceutically acceptable salt thereof, a composition containing the compound shown as a formula (1), (2), (3) and (4) and/or pharmaceutically acceptable salt thereof, a preparation method and application thereof as an Axl inhibitor in preparation of antitumor drugs.

Description

Axl inhibitors

The present application claims priority from chinese application CN202110518860.4, with application date 2021, 5-12. The present application refers to the entirety of the above-mentioned chinese application.

Technical Field

The invention relates to the field of pharmaceutical chemistry, in particular to a novel compound with an Axl kinase inhibition effect, a preparation method thereof and application of the novel compound in preparation of antitumor drugs.

Background

Receptor Tyrosine Kinases (RTKs) sense extracellular cell signals, activate cytoplasmic and nuclear signal transduction pathways, and regulate cell survival, growth, differentiation, adhesion, and migration. Aberrant expression or activation of RTKs is involved in transformation with cancer cells, 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 a ligand, a transmembrane domain that mediates signaling, and a conserved intracellular kinase domain. Axl and MER are overexpressed in various leukemia and various solid tumor cells, promoting survival, metastasis and resistance of cancer cells. In addition to cancer cells, AXL is expressed in vascular epithelial and smooth cells in tumor microenvironments (Korshunov et al, circ. Res.2006,98,1446), as well as in a variety of immune cells including macrophages, dendritic cells, natural killer cells, etc., promoting angiogenesis, immunosuppression and immune escape of tumor cells (shirif 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 follows that AXL is involved in tumor development and progression through multiple mechanisms.

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

Therefore, the development of small molecule inhibitors of highly active Axl kinase is of great clinical value.

WO2015012298A1 reports compound A having Axl inhibitory activity (example 5 in WO2015012298A 1). Clinical phase I experiments of the compound for the treatment of octenib resistant non-small-cell lung cancer (osimerinib-resistant non-small-cell lung cancer) are underway;

disclosure of Invention

The present invention provides a compound represented by the formula (1) -formula (4) or each isomer, each crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof:

it is another object of the present invention to provide a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent and/or excipient, and as an active ingredient a compound of the present invention, or each isomer, each crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof.

A further object of the present invention is to provide the use of a compound of the present invention, or each isomer, each crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof or a pharmaceutical composition as described above, for the manufacture of a medicament for the treatment, modulation or prevention of diseases associated with Axl protein.

Still another object of the present invention is to provide a method for treating, modulating or preventing diseases associated with Axl protein, comprising administering to a subject a therapeutically effective amount of a compound of the present invention, or each isomer, each crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition thereof.

Through synthesis and careful study of a variety of novel compounds involved in having Axl inhibitory effect, the inventors have found that among the compounds represented by formulas (1), (2), (3), (4), compound (1) has unexpected improvement in PK properties and improvement in Axl inhibitory activity as compared with compound a.

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 the compounds

By "pharmaceutically acceptable" is meant herein a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and which is relatively non-toxic, e.g., administration of a material to an individual does not cause an undesired biological effect or interact in a deleterious manner with any of the components thereof in which it is contained.

The term "pharmaceutically acceptable salt" refers to a form of a compound that does not cause significant irritation to the organism to which it is administered, and does not abrogate the biological activity and properties of the compound. In certain specific aspects, pharmaceutically acceptable salts are obtained by reacting a compound of the present invention with an acid, such as an inorganic acid, e.g., hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, and the like, an organic acid, e.g., 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, and the like, or an acidic amino acid, e.g., aspartic acid, glutamic acid.

References to pharmaceutically acceptable salts are understood to include solvent-added forms or crystalline forms, particularly solvates or polymorphs. Solvates contain a stoichiometric or non-stoichiometric amount of solvent 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 of the invention are conveniently prepared or formed in accordance with the methods described herein. The hydrates of the compounds of the present invention are conveniently prepared by recrystallisation from a water/organic solvent mixture, using organic solvents including, but not limited to, tetrahydrofuran, acetone, ethanol or methanol. Furthermore, the compounds mentioned herein can exist in unsolvated and solvated forms. In summary, for the purposes of the compounds and methods provided herein, solvated forms are considered to correspond to unsolvated forms.

In other embodiments, the compounds of the present invention are prepared in different forms, including, but not limited to, amorphous, crushed, and nano-sized forms. Furthermore, the compounds of the present invention include crystalline forms and may also be polymorphic forms. Polymorphs include different lattice arrangements of the same elemental composition of the compound. Polymorphs typically 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 crystalline form to dominate.

In another aspect, the compounds of the present invention may exhibit chiral centers and/or axial chiralities and thus occur as racemates, racemic mixtures, single enantiomers, diastereomeric compounds and single diastereomeric forms, and cis-trans isomers. Each chiral center or axial chiral will independently produce 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 non-natural proportions of atomic isotopes on one or more of the atoms comprising the compounds. For example, compounds can be labeled with radioisotopes, such as tritium @, for example ³ H) Iodine-125% ¹²⁵ I) And C-14% ¹⁴ C) A. The invention relates to a method for producing a fibre-reinforced plastic composite For another example, deuterium can be substituted for a hydrogen atom to form a deuterated compound, and the bond between deuterium and carbon is stronger than the bond between normal hydrogen and carbon, and generally deuterated drugs have the advantages of reducing toxic side effects, increasing drug stability, enhancing therapeutic effects, prolonging in vivo half-life of drugs, and the like, compared to non-deuterated drugs. All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

Terminology

The terms used in the present application, including the specification and claims, are defined as follows, unless otherwise indicated. It must be noted that, in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Conventional methods of mass spectrometry, nuclear magnetism, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology are used, if not otherwise indicated. In this application, the use of "or" and "means" and/or "unless otherwise indicated.

Specific pharmaceutical and medical terminology

The term "acceptable" as used herein, means that a prescription component or active ingredient does not unduly adversely affect the health of the general therapeutic objective.

The terms "treat," "course of treatment," or "therapy" as used herein include alleviation, inhibition, or amelioration of symptoms or conditions of a disease; inhibit the occurrence of complications; improving or preventing underlying metabolic syndrome; inhibiting the occurrence of a disease or condition, such as controlling the progression of a disease or condition; alleviating a disease or symptom; causing the disease or symptom to subside; alleviating complications caused by diseases or symptoms, or preventing or treating signs caused by diseases or symptoms. As used herein, a compound or pharmaceutical composition, upon administration, may result in an improvement in a disease, symptom, or condition, particularly an improvement in severity, delay of onset, delay of progression, or decrease in duration of the condition. Whether stationary or temporary, continuous or intermittent, may be due to or associated with administration.

"active ingredient" refers to the compounds of the present invention, as well as pharmaceutically acceptable inorganic or organic salts of the compounds of the present invention. The compounds of the invention may contain one or more asymmetric centers (chiral centers or axial chiralities) and thus appear as racemates, racemic mixtures, single enantiomers, diastereomeric compounds and single diastereomers. Asymmetric centers that may be present depend on the nature of the various substituents on the molecule. Each such asymmetric center will independently produce 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 "pharmaceutical (medicine or medicament)" are used interchangeably herein and refer to a compound or composition capable of inducing a desired pharmaceutical and/or physiological response through local and/or systemic effects when administered to an individual (human or animal).

The term "administration (administered, administering or administeration)" as used herein refers to the administration of the compound or composition directly, or the administration of a prodrug (pro), derivative (derivative), or analog (analog) of the active compound, and the like.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain standard deviations found in their respective testing measurements. 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 average value, as determined by one of ordinary skill in the art. Except in the experimental examples, or where otherwise explicitly indicated, all ranges, amounts, values, and percentages used herein (e.g., to describe amounts of materials, lengths of time, temperatures, operating conditions, ratios of amounts, and the like) are to be understood to be modified by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present specification and attached claims are approximations that may vary depending upon the desired properties. At least these numerical parameters should be construed as indicating the number of significant digits and by applying ordinary rounding techniques.

Unless defined otherwise herein, the meanings of scientific and technical terms used herein are the same as commonly understood by one of ordinary skill in the art. Furthermore, as used in this specification, the singular noun encompasses the plural version of the noun without conflict with the context; plural nouns as used also encompasses singular versions of the noun.

Therapeutic use

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

Cancers that may be treated with the compounds of the present invention include, but are not limited to, hematological malignancies (leukemias, lymphomas, myelomas including multiple myeloma, myelodysplastic syndrome, and myeloproliferative last name syndrome) and solid tumors (carcinomas such as prostate, breast, lung, colon, pancreas, kidney, ovary, and soft tissue carcinomas and osteosarcomas, as well as stromal tumors), among others.

Route of administration

The compounds of the present invention and pharmaceutically acceptable salts thereof can be formulated into a variety of formulations comprising a safe and effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof in combination with a pharmaceutically acceptable excipient or carrier. Wherein "safe, effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. The safe and effective amount of the compound is determined according to the specific conditions such as age, illness and treatment course of the subject.

"pharmaceutically acceptable excipient or carrier" means: one or more compatible solid or liquid filler or gel materials which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "compatible" as used herein means that the components of the composition are capable of blending with and between the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable excipients or carrier moieties are cellulose and its derivatives (e.g. sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g. stearic acid magnesium stearate), calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyalcohol (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), and emulsifying agent (such as) Wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizing agents, antioxidants, preservatives, pyrogen-free water and the like.

The compounds of the present invention may 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 the following ingredients: (a) Fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) Binders, for example, hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, e.g., glycerin; (d) Disintegrants, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) a slow solvent, such as paraffin; (f) an absorption accelerator, e.g., a quaternary amine compound; (g) Wetting agents, such as cetyl alcohol and glycerol monostearate; (h) an adsorbent, for example, kaolin; and (i) a lubricant, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be released in a delayed manner in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. The active compound may also be in the form of microcapsules with one or more of the above excipients, if desired.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of these substances and the like.

In addition to these inert diluents, the compositions can also include 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 as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar-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 nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms of the compounds of the present invention for topical administration 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 invention may be administered alone or in combination with other pharmaceutically acceptable compounds. When a pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is applied to a mammal (e.g., a human) in need of treatment, wherein the dose at the time of administration is a pharmaceutically effective dose, and the daily dose is usually 1 to 2000mg, preferably 50 to 1000mg, for a human having a body weight of 60 kg. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

The above-mentioned features of the invention, or of the embodiments, may be combined in any desired manner. All of the features disclosed in this specification may be combined with any combination of the features disclosed in this specification, and the various features disclosed in this specification may be substituted for any alternative feature serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the disclosed features are merely general examples of equivalent or similar features.

Detailed Description

The details of the various specific aspects, features and advantages of the above-described compounds, methods, pharmaceutical compositions will be set forth in the following description in order to provide a thorough understanding of the present invention. It is to be understood that the detailed description and examples, which follow, describe specific embodiments for reference only. Various changes and modifications to the present invention will become apparent to those skilled in the art upon reading the present description, and such equivalents fall within the scope of the present application.

In all of the embodiments described herein, the present invention, ¹ H-NMR was recorded on a Varian Mercury 400 Nuclear magnetic resonance apparatus, chemical shifts being expressed as delta (ppm); the silica gel for separation is not illustrated as 200-300 meshes, and the ratio of the eluents is volume ratio.

The invention adopts the following abbreviations: CDCl ₃ Represents deuterated chloroform; etOAc represents ethyl acetate; hexane represents n-Hexane; HPLC means high performance liquid chromatography; meCN represents acetonitrile; DCM represents dichloromethane; DIPEA stands for diisopropylethylamine; DMF represents N, N-dimethylformamide; DMAP represents 4- (dimethylamino) pyridine; DMSO represents dimethylsulfoxide; HATU represents O- (7-azabenzotriazol-1-yl) -NNN 'N' -tetramethylurea hexafluorophosphate; hr represents hours; min represents minutes; k (K) ₂ CO ₃ Represents potassium carbonate; min represents minutes; meOH represents methanol; MS stands for mass spectrum; NMR represents nuclear magnetic resonance; pd/C represents palladium on carbon; POCl (Point of care testing) ₃ Represents phosphorus oxychloride; TEA represents triethylamine; TLC stands for thin layer chromatography.

EXAMPLE 1 Synthesis of Compound 1

Step 1: synthesis of Compound int_1-2:

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

ESI-MS m/z:242[M+H] ⁺

Step 2: synthesis of Compound int_1-3:

to a 50mL single flask were added int_1-2 (211 mg,0.86 mmol), DMF (5 mL), DIPEA (1.5 mL) and 6-nitropyridin-3-ol (121 mg,0.86 mmol), the mixture was argon replaced and then warmed to 100deg.C and stirred for 72 hours, after LC-MS detection of the reaction was essentially complete, ethyl acetate (10 mL), water (10 mL) was added to the mixture, stirred, the separated aqueous phase was extracted with ethyl acetate (10 mL), and the combined organic phases were washed sequentially 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, 76% yield). ESI-MS m/z 346[ M+H ]] ⁺

Step 3: synthesis of Compound int_1-4:

to a 100mL single vial was added int_1-3 (228 mg, crude, 0.66 mmol), meOH (10 mL), DCM (1 mL), 10% palladium on carbon (50 mg, wet=55%) and after three hydrogen substitutions, the hydrogenation was stirred at room temperature and atmospheric pressure. After 72 hours, LC-MS detection reaction was complete, the mixture was filtered with celite, and the filtrate was concentrated to give the product (220 mg, yield: 100%).

ESI-MS m/z:316[M+H] ⁺

Step 4: synthesis of Compound 1:

to a 50mL single flask were added int_1-4 (93 mg, 0.025 mmol), DMF (5 mL), DIPEA (114 mg,0.885 mmol), HATU (170 mg,0.442 mmol) and int_1-5 (84 mg, 0.025 mmol), the mixture was argon replaced and then warmed to 60℃and stirred for 4 hours, after which the LC-MS detection reaction was essentially complete, and the mixture was purified by preparative LC-MS to give 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] ⁺

EXAMPLE 2 Synthesis of Compound 2

Step 1: synthesis of Compound int_2-2:

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

ESI-MS m/z:242[M+H] ⁺

Step 2: synthesis of Compound int_2-3:

to a 50mL single flask were added int_2-2 (208 mg,0.86 mmol), DMF (5 mL), DIPEA (1.5 mL) and 6-nitropyridin-3-ol (121 mg,0.86 mmol), the mixture was replaced with argon and then allowed to warm to 100deg.C and stirred for 72 hours, after LC-MS detection was essentially complete, ethyl acetate (10 mL), water (10 mL) was added to the mixture, stirred, the separated aqueous phase was extracted with ethyl acetate (10 mL), and the combined organic phases were washed sequentially 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, 76% yield). ESI-MS m/z 346[ M+H ]] ⁺

Step 3: synthesis of Compound int_2-4:

to a 100mL single vial was added int_2-3 (220 mg, crude, 0.66 mmol), meOH (10 mL), DCM (1 mL), 10% palladium on carbon (50 mg, wet=55%) and after three hydrogen substitutions, the hydrogenation was stirred at room temperature and atmospheric pressure. After 72 hours, LC-MS detection reaction was complete, the mixture was filtered with celite, and the filtrate was concentrated to give the product (220 mg, yield: 100%).

ESI-MS m/z:316[M+H] ⁺

Step 4: synthesis of Compound 2:

to a 50mL single flask was added int_2-4 (100 mg,0.317 mmol), DMF (5 mL), DIPEA (114 mg,0.885 mmol), HATU (170 mg,0.442 mmol) and int_1-5 (84 mg,0.295 mmol), the mixture was replaced with argon and then warmed to 60℃and stirred for 4 hours, and after the LC-MS detection reaction was substantially complete, the mixture was purified by preparative LC-MS to give 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] ⁺ .

EXAMPLE 3 Synthesis of Compound 3

Step 1: synthesis of Compound int_3-2:

to a 100ml single-port flask, 40 ml of nitric acid (64%) was added dropwise 1-fluoro-2, 3-dimethoxybenzene (8 g,51.23 mmol) under ice bath, followed by stirring under ice bath for 15 minutes, and then naturally heating to room temperature and stirring continued for 15 minutes. After the completion of the TLC monitoring, 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, and the filtrate was concentrated under reduced pressure, and the residue was subjected to column chromatography to give the objective 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: synthesis of Compound int_3-3:

int_3-2 (6 g,29.83 mmol), 10% palladium on carbon (600 mg, wet=55%) was suspended in methanol (150 mL) at room temperature. After three hydrogen substitutions, the reaction was stirred at room temperature and pressure overnight. LC-MS detects that a large amount of products are generated, the mixed solution is filtered by aid of diatomite, and the filtrate is concentrated to obtain the target product (5 g, yield 97% and gray solid).

ESI-MS m/z:172[M+H] ⁺

Step 3: 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. Reflux stirring for 1 hour under the protection of argon. After the reaction was completed by LC-MS, the mixture was cooled to room temperature, and then concentrated under reduced pressure, and the residue was subjected to column chromatography to give the objective product (10 g, yield 98%, brown liquid).

Step 4: synthesis of Compound int_3-6:

int_3-5 (10.2 g,29.88 mmol) was suspended in diphenyl ether (100 mL) at room temperature and refluxed at 250℃for 1 hour. After the completion of the reaction by LC-MS monitoring, the insoluble matter was collected by filtration after cooling to room temperature to give the objective product (2.6 g, yield 30%, brown solid). The crude product was used directly in the next reaction.

ESI-MS m/z:296[M+H] ⁺

Step 5: synthesis of Compound int_3-7:

suspending int_3-6 (3 g,10.16 mmol) in ethanol (20 mL) at room temperature, adding sodium hydroxide aqueous solution (4M, 80 mL), stirring the reaction solution at 90deg.C for 2 hr, LC-MS monitoring the reaction, cooling to room temperature, adjusting pH to 1 with concentrated hydrochloric acid, collecting solid, washing with cold dilute hydrochloric acid, and drying to obtain the targetThe product (2.6 g, yield: 95%, tan solid). ESI-MS m/z 268[ M+H ]] ⁺

Step 6: synthesis of Compound int_3-8:

int_3-6 (2.6 g,9.73 mmol) was suspended in diphenyl ether (120 mL) at room temperature and refluxed at 250℃for 1 hour. After the reaction was completed by LC-MS, cooled to room temperature, insoluble matters were collected by filtration to obtain the objective 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 oxide trichloride (15 mL) at room temperature. Stirring at 100deg.C for 1 hr, LC-MS monitoring the reaction, concentrating under reduced pressure to remove excessive phosphine oxide trichloride, adding the residue into a large amount of water, and stirring for 10 min. The organic phase was dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure, and the residue was subjected to column chromatography to give the objective 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:

to a 50mL single flask was added int_3-9 (300 mg,1.24 mmol), int_3-10 (260.7 mg,1.24 mmol), chlorobenzene (12 mL), DMAP (227 mg,1.86 mmol), after argon displacement of the mixture, the reaction was stirred for 12 hours at 130 ℃, after the LC-MS detection reaction was substantially complete, ethyl acetate (10 mL), water (10 mL), stirring, separation, extraction of the aqueous phase with ethyl acetate (10 mL), and sequential washing of the combined organic phases with saturated sodium bicarbonate solution (10 mL), saturated sodium chloride solution (10 mL), drying over anhydrous sodium sulfate, filtration, and concentration gave a brown oil (68 mg, 17.5% yield).

ESI-MS m/z:316[M+H] ⁺

Step 9: synthesis of Compound 3:

to a 50mL single flask were added int_3-11 (5 mg,0.17 mmol), DMF (5 mL), DIPEA (66 mg,0.5 mmol), HATU (100 mg,0.34 mmol) and int_1-5 (97 mg,0.34 mmol), and after argon displacement of the mixture, the reaction was allowed to warm to 60℃and stirred for 4 hours, after which the LC-MS detection reaction was substantially complete, the mixture was purified by preparative LC-MS to give 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] ⁺

EXAMPLE 4 Synthesis of Compound 4

Step 1: synthesis of Compound int_4-2:

to a 50mL single flask were added int_4-1 (210 mg,0.86 mmol), DMF (5 mL), DIPEA (1.5 mL) and 6-nitropyridin-3-ol (121 mg,0.86 mmol), the mixture was argon replaced and then warmed to 100deg.C and stirred for 72h, after LC-MS detection of the reaction was essentially complete, ethyl acetate (10 mL), water (10 mL), stirring, separation, extraction of the aqueous phase with ethyl acetate (10 mL), and sequential washing of the combined organic phases with saturated sodium bicarbonate solution (10 mL), saturated sodium chloride solution (10 mL), drying over anhydrous sodium sulfate, filtration and concentration gave an oil (198 mg, 68% yield).

ESI-MS m/z:346[M+H] ⁺

Step 2: synthesis of Compound int_4-3:

to a 100mL single vial was added int_4-2 (198mg, 0.57 mmol), meOH (10 mL), DCM (1 mL), 10% palladium on carbon (50 mg, wet=55%) and after three hydrogen substitutions, the hydrogenation was stirred at room temperature and atmospheric pressure. After 72h, LC-MS detection reaction was complete, the mixture was filtered with celite, and the filtrate was concentrated to give the product (180 mg, yield: 100%).

ESI-MS m/z:316[M+H] ⁺

Step 4: synthesis of Compound 4:

to a 50mL single flask was added int_4-3 (100 mg,0.317 mmol), DMF (5 mL), DIPEA (114 mg,0.885 mmol), HATU (170 mg,0.442 mmol) and int_1-5 (84 mg, 0.025 mmol), the mixture was argon replaced and then warmed to 60℃and stirred for 4h, after the LC-MS detection reaction was essentially complete, the mixture was purified by preparative LC-MS to give the product (29 mg, yield: 15.8%).

ESI-MS m/z:581[M+H] ⁺

EXAMPLE 5 in vitro Axl enzyme Activity inhibition assay for Compounds of the invention

The compound diluted with DMSO gradient was mixed with Axl or c-Met recombinant protein and after 10 minutes at room temperature, biotin-labeled TK substrate (TK) and ATP were added. After 40 minutes of room temperature reaction, sa-XL 665 and Crytate labeled TK antibodies were added and fluorescence intensities at 615 and 665nm were detected after 1 hour of room temperature incubation. The ratio of 665nm and 615 nm fluorescence intensities was calculated. Compound IC was calculated compared to DMSO control ₅₀ . The results are shown in Table 1 below.

TABLE 1 inhibitory Activity of the compounds of the invention on recombinant protein Axl

Compounds of formula (I)	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

++ + representing IC ₅₀ Less than or equal to 50nM

++ means IC ₅₀ 50nM to 100nM

+ represents IC ₅₀ Greater than 100nM.

-representing activity unmeasured

From the data in Table 1, the compounds of the present invention have a better inhibitory activity on the enzymatic activity of recombinant protein Axl. And compound 1 has an unexpected increase in inhibitory activity against c-Met enzyme as compared with compound A.

EXAMPLE 6 in vitro antiproliferative Activity of the Compounds of the invention against BaF3-Axl cells

10000 cells of BaF3 (BaF 3-Axl) over-expressing Axl were seeded in 96-well plates, and after 3 days of treatment, the intracellular ATP levels were measured with CTL-PLUS, followed by addition of compounds diluted in a gradient with DMSO. The percent inhibition of cell growth by the compound and IC were calculated as compared to DMSO group ₅₀ The results are shown in Table 2 below.

TABLE 2 antiproliferative Activity of the inventive compounds against BaF3-Axl cells

Compounds of formula (I)	BaF3-Axl cell antiproliferative activity IC 50 (nM)
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 compounds of the present invention have stronger antiproliferative activity on BaF3/Axl cells, but that both compound 2 and compound 3 have weaker antiproliferative activity on BaF3/Axl cells than compound 1. This result shows that in compounds 1,2,3 the position of the fluorine atom substitution has an unexpected effect on the activity of the compounds. And compound 1 has an unexpected increase in anti-BaF 3/Axl proliferation activity compared to compound A.

EXAMPLE 7 in vivo pharmacokinetic experiments of Compounds of the invention

CD-1 female mice 7 to 10 weeks old were selected and dosed intravenously and orally at 2mg/Kg and 10mg/Kg, respectively. Mice were fasted for at least 12 hours prior to dosing and fed was resumed 4 hours after dosing, with free water throughout the experiment. The day of the experiment, i.e. the animals of the intravenous group were given the corresponding compounds by single injection via the tail vein in a volume of 10ml/Kg. Animals in the oral group were given the corresponding compounds by a single injection by gavage, the administration volume being 10ml/Kg. Animals were weighed prior to dosing and dosing volumes were calculated from body weight. The sample collection time is as follows: 0.083 (injection group), 0.167,0.5,1,2,4,8, 24h. About 200uL of whole blood was collected through the submandibular venous plexus at each time point for preparation of plasma for concentration determination by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). All animals were CO-processed after collection of PK samples at the last time point ₂ The anesthesia is euthanized. By Phoenix WinNonlin ^TM Non-compartmental model of version8.3 (Certara) pharmacokinetic software plasma concentrations were processed and pharmacokinetic parameters were calculated using a linear log trapezoidal method. The in vivo pharmacokinetic results are shown in table 3 below.

TABLE 3 results of in vivo pharmacokinetic evaluation of the Compounds of the invention

As can be seen from the data in table 3, in the mouse pharmacokinetic experiment, the Vdss of compound 1 of the present invention is increased, the in vivo clearance is decreased, the half-life is prolonged, and the AUC is increased compared with compound a. Compared to compound a, compound 1 has unexpected improvements in pharmacokinetic properties.

While particular embodiments of the present invention have been described above, it will be appreciated by those skilled in the art that these are merely illustrative, and that many changes and modifications may be made to these embodiments without departing from the principles and spirit of the invention. Accordingly, the scope of the invention is defined by the appended claims.

Claims

A compound represented by formula (1) -formula (4) or each isomer, each crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof:
a pharmaceutical composition comprising a pharmaceutically acceptable excipient or carrier and, as active ingredient, a compound of claim 1, or each isomer, each crystal form, a pharmaceutically acceptable salt, hydrate or solvate thereof.
Use of a compound according to claim 1, or each isomer, each crystal form, pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition according to claim 2, in the manufacture of a medicament for the treatment of a disease associated with Axl protein.