Cyclic compound as JAK2 inhibitor and application thereof

Abstract

The invention provides a macrocyclic compound with a JAK2 (Janus kinase 2) inhibition effect. The invention also provides a pharmaceutical composition containing the compound and application of the pharmaceutical composition in treatment of JAK2-mediated related diseases. The compound provided by the invention is novel in structure, has an excellent JAK kinase inhibitor effect, and has better selective inhibition on JAK2. Therefore, the compound disclosed by the invention is higher in safety, so that a brand new material basis is laid for development of immune inflammation and antitumor drugs. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry. Specifically, the present invention relates to novel JAK2 inhibitors and their applications. Background Art

[0002] The JAK family has four members: JAK1, JAK2, TYK2, and JAK3. The amino acid composition of the family members exceeds 1000, and the molecular weight is between 120 - 140 kDa, belonging to a relatively large family among kinases. JAKs have two almost identical phosphotransfer domains, one with kinase activity and the other regulating the kinase activity of the former. The four members of the JAK family have become potential drug targets for treating different types of diseases, and they are involved in activating signal transduction between cytokine receptors. Different cytokine receptors send signals through specific JAKs. Each receptor is composed of multiple subunits, and each subunit binds to only one JAK protein.

[0003] JAK is involved in mediating multiple signal pathways, including the JAK - STAT pathway, RAS - RAF - MEK - ERK pathway, and P13K - AKT pathway, etc. Among them, the JAK / STAT signaling pathway is the most important signal pathway mediated by JAK in the body. This pathway is generally expressed in the body and is currently a proven signal pathway related to immune regulation. The JAK / STAT signaling pathway mainly consists of JAK kinases, STAT proteins, and tyrosine kinase - related receptors, and is relatively conserved in evolution. Multiple key factors transmit inter - cellular signals through this signal pathway, including IL - 2 - 7, Growth Hormone (GH), Interferon (IFNs), Epidermal Growth Factor (EGF), Platelet - derived Growth Factor (PDGF), etc.

[0004] Myeloproliferative Neoplasm (MPN) is a clonal hematopoietic stem cell disease characterized by the over - production of one or more types of myeloid cells. Among all the mutations driving MPN, JAK2V617F is the most common. This mutation occurs in the JH2 pseudokinase domain, where valine at position 617 is replaced by phenylalanine. After the JAK2 V617F mutation occurs, the JAK / STAT pathway will be over - activated, leading to changes in the levels of platelets and megakaryocytes in the body, and further increasing the thrombus risk in MPN patients. The clinical symptoms of these patients include splenomegaly, extramedullary hematopoiesis, local limb swelling and pain, anemia, fatigue, and motor dysfunction, etc., resulting in a low quality of life for the patients.

[0005] JAK plays a pivotal role in JAK-STAT signal transduction. Therefore, targeting JAKs can be an effective approach for treating such diseases. Among the more than a dozen JAK inhibitors currently on the market, three have black box warnings in their product labels. The label of tofacitinib shows risks of thrombosis and death. Baricitinib and upadacitinib indicate risks of thrombosis, infection, and malignancy. Therefore, non-selective inhibition of the JAK family poses potential safety risks, and selective JAK inhibitors still have great development significance and clinical needs. JAK2 is an important target related to blood diseases and is associated with the production of red blood cells and platelets. Currently, only two drugs, fedratinib and pacritinib, are available on the market as selective JAK2 inhibitors. Fedratinib has received a black box warning from the FDA due to its potentially fatal encephalopathy. Pacritinib was launched in 2022, but its safety and efficacy still require further clinical verification.

[0006] Therefore, the research and development of selective JAK2 inhibitors still have broad clinical application prospects and market space. Summary of the Invention

[0007] The object of the present invention is to provide a JAK inhibitor. The JAK inhibitor of the present invention has a selective inhibitory effect on JAK2, thereby significantly improving its safety.

[0008] In a first aspect, the present invention provides a compound of formula I or its stereoisomers or optical isomers, pharmaceutically acceptable salts, prodrugs or solvates,

[0009]

[0010] wherein,

[0011] R 1 is selected from hydrogen, halogen, substituted or unsubstituted C 1 -C 10 alkyl, hydroxyl, substituted or unsubstituted C 1 -C 10 alkoxy, nitro, amino, substituted or unsubstituted amino C1-3 acyl, substituted or unsubstituted aminosulfonyl, substituted or unsubstituted C 1 -C 3 alkylcarbonyl, substituted or unsubstituted hydroxy C1-3 acyl (preferably hydroxyacetyl), substituted or unsubstituted C 1 -C 3 alkoxycarbonyl;

[0012] n is selected from 0, 1, 2 or 3;

[0013] R 2Selected from the group: hydrogen, halogen, substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 1 -C 10 acyl;

[0014] R 3 Selected from the group: hydrogen, halogen, hydroxyl, amino, substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 1 -C 10 alkoxy, substituted (preferably C 1 -C 10 alkyl or alkoxy-substituted) or unsubstituted 4- to 6-membered nitrogen- or oxygen-containing heterocycle;

[0015] R 4 Selected from the group: hydrogen, substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted 4- to 6-membered heterocycle containing 1, 2 or 3 heteroatoms independently selected from N, O or S;

[0016] X 1 is a C3-C9 alkylene or alkenylene group, said alkylene or alkenylene group containing a heteroatom independently selected from N, O or S.

[0017] In a specific embodiment, R 1 is selected from hydrogen, halogen, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 1 -C 6 alkoxy, substituted or unsubstituted sulfamoyl;

[0018] n is selected from 0, 1 or 2;

[0019] R 2 is selected from the group: hydrogen, halogen, substituted or unsubstituted C 1 -C 6 alkyl;

[0020] R 3 is selected from the group: hydrogen;

[0021] R 4 is selected from the group: hydrogen, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted 4- to 6-membered heterocycle containing 1 or 2 heteroatoms independently selected from N or O;

[0022] X 1 is selected from the group:

[0023]

[0024] R 5 is a substituted or unsubstituted C 1 -C 3 alkyl;

[0025] l, m, o, r are each independently 0, 1 or 2;

[0026] p, q are each independently 0, 1, 2 or 3.

[0027] In a specific embodiment, X 1 is selected from

[0028]

[0029] R 4 is selected from hydrogen, hydroxyl, amino, methyl, methoxy and the following structural units:

[0030]

[0031] In a specific embodiment,

[0032] R 4 is selected from: X 1 is selected from:

[0033]

[0034] In a specific embodiment, the compound is as shown in Formula II,

[0035]

[0036] wherein,

[0037] R 1 is selected from hydrogen, halogen, halogenated or unsubstituted C 1 -C 6 alkyl;

[0038] R 2 is selected from the group consisting of: hydrogen, substituted or unsubstituted C 1 -C 6 alkyl;

[0039] R 3 is: hydrogen;

[0040] R 4 is: X 1 is: In a specific embodiment, the compound is selected from the group consisting of:

[0041]

[0042]

[0043] Preferably, the compound is:

[0044]

[0045] In a preferred embodiment, the compound has a selective inhibitory effect on JAK2.

[0046] In a second aspect, the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition contains the compound described in the first aspect or its stereoisomer or optical isomer, or its pharmaceutically acceptable salt, prodrug or solvate, and a pharmaceutically acceptable carrier or excipient.

[0047] In a preferred embodiment, the pharmaceutical combination is used for preparing a JAK2 inhibitor.

[0048] In a preferred embodiment, the JAK2 inhibitor is a drug for preventing or treating JAK2-mediated diseases.

[0049] In a preferred embodiment, the JAK2-mediated diseases are myelodysplastic syndrome (MDS), polycythemia vera (AV), eosinophilia, tumors, inflammatory diseases or infections caused by bacteria, viruses or fungi;

[0050] Preferably, the tumors are selected from the group consisting of: myeloproliferative carcinoma (MPN), melanoma, lung cancer, kidney cancer, ovarian cancer, prostate cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, pediatric solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvic cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal cord tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma; and / or

[0051] The inflammatory diseases are selected from the group consisting of: rheumatoid arthritis, ankylosing spondylitis, autoimmune hemolytic anemia, arthritis, myasthenia gravis, systemic lupus erythematosus, pernicious anemia, polymyositis; and / or

[0052] The virus is selected from the group consisting of: hepatitis virus (hepatitis A, B and C), herpes virus, influenza virus, adenovirus, coronavirus, measles virus, dengue virus, poliovirus, rabies virus; and / or

[0053] The bacterium is selected from the group consisting of: chlamydia, rickettsia, mycobacterium, staphylococcus, pneumococcus, cholera, tetanus; and / or

[0054] The fungus is selected from the group consisting of: candida, aspergillus, blastomyces dermatitidis.

[0055] In a third aspect, the present invention provides the use of the compound described in the first aspect, or its stereoisomer or optical isomer, or its pharmaceutically acceptable salt, prodrug or solvate, characterized in that it is used for the preparation of a JAK2 inhibitor.

[0056] In a specific embodiment, the JAK2 inhibitor is a drug for preventing or treating JAK2-mediated diseases.

[0057] In a specific embodiment, the JAK2-mediated diseases are myelodysplastic syndrome (MDS), polycythemia vera (AV), eosinophilia, tumors, inflammatory diseases or infections caused by bacteria, viruses or fungi;

[0058] Preferably, the tumor is selected from the group consisting of: myeloproliferative carcinoma (MPN), melanoma, lung cancer, kidney cancer, ovarian cancer, prostate cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, pediatric solid tumors, lymphocytic lymphoma, bladder cancer, renal or ureteral cancer, renal pelvic cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal cord tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T cell lymphoma; and / or

[0059] The inflammatory diseases are selected from the group consisting of: rheumatoid arthritis, ankylosing spondylitis, autoimmune hemolytic anemia, arthritis, myasthenia gravis, systemic lupus erythematosus, pernicious anemia, polymyositis; and / or

[0060] The virus is selected from the group consisting of: hepatitis virus (hepatitis A, B and C), herpes virus, influenza virus, adenovirus, coronavirus, measles virus, dengue virus, poliovirus, rabies virus; and / or

[0061] The bacteria are selected from the group consisting of: Chlamydia, Rickettsia, Mycobacterium, Staphylococcus, Streptococcus pneumoniae, cholera, tetanus; and / or

[0062] The fungi are selected from the group consisting of: Candida, Aspergillus, Blastomyces dermatitidis.

[0063] In a fourth aspect, the present invention provides a JAK2 inhibitor, which is characterized by containing the compound described in the first aspect or its stereoisomer or optical isomer, or its pharmaceutically acceptable salt, prodrug or solvate.

[0064] In a fifth aspect, the present invention provides a method for preventing or treating JAK2-mediated diseases, the method comprising the step of administering a prophylactically or therapeutically effective amount of the compound described in the first aspect or its stereoisomer or optical isomer, or its pharmaceutically acceptable salt, prodrug or solvate, or the pharmaceutical combination described in the second aspect to a subject in need thereof.

[0065] In a preferred embodiment, the JAK2-mediated disease is myelodysplastic syndrome (MDS), polycythemia vera (AV), eosinophilia, tumor, inflammatory disease or infection caused by bacteria, virus or fungi;

[0066] Preferably, the tumor is selected from the group consisting of: myeloproliferative carcinoma (MPN), melanoma, lung cancer, kidney cancer, ovarian cancer, prostate cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, pediatric solid tumor, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvic cancer, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis, spinal cord tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T cell lymphoma; and / or

[0067] The inflammatory disease is selected from the group consisting of: rheumatoid arthritis, ankylosing spondylitis, autoimmune hemolytic anemia, arthritis, myasthenia gravis, systemic lupus erythematosus, pernicious anemia, polymyositis; and / or

[0068] The virus is selected from the group consisting of: hepatitis virus (hepatitis A, B and C), herpes virus, influenza virus, adenovirus, coronavirus, measles virus, dengue virus, poliovirus, rabies virus; and / or

[0069] The bacteria are selected from the following group: Chlamydia, Rickettsia, Mycobacterium, Staphylococcus, Streptococcus pneumoniae, cholera, tetanus; and / or

[0070] The fungi are selected from the following group: Candida, Aspergillus, Blastomyces dermatitidis.

[0071] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 shows the results of blood routine analysis, where (A) is the proportion of reticulocytes and (B) is the hematocrit. In the figure, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns > 0.05 compared with the model; #P < 0.0001 compared with the control; ^^P < 0.01 compared with L3 100 mg / kg;

[0073] Figure 2 shows the flow cytometry analysis results of erythroid progenitor cells, that is, the flow cytometry statistical charts of the spleen (A) and bone marrow (B). In the figure, P < 0.05, ***P < 0.001, ****P < 0.0001 compared with the model; #P < 0.0001 compared with the control; ^P < 0.05, ^^^P < 0.001, ^^^^P < 0.0001 compared with L3 100 mg / kg;

[0074] Figure 3 shows the splenomegaly of mice, where (A) is the representative picture of the spleen and (B) is the statistical chart of spleen weight.

[0075] In the figure, ****P < 0.0001 compared with the model; #P < 0.0001 compared with the control; ^^^^P < 0.0001 compared with L3 100 mg / kg;

[0076] Figure 4 Shows the weight change of mice after administration. DETAILED DESCRIPTION OF THE INVENTION

[0077] Through extensive and in-depth research, the inventors discovered the potential use of aminopyrimidine macrocyclic structures as JAK inhibitors, and finally discovered a series of novel macrocyclic compounds that can selectively inhibit JAK2 kinase. This inhibitor has a selective inhibitory effect on JAK2, thus having clinical significance and application prospects and providing a new material basis for the development of immunoinflammatory and anti-tumor drugs. The present invention was completed on this basis.

[0078] Term Definitions

[0079] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention pertains. For the convenience of understanding the present invention, the relevant terms related to the present invention are defined as follows, but the scope of the present invention is not limited to these specific definitions.

[0080] As used herein, "JAK2" refers to Janus kinase 2, which is a cytoplasmic non-receptor type soluble protein tyrosine kinase. JAK-STAT is the Janus kinase-signal transducer and activator of transcription pathway, which is a hot topic in the current field of cytokine research.

[0081] As used herein, "alkyl" refers to a straight-chain or branched-chain saturated group composed of carbon atoms and hydrogen atoms. For example, "C 1 -C 10 alkyl" refers to a saturated branched-chain or straight-chain alkyl with a carbon chain length of 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, and more preferably 1 to 3 carbon atoms. Examples of alkyl include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, heptyl, pentyl, and the like.

[0082] As used herein, "alkoxy" refers to an oxygen group substituted by an alkyl group. In a specific embodiment, the alkoxy used herein is an alkoxy with a length of 1 to 10 carbon atoms, and more preferably an alkoxy with a length of 1 to 4 carbon atoms. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, etc. In a further embodiment, the alkoxy can be a substituted alkoxy, for example, a halogen-substituted alkoxy. In a specific embodiment, a halogen-substituted C 1 -C 3 alkoxy is preferred.

[0083] As used herein, "halogen" refers to fluorine, chlorine, bromine, and iodine. In a preferred embodiment, the halogen is fluorine or chlorine.

[0084] As used herein, "halogenated" refers to fluorination, chlorination, bromination, and iodination.

[0085] As used herein, "substituted or unsubstituted" or "optionally substituted" means that the substituent modified by this term can optionally be substituted by 1 to 5 (for example, 1, 2, 3, 4, or 5) substituents selected from the following: halogen, C1-4 aldehyde group, C1-6 straight-chain or branched-chain alkyl, halogen-substituted C1-6 straight-chain or branched-chain alkyl (such as trifluoromethyl), C1-6 alkoxy, halogen-substituted C1-6 alkoxy (such as trifluoromethoxy), cyano, nitro, amino, hydroxyl, hydroxymethyl, carboxyl, ethoxycarbonyl, N(CH3), and C1-4 acyl group.

[0086] Active ingredient

[0087] As used herein, the "compounds of the present invention" refers to the compounds represented by formula (I), and also includes various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compounds of formula (I).

[0088]

[0089] wherein R 1 、R 2 、R 3 、R 4 and X 1 are as described above.

[0090] Based on the teachings of the present invention and common general knowledge in the art, those skilled in the art will understand that each group in the compounds of the present invention can be further substituted to obtain derivatives that can have the same or similar activities as the compounds specifically disclosed in the present invention. Each group in the compounds of the present invention can be substituted by various conventional substituents in the art, as long as such substitution does not violate the rules of chemical synthesis or valence rules.

[0091] The term "substituted" as used herein means that one or more hydrogen atoms on a specific group are replaced by specific substituents. The specific substituents can be the substituents described correspondingly in the foregoing, or the specific substituents appearing in each embodiment or the conventional substituents in the art. Therefore, in the present invention, the substituents in the general formula can also be independently the corresponding groups in the specific compounds in the embodiments; that is, the present invention includes both the combinations of the substituents in the above general formula and the combinations of some of the substituents shown in the general formula and other specific substituents appearing in the embodiments. It is not difficult for those skilled in the art to prepare compounds having such combinations of substituents and to detect the activities of the obtained compounds based on the conventional technical means in the art.

[0092] The term "pharmaceutically acceptable salt" as used herein refers to the salts formed by the compounds of the present invention with acids or bases that are suitable for use as drugs. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts is the salts formed by the compounds of the present invention with acids. Acids suitable for forming salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, etc., organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, benzene sulfonic acid, etc.; and acidic amino acids such as aspartic acid and glutamic acid.

[0093] Unless otherwise specified, the structural formulas described in this invention are intended to include all isomeric forms (such as enantiomers, diastereomers and geometric isomers (or conformational isomers)): for example, the R and S configurations containing asymmetric centers, the (Z) and (E) isomers of double bonds, etc. Therefore, the individual stereochemical isomers of the compounds of this invention or mixtures of their enantiomers, diastereomers or geometric isomers (or conformational isomers) are all within the scope of this invention.

[0094] As used herein, the term "tautomer" means that structural isomers with different energies can interconvert over a low energy barrier. For example, proton tautomers (i.e., prototropy) include interconversion through proton migration, such as 1H-indazole and 2H-indazole. Valence tautomers include interconversion through some reorganization of bonding electrons.

[0095] As used herein, the term "solvate" refers to a complex formed by the coordination of a compound of this invention with solvent molecules in a specific ratio.

[0096] As used herein, the term "hydrate" refers to a complex formed by the coordination of a compound of this invention with water.

[0097] Pharmaceutical Compositions and Administration Methods

[0098] Since the compounds of this invention have excellent inhibitory activity against JAK kinases, the compounds of this invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, as well as pharmaceutical compositions containing the compounds of this invention as the main active ingredient can be used for the prevention and / or treatment (stabilization, alleviation or cure) of JAK kinase-related diseases.

[0099] The pharmaceutical compositions of this invention contain the compounds of this invention within a safe and effective amount range and pharmaceutically acceptable excipients or carriers. The "safe and effective amount" herein refers to: an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Generally, the pharmaceutical composition contains 1 - 2000 mg of the compound of this invention / dose, more preferably, 10 - 200 mg of the compound of this invention / dose. Preferably, the "dose" is a capsule or a tablet.

[0100] "Pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be admixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers are cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0101] There is no particular limitation on the mode of administration of the compounds or pharmaceutical compositions of the present invention. Representative modes of administration include (but are not limited to): oral, parenteral (intravenous, intramuscular or subcutaneous).

[0102] 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 calcium phosphate, or is admixed with the following components: (a) fillers or bulking agents, such as starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, such as hydroxypropylmethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, such as glycerol; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, such as paraffin wax; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain buffering agents.

[0103] Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and casings, 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 can be delayed in a manner such that release occurs in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric materials and wax-like substances. If necessary, the active compound can also be in the form of microcapsules with one or more of the above excipients.

[0104] 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 conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3 - butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, etc.

[0105] In addition to these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifying agents and suspending agents, sweetening agents, flavoring agents and fragrances.

[0106] In addition to the active compound, the suspension may contain suspending agents, for example, ethoxylated isooctadecanol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum monostearate and agar or mixtures of these substances, etc.

[0107] Compositions for parenteral injection may contain physiologically acceptable sterile aqueous or non - aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for re - dissolving into sterile injectable solutions or dispersions. Suitable aqueous and non - aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0108] The compounds of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds.

[0109] When administered in combination, the pharmaceutical composition further comprises one or more (2, 3, 4, or more) other pharmaceutically acceptable compounds. One or more of the other pharmaceutically acceptable compounds can be administered simultaneously with, separately from, or sequentially to the compound of the present invention.

[0110] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal in need of treatment (such as a human), wherein the dosage during administration is a pharmaceutically effective dosage. For a person weighing 60 kg, the daily dosage is usually 1 - 2000 mg, preferably 20 - 500 mg. Of course, the specific dosage should also consider factors such as the route of administration and the health status of the patient, which are all within the scope of the skills of a skilled physician.

[0111] Preparation Method

[0112] The compounds of the present invention can be prepared according to conventional routes or methods, or can be obtained according to the methods or routes described herein.

[0113] Intermediate Synthesis Route 1:

[0114]

[0115] Scheme-1. Synthetic route of intermediate 4a-t a

[0116] a Reagents and conditions: (a) Cs 2 CO 3 , DMF, 100 °C, 8 h; (b) NaBH 4 , THF, 0 °C - r.t., 30 min. (c) NaH, DMF, 0 °C, 2 h;

[0117] Synthetic route 2 of the target compound:

[0118]

[0119] Scheme-2. Synthetic route of L1-15, B1-4, Z1 a

[0120] a Reagents and conditions: (a) Cs 2 CO 3 , Xantphos, Pd 2 (dba) 3 , DMF, 130 °C, 50 W, 0.5 h, 30%; (b) N 2 H 4 ·H 2 O, FeCl 3 , EtOH, 80 °C, 2 h; (c) TsOH·H 2 O, n-butanol, 105 °C, 2 h.

[0121] The advantages of the present invention are as follows:

[0122] 1. The compounds of the present invention have a novel structure and excellent JAK kinase inhibitor activity;

[0123] 2. The compounds of the present invention have better selective inhibition of JAK2;

[0124] 3. The compounds of the present invention have higher safety; and

[0125] 4. The compounds of the present invention lay a new material foundation for the development of immunoinflammatory and anti-tumor drugs, thus having clinical significance and application prospects,

[0126] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are by weight. The experimental materials and reagents used in the following embodiments can be obtained from commercial channels without special instructions.

[0127] In the following embodiments, "reflux" means reflux.

[0128] Embodiment

[0129] Example 1 Preparation of Compounds L1-15, B1-4

[0130] Synthesis of 5-Nitro-2-(2-(1-pyrrolidinyl)ethoxy)benzaldehyde (2a)

[0131]

[0132] Put 2-Hydroxy-5-nitrobenzaldehyde (4.00 g, 23.90 mmol), N-(2-Chloroethyl)pyrrolidine hydrochloride (5.70 g, 33.50 mmol), and cesium carbonate (11.70 g, 35.90 mmol) into a 250 mL two-necked flask, and N 2 Replace three times. Then add anhydrous DMF (30 mL) solution and reflux at 100 °C. Monitor the progress of the reaction by TLC (DCM:CH 2 Cl 2 = 40:1). After 10 h, extract with ethyl acetate three times, combine the organic solvents, and dry over anhydrous sodium sulfate. After removing the solvent by rotary evaporation, it is directly used for the next step.

[0133] Synthesis of 5-Nitro-2-(2-(1-piperidinyl)ethoxy)benzaldehyde (2b)

[0134]

[0135] Refer to the synthesis steps of 2a and directly use it for the next step.

[0136] Synthesis of 5-Nitro-2-(2-(1-morpholinyl)ethoxy)benzaldehyde (2c)

[0137]

[0138] Refer to the synthesis steps of 2a to obtain 0.40 g of yellow oil, with a yield of 62.6%.

[0139] 1 H NMR (400 MHz, DMSO-d 6)δ 10.34 (s, 1H), 8.49 (dd, J = 9.2, 3.0 Hz, 1H), 8.41 (d, J = 3.0 Hz, 1H), 7.51 (d, J = 9.2 Hz, 1H), 4.42 (t, J = 5.6 Hz, 1H), 3.56 (t, J = 4.7 Hz, 2H), 2.89 (s, 1H), 2.81 (t, J = 5.7 Hz, 2H), 2.73 (s, 1H), 2.48 (s, 2H).

[0140] Synthesis of 5-nitro-2-methoxybenzaldehyde (2d)

[0141]

[0142] Refer to the synthesis procedure of 2a, used directly in the next step without purification.

[0143] Synthesis of (5-nitro-2-(2-(1-pyrrolidinyl)ethoxy)phenyl)methanol (3a)

[0144]

[0145] Dissolve 5-nitro-2-(2-(pyrrolidin-1-yl)ethoxy)benzaldehyde (1.00 g, 3.79 mmol) in methanol (15 mL), and quickly add sodium borohydride (0.14 g, 3.79 mmol) in several portions under ice bath conditions. Then react at room temperature, and monitor the reaction progress by TLC (DCM:CH 2 Cl 2 = 40:1). After half an hour, the reaction is complete. After removing most of the methanol by rotary evaporation, extract with dichloromethane and dry over anhydrous sodium sulfate. After removing the solvent by rotary evaporation, purify the residue by silica gel column chromatography (DCM:CH 2 Cl 2 = 50:1) to obtain a yellow solid.

[0146] 1 1H NMR (400 MHz, DMSO-d 6 ) δ 8.25–8.22 (m, 1H), 8.14 (dd, J = 9.0, 3.0 Hz, 1H), 7.20 (d, J = 9.1 Hz, 1H), 5.44 (t, J = 5.3 Hz, 1H), 4.52 (d, J = 4.3 Hz, 2H), 4.23 (t, J = 5.7 Hz, 2H), 2.69 (t, J = 5.7 Hz, 2H), 2.43 (t, J = 5.3 Hz, 4H), 1.51–1.44 (m, 4H).

[0147] Synthesis of 5-nitro-2-(2-(1-piperidinyl)ethoxy)benzaldehyde (3b)

[0148]

[0149] Refer to the synthesis procedure of 3a. Without purification, it was directly used for the next step.

[0150] Synthesis of 5-nitro-2-(2-(1-morpholinyl)ethoxy)benzaldehyde (3c)

[0151]

[0152] Refer to the synthesis procedure of 3a to obtain 0.40 g of yellow oil, with a yield of 62.6%.

[0153] 1 H NMR (600 MHz, DMSO-d 6 ) δ 8.20 - 8.16 (m, 2H), 7.24 - 7.19 (m, 1H), 7.09 - 7.03 (m, 2H), 6.88 (t, J = 1.8 Hz, 1H), 5.87 - 5.83 (m, 2H), 4.50 (d, J = 1.8 Hz, 2H), 4.44 (s, 2H), 4.26 - 4.21 (m, 2H), 4.12 - 4.08 (m, 2H), 4.02 (q, J = 1.8 Hz, 2H), 3.75 (s, 3H), 2.81 (t, J = 5.7 Hz, 2H), 2.53 (s, 4H), 1.67 - 1.63 (m, 4H).

[0154] Synthesis of 5-nitro-2-methoxybenzaldehyde (3d)

[0155]

[0156] Refer to the synthesis procedure of 3a. Without purification, it was directly used for the next step.

[0157] Synthesis of compound (4a)

[0158]

[0159] Weigh compound 3a (0.90 g, 3.38 mmol) and place it in a 25 mL round-bottom flask. After adding 60% NaH (161 mg, 4.05 mmol), quickly displace the air with nitrogen three times. Under an ice bath, add anhydrous DMF (15 mL) to dissolve it. A large number of bubbles are generated. Stir for 1 h while maintaining the ice bath. After 1 h, use a syringe to aspirate compound 5a (1.22 g, 4.05 mmol) dissolved in anhydrous DMF and slowly add it dropwise. After adding, transfer it to room temperature for reaction. After 1 h, monitor the reaction by TLC until it is complete. Slowly add water to the reaction solution to quench it, extract it three times with ethyl acetate, combine the organic phases, wash the organic layer three times with water and saturated brine in sequence, dry it over anhydrous sodium sulfate, filter it by suction, and remove the solvent by rotary evaporation to obtain a yellow oil. Purify it by silica gel column chromatography to obtain 800 mg of yellow oil, with a yield of 39.1%.

[0160] 1 H NMR(400MHz,DMSO-d 6 )δ8.23(d,J=7.5Hz,2H),7.49(d,J=8.4Hz,1H),7.43(s,1H),7.27(s,1H),7.24(s,1H),4.65(s,2H),4.61(s,2H),4.33(t,J=5.4Hz,2H),3.09(s,2H),2.78(s,4H),1.73(s,4H).LC-MS(ESI):m / z:453.17[M+H] + .

[0161] Synthesis of compound (4b)

[0162]

[0163] Refer to the synthesis procedure of 4a to obtain 1.03 g of yellow oil, with a yield of 32.5%.

[0164] 1 H NMR(600MHz,DMSO-d 6 )δ8.20(d,J=7.6Hz,2H),7.70(dd,J=6.8,2.1Hz,1H),7.43(ddd,J=8.5,4.9,2.1Hz,1H),7.37(t,J=8.7Hz,1H),7.25-7.20(m,1H),4.61(s,2H),4.55(s,2H),4.24(t,J=5.6Hz,2H),2.82(t,J=5.6Hz,2H),1.67-1.61(m,J=3.8,3.2Hz,4H).LC-MS(ESI):m / z:453.17[M+H] + .

[0165] Synthesis of Compound (4c)

[0166]

[0167] Referring to the synthesis procedure of 4a, 870 mg of yellow oil was obtained with a yield of 32.1%.

[0168] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.25 (d, J = 7.2 Hz, 2H), 7.48 (d, J = 8.1 Hz, 1H), 7.53 (s, 1H), 7.37 (s, 1H), 7.27 (s, 1H), 4.66 (s, 2H), 4.63 (s, 2H), 4.37 (t, J = 5.5 Hz, 2H), 3.08 (s, 2H), 2.78 (s, 4H), 1.75 (s, 4H). LC-MS (ESI): m / z: 469.07 [M+H] + .

[0169] Synthesis of Compound (4d)

[0170]

[0171] Referring to the synthesis procedure of 4a, 650 mg of yellow oil was obtained with a yield of 30.1%.

[0172] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.20 (d, J = 7.1 Hz, 2H), 7.44 (d, J = 8.2 Hz, 1H), 7.51 (s, 1H), 7.33 (s, 1H), 7.21 (s, 1H), 4.62 (s, 2H), 4.61 (s, 2H), 4.32 (t, J = 5.2 Hz, 2H), 3.01 (s, 2H), 2.72 (s, 4H), 1.71 (s, 4H). LC-MS (ESI): m / z: 512.98 [M+H] + .

[0173] Synthesis of Compound (4e)

[0174]

[0175] Referring to the synthesis procedure of 4a, 600 mg of yellow oil was obtained with a yield of 35.1%.

[0176] 1 H NMR (400 MHz, DMSO-d 6) δ 8.24 - 8.17 (m, 2H), 7.34 (s, 2H), 7.26 - 7.21 (m, 1H), 7.18 (s, 1H), 4.58 (s, 2H), 4.55 (s, 2H), 4.24 (t, J = 5.6 Hz, 2H), 2.82 (t, J = 5.6 Hz, 2H), 2.30 (s, 3H), 1.70 - 1.58 (m, 4H). LC-MS (ESI): m / z: 449.13 [M + H] + .

[0177] Synthesis of compound (4f)

[0178]

[0179] Referring to the synthesis procedure of 4a, 530 mg of yellow oil was obtained with a yield of 30.1%.

[0180] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.19 (d, J = 3.0 Hz, 2H), 7.24 (d, J = 8.9 Hz, 1H), 7.13 (s, 1H), 7.08 (s, 1H), 6.94 (s, 1H), 4.59 (s, 2H), 4.56 (s, 2H), 4.24 (d, J = 5.5 Hz, 2H), 3.76 (s, 3H), 2.83 (d, J = 5.7 Hz, 2H), 2.54 (s, 4H), 1.65 (s, 4H). LC-MS (ESI): m / z: 465.19 [M + H] + .

[0181] Synthesis of compound (4g)

[0182]

[0183] Referring to the synthesis procedure of 4a, 1.20 g of brown oil was obtained with a yield of 56.7%.

[0184] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.23 - 8.16 (m, 2H), 7.88 (s, 2H), 7.72 (s, 1H), 7.23 (d, J = 8.9 Hz, 1H), 4.71 (s, 2H), 4.60 (s, 2H), 4.23 (t, J = 5.6 Hz, 2H), 2.80 (t, J = 5.6 Hz, 2H), 2.47 (d, J = 6.6 Hz, 4H), 1.62 (m, J = 3.0 Hz, 4H). LC-MS (ESI): m / z: 503.18 [M + H] + .

[0185] Synthesis of Compound (4h)

[0186]

[0187] Referring to the synthesis procedure of 4a, 1.02 g of yellow oil was obtained with a yield of 55.1%.

[0188] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.20 - 8.14 (m, 2H), 7.53 - 7.52 (m, 1H), 7.47 - 7.46 (m, 1H), 7.36 - 7.28 (m, 2H), 7.18 (d, J = 8.7 Hz, 1H), 4.60 (s, 2H), 4.53 (s, 2H), 4.20 (t, J = 5.6 Hz, 2H), 2.78 (t, J = 5.6 Hz, 2H), 2.48 - 2.44 (m, 4H), 1.63 - 1.60 (m, 4H). LC-MS (ESI): m / z: 435.15 [M+H] + .

[0189] Synthesis of Compound (4i)

[0190]

[0191] Referring to the synthesis procedure of 4a, 248 mg of brownish-yellow oily liquid was obtained with a yield of 74.0%.

[0192] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.21 (d, J = 8.0 Hz, 2H), 7.57 (t, J = 1.8 Hz, 1H), 7.51 (dt, J = 7.6, 1.7 Hz, 1H), 7.39 (dt, J = 7.6, 1.5 Hz, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.28–7.23 (m, 1H), 4.64 (s, 2H), 4.57 (s, 2H), 4.27 (t, J = 5.6 Hz, 2H), 3.54–3.49 (m, 4H), 2.72 (t, J = 5.5 Hz, 2H), 2.47–2.42 (m, 4H). LC-MS (ESI): m / z: 451.10 [M+H] + .451.10.

[0193] Synthesis of Compound (4j)

[0194]

[0195] Referring to the synthesis procedure of 4a, 1.5 g of yellow oil was obtained with a yield of 53.1%.

[0196] 1 H NMR (400 MHz, DMSO-d6) δ 8.22 - 8.17 (m, 2H), 7.56 (t, J = 1.8 Hz, 1H), 7.52 - 7.48 (m, 1H), 7.40 - 7.36 (m, 1H), 7.36 - 7.32 (m, 1H), 7.24 (d, J = 8.8 Hz, 1H), 4.63 (s, 2H), 4.55 (s, 2H), 4.24 (t, J = 5.8 Hz, 2H), 2.67 (t, J = 5.8 Hz, 2H), 2.40 (t, J = 5.4 Hz, 4H), 1.46 - 1.39 (m, 4H), 1.37 - 1.30 (m, 2H).

[0197] Synthesis of compound (4k)

[0198]

[0199] Referring to the synthesis procedure of 4a, 390 mg of brown oil was obtained with a yield of 46.4%.

[0200] 1 H NMR (400 MHz, DMSO-d6) δ 8.22 (s, 1H), 7.58 (t, J = 1.8 Hz, 1H), 7.52 - 7.48 (m, 1H), 7.42 - 7.37 (m, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.31 - 7.27 (m, 1H), 7.25 - 7.22 (m, 1H), 4.64 (s, 2H), 4.57 (s, 2H), 3.94 (s, 3H).

[0201] Synthesis of compound (4l)

[0202]

[0203] Referring to the synthesis procedure of 4a, 900 mg of brown oil was obtained with a yield of 50.3%.

[0204] 11H NMR (400 MHz, Chloroform-d) δ 8.34 (d, J = 2.8 Hz, 1H), 8.20 (dd, J = 9.0, 2.9 Hz, 1H), 7.55 (s, 1H), 7.44 (d, J = 7.9 Hz, 1H), 7.32 (d, J = 7.6 Hz, 1H), 7.23 (d, J = 7.7 Hz, 1H), 6.92 (d, J = 9.0 Hz, 1H), 4.62 (s, 2H), 4.58 (s, 2H), 3.94 (s, 3H). LC-MS (ESI): m / z: 352.10 [M+H] + .

[0205] Synthesis of compound (4m)

[0206]

[0207] Referring to the synthesis procedure of 4a, 490 mg of brown oil was obtained with a yield of 40.4%.

[0208] 1 1H NMR (400 MHz, Chloroform-d) δ 8.33 (d, J = 2.9 Hz, 1H), 8.20 (dd, J = 9.0, 2.9 Hz, 1H), 7.30 (s, 1H), 7.19 (d, J = 8.1 Hz, 1H), 7.05 (d, J = 10.7 Hz, 1H), 6.92 (d, J = 9.0 Hz, 1H), 4.60 (s, 2H), 4.58 (s, 2H), 4.23 (t, J = 5.8 Hz, 2H), 3.73 - 3.66 (m, 4H), 2.84 (t, J = 5.7 Hz, 2H), 2.57 (t, J = 4.7 Hz, 4H). LC-MS (ESI): m / z: 469.15 [M+H] + .

[0209] Synthesis of compound (4n)

[0210]

[0211] Referring to the synthesis procedure of 4a, 1.06 g of brown oil was obtained with a yield of 63.2%.

[0212] 1 1H NMR (400 MHz, DMSO-d 6)δ 8.23 - 8.16 (m, 2H), 7.88 (s, 2H), 7.72 (s, 1H), 7.23 (d, J = 8.9 Hz, 1H), 4.71 (s, 2H), 4.60 (s, 2H), 4.23 (t, J = 5.6 Hz, 2H), 2.40 (t, J = 5.4 Hz, 4H), 1.46 - 1.39 (m, 4H), 1.37 - 1.30 (m, 2H).

[0213] Synthesis of compound (5a)

[0214]

[0215] Weigh compound 4a (250 mg, 0.55 mmol), 4 - amino - 2 - chloro - 5 - methylpyrimidine (79 mg, 0.55 mmol), cesium carbonate (536 mg, 1.65 mmol), Xantphos (64 mg, 0.11 mmol) and Pd 2 (dba) 3 (50 mg, 0.05 mmol) and add them successively into a 50 mL microwave tube. Add anhydrous DMF (15 mL) to dissolve, and perform N 2 displacement for 3 times. Set the microwave reaction conditions: temperature 130 °C, time 0.5 h, heating power 85 W. After the microwave heating is completed, monitor the reaction by TLC (DCM:MeOH = 20:1). Extract with ethyl acetate three times, combine the organic phases. Wash the organic layer with water and saturated brine three times successively, dry over anhydrous sodium sulfate, filter by suction, and remove the solvent by rotary evaporation to obtain a yellow oil. Purify the crude product by silica gel column chromatography (DCM:MeOH = 40:1) to obtain 60 mg of a yellow oil, with a yield of 21.2%.

[0216] 1 H NMR (400 MHz, DMSO - d 6 )δ 9.06 (s, 1H), 8.24 (d, J = 2.8 Hz, 1H), 8.20 (dd, J = 9.0, 2.9 Hz, 1H), 8.10 (s, 1H), 7.66 (s, 1H), 7.56 (s, 1H), 7.24 (d, J = 9.0 Hz, 1H), 6.92 (d, J = 8.8 Hz, 1H), 4.65 (s, 2H), 4.59 (s, 2H), 4.23 (t, J = 5.5 Hz, 2H), 2.78 (d, J = 5.5 Hz, 2H), 2.47 (s, 4H), 2.19 (s, 3H), 1.61 (s, 4H). LC - MS (ESI): m / z: 516.20 [M + H] + .

[0217] Synthesis of compound (5b)

[0218]

[0219] Referring to the synthesis procedure of 5a, 600 mg of yellow oil was obtained with a yield of 35.3%.

[0220] 1 H NMR (600 MHz, DMSO-d 6 ) δ 8.96 (s, 1H), 8.23 (d, J = 2.9 Hz, 1H), 8.20 (dd, J = 9.0, 3.0 Hz, 1H), 8.04 (s, 1H), 7.44 (d, J = 8.1 Hz, 1H), 7.30 (d, J = 8.7 Hz, 2H), 7.24 (d, J = 9.1 Hz, 1H), 4.64 (s, 2H), 4.59 (s, 2H), 4.24 (t, J = 5.7 Hz, 2H), 2.81 (t, J = 5.6 Hz, 2H), 2.48 (s, 4H), 2.15 (s, 3H), 1.62 (s, 4H). LC-MS (ESI): m / z: 516.20 [M+H] + .

[0221] Synthesis of compound (5c)

[0222]

[0223] Referring to the synthesis procedure of 5a, 150 mg of yellow oil was obtained with a yield of 25.3%.

[0224] 1 H NMR (400 MHz, Chloroform-d) δ 8.33 (s, 1H), 8.17 (dd, J = 9.0, 2.9 Hz, 1H), 8.02 (s, 1H), 7.80 (s, 1H), 7.46 (s, 1H), 7.10 (s, 1H), 6.93 (s, 1H), 6.82 (s, 1H), 4.62 (s, 2H), 4.60 (s, 2H), 4.27 (t, J = 5.6 Hz, 2H), 3.01 (t, J = 5.7 Hz, 2H), 2.71 (s, 4H), 2.19 (s, 3H), 1.80 (s, 4H). LC-MS (ESI): m / z: 532.17 [M+H] + .

[0225] Synthesis of compound (5d)

[0226]

[0227] Referring to the synthesis procedure of 5a, 110 mg of yellow oil was obtained with a yield of 21.3%.

[0228] 11H NMR (400 MHz, Chloroform-d) δ 8.35 (d, J = 2.9 Hz, 1H), 8.18 (dd, J = 9.0, 2.9 Hz, 1H), 8.03 (s, 1H), 7.92 (s, 1H), 7.52 (s, 1H), 6.92 (d, J = 9.1 Hz, 1H), 6.69 (s, 1H), 4.63 (s, 2H), 4.61 (s, 2H), 4.27 (t, J = 5.8 Hz, 2H), 3.01 (t, J = 5.6 Hz, 2H), 2.70 (s, 4H), 2.20 (s, 3H), 1.81 (s, 4H). LC-MS (ESI): m / z: 576.10 [M+H] + .

[0229] Synthesis of compound (5e)

[0230]

[0231] Referring to the synthesis procedure of 5a, 130 mg of yellow oil was obtained with a yield of 19.3%.

[0232] 1 1H NMR (400 MHz, DMSO-d 6 ) δ 8.83 (s, 1H), 8.23 (d, J = 3.0 Hz, 1H), 8.19 (dd, J = 9.0, 2.9 Hz, 1H), 8.03 (s, 1H), 7.50 (d, J = 2.3 Hz, 1H), 7.41 (s, 1H), 7.24 (s, 1H), 7.22 (s, 1H), 4.61 (s, 2H), 4.57 (s, 2H), 4.23 (t, J = 5.6 Hz, 2H), 2.79 (t, J = 5.7 Hz, 2H), 2.47 (d, J = 5.5 Hz, 4H), 2.31 (s, 4H), 2.16 (s, 3H), 1.61 (h, J = 3.1 Hz, 4H). LC-MS (ESI): m / z: 512.20 [M+H] + .

[0233] Synthesis of compound (5f)

[0234]

[0235] Referring to the synthesis procedure of 5a, 110 mg of yellow oil was obtained with a yield of 22.3%.

[0236] 1 1H NMR (400 MHz, DMSO-d 6)δ8.84(s,1H),8.25(d,J=3.0Hz,1H),8.20(dd,J=9.0,3.0Hz,1H),8.05(s,1H),7.34(d,J=2.3Hz,1H),7.30(s,1H),7.25(s,1H),7.22(s,1H),4.61(s,2H),4.58(s,2H),4.24(t,J=5.6Hz,2H),3.76(s,3H),2.82(t,J=5.6Hz,2H),2.45(s,4H),2.17(s,3H),1.64-1.60(m,4H).LC-MS(ESI):m / z:528.22[M+H] + .

[0237] Synthesis of compound (5g)

[0238]

[0239] Referring to the synthesis procedure of 5a, 120 mg of yellow oil was obtained with a yield of 20.1%.

[0240] 1 H NMR(400MHz,DMSO-d 6 )δ9.14(s,1H),8.25(d,J=2.9Hz,1H),8.20(dd,J=9.1,3.0Hz,1H),8.13(s,1H),8.07(d,J=7.5Hz,2H),7.41(s,1H),7.25(s,1H),4.74(s,2H),4.63(s,2H),4.24(t,J=5.6Hz,2H),2.80(t,J=5.6Hz,2H),2.49(s,4H),2.19(s,3H),1.60(s,4H).LC-MS(ESI):m / z:566.15[M+H] + .

[0241] Synthesis of compound (5h)

[0242]

[0243] Referring to the synthesis procedure of 5a, 100 mg of yellow oil was obtained with a yield of 10.1%.

[0244] 11H NMR (400 MHz, Chloroform-d) δ 8.35 (d, J = 2.8 Hz, 1H), 8.18 (dd, J = 9.0, 3.0 Hz, 1H), 8.13 (d, J = 5.8 Hz, 1H), 7.67 (dd, J = 12.2, 7.1 Hz, 1H), 7.57–7.50 (m, 1H), 7.46 (dd, J = 7.5, 2.8 Hz, 1H), 7.39 (d, J = 7.6 Hz, 1H), 7.33 (s, 1H), 7.23 (d, J = 7.5 Hz, 1H), 6.92 (d, J = 9.1 Hz, 1H), 4.65 (s, 2H), 4.64 (s, 2H), 4.23 (t, J = 5.9 Hz, 2H), 2.95 (t, J = 5.8 Hz, 2H), 2.64 (s, 4H), 1.25 (s, 4H). LC-MS (ESI): m / z: 484.20 [M+H] + .

[0245] Synthesis of compound (5i)

[0246]

[0247] Referring to the synthesis procedure of 5a, 108 mg of yellow oil was obtained with a yield of 21.3%.

[0248] 1 1H NMR (400 MHz, DMSO-d 6 ) δ 10.05 (s, 1H), 8.31 (s, 1H), 8.25 (d, J = 2.9 Hz, 1H), 8.20 (dd, J = 9.0, 3.0 Hz, 1H), 7.74 (s, 1H), 7.66 (s, 1H), 7.37 (d, J = 7.9 Hz, 1H), 7.24 (d, J = 9.1 Hz, 1H), 7.14 (d, J = 7.6 Hz, 1H), 4.66 (s, 2H), 4.58 (s, 2H), 4.23 (t, J = 5.6 Hz, 2H), 2.78 (d, J = 5.7 Hz, 2H), 2.47 (s, 4H), 1.61 (s, 4H). LC-MS (ESI): m / z: 502.15 [M+H] + .

[0249] Synthesis of compound (5j)

[0250]

[0251] Referring to the synthesis procedure of 5a, 120 mg of yellow solid was obtained with a yield of 25.3%.

[0252] 11H NMR (400 MHz, DMSO-d 6 ) δ 10.03 (s, 1H), 8.32 (d, J = 3.5 Hz, 1H), 8.25 (d, J = 2.9 Hz, 1H), 8.20 (dd, J = 9.0, 3.0 Hz, 1H), 7.75 (s, 1H), 7.65 (d, J = 6.9 Hz, 1H), 7.38 (d, J = 7.9 Hz, 1H), 7.25 (d, J = 9.0 Hz, 1H), 7.14 (d, J = 7.7 Hz, 1H), 4.66 (s, 2H), 4.58 (s, 2H), 4.26 (t, J = 5.6 Hz, 2H), 3.49 (t, J = 4.6 Hz, 4H), 2.70 (t, J = 5.6 Hz, 2H), 2.42 (t, J = 4.6 Hz, 4H). LC-MS (ESI): m / z: 518.20 [M+H] + .

[0253] Synthesis of compound (5k)

[0254]

[0255] Referring to the synthesis procedure of 5a, 95 mg of yellow oil was obtained with a yield of 20.1%.

[0256] 1 1H NMR (400 MHz, DMSO-d 6 ) δ 10.04 (s, 1H), 8.32 (d, J = 3.4 Hz, 1H), 8.24 (d, J = 2.9 Hz, 1H), 8.19 (dd, J = 9.0, 2.9 Hz, 1H), 7.74 (s, 1H), 7.65 (d, J = 8.1 Hz, 1H), 7.38 (t, J = 7.9 Hz, 1H), 7.24 (d, J = 9.1 Hz, 1H), 7.14 (d, J = 7.6 Hz, 1H), 4.65 (s, 2H), 4.57 (s, 2H), 4.23 (t, J = 5.7 Hz, 2H), 2.67 (t, J = 5.7 Hz, 2H), 2.39 (s, 4H), 1.42 (m, J = 5.5 Hz, 4H), 1.30 (s, 2H). LC-MS (ESI): m / z: 516.20 [M+H] + .

[0257] Synthesis of compound (5l)

[0258]

[0259] Referring to the synthesis procedure of 5a, 105 mg of yellow oil was obtained with a yield of 22.1%.

[0260] 1 H NMR (400 MHz, Chloroform-d) δ 8.37 (s, 1H), 8.20 (d, J = 12.1 Hz, 1H), 8.06 (s, 1H), 7.68 (d, J = 8.1 Hz, 1H), 7.61 (s, 1H), 7.41 (t, J = 7.9 Hz, 1H), 7.08 (s, 1H), 6.91 (d, J = 9.1 Hz, 1H), 4.69 (s, 2H), 4.62 (s, 2H), 3.93 (s, 3H). LC-MS (ESI): m / z: 419.08 [M+H] + .

[0261] Synthesis of compound (5m)

[0262]

[0263] Referring to the synthesis procedure of 5a, 115 mg of yellow oil was obtained with a yield of 25.1%.

[0264] 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.01 (s, 1H), 8.24 (s, 1H), 8.21 (d, J = 5.9 Hz, 1H), 8.10 (s, 1H), 7.63 (d, J = 11.3 Hz, 1H), 7.55 (s, 1H), 7.25 (d, J = 9.0 Hz, 1H), 6.93 (d, J = 9.2 Hz, 1H), 4.66 (s, 2H), 4.59 (s, 2H), 4.26 (t, J = 5.4 Hz, 2H), 3.49 (t, J = 4.5 Hz, 4H), 2.71 (d, J = 5.6 Hz, 2H), 2.44 (s, 4H), 2.18 (s, 3H). LC-MS (ESI): m / z: 532.17 [M+H] + .

[0265] Synthesis of compound (5n)

[0266]

[0267] Referring to the synthesis procedure of 5a, 110 mg of yellow oil was obtained with a yield of 22.1%.

[0268] 11H NMR (400 MHz, Chloroform-d) δ 8.35 (d, J = 2.4 Hz, 1H), 8.18 (dd, J = 9.0, 2.9 Hz, 1H), 8.05 (s, 1H), 7.97 (s, 1H), 7.85 (s, 1H), 7.37 (s, 1H), 6.94 (s, 1H), 6.92 (s, 1H), 4.70 (s, 2H), 4.65 (s, 2H), 4.26 (t, J = 5.9 Hz, 2H), 2.84 (t, J = 5.9 Hz, 2H), 2.55 (s, 6H), 2.22 (s, 3H), 1.42 (s, 4H). LC-MS (ESI): m / z: 580.20 [M+H] + .

[0269] Synthesis of compound (5o)

[0270]

[0271] Referring to the synthetic procedure of 5a, 125 mg of yellow oil was obtained with a yield of 21.1%.

[0272] 1 1H NMR (400 MHz, DMSO-d 6 ) δ 8.39 (s, 1H), 8.27 (d, J = 2.9 Hz, 1H), 8.22 (dd, J = 9.0, 2.9 Hz, 1H), 8.12 (s, 1H), 8.08 (s, 1H), 7.45 (s, 1H), 7.25 (d, J = 9.1 Hz, 1H), 4.75 (s, 2H), 4.67 (s, 2H), 4.34 (s, 2H), 2.89 (s, 2H), 2.73 (s, 4H), 1.70 (s, 4H). LC-MS (ESI): m / z: 570.20 [M+H] + .

[0273] Synthesis of 2-chloro-5-methyl-N-(3-(((2-(2-morpholinoethoxy)-5-nitrobenzyl)oxy)methyl)phenyl)pyrimidin-4-amine (5p)

[0274]

[0275] Referring to the synthetic procedure of 5a, 80 mg of yellow solid was obtained with a yield of 30%.

[0276] 1 1H NMR (400 MHz, DMSO-d 6)δ8.89(s,1H),8.24(d,J=2.9Hz,1H),8.20(dd,J=9.0,3.0Hz,1H),8.04(d,J=1.0Hz,1H),7.66(t,J=1.9Hz,1H),7.64–7.60(m,1H),7.37(t,J=7.8Hz,1H),7.25(d,J=9.1Hz,1H),7.14–7.10(m,1H),4.65(s,2H),4.58(s,2H),4.26(t,J=5.6Hz,2H),3.51–3.48(m,4H),2.70(t,J=5.6Hz,2H),2.45–2.40(m,4H),2.17(d,J=1.0Hz,3H).LC-MS(ESI):m / z:514.20[M+H] + .

[0277] Synthesis of 2-chloro-5-methyl-N-(3-((5-nitro-2-(2-(piperidin-1-yl)ethoxy)benzyl)oxy)methyl)phenyl)pyrimidin-4-amine (5q)

[0278]

[0279] Referring to the synthetic procedure of 5a, 250 mg of yellow oil was obtained with a yield of 22.9%.

[0280] 1 H NMR(400MHz,DMSO-d6)δ8.88(s,1H),8.24(d,J=3.0Hz,1H),8.19(dd,J=9.0,3.0Hz,1H),8.04(s,1H),7.65(d,J=2.0Hz,1H),7.62(dd,J=8.0,2.2Hz,1H),7.36(t,J=7.8Hz,1H),7.24(d,J=9.0Hz,1H),7.11(d,J=7.5Hz,1H),4.65(s,2H),4.58(s,2H),4.26(t,J=5.8Hz,2H),2.70(t,J=11.4Hz,2H),2.43(s,4H),2.16(s,3H),1.46-1.39(m,4H),1.35-1.28(m,2H).LC-MS(ESI):m / z:512.20[M+H]+.

[0281] Synthesis of 2-chloro-5-methyl-N-(3-(2-(5-nitro-2-(2-(pyrrolidin-1-yl)ethoxy)phenoxy)phenyl)pyrimidin-4-amine (5r)

[0282]

[0283] Referring to the synthesis procedure of 5a, 350 mg of yellow oil was obtained with a yield of 27.9%.

[0284] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.93 (s, 1H), 8.02 (s, 1H), 7.86 (dd, J = 8.9, 2.1 Hz, 1H), 7.74 (d, J = 2.1 Hz, 1H), 7.58 (s, 1H), 7.51 (d, J = 7.4 Hz, 1H), 7.28 (t, J = 7.8 Hz, 1H), 7.16 (d, J = 8.9 Hz, 1H), 7.10 (d, J = 7.4 Hz, 1H), 4.32 (t, J = 6.3 Hz, 2H), 4.15 (t, J = 5.6 Hz, 2H), 3.06 (t, J = 6.2 Hz, 2H), 2.75 (t, J = 5.6 Hz, 2H), 2.48 - 7.44 (m, 4H), 2.16 (s, 3H), 1.64 - 1.60 (m, 4H). LC-MS: m / z: 498.20 [M+H] + .

[0285] Synthesis of 2-chloro-N-(3-((2-methoxy-5-nitrobenzyl)oxy)methyl)phenyl)-5-methylpyrimidin-4-amine (5s)

[0286]

[0287] Referring to the synthesis procedure of 5a, 330 mg of white solid was obtained with a yield of 29.9%.

[0288] 1 H NMR (400 MHz, DMSO-d6) δ 8.90 (s, 1H), 8.23 (d, J = 8.5 Hz, 2H), 8.04 (d, J = 1.1 Hz, 1H), 7.68 - 7.58 (m, 2H), 7.37 (t, J = 7.8 Hz, 1H), 7.23 (d, J = 8.8 Hz, 1H), 7.13 (d, J = 7.5 Hz, 1H), 4.66 (s, 2H), 4.59 (s, 2H), 3.92 (s, 3H), 2.17 (s, 3H). LC-MS (ESI): m / z: 415.10 [M+H]+.

[0289] Synthesis of 2-chloro-N-(3-((5-nitrobenzyl)oxy)methyl)phenyl)-5-methylpyrimidin-4-amine (5t)

[0290]

[0291] Referring to the synthesis procedure of 5a, 298 mg of yellow oil was obtained with a yield of 28.9%.

[0292] 1 1H NMR (400 MHz, DMSO-d6) δ 8.88 (s, 1H), 8.22 (t, J = 2.0 Hz, 1H), 8.19 - 8.14 (m, 1H), 8.05 (d, J = 1.0 Hz, 1H), 7.87 - 7.81 (m, 1H), 7.69 - 7.64 (m, 2H), 7.63 - 7.59 (m, 1H), 7.37 (t, J = 7.8 Hz, 2H), 7.14 - 7.09 (m, 1H), 4.71 (s, 2H), 4.61 (s, 2H), 2.17 (d, J = 1.0 Hz, 4H). LC-MS (ESI): m / z: 385.20 [M+H]+.

[0293] 5 5 -fluoro-3 5 -methyl-1 4 Synthesis of 5-fluoro-3-methyl-1-(2-(pyrrolidin-1-yl)ethoxy)-7-oxo-2,4-diazabicyclo[3.2.4]non-1,5(1,3)-dibenzene (L1)

[0294]

[0295] Weigh 2-chloro-5-methyl-N-(5-fluoro-3-((5-nitro-2-(2-(pyrrolidin-1-yl)ethoxy)benzyloxy)phenyl)pyrimidin-4-amine (200 mg, 0.38 mmol), reduced iron powder (106 mg, 1.90 mmol) and ammonium chloride (205 mg, 3.80 mmol) and place them in a 50 mL two-necked flask. Add 80% ethanol (15 mL) to dissolve, and 2 replace the air three times with N2, and react at 80 °C. After 4 h, monitor the completion of the reaction by TLC (DCM:MeOH = 10:1). Filter the iron powder through diatomaceous earth, wash the filter cake three times with anhydrous ethanol and ethyl acetate, evaporate the solvent under reduced pressure, extract three times with ethyl acetate, combine the organic layers, wash the organic layers three times with water and saturated brine in sequence, dry over anhydrous sodium sulfate, filter by suction, and evaporate the solvent to obtain a yellow oil. The crude product can be directly used for the next step without purification. LC-MS (ESI): m / z: 486.20 [M+H] + .

[0296] Weigh N-(5-fluoro-3-((5-amino-2-(2-(pyrrolidin-1-yl)ethoxy)benzyloxy)phenyl)-2-chloro-5-methylpyrimidin-4-amine (120 mg, 0.24 mmol) and p-toluenesulfonic acid (83 mg, 0.48 mmol) and add them to a 250 mL single-necked flask. Add n-butanol (120 mL) to dissolve, react at 100 °C, and monitor the reaction by TLC after 2 h. After cooling to room temperature, rotary evaporate the n-butanol, adjust the pH to 8 - 9 with saturated sodium bicarbonate solution, extract three times with dichloromethane, combine the organic phases, wash the organic layer three times with water and saturated brine in sequence, dry over anhydrous sodium sulfate, filter by suction, and rotary evaporate the solvent to obtain a brown oil. Purify the crude product by silica gel column chromatography (DCM:MeOH = 15:1) to obtain 40 mg of a light yellow solid with a yield of 37.3%.

[0297] 1 H NMR(600MHz,DMSO-d 6 )δ8.92(s,1H),8.58(s,1H),8.24–8.21(m,2H),7.89(s,1H),7.12(d,J=11.1Hz,1H),6.95(dd,J=8.7,2.7Hz,1H),6.85(d,J=8.7Hz,1H),6.79(d,J=9.3Hz,1H),4.48(s,2H),4.43(s,2H),4.03(t,J=5.8Hz,2H),2.80(t,J=5.9Hz,2H),2.56(s,4H),2.12(s,3H),1.69(s,4H). 13 C NMR(151MHz,DMSO-d 6 )δ163.59,161.00,158.80,157.68(d,J C-F =259.7Hz),151.84,141.47(d,J C-F =10.6Hz),140.59(d,J C-F =9.1Hz),134.48,126.96,123.76,120.59,119.54,112.35,108.44(d,J C-F =22.6Hz),107.06(d,J C-F =25.6Hz),105.72,70.48,67.67,65.47,54.34,54.10,23.20,13.66.HRMS(ESI):(m / z):[M+H] + calcd for C 25 H 29 FN 5 O 2,450.2305; found: 450.2307.

[0298] 5 6 -fluoro-3 5 -methyl-1 4 - (2-(pyrrolidin-1-yl)ethoxy)-7-oxo-2,4-diaza-3(2,4)-pyrimidine-1,5(1,3)-dibenzocyclooctane (L2) synthesis

[0299]

[0300] Referring to the synthesis procedure of L1, 150 mg of light yellow solid was obtained with a yield of 31.2%.

[0301] 1 H NMR (600 MHz, DMSO-d 6 ) δ 8.90 (s, 1H), 8.16 (d, J = 2.7 Hz, 1H), 8.14 (d, J = 8.0 Hz, 1H), 8.08 (s, 1H), 7.88 (s, 1H), 7.19 (dd, J = 10.5, 8.3 Hz, 1H), 7.10 (s, 1H), 6.90 (dd, J = 8.7, 2.8 Hz, 1H), 6.81 (d, J = 8.7 Hz, 1H), 4.49 (s, 2H), 4.45 (s, 2H), 4.00 (t, J = 5.9 Hz, 2H), 2.78 (t, J = 5.8 Hz, 2H), 2.55 (s, 4H), 2.10 (s, 3H), 1.69 (p, J = 3.0 Hz, 4H). 13 C NMR (151 MHz, DMSO-d 6 ) δ 159.09, 157.56 (d, J C-F = 259.7 Hz), 155.10, 153.48, 150.28, 134.04, 134.01, 129.09, 126.92 (d, J C-F = 12.0 Hz), 126.21, 124.85 (d, J C-F = 9.0 Hz), 121.65, 118.84, 114.63 (d, J C-F = 21.1 Hz), 112.14, 104.44, 70.70, 67.62, 64.70, 54.37, 54.08, 23.18, 13.20. HRMS (ESI): (m / z): [M+H] + calcd for C 25 H 29 FN 5 O 2, 450.2305; found: 450.2304.

[0302] 5 5 -Chloro-3 5 -Methyl-1 4 Synthesis of 5-Chloro-3-Methyl-1-(2-(pyrrolidin-1-yl)ethoxy)-7-oxo-2,4-diaza-3(2,4)-pyrimido-1,5(1,3)-dibenzocyclooctane (L3)

[0303]

[0304] Referring to the synthesis procedure of L1, 55 mg of light yellow solid was obtained with a yield of 31.1%.

[0305] 1 H NMR (600 MHz, DMSO-d 6 ) δ 8.93 (s, 1H), 8.49 (s, 1H), 8.33 (s, 1H), 8.21 (d, J = 2.7 Hz, 1H), 7.89 (s, 1H), 7.32 (s, 1H), 7.02 (s, 1H), 6.94 (dd, J = 8.7, 2.7 Hz, 1H), 6.86 (d, J = 8.7 Hz, 1H), 4.49 (s, 2H), 4.44 (s, 2H), 4.03 (t, J = 5.8 Hz, 2H), 2.82 (s, 2H), 2.58 (s, 4H), 2.10 (s, 3H), 1.69 (s, 4H). 13 C NMR (151 MHz, DMSO-d 6 ) δ 158.73, 158.53, 156.83, 150.77, 141.27, 140.63, 133.92, 132.08, 125.93, 122.64, 122.41, 121.55, 120.00, 119.52, 112.31, 104.93, 70.39, 65.49, 54.31, 54.08, 48.60, 23.17, 13.49. HRMS (ESI): (m / z): [M+H] + calcd for C 25 H 29 ClN 5 O 2 , 466.2010; found: 466.2009.

[0306] 5 5 -Bromo-3 5 -Methyl-1 4Synthesis of -(2-(Pyrrolidin-1-yl)ethoxy)-7-oxo-2,4-diaza-3(2,4)-pyrimido-1,5(1,3)-dibenzocyclooctane (L4)

[0307]

[0308] Referring to the synthesis procedure of L1, 50 mg of light yellow solid was obtained with a yield of 32.2%.

[0309] 1 H NMR (600 MHz, DMSO-d 6 ) δ 8.95 (s, 1H), 8.51 (s, 1H), 8.36 (s, 1H), 8.22 (d, J = 2.7 Hz, 1H), 7.90 (s, 1H), 7.47 (s, 1H), 7.16 (s, 1H), 6.96 (dd, J = 8.7, 2.7 Hz, 1H), 6.87 (d, J = 8.7 Hz, 1H), 4.50 (s, 2H), 4.44 (s, 2H), 4.11 (d, J = 5.7 Hz, 2H), 3.01 (s, 2H), 2.78 (s, 4H), 2.10 (s, 3H), 1.77 (s, 4H). 13 C NMR (151 MHz, DMSO-d 6 ) δ 158.68, 158.47, 156.82, 150.54, 141.42, 140.80, 134.07, 129.15, 125.89, 124.42, 122.88, 122.69, 120.45, 119.48, 112.31, 104.97, 70.28, 65.49, 53.95, 53.88, 48.58, 23.04, 13.47. HRMS (ESI): (m / z): [M+H] + calcd for C 25 H 29 BrN 5 O 2 , 510.1505; found: 510.1504.

[0310] 5 5 -Methyl-3 5 -Methyl-1 4 Synthesis of 5-Methyl-3-Methyl-1-(2-(pyrrolidin-1-yl)ethoxy)-7-oxo-2,4-diaza-3(2,4)-pyrimido-1,5(1,3)-dibenzocyclooctane (L5)

[0311]

[0312] Referring to the synthesis procedure of L1, 30 mg of yellow oil was obtained with a yield of 32.3%.

[0313] 1 H NMR(600MHz,DMSO-d 6 )δ8.86(s,1H),8.35(s,1H),8.32(d,J=2.7Hz,1H),8.04(s,1H),7.84(s,1H),7.02(s,1H),6.93(dd,J=8.6,2.7Hz,1H),6.84(d,J=8.7Hz,1H),6.80(s,1H),4.45(s,2H),4.42(s,2H),4.10(s,2H),3.01(s,2H),2.79(s,4H),2.27(s,3H),2.09(s,3H),1.77(s,4H). 13 C NMR(151MHz,DMSO)δ159.07,158.47,156.31,150.28,139.59,138.08,137.15,134.31,126.10,123.33,122.56,122.02,121.61,118.98,112.24,104.68,71.16,65.06,53.94,53.86,40.06,23.05,20.96,13.59.HRMS(ESI):(m / z):[M+H] + calcdfor C 26 H 32 N 5 O 2 ,446.2556;found:446.2557.

[0314] 5 5 -Methoxy-3 5 -Methyl-1 4 -(2-(Pyrrolidin-1-yl)ethoxy)-7-oxo-2,4-diaza-3(2,4)-pyrimidine-1,5(1,3)-dibenzocyclooctane (L6) synthesis

[0315]

[0316] Referring to the synthesis procedure of L1, 45 mg of light yellow solid was obtained with a yield of 35.4%.

[0317] 1 H NMR(600MHz,DMSO-d 6)δ8.88(s,1H),8.35(d,J=4.6Hz,2H),7.90(s,1H),7.86(s,1H),6.94(d,J=8.7Hz,1H),6.85(d,J=8.7Hz,1H),6.82(s,1H),6.57(s,1H),4.48(s,2H),4.43(s,2H),4.08(t,J=5.5Hz,2H),3.74(s,3H),2.97(s,2H),2.74(s,4H),2.10(s,3H),1.75(s,4H). 13 C NMR(151MHz,DMSO-d 6 )δ159.19,159.03,158.48,156.44,150.40,140.74,139.39,134.26,126.11,122.67,119.06,116.99,112.28,108.30,106.27,104.75,71.22,68.26,65.21,55.09,54.03,40.57,23.08,13.58.HRMS(ESI):(m / z):[M+H] + calcdfor C 26 H 32 N 5 O 3 ,462.2505;found:462.2503.

[0318] 5 5 -Trifluoromethyl-3 5 -Methyl-1 4 -(2-(Pyrrolidin-1-yl)ethoxy)-7-oxo-2,4-diaza-3(2,4)-pyrimidine-1,5(1,3)-dibenzocyclooctane (L7) Synthesis

[0319]

[0320] Referring to the synthesis procedure of L1, 65 mg of light yellow solid was obtained with a yield of 32.2%.

[0321] 1 H NMR(600MHz,DMSO-d 6)δ8.96(s,1H),8.69(d,J=42.4Hz,2H),8.18(s,1H),7.92(s,1H),7.62(s,1H),7.29(s,1H),6.96(s,1H),6.87(s,1H),4.53(s,4H),4.07(s,2H),2.89(s,2H),2.66(s,4H),2.12(s,3H),1.78(s,4H). 13 C NMR(151MHz,MeOD-d 4 :DMSO-d 6 =1:1)δ159.08,158.00,153.41(d,J C-F =478.6Hz),139.69,138.46,133.00,130.58,130.37,127.91,124.90,124.78,122.79,120.08,118.77(d,J C-F =3.0Hz),116.98(d,J C-F =3.0Hz),111.55,105.33,69.81,66.44,65.47,54.34,54.24,22.76,12.39.HRMS(ESI):(m / z):[M+H] + calcd for C 26 H 29 F 3 N 5 O 2 ,500.2273;found:500.2275.

[0322] 1 4 -(2-(Pyrrolidin-1-yl)ethoxy)-7-oxo-2,4-diaza-3(2,4)-pyrimidine-1,5(1,3)-dibenzocyclooctane (L8) synthesis

[0323]

[0324] Referring to the synthesis procedure of L1, 20 mg of light yellow solid was obtained with a yield of 22.2%.

[0325] 1 H NMR(600MHz,DMSO-d 6)δ9.55(s,1H),9.05(s,1H),8.65(s,1H),8.36(d,J=2.6Hz,1H),7.97(d,J=5.7Hz,1H),7.21(t,J=7.7Hz,1H),6.99(d,J=8.7Hz,1H),6.96(d,J=7.9Hz,1H),6.90(s,1H),6.89(s,1H),6.18(d,J=5.6Hz,1H),4.56(s,2H),4.46(s,2H),4.12(s,2H),3.01(s,2H),2.78(s,4H),1.76(s,4H). 13 C NMR(151MHz,DMSO-d 6 )δ160.46,159.88,156.48,150.75,139.95,138.98,133.95,128.19,126.50,123.01,122.29,121.16,119.80,118.68,112.26,98.13,71.49,65.80,54.96,53.98,53.92,23.05.HRMS(ESI):(m / z):[M+H] + calcd for C 24 H 28 N 5 O 2 ,418.2243;found:418.2242.

[0326] 3 5 -fluoro-1 4 -(2-(pyrrol-1-yl)ethoxy)-7-oxo-2,4-diaza-3(2,4)-pyrimido-1,5(1,3)-dibenzocyclooctane (L9) synthesis

[0327]

[0328] Referring to the synthesis procedure of L1, 25 mg of light yellow solid was obtained with a yield of 35.2%.

[0329] 1 H NMR(600MHz,DMSO-d 6)δ9.44(s,1H),9.10(s,1H),8.45(s,1H),8.26(d,J=2.8Hz,1H),8.07(s,1H),7.25(s,1H),7.18(d,J=7.8Hz,1H),6.98(d,J=7.3Hz,1H),6.97–6.94(m,1H),6.86(d,J=8.7Hz,1H),4.51(s,2H),4.47(s,2H),4.04(t,J=5.8Hz,2H),2.82(s,2H),2.58(s,4H),1.70(s,4H). 13 C NMR(151MHz,DMSO-d 6 )δ155.94(d,J C-F =3.0Hz),150.84,149.29(d,J C-F =10.5Hz),141.61(d,J C-F =21.1Hz),140.88,139.25,138.82,138.79,133.86,128.18,126.41,123.22,122.46,121.27(d,J C-F =268.7Hz),119.53,112.24,71.30,65.50,54.29,54.07,23.17,23.07.HRMS(ESI):(m / z):[M+H] + calcd forC 24 H 27 FN 5 O 2 ,436.2149;found:436.2151.

[0330] 3 5 -Fluoro-1 4 -(2-(Morpholin-1-yl)ethoxy)-7-oxo-2,4-diaza-3(2,4)-pyrimidine-1,5(1,3)-dibenzocyclooctane (L10) Synthesis

[0331]

[0332] Referring to the synthesis procedure of L1, 40 mg of light yellow solid was obtained, with a yield of 37.2%.

[0333] 1 H NMR(600MHz,DMSO-d 6)δ9.44(s,1H),9.09(s,1H),8.44(s,1H),8.24(d,J=2.7Hz,1H),8.06(s,1H),7.24(t,J=7.7Hz,1H),7.18(d,J=6.7Hz,1H),6.98(d,J=7.4Hz,1H),6.95(dd,J=8.7,2.8Hz,1H),6.87(d,J=8.7Hz,1H),4.51(s,2H),4.47(s,2H),4.03(t,J=5.7Hz,2H),3.57(t,J=4.6Hz,4H),2.66(t,J=5.7Hz,2H),2.47(s,4H). 13 C NMR(151MHz,DMSO-d 6 )δ155.96(d,J C-F =3.0Hz),150.89,149.31(d,J C-F =10.5Hz),141.70,141.57,140.90,139.28,138.81(d,J C-F =4.5Hz),133.90,127.35(d,J C-F =261.2Hz),123.23,122.50,122.23,120.40,119.59,112.47,71.31,66.53,66.28,65.52,57.10,53.69.HRMS(ESI):(m / z):[M+H] + calcd for C 24 H 27 FN 5 O 3 ,452.2098;found:452.2099.

[0334] 3 5 -Fluoro-1 4 -(2-(Piperidin-1-yl)ethoxy)-7-oxo-2,4-diaza-3(2,4)-pyrimido-1,5(1,3)-dibenzocyclooctane (L11) Synthesis

[0335]

[0336] Referring to the synthesis procedure of L1, 60 mg of light yellow solid was obtained with a yield of 33.2%.

[0337] 1 H NMR(600MHz,DMSO-d 6) δ 9.45 (s, 1H), 9.11 (s, 1H), 8.44 (s, 1H), 8.25 (d, J = 2.7 Hz, 1H), 8.07 (d, J = 3.9 Hz, 1H), 7.25 (t, J = 7.7 Hz, 1H), 7.19 (d, J = 5.8 Hz, 1H), 7.00–6.93 (m, 2H), 6.87 (d, J = 8.7 Hz, 1H), 4.51 (s, 2H), 4.47 (s, 2H), 4.09 (s, 2H), 2.80 (s, 2H), 2.61 (s, 4H), 1.56 (s, 4H), 1.40 (s, 2H). 13 13C NMR (151 MHz, DMSO-d 6 ) δ 155.93 (d, J C-F = 3.0 Hz), 150.73, 149.28 (d, J C-F = 10.5 Hz), 141.61 (d, J C-F = 19.6 Hz), 140.90, 139.27, 138.82, 138.72, 133.96, 128.20, 126.39, 123.26, 122.48, 121.34 (d, J C-F = 288.4 Hz), 119.58, 112.36, 71.26, 65.89, 65.51, 56.82, 53.99, 24.85, 23.32. HRMS (ESI): (m / z): [M + H] + calcd for C 25 H 29 FN 5 O 2 , 450.2305; found: 450.2306.

[0338] 3 5 -Fluoro-1 4 -Methoxy-7-oxo-2,4-diaza-3(2,4)-pyrimidine-1,5(1,3)-dibenzocyclooctane (L12) Synthesis

[0339]

[0340] Referring to the synthesis procedure of L1, 50 mg of light yellow solid was obtained with a yield of 32.2%.

[0341] 1 1H NMR (600 MHz, DMSO-d 6)δ9.42(s,1H),9.09(s,1H),8.45(s,1H),8.25(d,J=2.7Hz,1H),8.07(d,J=3.9Hz,1H),7.25(t,J=7.7Hz,1H),7.18(d,J=8.0Hz,1H),6.98(d,J=6.0Hz,2H),6.86(d,J=8.7Hz,1H),4.50(s,2H),4.47(s,2H),3.73(s,3H). 13 C NMR(151MHz,DMSO-d 6 )δ155.96(d,J C-F =3.0Hz),151.72,149.28(d,J C-F =10.5Hz),141.62(d,J C-F =19.6Hz),140.08(d,J C-F =246.1Hz),138.81,138.72,133.70,128.21,125.89,123.30,122.49,122.35,120.35,119.55,110.86,71.30,65.52,55.62.HRMS(ESI):(m / z):[M+H] + calcd for C 19 H 18 FN 4 O 2 ,353.1414;found:353.1416.

[0342] 5 5 -Fluoro-3 5 -methyl-1 4 -(2-(morpholin-1-yl)ethoxy)-7-oxo-2,4-diaza-3(2,4)-pyrimidine-1,5(1,3)-dibenzocyclooctane (L13) Synthesis

[0343]

[0344] Referring to the synthesis procedure of L1, 40 mg of light yellow solid was obtained with a yield of 33.2%.

[0345] 1 H NMR(600MHz,DMSO-d 6)δ8.94(s,1H),8.46(s,1H),8.24(s,2H),7.90(s,1H),7.05(d,J=11.0Hz,1H),6.95(dd,J=8.7,2.7Hz,1H),6.87(d,J=8.7Hz,1H),6.80(d,J=9.2Hz,1H),4.49(s,2H),4.44(s,2H),4.04(t,J=5.7Hz,2H),3.57(t,J=4.6Hz,4H),2.67(t,J=5.7Hz,2H),2.47(s,4H),2.11(s,3H). 13 C NMR(151MHz,DMSO-d 6 )δ162.58,160.99,158.72,157.67(d,J C-F =262.7Hz),150.83,141.37(d,J C-F =10.5Hz),140.64(d,J C-F =9.0Hz),133.97,125.94,122.78,119.78,119.51,112.50,108.46(d,J C-F =21.1Hz),106.95(d,J C-F =24.1Hz),104.86,70.45,66.55,66.25,65.47,57.09,53.67,13.50.HRMS(ESI):(m / z):[M+H] + calcd for C 25 H 29 FN 5 O 3 ,466.2254;found:466.2253.

[0346] 5 5 -Trifluoromethyl-3 5 -Methyl-1 4 -(2-(Piperidin-1-yl)ethoxy)-7-oxo-2,4-diaza-3(2,4)-pyrimido-1,5(1,3)-dibenzocyclooctane (L14) Synthesis

[0347]

[0348] Referring to the synthesis procedure of L1, 45 mg of light yellow solid was obtained with a yield of 31.2%.

[0349] 1 H NMR(600MHz,DMSO-d 6)δ8.97(s,1H),8.67(d,J=19.7Hz,2H),8.17(s,1H),7.92(s,1H),7.62(s,1H),7.29(s,1H),6.95(s,1H),6.89(s,1H),4.52(s,4H),4.09(s,2H),3.34(s,6H),2.12(s,3H),1.56(s,4H),1.41(s,2H). 13 C NMR(151MHz,DMSO-d 6 )δ159.03,158.95,157.34,151.20,141.07,140.45,134.31,129.26(d,J C-F =31.7Hz),127.45,124.69(d,J C-F =502.8Hz),125.53,123.72,120.13,118.30,117.31,112.87,105.39,70.75,66.00,57.48,54.57,48.98,25.69,24.01,13.84.HRMS(ESI):(m / z):[M+H] + calcd forC 27 H 31 F 3 N 5 O 2 ,514.2430;found:514.2431.

[0350] 5 5 -Trifluoromethyl-3 5 -fluoro-1 4 -(2-(Pyrrolidin-1-yl)ethoxy)-7-oxo-2,4-diaza-3(2,4)-pyrimido-1,5(1,3)-dibenzocyclooctane (L15) Synthesis

[0351]

[0352] Referring to the synthesis procedure of L1, 50 mg of light yellow solid was obtained with a yield of 32.3%.

[0353] 1 H NMR(600MHz,DMSO-d 6)δ9.68(s,1H),9.18(s,1H),8.84(s,1H),8.15(d,J=2.7Hz,1H),8.14(d,J=3.7Hz,1H),7.59(s,1H),7.31(s,1H),6.97(dd,J=8.7,2.7Hz,1H),6.90(d,J=8.7Hz,1H),4.58(s,2H),4.55(s,2H),4.06(t,J=5.7Hz,2H),2.85(s,2H),2.61(s,4H),1.71(s,4H). 13 C NMR(151MHz,DMSO-d 6 )δ156.47(d,J C-F =3.0Hz),151.55,149.43(d,J C-F =10.5Hz),142.67(d,J C-F =19.6Hz),141.30,141.20,140.20,139.67,134.14,129.49(d,J C-F =31.7Hz),126.77,124.60(d,J C-F =520.9Hz),124.56(d,J C-F =273.3Hz),120.59,118.49(d,J C-F =3.0Hz),116.88(d,J C-F =4.5Hz),112.78,71.06,67.83,66.38,55.64,54.51,24.32.HRMS(ESI):(m / z):[M+H] + calcd for C 25 H 26 F 4 N 5 O 2 ,504.2023;found:504.2025.

[0354] Synthesis of 6-methyl-18-[2-(morpholin-1-yl)ethoxy]-15-oxa-2,4,8,23-tetraazatricyclo[15.3.1.1 3 ,7 .1 9,13 triazatricyclo[15.3.1.1 21 ,3,5,7 23 ,9 22 ,10,12,17,19-nonadecane(B1)

[0355]

[0356] Referring to the synthesis procedure of L1, 15 mg of pale yellow solid was obtained with a yield of 75.0%.

[0357] 1 H NMR (600 MHz, DMSO-d 6 ) δ 8.86 (s, 1H), 8.36 (s, 1H), 8.29 (d, J = 2.5 Hz, 2H), 7.86 (s, 1H), 7.25 (t, J = 7.7 Hz, 1H), 7.16 (dd, J = 8.2, 2.2 Hz, 1H), 6.98 (d, J = 7.5 Hz, 1H), 6.92 (dd, J = 8.7, 2.7 Hz, 1H), 6.85 (d, J = 8.7 Hz, 1H), 4.46 (d, J = 1.8 Hz, 4H), 4.03 (t, J = 5.7 Hz, 2H), 3.58 (t, J = 4.6 Hz, 4H), 2.67 (t, J = 5.7 Hz, 2H), 2.49–2.45 (m, 4H), 2.10 (s, 3H). 13 C NMR (151 MHz, DMSO-d 6 ) δ 159.46, 159.00, 156.88, 151.05, 140.19, 138.72, 134.61, 128.51, 126.66, 125.02, 122.97, 122.86, 121.40, 119.56, 112.95, 105.09, 71.55, 67.03, 66.72, 65.54, 57.57, 54.15, 40.52, 14.02, 13.95. HRMS (ESI): (m / z): [M + H] + calcd for C 25 H 30 N 5 O 3 , 448.2349; found 448.2350. HPLC purity: 96.84%, retention time = 1.46 min.

[0358] 3 5 -Methyl-1 4 -(2-(piperidin-1-yl)ethoxy)-7-oxa-2,4-diazabicyclo[3.2.4]non-1,5(1,3)-dibenzocyclooctane (B2) Synthesis

[0359]

[0360] Referring to the synthesis procedure of L1, 76 mg of light yellow solid was obtained with a yield of 36.3%.

[0361] 1 H NMR (600 MHz, DMSO-d 6 ) δ 8.85 (s, 1H), 8.36 (s, 1H), 8.29 (d, J = 2.0 Hz, 1H), 8.28 (d, J = 2.7 Hz, 1H), 7.86 (s, 1H), 7.25 (t, J = 7.7 Hz, 1H), 7.16 (dd, J = 8.1, 2.2 Hz, 1H), 7.00–6.96 (m, 1H), 6.92 (dd, J = 8.7, 2.7 Hz, 1H), 6.84 (d, J = 8.7 Hz, 1H), 4.46 (d, J = 2.5 Hz, 4H), 4.01 (t, J = 5.8 Hz, 2H), 2.66 (t, J = 5.8 Hz, 2H), 2.49–2.41 (m, 4H), 2.10 (s, 3H), 1.53–1.48 (m, 4H), 1.41–1.36 (m, 2H). 13 C NMR (151 MHz, CDCl 3 ) δ 164.23, 163.76, 155.82, 144.95, 143.49, 139.33, 133.25, 131.41, 129.75, 127.69, 126.15, 124.33, 117.66, 109.84, 76.32, 71.83, 70.30, 62.57, 59.58, 45.27, 30.74, 29.04, 18.77. HRMS (ESI): (m / z): [M + H] + calcd for C 26 H 32 N 5 O 2 , 446.2556; found 446.2555. HPLC purity: 96.46%, retention time = 1.10 min.

[0362] 1 4 -Methoxy-3 5 -Methyl-7-oxa-2,4-diaza-3(2,4)-pyrimido-1,5(1,3)-dibenzocyclooctane (B3) Synthesis

[0363]

[0364] Referring to the synthesis procedure of L1, 36 mg of light yellow solid was obtained with a yield of 30.3%.

[0365] 1 H NMR (600 MHz, DMSO-d 6)δ8.90(s,1H),8.42(s,1H),8.29–8.23(m,2H),7.86(s,1H),7.26(t,J=7.7Hz,1H),7.16(dd,J=7.9,2.2Hz,1H),6.98(d,J=7.4Hz,1H),6.95(dd,J=8.7,2.8Hz,1H),6.83(d,J=8.7Hz,1H),4.45(s,4H),3.72(s,3H),2.10(s,3H). 13 C NMR(151MHz,DMSO-d 6 )δ159.52,158.70,151.99,140.07,138.62,134.23,128.53,126.05,125.17,123.11,123.07,121.45,119.65,111.30,105.19,71.53,65.56,56.08,40.52,14.01,13.98.HRMS(ESI):(m / z):[M+H] + calcd for C 20 H 21 N 4 O 2 ,349.1665;found349.1667.HPLC purity:99.53%,retention time=1.49min.

[0366] 3 5 Synthesis of 3-Methyl-7-oxa-2,4-diaza-3(2,4)-pyrimidine-1,5(1,3)-dibenzocyclooctane (B4)

[0367]

[0368] Referring to the synthesis procedure of L1, 55 mg of light yellow solid was obtained with a yield of 42.9%.

[0369] 1 H NMR(600MHz,DMSO-d 6)δ9.12(s,1H),8.51–8.48(m,1H),8.45(s,1H),8.25(d,J=2.0Hz,1H),7.90(s,1H),7.28(t,J=7.7Hz,1H),7.19(dd,J=7.8,2.2Hz,1H),7.13(t,J=7.7Hz,1H),7.01(d,J=7.5Hz,1H),6.98(dd,J=7.9,2.3Hz,1H),6.79(d,J=7.3Hz,1H),4.48(s,2H),4.38(s,2H),2.13(s,3H). 13 C NMR(151MHz,DMSO-d 6 )δ159.57,158.71,156.58,141.36,140.16,139.05,138.85,128.59,128.38,125.14,123.43,121.86,120.97,120.47,118.48,105.67,71.20,70.66,40.52,14.04.HRMS(ESI):(m / z):[M+H] + calcd forC 19 H 19 N 4 O,319.1559;found 319.1557.HPLC purity:95.75%,retention time=2.71min.

[0370] Experimental Example 2 Molecular-Level Activity Test of JAK2 Inhibitor

[0371] Experimental Principle:

[0372] JAK2 can catalyze the transfer of a phosphate group of adenosine triphosphate (ATP) to a polypeptide substrate, and the polypeptide substrate is labeled with two fluorophores, coumarin and fluorescein. Based on the fluorescence energy resonance transfer (FRET) method, the reaction catalyzed by JAK2 on ATP causes the two fluorophores to approach. The donor (coumarin) is excited at 400 nM, part of the energy is released, and the emission wavelength is 445 nM. Another part of the energy is transferred to fluorescein, and the emission wavelength is 520 nM. Different compounds have different inhibitory degrees on JAK2, resulting in different degrees of substrate phosphorylation. Therefore, by measuring the ratio of the percentage of enzyme-catalyzed substrate phosphorylation, the inhibition rate of different compounds is calculated.

[0373] Experimental Method:

[0374] Add 2.5 μL of the test compound, 5 μL of the kinase / peptide substrate mixture, 2.5 μL of the ATP solution, and 10 μL of the reaction system into a 384-well plate. Vortex for 30 s to mix well, and incubate at room temperature for 1 h. Add 5 μL of the protease, vortex 15 μL of the reaction system for 30 s to mix well, and incubate at room temperature for 1 h. Add 5 μL of the stop reagent, vortex 20 μL of the total volume reaction system for 30 s to mix well, and detect the fluorescence signal using a microplate reader. The excitation wavelength is 400 nm, and the emission wavelengths are 445 nm and 520 nm respectively. Determine the inhibition rate of the compound at 7 concentration gradients, and calculate the IC 50 value of each compound by fitting the curve with Origin8.0. During the experiment, a positive control is used to confirm the feasibility of the reaction system, and three parallels are set for each experiment. Fedratinib is used as the positive control during the experiment, and at least three parallels are set for each experiment.

[0375] Table 1. Test results of the inhibitory activity of compounds against JAK2

[0376] Compound <![CDATA[IC 50 / nM]]> Fedratinib 3.38±0.06 L1 33.38±3.70 L2 78.40±6.42 L3 54.70±6.99 L4 156.0±27.27 L5 49.79±4.120 L6 61.71±9.719 L7 67.80±0.43 L8 841.10±71.80 L9 22.66±1.98 L10 115.60±8.95 L11 499.60±20.85 L12 444.90±58.55 L13 344.10±39.90 L14 278.60±32.25 L15 360.70±25.35

[0377] Experimental Example 3. Testing the anti-cell proliferation activity of JAK2 inhibitors by CCK-8 method

[0378] Detection principle:

[0379] WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt), which is reduced by dehydrogenases in cells to a highly water-soluble yellow formazan product under the action of the electron carrier 1-methoxy-5-methylphenazinium sulfate (1-Methoxy PMS). The number of formazan products generated is proportional to the number of living cells. Therefore, the cell survival rate can be indirectly calculated by measuring the light absorption value at 450 nm. Determine the half-maximal inhibitory concentration (IC

[0380] Experimental method:

[0381] Culture cells using RPMI medium containing 10% FBS. Add 100 μL per well and inoculate 3500 cells into a 96-well plate. Place it in an incubator at 37 °C and 5% CO 2 for 24 h. When the cell confluence reaches 50%-70%, add drugs at different concentration gradients respectively, and continue to incubate for 72 h. Then add 10 μL of CCK-8 to each well, vortex to mix well, incubate at 37 °C for 2 h. Finally, measure the light absorption value of each well at a wavelength of 450 nm. Finally, calculate the IC 50 value using origin software.

[0382] Table 2. Evaluation of the anti-proliferative activities of compounds against HEL and SET-2 cells

[0383]

[0384] Experimental Example 4. In vivo pharmacodynamic evaluation of JAK2 inhibitor L3

[0385] 1. Establishment of a rhEPO-induced polycythemia model

[0386] Detection principle:

[0387] According to literature reports, the rhEPO-induced polycythemia mouse model is highly relevant to human PV disease. By administering rhEPO to mice, the expansion of erythroid progenitor cells in the body can be achieved, resulting in polycythemia, thus forming a secondary polycythemia model, and then evaluating whether JAK2 inhibitor L3 has a therapeutic and preventive effect on polycythemia.

[0388] Experimental method:

[0389] Balb / c female mice aged 8 - 10 weeks and weighing 18 - 20 g were randomly divided into a control group, a model group, and a model + drug administration group. They were subcutaneously injected with rhEPO daily for modeling and orally administered drugs simultaneously for 4 days. During the experiment, Fedratinib and Pacritinib were used as positive controls.

[0390] 2. JAK2 inhibitor L3 inhibits erythropoiesis in the rhEPO-induced PV mouse model

[0391] The polycythemia induced by rhEPO can show the same pathological changes as PV disease, such as an increase in the number of reticulocytes, an increase in hematocrit, splenomegaly, etc. According to Figure 1, it can be seen that the proportion of reticulocytes and hematocrit in the model group increased significantly compared with the control group, proving that the rhEPO-induced PV model was successfully constructed. Figure 1A and Figure 1B After administration of L3, the proportion of reticulocytes and hematocrit both showed a significant decrease and had a certain dose-dependence. At a dose of 100 mg / kg, the proportion of reticulocytes and hematocrit reached 10.26% and 48.03% respectively. In terms of inhibiting the proportion of reticulocytes, the effect was equivalent to that of 120 mg / kg Fedratinib and better than that of 120 mg / kg Pacritinib.

[0392] 3. JAK2 inhibitor L3 inhibits the generation of erythroid progenitor cells in the spleen and bone marrow of the rhEPO-induced PV mouse model

[0393] The spleen and bone marrow are the main hematopoietic organs in the human body. The onset of PV can cause a large expansion of red blood cells in the spleen and bone marrow. The spleen and bone marrow cells of mice were extracted and analyzed by flow cytometry. The results are shown in Figure 2. After induction with rhEPO, the percentage of Ter119 / CD71 positive cells in the spleen and bone marrow of the model group increased significantly compared with the control group. After administration of L3, the proportion of Ter119 / CD71 positive cells in the spleen and bone marrow showed a downward trend and had a certain dose-dependence. At a dose of 100 mg / kg, the number of Ter119 / CD71 positive cells in the spleen and bone marrow was less than that in the positive drug group. In summary, L3 can significantly inhibit the expansion of Te119 / CD71 red blood cells in the spleen and bone marrow of the PV mouse model induced by rhEPO.

[0394] 4. JAK2 inhibitor L3 inhibits splenomegaly in the PV mouse model induced by rhEPO

[0395] Since rhEPO induces erythropoiesis, the spleen becomes enlarged due to excessive extramedullary hematopoiesis. As shown in Figure 3, compared with the control group, the size of the spleen in the model group increased significantly. With the increase of the L3 administration dose, the spleen decreased significantly, showing a certain dose-dependence. In summary, L3 can inhibit splenomegaly caused by extramedullary hematopoiesis in the PV model induced by rhEPO.

[0396] 5. JAK2 inhibitor L3 has a certain safety profile in the PV mouse model induced by rhEPO

[0397] The body weights of the mice were recorded starting from two days before the start of dosing. During the experiment, the body weights of the mice in each group remained at about 19 g, without an obvious upward or downward trend.

[0398] Therefore, the safety of compound L3 is comparable to that of the marketed JAK2 inhibitors.

[0399] 6. Pharmacokinetic study of JAK2 inhibitor L3

[0400] The inventors further studied the pharmacokinetic properties of JAK inhibitor L3. Finally, it was found that JAK2 inhibitor L3 also has good pharmacokinetic properties.

[0401] All documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. A compound of formula I or a stereoisomer or optical isomer, pharmaceutically acceptable salt, prodrug or solvate thereof, In the formula, R 1 is selected from hydrogen, halogen, substituted or unsubstituted C1-C 10 Alkyl, hydroxy, substituted or unsubstituted C1-C 10 Alkoxy, nitro, amino, substituted or unsubstituted aminoC1-3 acyl, substituted or unsubstituted aminosulfonyl, substituted or unsubstituted C1-C3 alkylformyl, substituted or unsubstituted hydroxyC1-3 acyl (preferably hydroxyacetyl), substituted or unsubstituted C1-C3 alkoxyformyl; n is selected from 0, 1, 2 or 3; R 2 Selected from the group consisting of hydrogen, halogen, substituted or unsubstituted C1-C 10 Alkyl, substituted or unsubstituted C1-C 10 acyl group; R 3 Selected from the group consisting of hydrogen, halogen, hydroxy, amino, substituted or unsubstituted C1-C 10 Alkyl, substituted or unsubstituted C1-C 10 Alkoxy, substituted (preferably C1-C 10 Alkyl or alkoxy substituted) or unsubstituted 4- to 6-membered nitrogen- or oxygen-containing heterocyclic ring; R 4 Selected from the group consisting of hydrogen, substituted or unsubstituted C1-C 10 Alkyl, substituted or unsubstituted 4-6 membered heterocyclic ring containing 1, 2 or 3 heteroatoms independently selected from N, O or S; X 1 is a C3-C9 alkylene or alkenylene group containing a heteroatom independently selected from N, O or S.

2. The compound according to claim 1 or its stereoisomer or optical isomer, or its pharmaceutically acceptable salt, prodrug or solvate, characterized in that: R 1 is selected from hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted aminosulfonyl; n is selected from 0, 1 or 2; R 2 Selected from the group consisting of hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl; R 3 Selected from the group consisting of: hydrogen; R 4 Selected from the following group: hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted 4-6 membered heterocyclic ring containing 1 or 2 heteroatoms independently selected from N or O; X 1 Select from the following group: R 5 is a substituted or unsubstituted C1-C3 alkyl group; l, m, o, r are each independently 0, 1 or 2; p and q are each independently 0, 1, 2 or 3.

3. The compound according to claim 2, or its stereoisomer or optical isomer, or its pharmaceutically acceptable salt, prodrug or solvate, characterized in that: Where, X 1 Selected from R 4 Selected from hydrogen, hydroxyl, amino, methyl, methoxy and the following structural units:

4. The compound according to claim 3, or its stereoisomer or optical isomer, or its pharmaceutically acceptable salt, prodrug or solvate, characterized in that: R 4 Selected from: X1 is selected from:

5. The compound according to claim 4, or its stereoisomer or optical isomer, or its pharmaceutically acceptable salt, prodrug or solvate, characterized in that: The compound is as shown in Formula II, In the formula, R 1 Selected from hydrogen, halogen, halogenated or unsubstituted C1-C6 alkyl; R 2 Selected from the group consisting of hydrogen, substituted or unsubstituted C1-C6 alkyl; R 3 For: Hydrogen; R 4 for: X 1 for:

6. The compound according to claim 1 or its stereoisomer or optical isomer, or its pharmaceutically acceptable salt, prodrug or solvate, characterized in that: The compound is selected from the group consisting of: Preferably, the compound is:

7. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the compound according to any one of claims 1 to 6 or its stereoisomer or optical isomer, or a pharmaceutically acceptable salt, prodrug or solvate thereof, and a pharmaceutically acceptable carrier or excipient.

8. Use of the compound or its stereoisomer or optical isomer, or its pharmaceutically acceptable salt, prodrug or solvate according to any one of claims 1 to 6, characterized in that: Used to prepare JAK2 inhibitors.

9. The use according to claim 8, characterized in that The JAK2 inhibitor is a drug for preventing or treating JAK2-mediated diseases.

10. The use according to claim 9, characterized in that The JAK2-mediated disease is myelodysplastic syndrome (MDS), polycythemia vera (AV), eosinophilia, tumor, inflammatory disease or infection caused by bacteria, virus or fungus; Preferably, the tumor is selected from the group consisting of myeloproliferative carcinoma (MPN), melanoma, lung cancer, kidney cancer, ovarian cancer, prostate cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, pediatric solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axonoma, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma; and / or The inflammatory disease is selected from the group consisting of rheumatoid arthritis, ankylosing spondylitis, autoimmune hemolytic anemia, arthritis, myasthenia gravis, systemic lupus erythematosus, pernicious anemia, polymyositis; and / or The virus is selected from the group consisting of hepatitis virus (type A, B and C), herpes virus, influenza virus, adenovirus, coronavirus, measles virus, dengue virus, polio virus, rabies virus; and / or The bacteria is selected from the group consisting of Chlamydia, Rickettsia, Mycobacterium, Staphylococcus, Pneumococcus, Cholera, Tetanus; and / or The fungus is selected from the group consisting of Candida, Aspergillus, and Buddella dermatitidis.

11. A JAK2 inhibitor, characterized in that A compound according to any one of claims 1 to 6 or a stereoisomer or optical isomer thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof.

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