New JAK2 selective inhibitor, and preparation method therefor and use thereof

Abstract

The present application belongs to the technical field of biomedicine, and specifically relates to a new JAK2 selective inhibitor. The JAK2 selective inhibitor comprises a compound as represented by formula I or a pharmaceutically acceptable salt thereof.

Description

A novel selective JAK2 inhibitor, its preparation method and uses

[0001] Priority information

[0002] This application claims priority and benefit to patent application 202411955586.7, filed with the China National Intellectual Property Administration on December 28, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application belongs to the field of biomedical technology, specifically relating to a novel selective JAK2 inhibitor, its preparation method, and its uses. Background Technology

[0004] Protein kinases have become major pathogenic factors in many diseases, among which the JAK family of cellular protein tyrosine kinases (including Jak1, Jak2, Jak3, and Tyk2) plays a crucial role in cytokine signaling. From gain-of-function expression or mutation analysis, JAK1 and JAK3 are more related to immune regulation, while JAK2 is directly related to the production of erythrocytes and platelets. From loss-of-function analysis, loss of JAK1 and JAK2 function causes lethality in mouse embryos. No diseases related to JAK1 and JAK2 loss of function have been found in humans, perhaps indirectly indicating the importance of JAK1 / 2 physiological functions. JAK3 loss of function causes severe comprehensive immunodeficiency, which is the basis for targeting JAK3 to regulate autoimmune-related diseases mentioned later. There is less research on the function of Tyk2, but it has been reported to cause defects related to intrinsic immunity. JAK kinases sense extracellular signals, such as interferon, interleukin, and growth factors, by binding to receptors and transmitting information to STATs. Cytokines activate JAK upon binding to their receptors, which then phosphorylate the cytokine receptors, activating the signal transducers and activators of transcription (STAT) family. In recent years, the therapeutic potential of JAK inhibitors has focused on diseases affecting various immune system pathologies, including atopic dermatitis, cell-mediated hypersensitivity reactions (allergic contact dermatitis, allergic pneumonia), systemic lupus erythematosus (SLE), rheumatoid arthritis, psoriasis, and transplantation (graft rejection, graft-versus-host disease). JAK kinase inhibitors such as ruxolitinib and tofacitinib are already marketed for the treatment of diseases such as myelofibrosis and rheumatoid arthritis.

[0005] Currently, although a series of JAK inhibitors have been disclosed, there is still a need to develop new JAK inhibitor compounds with better efficacy and fewer side effects, especially selective JAK2 inhibitors. Summary of the Invention

[0006] In order to overcome the problems existing in the prior art, the purpose of this application is to provide a novel selective JAK2 inhibitor.

[0007] To achieve the above objectives and other related objectives, this application adopts the following technical solution:

[0008] In a first aspect of this application, a JAK2 selective inhibitor is provided. According to embodiments of this application, the JAK2 selective inhibitor has a compound as shown in Formula I or a pharmaceutically acceptable salt thereof:

[0009] Wherein, R1 is deuterated methyl, isobutyl, deuterated isobutyl-d9, 2,2,2-trifluoroethyl, ethyl, propyl or cyclopropyl;

[0010] R2 is hydrogen or methoxy;

[0011] R3 is

[0012] R4 is hydrogen or

[0013] When R1 is isobutyl, R2 cannot be hydrogen, and / or R4 cannot be hydrogen.

[0014] This application provides a selective JAK2 inhibitor. According to embodiments of this application, the selective JAK2 inhibitor has a compound as shown in Formula I or a pharmaceutically acceptable salt thereof:

[0015] Wherein: R1 is deuterated methyl, isobutyl, deuterated isobutyl-d9, 2,2,2-trifluoroethyl, ethyl, propyl or cyclopropyl;

[0016] R2 is hydrogen or methoxy;

[0017] R3 is

[0018] R4 is hydrogen or

[0019] When R2 is hydrogen, and when R1 is isobutyl, R2 cannot be hydrogen, R4 cannot be hydrogen, and R3 cannot be hydrogen.

[0020] According to embodiments of this application, the above-mentioned selective JAK2 inhibitor may further include at least one of the following technical features:

[0021] According to an embodiment of this application, R3 is

[0022] According to embodiments of this application, R1 is deuterated isobutyl-d9, and R3 is... When R4 is not H.

[0023] According to embodiments of this application, R1 is deuterated isobutyl-d9 or 2,2,2-trifluoroethyl, and R3 is... When R2 is methoxy and R4 is They do not exist simultaneously.

[0024] According to embodiments of this application, the compound represented by Formula I, but not limited to the following structures:

[0025] In a second aspect of this application, a method for preparing the JAK2 selective inhibitor described in the first aspect is provided. According to embodiments of this application, the JAK2 selective inhibitor has a compound as shown in Formula I or a pharmaceutically acceptable salt thereof, and the method comprises the following steps:

[0026] (1) Intermediate 1 and intermediate 2 are combined by a condensation reaction to obtain intermediate 3;

[0027] (2) Intermediate 3 is oxidized to obtain intermediate 4;

[0028] (3) Intermediate 4 was reduced to obtain intermediate 5;

[0029] (4) The hydroxyl groups of intermediate 5 are activated and then undergo a cyclization reaction to obtain general intermediate 6;

[0030] (5) The final product I was prepared by the condensation reaction of intermediate 6 and intermediate 7.

[0031] The definitions of R1, R2, R3, and R4 are as shown in the JAK2 selective inhibitors described in the first aspect.

[0032] According to embodiments of this application, the above method may further include at least one of the following technical features:

[0033] According to embodiments of this application, the pharmaceutically acceptable salt of the compound represented by Formula I is selected from salts formed with inorganic acids or inorganic acids.

[0034] According to an embodiment of this application, the condensation reaction is carried out at 140°C to 160°C.

[0035] According to embodiments of this application, the solvent for the condensation reaction is selected from DMSO (dimethyl sulfoxide).

[0036] According to embodiments of this application, the oxidation is achieved using an oxidizing agent.

[0037] According to an embodiment of this application, the reduction is achieved using a reducing agent.

[0038] According to an embodiment of this application, the hydroxyl activation of intermediate 5 is achieved by MsCl (methanesulfonyl chloride).

[0039] According to embodiments of this application, the cyclization reaction is carried out under catalytic conditions.

[0040] According to embodiments of this application, the inorganic acid includes hydrochloric acid, sulfuric acid, hydrobromic acid, nitric acid, or phosphoric acid.

[0041] According to embodiments of this application, the inorganic acid includes formic acid, acetic acid, trifluoroacetic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, fumaric acid, oxalic acid, maleic acid, tartaric acid, citric acid, or succinic acid.

[0042] According to an embodiment of this application, the condensation reaction is carried out at 150°C.

[0043] According to embodiments of this application, the oxidant is selected from potassium persulfate.

[0044] According to an embodiment of this application, the oxidation is carried out at room temperature.

[0045] According to an embodiment of this application, the reducing agent is selected from sodium borohydride.

[0046] According to an embodiment of this application, the reduction is performed at room temperature.

[0047] According to embodiments of this application, the catalyst is selected from DBU (1,8-diazabicyclo[5.4.0]undec-7-ene).

[0048] In a third aspect of this application, a composition is provided. According to embodiments of this application, the composition comprises the JAK2 selective inhibitor described in the first aspect, or the JAK2 selective inhibitor prepared by the method described in the second aspect.

[0049] According to embodiments of this application, the composition may further include pharmaceutically acceptable excipients, carriers, and mediators.

[0050] In one optional embodiment of this application, pharmaceutically acceptable excipients refer to pharmaceutical excipients conventional in the pharmaceutical field, such as: diluents, excipients, fillers (e.g., starch, sucrose, lactose, microcrystalline cellulose, etc.), binders (e.g., cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone), wetting agents (e.g., glycerin), disintegrants (e.g., sodium carboxymethyl starch, hydroxypropyl cellulose, cross-linked carboxymethyl cellulose, agar, calcium carbonate, and sodium bicarbonate), absorption enhancers (e.g., quaternary ammonium compounds); surfactants (e.g., hexadecyl alcohol, sodium dodecyl sulfate), etc., and other excipients such as flavoring agents, sweeteners, etc. may also be added.

[0051] In one alternative embodiment of this application, a pharmaceutically acceptable carrier refers to a drug carrier conventional in the pharmaceutical field, such as: adsorbent carriers (e.g., kaolin and soap clay), lubricants (e.g., talc, calcium and magnesium stearate, micronized silica gel and polyethylene glycol), etc.

[0052] In one alternative embodiment of this application, pharmaceutically acceptable mediators refer to pharmaceutical mediators conventional in the pharmaceutical field, such as creams, gels, emulsions, solutions (e.g., water) and liposomes.

[0053] In one alternative embodiment of this application, examples of suitable pharmaceutically acceptable carriers, excipients, and mediators are well known in the art. Pharmaceutical compositions comprising such carriers, excipients, and mediators can be formulated using known conventional methods.

[0054] In some alternative embodiments, the pharmaceutical composition of this application may also contain other active ingredients for treatment.

[0055] The pharmaceutical composition of this application can be administered via various routes, such as enterically, orally (e.g., pills, tablets, sublingual, sublingual, disintegrant, capsules, films, liquid solutions or suspensions, powders, solid crystals or liquids), rectally (e.g., suppositories, enemas), by injection (e.g., intravenous, subcutaneous, intramuscular, intraperitoneal, intradermal), by inhalation (e.g., intrabronchial), topically, vaginally, on the skin, or intranasally. Preferably, the pharmaceutical composition of this application is in the form of a lyophilized formulation or an aqueous solution. The clinical dosing regimen will be determined by the attending physician and clinical factors. As is known in the medical field, the dosage for any given patient depends on many factors, including the patient's physique, body surface area, age, the drug to be administered, sex, time and route of administration, general health, and other concurrently administered drugs. The pharmaceutical composition of this application can be administered topically or systemically. Preferably, it can be administered intravenously or subcutaneously. The pharmaceutical composition of this application can also be administered directly to the target site, for example, by targeted administration to internal or external target sites.

[0056] Various dosage forms of the compositions of this application can be prepared using methods conventional in the medical field, wherein the content of the active ingredient is 0.1% to 99.5% (by weight), for example 0.1%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 15.0%, and 20.0%. 25.0%, 30.0%, 35.0%, 40.0%, 45.0%, 50.0%, 55.0%, 60.0%, 65.0%, 70.0%, 75.0%, 80.0%, 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0%, 99.0%, 99.5%, or any two of these values, are used as the range of values ​​between the endpoints.

[0057] The dosage of this application may vary depending on the route of administration, the patient's age, weight, the type and severity of the disease being treated, etc., and the daily dose is 0.001-30 mg / kg body weight (oral) or 0.005-30 mg / kg body weight (injection).

[0058] Compared with existing technologies, the compounds, stereoisomers and pharmaceutically acceptable salts provided in this application have better malignant nerve kinase inhibitory activity, and their selectivity for JAK2 inhibition targets is significantly better than existing compounds. They have the potential to be developed into better selective JAK2 inhibitors and have broad application prospects.

[0059] In a fourth aspect of this application, a JAK2 selective inhibitor as described in the first aspect, a JAK2 selective inhibitor prepared by the method described in the second aspect, or a composition described in the third aspect is provided for the prevention or treatment of JAK-related diseases.

[0060] In a fifth aspect of this application, the use of the JAK2 selective inhibitor as described in the first aspect, the JAK2 selective inhibitor prepared by the method described in the second aspect, or the composition described in the third aspect in the preparation of a medicament for the prevention or treatment of JAK-related diseases is provided.

[0061] In a sixth aspect of this application, the use of a JAK2 selective inhibitor as described in the first aspect, a JAK2 selective inhibitor prepared by the method described in the second aspect, or a composition described in the third aspect in the prevention or treatment of JAK-related diseases is provided.

[0062] In a seventh aspect of this application, a method for preventing or treating JAK-related diseases is provided. According to embodiments of this application, the method comprises administering to a subject a pharmaceutically acceptable dose of the JAK2 selective inhibitor described in the first aspect, the JAK2 selective inhibitor prepared by the method described in the second aspect, or the composition described in the third aspect.

[0063] According to embodiments of this application, the uses described in the fourth, fifth, and sixth aspects above, as well as the method described in the seventh aspect, may further include at least one of the following technical features:

[0064] According to embodiments of this application, the JAK-related diseases include autoimmune diseases, inflammatory diseases, pain disorders, respiratory diseases, airway diseases, lung diseases, pneumonia and injuries, pulmonary hypertension, gastrointestinal diseases, allergic diseases, infectious diseases, trauma and tissue damage diseases, fibrotic diseases, eye diseases, joint diseases, muscle diseases, bone diseases, skin diseases, kidney diseases, hematopoietic system diseases, liver diseases, oral diseases, metabolic diseases, heart diseases, vascular diseases, neuroinflammatory diseases, neurodegenerative diseases, sepsis, genetic diseases, or cancer.

[0065] The inflammatory and autoimmune diseases mentioned include systemic lupus erythematosus, lupus nephritis, arthritis, psoriasis, Crohn's disease, rheumatoid arthritis, ulcerative colitis, atopic dermatitis, gout, alopecia areata, vitiligo, hidradenitis suppurativa, type I diabetes, chronic kidney disease, acute kidney injury, chronic obstructive pulmonary disease, asthma, bronchitis, or graft-versus-host disease.

[0066] The cancers mentioned include breast cancer, lung cancer, prostate cancer, bile duct cancer, bone cancer, bladder cancer, head and neck cancer, kidney cancer, liver cancer, gastrointestinal cancer, esophageal cancer, ovarian cancer, pancreatic cancer, skin cancer, testicular cancer, thyroid cancer, uterine cancer, cervical cancer, vaginal cancer, leukemia, myelofibrosis, multiple myeloma, or lymphoma.

[0067] The compounds of this application can be used to treat related cancers and other diseases through oral administration, injection, or other methods in the course of disease treatment. When used orally, they can be prepared into conventional solid dosage forms such as tablets, powders, or capsules; when used by injection, they can be prepared into injection solutions or lyophilized powder for injection. Detailed Implementation

[0068] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0069] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0070] It should be noted that the structural and chemical formula descriptions in the embodiments or implementations of this application are intended to cover all alternatives, modifications, and equivalent technical solutions, all of which are within the scope of this application as defined in the claims. Those skilled in the art will recognize that many similar or equivalent methods and materials can be used to practice this application. This application is by no means limited to the methods and materials described herein. In the event that one or more of the cited documents, patents, and similar materials differ from or contradict this application (including but not limited to defined terminology, application of terminology, described techniques, etc.), this application shall prevail.

[0071] It should be further appreciated that some features of this application, for clarity, have been described in multiple independent embodiments or implementations, but may also be provided in combination in a single embodiment or implementation. Conversely, various features of this application, for the sake of brevity, have been described in a single embodiment or implementation, but may also be provided individually or in any suitable sub-combination.

[0072] Unless otherwise stated, the technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains, and unless otherwise stated, all patent publications cited in the entirety of this application are incorporated herein by reference.

[0073] Unless otherwise stated, the following definitions will apply in this application. For the purposes of this application, chemical elements are defined according to the periodic table.

[0074] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this application but do not exclude other contents.

[0075] In this document, the compounds of this application also include isotopically labeled compounds of this application that are identical to those compounds described herein except that one or more atoms are replaced by atoms with atomic masses or mass numbers different from those of naturally common atomic masses or mass numbers. Exemplary isotopes that may also be introduced into the compounds of this application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as... 2 H, 3 H, 13 C 14 C15 N、 16 O、 17 O、 31 P, 32 P, 36 S, 18 F and 37 Cl.

[0076] Compounds of this application containing other isotopes of the aforementioned isotopes and / or other atoms, as well as pharmaceutically acceptable salts of said compounds, are included within the scope of this application. Isotope-labeled compounds of this application, such as radioactive isotopes, are also included. 3 H and 14 The incorporation of tritium into the compounds of this application can be used for drug and / or substrate tissue distribution analysis. Due to its ease of preparation and detection, tritium-substituted compounds... 3 H, and carbon-14, i.e. 14 C isotopes are particularly preferred. In addition, heavier isotopes, such as deuterium, are used. 2 H substitution can offer therapeutic advantages stemming from greater metabolic stability, such as increased in vivo half-life or reduced dose requirements. Therefore, it may be preferred in some cases.

[0077] The compounds of this application may contain asymmetric or chiral centers, and thus exist in different stereoisomeric forms. It is contemplated that all stereoisomers of the compounds of this application, including but not limited to diastereomers, enantiomers, and atropisomers, and mixtures thereof such as racemic mixtures, are also included within the scope of this application. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of polarized light. When describing optically active compounds, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule with respect to the chiral centers (or multiple chiral centers) in the molecule. The prefixes d and l or (+) and (-) are symbols used to specify the rotation of plane-polarized light caused by the compound, where (-) or l indicates that the compound is levorotatory. Compounds with the prefix (+) or d are dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. Specific stereoisomers can also be called enantiomers, and mixtures of such isomers are usually referred to as mixtures of enantiomers. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which may occur when there is no stereoselectivity or stereospecificity in a chemical reaction or method.

[0078] Depending on the choice of raw materials and methods, the compounds of this application may exist as one or a mixture of possible isomers, for example, as pure optical isomers, or as mixtures of isomers, such as as racemic and non-corresponding isomer mixtures, depending on the number of asymmetric carbon atoms. Optically active (R)- or (S)- isomers can be prepared using chiral synthons or chiral formulations, or resolved using conventional techniques. If the compound contains a double bond, the substituent may be E or Z configuration; if the compound contains a disubstituted cycloalkyl group, the cycloalkyl substituent may be cis or trans (cis- or trans-) configuration.

[0079] The compounds of this application may contain asymmetric or chiral centers, and thus exist in different stereoisomer forms. It is contemplated that all stereoisomer forms of the compounds of this application, including but not limited to diastereomers, enantiomers, atropisomers, and geometric (or conformational) isomers and mixtures thereof, such as racemic mixtures, are within the scope of this application.

[0080] Unless otherwise stated, the structures described in this application also represent all isomers including this structure (e.g., enantiomers, diastereotropic atropisomers, and geometric (or conformational) forms; for example, R and S configurations of each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers). Therefore, individual stereochemical isomers of the compounds of this application, as well as mixtures of enantiomers, diastereomeric mixtures, and mixtures of geometric isomers (or conformational isomers), are all within the scope of this application.

[0081] Any asymmetric atom (e.g., carbon) in the compounds of this application may exist in a racemic or enantiomerically enriched form, such as (R)-, (S)-, or (R,S)- configuration. In some embodiments, each asymmetric atom has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% enantiomeric excess in the (R)- or (S)- configuration. If possible, substituents on atoms having unsaturated double bonds may be present in cis-(Z)- or trans-(E)- form.

[0082] Therefore, as described in this application, the compounds of this application may exist in the form of one of the possible isomers, rotational isomers, tautomers, tautomers or mixtures thereof, for example, in the form of essentially pure geometric (cis or trans) isomers, diastereomers, optical isomers (enantiomers), racemates or mixtures thereof.

[0083] Any mixture of isomers can be separated into pure or substantially pure geometric or optical isomers, diastereomers, racemates, for example by chromatography and / or stepwise crystallization, based on the physicochemical differences of the components.

[0084] Racemic derivatives of any resulting end product or intermediate can be separated into optical enantiomers using known methods familiar to those skilled in the art, such as by separating salts of their diastereomers. Racemic products can also be separated by chiral chromatography, such as high-performance liquid chromatography (HPLC) using chiral adsorbents. In particular, enantiomers can be prepared via asymmetric synthesis.

[0085] In this document, the terms "tautomer" or "tautomer form" refer to structural isomers with different energies that can interconvert through a low energy barrier. If tautomerization is possible (e.g., in solution), chemical equilibrium can be achieved in the tautomer. For example, proton tautomers (also known as prototropic tautomers) involve interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers involve interconversions via the rearrangement of some bonding electrons. Unless otherwise stated, all tautomer forms of the compounds in this application are within the scope of this application.

[0086] In this document, the term "pharmaceutically acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammals to which it is treated.

[0087] In this document, the term "pharmaceutically acceptable salt" refers to the organic and inorganic salts of the compounds of this application. Pharmaceutically acceptable salts are those well known and documented in the art. Salts formed from pharmaceutically acceptable non-toxic acids include, but are not limited to, inorganic acid salts and organic acid salts formed by reaction with amino groups, or obtained by other methods described in the literature, such as ion exchange.

[0088] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0089] In this document, the term "pharmaceuticalally acceptable excipient" includes any solvent, dispersion medium, coating material, surfactant, antioxidant, preservative (e.g., antibacterial, antifungal), isotonic agent, salt, drug stabilizer, binder, excipient, dispersant, lubricant, sweetener, flavoring agent, colorant, or combination thereof, all of which are known to those skilled in the art (as described in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Except in cases where any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is covered.

[0090] In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The compounds or pharmaceutical compositions of this application may be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are foreseeable, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but this application is not limited to these exemplified routes of administration.

[0091] In this document, the term "treatment" refers to the administration of a drug or compound to an individual to achieve a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a condition in an individual who is susceptible but has not yet been diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, relieve, improve, reduce, or inhibit the individual's disease, including but not limited to administration of a drug containing a compound described herein to an individual in need.

[0092] This application provides a pharmaceutically acceptable salt of a compound as shown in Formula I:

[0093] Wherein, R1 is deuterated methyl, isobutyl, deuterated isobutyl-d9, 2,2,2-trifluoroethyl, ethyl, propyl or cyclopropyl; R2 is hydrogen or methoxy.

[0094] R3 is

[0095] R4 is hydrogen or When R2 is hydrogen, and when R1 is isobutyl, R2 cannot be hydrogen, R4 cannot be hydrogen, and R3 cannot be hydrogen.

[0096] Typical compounds of this application include, but are not limited to, the following compounds:

[0097] The second objective of this application is to provide a method for synthesizing the above-mentioned compounds:

[0098] (1) Intermediate 1 and intermediate 2 are combined by a condensation reaction to obtain intermediate 3;

[0099] (2) Intermediate 3 is oxidized to obtain intermediate 4;

[0100] (3) Intermediate 4 was reduced to obtain intermediate 5;

[0101] (4) The hydroxyl groups of intermediate 5 are activated and then undergo a cyclization reaction to obtain general intermediate 6;

[0102] (5) The final product I was prepared by the condensation reaction of intermediate 6 and intermediate 7.

[0103] The definitions of each group in the above steps are as described above.

[0104] The third objective of this application is to provide the use of the compound as a novel selective JAK inhibitor in the preparation of a medicament for the prevention or treatment of JAK-related diseases, specifically for the prevention or treatment of the following diseases: autoimmune diseases, inflammatory diseases, pain disorders, respiratory diseases, airway diseases, lung diseases, pneumonia and injuries, pulmonary hypertension, gastrointestinal diseases, allergic diseases, infectious diseases, trauma and tissue damage diseases, fibrotic diseases, eye diseases, joint diseases, muscle diseases, bone diseases, skin diseases, kidney diseases, hematopoietic system diseases, liver diseases, oral diseases, metabolic diseases, heart diseases, vascular diseases, neuroinflammatory diseases, neurodegenerative diseases, sepsis, genetic diseases, or cancer.

[0105] The inflammatory and autoimmune diseases mentioned include systemic lupus erythematosus, lupus nephritis, arthritis, psoriasis, Crohn's disease, rheumatoid arthritis, ulcerative colitis, atopic dermatitis, gout, alopecia areata, vitiligo, hidradenitis suppurativa, type I diabetes, chronic kidney disease, acute kidney injury, chronic obstructive pulmonary disease, asthma, bronchitis, or graft-versus-host disease.

[0106] The cancers mentioned include breast cancer, lung cancer, prostate cancer, bile duct cancer, bone cancer, bladder cancer, head and neck cancer, kidney cancer, liver cancer, gastrointestinal cancer, esophageal cancer, ovarian cancer, pancreatic cancer, skin cancer, testicular cancer, thyroid cancer, uterine cancer, cervical cancer, vaginal cancer, leukemia, myelofibrosis, multiple myeloma, or lymphoma.

[0107] The compounds of this application can be used to treat related cancers and other diseases through oral administration, injection, or other methods in the course of disease treatment. When used orally, they can be prepared into conventional solid dosage forms such as tablets, powders, or capsules; when used by injection, they can be prepared into injection solutions or lyophilized powder for injection.

[0108] The fourth objective of this application is to provide a stereoisomer, geometric isomer, tautomer, or pharmaceutically acceptable salt thereof of the compound described in the first aspect.

[0109] A fifth objective of this application is to provide a composition comprising a therapeutically effective amount of the above-described compound, its stereoisomers, geometric isomers, tautomers, or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier.

[0110] Pharmaceutically acceptable salts, such as salts formed with inorganic acids and salts formed with organic acids. Non-limiting examples of salts formed with inorganic acids include, but are not limited to, salts formed with hydrochloric acid, sulfuric acid, hydrobromic acid, nitric acid, phosphoric acid, etc. Non-limiting examples of salts formed with organic acids include, but are not limited to, salts formed with formic acid, acetic acid, trifluoroacetic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, fumaric acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, etc.

[0111] The carriers mentioned refer to conventional carriers in the pharmaceutical field, such as: diluents, excipients such as water; binders such as cellulose derivatives, gelatin, polyvinylpyrrolidone, etc.; fillers such as starch; disintegrants such as calcium carbonate, sodium bicarbonate; in addition, other excipients such as flavoring agents and sweeteners may be added to the composition.

[0112] Various dosage forms of the compositions of this application can be prepared using conventional methods in the medical field, wherein the content of the active ingredient is 0.1% to 99.5% (by weight).

[0113] The dosage of this application may vary depending on the route of administration, the patient's age, weight, the type and severity of the disease being treated, etc., and the daily dose is 0.001-30 mg / kg body weight (oral) or 0.005-30 mg / kg body weight (injection).

[0114] The technical solution of this application will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of this application and should not be considered as specific limitations thereof. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0115] Example 1: Synthesis of Intermediate 6-1

[0116] Synthesis route:

[0117] Operating steps:

[0118] Step 1, Synthesis of Intermediate 3-1

[0119] Intermediate 1-1 (10 g, 52.8 mmol) and intermediate 2-1 (13 g, 52.8 mmol) were added to DMSO (100 mL) and reacted in a microwave-assisted reaction at 150 °C for 2 h. The reaction mixture was concentrated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography to give solid intermediate 3-1 (11.6 g, 55% yield). MS (ESI): m / s [M+H] + =398.5.

[0120] Step 2, Synthesis of Intermediate 4-1

[0121] Potassium persulfate (15.5 g, 50.2 mmol) was added to a DMF (100 mL) solution containing intermediate 3-1 (10 g, 25.1 mmol). The reaction mixture was stirred overnight at room temperature, and the reaction was monitored by HPLC until complete. The reaction mixture was concentrated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography to give solid intermediate 4-1 (7.3 g, 67% yield). MS (ESI): m / s [M+H] + =432.5.

[0122] Step 3, Synthesis of Intermediate 5-1

[0123] Sodium borohydride (1.75 g, 46.4 mmol) was added to a THF (100 mL) solution containing intermediate 4-1 (10 g, 23.2 mmol). The reaction mixture was stirred overnight at room temperature, and the reaction was monitored by TLC until complete. The reaction mixture was concentrated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography to give solid intermediate 5-1 (6.3 g, 70% yield). MS (ESI): m / s [M+H] + =390.5.

[0124] Step 4, Synthesis of Intermediate 6-1

[0125] To a solution of dichloromethane (100 mL) containing intermediate 5-1 (10 g, 25.7 mmol), methanesulfonyl chloride (5.9 g, 51.3 mmol) and triethylamine (5.2 g, 51.3 mmol) were added. The resulting reaction mixture was stirred at room temperature for 1 h, and the reaction was monitored for completeness by TLC. The reaction mixture was concentrated to dryness under reduced pressure, and DBU (3.9 g, 25.7 mmol) was added to a solution of DMF (100 mL). The mixture was stirred at 80 °C for 1 h, and the reaction was monitored for completeness by HPLC. The reaction mixture was concentrated to dryness under reduced pressure, and the residue was separated by silica gel column chromatography to obtain solid intermediate 6-1 (3.9 g, yield 41%). MS (ESI): m / s [M+H] + =390.5.

[0126] Example 2: Synthesis of Intermediate 6-2

[0127] Synthesis route:

[0128] Operating steps:

[0129] The operating procedures and purification methods were as described in Example 1, with an overall yield of 9.7%. MS (ESI): m / s [M+H] + =455.5.

[0130] Example 3: Synthesis of intermediate 6-3

[0131] Synthesis route:

[0132] Operating steps:

[0133] The operating procedures and purification methods were as described in Example 1, with an overall yield of 10.1%. MS (ESI): m / s [M+H] + =402.5.

[0134] Example 4: Synthesis of intermediate 6-4

[0135] Synthesis route:

[0136] Operating steps:

[0137] The operating procedures and purification methods were as described in Example 1, with an overall yield of 9.9%. MS (ESI): m / s [M+H] + =485.5.

[0138] Example 5: Synthesis of intermediate 6-5

[0139] Synthesis route:

[0140] Operating steps:

[0141] The operating procedures and purification methods were as described in Example 1, with an overall yield of 9.4%. MS (ESI): m / s [M+H] + =494.5.

[0142] Example 6: Synthesis of intermediate 6-6

[0143] Synthesis route:

[0144] Operating steps:

[0145] The operating procedures and purification methods were as described in Example 1, with an overall yield of 9.7%. MS (ESI): m / s [M+H] + =441.5.

[0146] Example 7: Synthesis of intermediates 6-7

[0147] Synthesis route:

[0148] Operating steps:

[0149] The operating procedures and purification methods were as described in Example 1, with an overall yield of 10.4%. MS (ESI): m / s [M+H] + =524.5.

[0150] Example 8: Synthesis of intermediates 6-8

[0151] Synthesis route:

[0152] Operating steps:

[0153] The operating procedures and purification methods were as described in Example 1, with an overall yield of 10.0%. MS (ESI): m / s [M+H] + =420.5.

[0154] Example 9: Synthesis of intermediates 6-9

[0155] Synthesis route:

[0156] Operating steps:

[0157] The operating procedures and purification methods were as described in Example 1, with an overall yield of 9.6%. MS (ESI): m / s [M+H] + =503.5.

[0158] Example 10: Synthesis of intermediates 6-10

[0159] Synthesis route:

[0160] Operating steps:

[0161] The operating procedures and purification methods were as described in Example 1, with an overall yield of 10.3%. MS (ESI): m / s [M+H] + =450.5.

[0162] Example 11: Synthesis of intermediate 6-11

[0163] Synthesis route:

[0164] Operating steps:

[0165] The operating procedures and purification methods were as described in Example 1, with an overall yield of 9.5%. MS (ESI): m / s [M+H] + =533.5.

[0166] Example 12: Synthesis of intermediates 6-12

[0167] Synthesis route:

[0168] Operating steps:

[0169] The operating procedures and purification methods were as described in Example 1, with an overall yield of 10.0%. MS (ESI): m / s [M+H] + =437.5.

[0170] Example 13: Synthesis of intermediates 6-13

[0171] Synthesis route:

[0172] Operating steps:

[0173] The operating procedures and purification methods were as described in Example 1, with an overall yield of 9.7%. MS (ESI): m / s [M+H] + =519.5.

[0174] Example 14: Synthesis of intermediates 6-14

[0175] Synthesis route:

[0176] Operating steps:

[0177] The operating procedures and purification methods were as described in Example 1, with an overall yield of 10.2%. MS (ESI): m / s [M+H] + =467.5.

[0178] Example 15: Synthesis of intermediates 6-15

[0179] Synthesis route:

[0180] Operating steps:

[0181] The operating procedures and purification methods were as described in Example 1, with an overall yield of 10.1%. MS (ESI): m / s [M+H] + =550.5.

[0182] Example 16: Synthesis of intermediates 6-16

[0183] Synthesis route:

[0184] Operating steps:

[0185] The operating procedures and purification methods were as described in Example 1, with an overall yield of 9.4%. MS (ESI): m / s [M+H] + =383.4.

[0186] Example 17: Synthesis of intermediates 6-17

[0187] Synthesis route:

[0188] Operating steps:

[0189] The operating procedures and purification methods were as described in Example 1, with an overall yield of 10.0%. MS (ESI): m / s [M+H] + =397.4.

[0190] Example 18: Synthesis of intermediates 6-18

[0191] Synthesis route:

[0192] Operating steps:

[0193] The operating procedures and purification methods were as described in Example 1, with an overall yield of 9.7%. MS (ESI): m / s [M+H] + =395.4.

[0194] Intermediates 6-1 to 6-18 in the compounds in the following examples were prepared according to the methods of Examples 1 to 18 above.

[0195] Example 19: Synthesis of Compound 1

[0196] Synthesis route:

[0197] Operating steps:

[0198] Compound 6-6 (10 g, 22.7 mmol) prepared according to Example 6 and intermediate 7-1 (7.0, 34.0 mmol) were added to trifluoroacetic acid (100 mL). The reaction mixture was heated to 100 °C and reacted for 12 h. The reaction was monitored by HPLC until complete. The reaction mixture was concentrated to dryness under reduced pressure, and the residue was separated by silica gel column chromatography to obtain compound 1 (9.1 g, yield 71%). MS (ESI): m / s [M+H] + =567.7.

[0199] Example 20: Synthesis of Compound 2

[0200] Synthesis route:

[0201] Operating steps:

[0202] The operating procedures and purification methods are the same as in Example 19, with a yield of 67%. MS (ESI): m / s [M+H] + =528.6.

[0203] Example 21: Synthesis of Compound 3

[0204] Synthesis route:

[0205] Operating steps:

[0206] The operating procedures and purification methods are the same as in Example 19, with a yield of 69%. MS (ESI): m / s [M+H] + =498.6.

[0207] Example 22: Synthesis of Compound 4

[0208] Synthesis route:

[0209] Operating steps:

[0210] The operating procedures and purification methods are the same as in Example 19, with a yield of 68%. MS (ESI): m / s [M+H] + =576.7.

[0211] Example 23: Synthesis of Compound 5

[0212] Synthesis route:

[0213] Operating steps:

[0214] The operating procedures and purification methods are the same as in Example 19, with a yield of 72%. MS (ESI): m / s [M+H] + =546.7.

[0215] Example 24: Synthesis of Compound 6

[0216] Synthesis route:

[0217] Operating steps:

[0218] The operating procedures and purification methods are the same as in Example 19, with a yield of 67%. MS (ESI): m / s [M+H] + =581.7.

[0219] Example 25: Synthesis of Compound 7

[0220] Synthesis route:

[0221] Operating steps:

[0222] The operating procedures and purification methods are the same as in Example 19, with a yield of 72%. MS (ESI): m / s [M+H] + =620.7.

[0223] Example 26: Synthesis of Compound 8

[0224] Synthesis route:

[0225] Operating steps:

[0226] The operating procedures and purification methods are the same as in Example 19, with a yield of 70%. MS (ESI): m / s [M+H] + =629.8.

[0227] Example 27: Synthesis of Compound 9

[0228] Synthesis route:

[0229] Operating steps:

[0230] The operating procedures and purification methods are the same as in Example 19, with a yield of 68%. MS (ESI): m / s [M+H] + =650.8.

[0231] Example 28: Synthesis of Compound 10

[0232] Synthesis route:

[0233] Operating steps:

[0234] The operating procedures and purification methods are the same as in Example 19, with a yield of 72%. MS (ESI): m / s [M+H] + =611.7.

[0235] Example 29: Synthesis of Compound 11

[0236] Synthesis route:

[0237] Operating steps:

[0238] The operating procedures and purification methods are the same as in Example 19, with a yield of 67%. MS (ESI): m / s [M+H] + =659.8.

[0239] Example 30: Synthesis of Compound 12

[0240] Synthesis route:

[0241] Operating steps:

[0242] The operating procedures and purification methods are the same as in Example 19, with a yield of 72%. MS (ESI): m / s [M+H] + =563.6.

[0243] Example 31: Synthesis of Compound 13

[0244] Synthesis route:

[0245] Operating steps:

[0246] The operating procedures and purification methods are the same as in Example 19, with a yield of 69%. MS (ESI): m / s [M+H] + =593.6.

[0247] Example 32: Synthesis of Compound 14

[0248] Synthesis route:

[0249] Operating steps:

[0250] The operating procedures and purification methods are the same as in Example 19, with a yield of 72%. MS (ESI): m / s [M+H] + =676.7.

[0251] Example 33: Synthesis of Compound 15

[0252] Synthesis route:

[0253] Operating steps:

[0254] The operating procedures and purification methods are the same as in Example 19, with a yield of 70%. MS (ESI): m / s [M+H] + =646.7.

[0255] Example 34: Synthesis of Compound 16

[0256] Synthesis route:

[0257] Operating steps:

[0258] The operating procedures and purification methods are the same as in Example 19, with a yield of 75%. MS (ESI): m / s [M+H] + =601.7.

[0259] Example 35: Synthesis of Compound 17

[0260] Synthesis route:

[0261] Operating steps:

[0262] The operating procedures and purification methods are the same as in Example 19, with a yield of 65%. MS (ESI): m / s [M+H] + =562.6.

[0263] Example 36: Synthesis of Compound 18

[0264] Synthesis route:

[0265] Operating steps:

[0266] The operating procedures and purification methods are the same as in Example 19, with a yield of 71%. MS (ESI): m / s [M+H] + =532.6.

[0267] Example 37: Synthesis of Compound 19

[0268] Synthesis route:

[0269] Operating steps:

[0270] The operating procedures and purification methods are the same as in Example 19, with a yield of 75%. MS (ESI): m / s [M+H] + =610.8.

[0271] Example 38: Synthesis of Compound 20

[0272] Synthesis route:

[0273] Operating steps:

[0274] The operating procedures and purification methods are the same as in Example 19, with a yield of 72%. MS (ESI): m / s [M+H] + =580.7.

[0275] Example 39: Synthesis of Compound 21

[0276] Synthesis route:

[0277] Operating steps:

[0278] The operating procedures and purification methods are the same as in Example 19, with a yield of 70%. MS (ESI): m / s [M+H] + =615.7.

[0279] Example 40: Synthesis of Compound 22

[0280] Synthesis route:

[0281] Operating steps:

[0282] The operating procedures and purification methods are the same as in Example 19, with a yield of 73%. MS (ESI): m / s [M+H] + =654.8.

[0283] Example 41: Synthesis of Compound 23

[0284] Synthesis route:

[0285] Operating steps:

[0286] The operating procedures and purification methods are the same as in Example 19, with a yield of 70%. MS (ESI): m / s [M+H] + =663.8.

[0287] Example 42: Synthesis of Compound 24

[0288] Synthesis route:

[0289] Operating steps:

[0290] The operating procedures and purification methods are the same as in Example 19, with a yield of 72%. MS (ESI): m / s [M+H] + =684.8.

[0291] Example 43: Synthesis of Compound 25

[0292] Synthesis route:

[0293] Operating steps:

[0294] The operating procedures and purification methods are the same as in Example 19, with a yield of 63%. MS (ESI): m / s [M+H] + =645.7.

[0295] Example 44: Synthesis of Compound 26

[0296] Synthesis route:

[0297] Operating steps:

[0298] The operating procedures and purification methods are the same as in Example 19, with a yield of 70%. MS (ESI): m / s [M+H] + =693.9.

[0299] Example 45: Synthesis of Compound 27

[0300] Synthesis route:

[0301] Operating steps:

[0302] The operating procedures and purification methods are the same as in Example 19, with a yield of 74%. MS (ESI): m / s [M+H] + =627.6.

[0303] Example 46: Synthesis of Compound 28

[0304] Synthesis route:

[0305] Operating steps:

[0306] The operating procedures and purification methods are the same as in Example 19, with a yield of 70%. MS (ESI): m / s [M+H] + =597.6.

[0307] Example 47: Synthesis of Compound 29

[0308] Synthesis route:

[0309] Operating steps:

[0310] The operating procedures and purification methods are the same as in Example 19, with a yield of 68%. MS (ESI): m / s [M+H] + =710.7.

[0311] Example 48: Synthesis of Compound 30

[0312] Synthesis route:

[0313] Operating steps:

[0314] The operating procedures and purification methods are the same as in Example 19, with a yield of 71%. MS (ESI): m / s [M+H] +=680.7.

[0315] Example 49: Synthesis of Compound 31

[0316] Synthesis route:

[0317] Operating steps:

[0318] The operating procedures and purification methods are the same as in Example 19, with a yield of 75%. MS (ESI): m / s [M+H] + =552.7.

[0319] Example 50: Synthesis of Compound 32

[0320] Synthesis route:

[0321] Operating steps:

[0322] The operating procedures and purification methods are the same as in Example 19, with a yield of 70%. MS (ESI): m / s [M+H] + =513.6.

[0323] Example 51: Synthesis of Compound 33

[0324] Synthesis route:

[0325] Operating steps:

[0326] The operating procedures and purification methods are the same as in Example 19, with a yield of 73%. MS (ESI): m / s [M+H] + =483.6.

[0327] Example 52: Synthesis of Compound 34

[0328] Synthesis route:

[0329] Operating steps:

[0330] The operating procedures and purification methods are the same as in Example 19, with a yield of 67%. MS (ESI): m / s [M+H] + =531.7.

[0331] Example 53: Synthesis of Compound 35

[0332] Synthesis route:

[0333] Operating steps:

[0334] The operating procedures and purification methods are the same as in Example 19, with a yield of 72%. MS (ESI): m / s [M+H]+ =614.8.

[0335] Example 54: Synthesis of Compound 36

[0336] Synthesis route:

[0337] Operating steps:

[0338] The operating procedures and purification methods are the same as in Example 19, with a yield of 70%. MS (ESI): m / s [M+H] + =561.7.

[0339] Example 55: Synthesis of Compound 37

[0340] Synthesis route:

[0341] Operating steps:

[0342] The operating procedures and purification methods are the same as in Example 19, with a yield of 72%. MS (ESI): m / s [M+H] + =635.7.

[0343] Example 56: Synthesis of Compound 38

[0344] Synthesis route:

[0345] Operating steps:

[0346] The operating procedures and purification methods are the same as in Example 19, with a yield of 69%. MS (ESI): m / s [M+H] + =644.8.

[0347] Example 57: Synthesis of Compound 39

[0348] Synthesis route:

[0349] Operating steps:

[0350] The operating procedures and purification methods are the same as in Example 19, with a yield of 71%. MS (ESI): m / s [M+H] + =566.7.

[0351] Example 58: Synthesis of Compound 40

[0352] Synthesis route:

[0353] Operating steps:

[0354] The operating procedures and purification methods are the same as in Example 19, with a yield of 70%. MS (ESI): m / s [M+H] + =605.7.

[0355] Example 59: Synthesis of Compound 41

[0356] Synthesis route:

[0357] Operating steps:

[0358] The operating procedures and purification methods are the same as in Example 19, with a yield of 73%. MS (ESI): m / s [M+H] + =661.7.

[0359] Example 60: Synthesis of Compound 42

[0360] Synthesis route:

[0361] Operating steps:

[0362] The operating procedures and purification methods are the same as in Example 19, with a yield of 69%. MS (ESI): m / s [M+H] + =596.7.

[0363] Example 61: Synthesis of Compound 43

[0364] Synthesis route:

[0365] Operating steps:

[0366] The operating procedures and purification methods are the same as in Example 19, with a yield of 71%. MS (ESI): m / s [M+H] + =548.6.

[0367] Example 62: Synthesis of Compound 44

[0368] Synthesis route:

[0369] Operating steps:

[0370] The operating procedures and purification methods are the same as in Example 19, with a yield of 75%. MS (ESI): m / s [M+H] + =631.6.

[0371] Example 63: Synthesis of Compound 45

[0372] Synthesis route:

[0373] Operating steps:

[0374] The operating procedures and purification methods are the same as in Example 19, with a yield of 70%. MS (ESI): m / s [M+H] + =578.6.

[0375] Example 64: Synthesis of Compound 46

[0376] Synthesis route:

[0377] Operating steps:

[0378] The operating procedures and purification methods are the same as in Example 19, with a yield of 68%. MS (ESI): m / s [M+H] + =494.6.

[0379] Example 65: Synthesis of Compound 47

[0380] Synthesis route:

[0381] Operating steps:

[0382] The operating procedures and purification methods are the same as in Example 19, with a yield of 72%. MS (ESI): m / s [M+H] + =508.6.

[0383] Example 66: Synthesis of Compound 48

[0384] Synthesis route:

[0385] Operating steps:

[0386] The operating procedures and purification methods are the same as in Example 19, with a yield of 70%. MS (ESI): m / s [M+H] + =506.6.

[0387] Biological testing

[0388] Test Example 1: Activity Test of JAK1, JAK2, and JAK3

[0389] Compound preparation:

[0390] The compound was dissolved in 100% DMSO to prepare a 10 mM stock solution, which was then frozen at -20°C.

[0391] Kinase reaction process:

[0392] (1) Prepare 1×Kinase buffer.

[0393] (2) Preparation of compound concentration gradients: The initial concentration of the test compound was 500 nM. It was diluted 100 times to a final concentration of 100% DMSO in a 384source plate. The compound was then diluted 3 times with Precision to obtain 12 concentrations. 250 nL of the 100-fold final concentration of the compound was transferred to the target plate OptiPlate-384F using an Echo 550 dispenser.

[0394] (3) Prepare a kinase solution with a final concentration of 2.5 times using 1×Kinase buffer.

[0395] (4) Add 10 μL of kinase solution at 2.5 times the final concentration to the compound wells and the positive control wells respectively; add 10 μL of 1×Kinase buffer to the negative control wells.

[0396] (5) Centrifuge at 1000 rpm for 30 seconds, shake the reaction plate to mix, and incubate at room temperature for 10 minutes.

[0397] (6) Prepare a mixed solution of ATP and Kinase substrate at a final concentration of 5 / 3 times using 1×Kinase buffer.

[0398] (7) Add 15 μL of a mixture of ATP and substrate at 5 / 3 times the final concentration to initiate the reaction.

[0399] (8) Centrifuge the 384-well plate at 1000 rpm for 30 seconds, shake to mix, and then incubate at room temperature for the corresponding time.

[0400] (9) Add 30 μL of the stop detection solution to stop the kinase reaction, centrifuge at 1000 rpm for 30 seconds, and shake to mix.

[0401] (10) Read the conversion rate using Caliper EZ Reader.

[0402] Data Analysis:

[0403] Calculation formula:

[0404] Where: Conversion%_sample is the conversion rate reading of the sample; Conversion%min: the mean value of the negative control wells, representing the conversion rate reading of wells without enzyme activity; Conversion%_max: the mean value of the positive control wells, representing the conversion rate reading of wells without compound inhibition.

[0405] Fitted dose-response curve:

[0406] Using the logarithm of concentration as the X-axis and the percentage inhibition rate as the Y-axis, dose-response curves were fitted using the log(inhibitor) vs. response-variable slope function of GraphPad Prism 5 to obtain the IC50 values ​​of each compound on enzyme activity. The calculation formula is as follows:

[0407] Y=Bottom+(Top-Bottom) / (1+10^((LogIC50-X)*HillSlope))

[0408] The experimental results are shown in Table 1.

[0409] Table 1 Results of compound enzyme experiments

[0410] Note: The above control compounds and the compounds in this application are all measured values ​​under the same experimental conditions.

[0411] Conclusion: The compounds in this application exhibit superior selectivity for the JAK2 target compared to the positive control fenzotinib, control compound 1, control compound 2, and control compound 3.

[0412] Test Example 2: Cell Proliferation Experiment

[0413] HEL92.1.7 Cell Proliferation Assay

[0414] Experimental steps:

[0415] (1)Planning

[0416] a. Digest and resuspend the cells, and count them using an automated cell counter;

[0417] b. Dilute the cell suspension to the desired density;

[0418] c. Seed 100 μL of cells into each well and incubate overnight at 37°C;

[0419] (2) Compound preparation

[0420] a. Prepare a solution of the compound at a final concentration of 200 times;

[0421] b. Dilute the compound with culture medium to prepare a final concentration three times higher. Add 50 μL of the compound to each well, with the well containing the same volume of DMSO as a control. Incubate at 37°C and 5% CO2 for 72 hours.

[0422] (3) Detection

[0423] a. Equilibrate the cell plate to room temperature;

[0424] b. Add 40 μL of Cell reagent to each well, shake for 2 minutes, and let stand for 10 minutes;

[0425] c. Use EnVision for detection.

[0426] Data Analysis:

[0427] (1) Calculate IC50 using GraphPad Prism 5.

[0428] (2)%Inh=(Max signal-Compound signal) / (Max signal-Min signal)×100%.

[0429] (3) Max signal is a positive control well containing only DMSO of the same volume as the compound.

[0430] (4) Min signal is the negative control well, containing only culture medium.

[0431] TF-1 cell proliferation experiment

[0432] (1) Cell plating

[0433] a. Prepare complete culture medium.

[0434] b. Revive the cells and culture them.

[0435] c. Centrifuge the cells, resuspend them, count them, plate them, and place the culture plates in a CO2 incubator overnight.

[0436] (2) Preparation and addition of compounds

[0437] a. Prepare a 10 mM stock solution of the compound using DMSO, dilute the 10 mM solution to the working concentration, and then gradually dilute it to obtain compounds with multiple concentration gradients.

[0438] b. Transfer 0.5 μL from the corresponding compound plate to the cell culture plate that has been cultured overnight.

[0439] c. Incubate at 37°C for 72 hours.

[0440] (3) Detection and Analysis

[0441] a. Prepare the CellTiter Glo assay reagent.

[0442] b. Add the test reagent to the culture plate, mix well, let stand, and read the plate.

[0443] The inhibition rate formula is (1 - (the value of the corresponding well - the average value of BLANK) / (the average value of DMSO control - the average value of BLANK)) * 100%)

[0444] The formula for the curve fitting tool (XL fit) is: Data Analysis: (XLfit software: Fit model: Dose response one site / f(x)205 [fit=(A+((BA) / (1+((C / x)^D))))])

[0445] The experimental results are shown in Table 2.

[0446] Table 2. Results of cell proliferation experiment

[0447] Note: The above control compounds and the compounds in this application are all measured values ​​under the same experimental conditions.

[0448] Conclusion: The compounds in this application have significant inhibitory activity against HEL92.1.7 and TF-1 proliferation, and their inhibitory activity is superior to that of the positive control phenotypeinib, control compound 1, control compound 2 and control compound 3.

[0449] Test Example 3: Pharmacokinetic Test of Compounds in this Application

[0450] Using SD rats as test animals, the drug concentration in plasma at different time points after oral administration of the control compound and the compound of this application was determined by LC / MS / MS to study the pharmacokinetic characteristics of the compound of this application in rats.

[0451] SD rats were sourced from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0452] Administration method: Single oral gavage

[0453] Dosage and concentration: 30 mg / kg; 3 mg / mL

[0454] Sampling points: 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h

[0455] Preparation and processing of standard curves and quality control samples: Take an appropriate amount of stock solution and dilute it with 50% acetonitrile water to prepare standard working solutions of 0.04, 0.10, 0.20, 0.40, 1.00, 2.00, and 4.00 μg / mL, and quality control working solutions of 0.10, 1.00, and 3.00 μg / mL. 47.5 μL of blank rat plasma was used to prepare standard curve working solution and quality control working solution by adding 2.50 μL of standard curve working solution and quality control working solution respectively. Standard curves and quality control samples with analyte concentrations of 2.00, 5.00, 10.00, 20.00, 50.00, 100.00, and 200.00 ng / mL were prepared. 200 μL of acetonitrile (containing 5 ng / mL of internal standard loratadine) was added to each sample. The mixture was vortexed for 3 min, centrifuged at 15000 rpm and 4℃ for 15 min, and 100 μL of the supernatant was collected for LC-MS / MS analysis. The experimental results were calculated using WinNonlin 8.0.

[0456] The pharmacokinetic parameters are shown in Table 3.

[0457] Table 3: Pharmacokinetic parameters of preferred compounds

[0458] Conclusion: The compound of this application exhibits favorable pharmacokinetic properties. Compared with the positive control fenzotinib, control compound 1, control compound 2 and control compound 3, the compound of this application has a longer half-life and a longer duration of action, demonstrating significant pharmacokinetic advantages.

[0459] Test Example 4: Acute toxicity test of the compound in this application

[0460] The compound of this application, as well as the positive control drug, were subjected to acute toxicity tests.

[0461] (1) Experimental scheme

[0462] ① Observe the toxic symptoms and mortality of animals after oral administration of the positive control drug and the compound of this application to ICR mice, and compare their acute toxicity.

[0463] ② Solvent preparation: Weigh an appropriate amount of Tween-80 and dilute it with deionized water to a concentration of 5% (g / v) Tween-80.

[0464] ③ Preparation of the drug: Weigh the required test samples and prepare suspensions with concentrations of 25.00, 50.00, 75.00, 100.00, 125.00 and 150.00 mg / mL (equivalent to 500, 1000, 1500, 2000, 2500 and 3000 mg / kg, respectively) using 5% Tween 80 solution.

[0465] ④ Route of administration: The test sample and the solvent control group (0.5% Tween-80) were administered orally.

[0466] ⑤ Dosage frequency: Single dose, with fasting required overnight before administration.

[0467] ⑥ Dosage volume: 20 mL / kg.

[0468] General symptom observation: On the day of administration, observe once at approximately 0.5, 1, 2, 4, and 6 hours after the first dose; during the observation period from day 2 to day 6, observe twice a day, once in the morning and once in the afternoon.

[0469] The observation content includes, but is not limited to: general condition, behavior, gait and posture, eyes, mouth, nose, gastrointestinal tract, skin and hair, and urogenital tract.

[0470] (2) Statistical Analysis

[0471] Weight data are expressed as mean ± standard deviation. Levene's test and one-way ANOVA were used for intergroup comparisons. If differences were found, Dunnett's t-test was used.

[0472] (3) Experimental Results

[0473] The compound of this application, along with the positive control, underwent acute toxicity testing as described above. The experimental results are shown in Table 4.

[0474] In the MTD trial, the tolerance of animals to the drug is examined. The maximum tolerated dose is the dose at which the animal is on the verge of death.

[0475] Table 4: Results of Acute Toxicity Experiments of the Compound and Positive Control Drug After a Single Oral Dosage in this Application

[0476] Note: MTD: Maximum Tolerable Dosage.

[0477] The results showed that the MTD (maximum tolerated dose) of the compounds in this application among the above-mentioned test substances was greater than 3000 mg / kg, and the acute toxicity was lower than that of the positive control drug.

[0478] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A JAK2 selective inhibitor, characterized in that, The JAK2 selective inhibitor has a compound as shown in Formula I or a stereoisomer, geometric isomer, tautomer, or pharmaceutically acceptable salt thereof, Wherein: R1 is deuterated methyl, isobutyl, deuterated isobutyl-d9, 2,2,2-trifluoroethyl, ethyl, propyl or cyclopropyl; R2 is hydrogen or methoxy; R3 is R4 is hydrogen or When R1 is isobutyl, R2 cannot be hydrogen, and / or R4 cannot be hydrogen.

2. A JAK2 selective inhibitor, characterized in that, The JAK2 selective inhibitor has a compound as shown in Formula I or a stereoisomer, geometric isomer, tautomer, or pharmaceutically acceptable salt thereof, Wherein: R1 is deuterated methyl, isobutyl, deuterated isobutyl-d9, 2,2,2-trifluoroethyl, ethyl, propyl or cyclopropyl; R2 is hydrogen or methoxy; R3 is R4 is hydrogen or when R1is isobutyl, R2cannot be hydrogen, R4cannot be hydrogen and R3cannot be 3. The JAK2 selective inhibitor according to claim 1 or 2, wherein The compound represented by Formula I satisfies the following condition: R3 is 4. The JAK2 selective inhibitor according to claim 1 or 2, wherein The compound of Formula I is selected from the group consisting of:

5. A process for the preparation of a JAK2 selective inhibitor according to any one of claims 1 to 4, characterized in that, The JAK2 selective inhibitor has a compound as shown in Formula I or a stereoisomer, geometric isomer, tautomer, or pharmaceutically acceptable salt thereof, and the method comprises the following steps: (1) Intermediate 1 and intermediate 2 are combined by a condensation reaction to obtain intermediate 3; (2) Intermediate 3 is oxidized to obtain intermediate 4; (3) Intermediate 4 was reduced to obtain intermediate 5; (4) The hydroxyl groups of intermediate 5 are activated and then undergo a cyclization reaction to obtain general intermediate 6; (5) The final product I was prepared by the condensation reaction of intermediate 6 and intermediate 7. The definitions of R1, R2, R3, and R4 are as follows: R1, R2, R3, and R4 are the JAK2 selective inhibitors as described in any one of claims 1 to 4.

6. The method of claim 5, wherein the JAK2 selective inhibitor is The method satisfies one or more of the following conditions: 1) The pharmaceutically acceptable salt of the compound represented by Formula I is selected from salts formed with or from inorganic acids; 2) The condensation reaction is carried out at 140℃~160℃; 3) The solvent for the condensation reaction is selected from DMSO (dimethyl sulfoxide); 4) The oxidation is achieved through an oxidizing agent; 5) The reduction is achieved through a reducing agent; 6) The hydroxyl group activation of intermediate 5 is achieved via MsCl (methanesulfonyl chloride); 7) The cyclization reaction is carried out under catalytic conditions.

7. The method of claim 6, wherein, The method satisfies one or more of the following conditions: 1) The inorganic acids include hydrochloric acid, sulfuric acid, hydrobromic acid, nitric acid, or phosphoric acid; The inorganic acids described in the JAK2 selective inhibitor 3) include formic acid, acetic acid, trifluoroacetic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, fumaric acid, oxalic acid, maleic acid, tartaric acid, citric acid, or succinic acid. 2) The condensation reaction was carried out at 150°C; 3) The oxidant is selected from potassium peroxymonosulfate; 4) The oxidation is carried out at room temperature; 5) The reducing agent is selected from sodium borohydride; 6) The reduction is carried out at room temperature; 7) The catalyst is selected from DBU (1,8-diazabicyclo[5.4.0]undec-7-ene).

8. A composition characterized in that, The composition comprises the JAK2 selective inhibitor according to any one of claims 1 to 4, or the JAK2 selective inhibitor prepared by the method according to any one of claims 5 to 7, and optionally pharmaceutically acceptable excipients, carriers, or mediators.

9. The JAK2 selective inhibitor according to any one of claims 1 to 4, the JAK2 selective inhibitor prepared by the method according to any one of claims 5 to 7, or the composition according to claim 8, for the prevention or treatment of JAK-related diseases, or having the following uses; 1) Prevention or treatment of JAK-related diseases; 2) Prepare drugs for the prevention or treatment of JAK-related diseases.

10. A method for preventing or treating a JAK-associated disease, comprising administering to a subject in need thereof a therapeutically effective amount of a compound according to any one of claims 1 to 9. The method includes: administering to the subject a pharmaceutically acceptable dose of the JAK2 selective inhibitor of any one of claims 1-4, the JAK2 selective inhibitor prepared by the method of any one of claims 5-7, or the composition of claim 8.

11. The use as claimed in claim 9 or the method as claimed in claim 10, characterised in that, The JAK-associated diseases include diseases of the autoimmune system, inflammatory diseases, pain diseases, diseases of the respiratory tract, diseases of the airway, diseases of the lung, inflammation and damage of the lung, pulmonary hypertension, diseases of the gastrointestinal tract, allergic diseases, infectious diseases, diseases of trauma and tissue damage, fibrotic diseases, diseases of the eye, diseases of the joint, diseases of the muscle, diseases of the bone, diseases of the skin, diseases of the kidney, diseases of the hematopoietic system, diseases of the liver, diseases of the oral cavity, metabolic diseases, diseases of the heart, diseases of the blood vessels, neuroinflammatory diseases, neurodegenerative diseases, sepsis, genetic diseases, or cancer.

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