1H-pyrazole derivatives as Syk and VEGFR2 dual-target inhibitors and their applications

Abstract

A class of dual inhibitors of spleen tyrosine kinase (Syk) and vascular endothelial growth factor 2 (VEGFR2), specifically discloses a compound represented by formula (I) and a pharmaceutically acceptable salt thereof, and use thereof in the preparation of drugs related to dual inhibitors of spleen tyrosine kinase (Syk) and vascular endothelial growth factor 2 (VEGFR2).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a divisional application of the Chinese patent application with application number 202180005288X, and the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to a dual-target inhibitor of spleen tyrosine kinase (Syk) and vascular endothelial growth factor 2 (VEGFR2), and specifically to a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and use thereof in the preparation of drugs related to the dual inhibitor of spleen tyrosine kinase (Syk) and vascular endothelial growth factor 2 (VEGFR2). Background Art

[0004] Protein kinases, the largest family of human kinases, include over 500 proteins. Spleen tyrosine kinase (Syk) is a member of the Syk family of tyrosine kinases and is a regulator of early B-cell development as well as mature B-cell activation, signaling, and survival.

[0005] Syk is a non-receptor tyrosine kinase that plays an important role in immunoreceptor-mediated and integrin-mediated signal transduction in a variety of cell types, including B cells, macrophages, monocytes, mast cells, eosinophils, basophils, neutrophils, dendritic cells, T cells, natural killer cells (natural killer cells), platelets and osteoclasts. The immunoreceptors described in this application include typical immunoreceptors and immunoreceptor-like molecules. Typical immunoreceptors include B-cell and T-cell antigen receptors and a variety of immunoglobulin receptors (Fc receptors). Immunoreceptor-like molecules are structurally related to immunoreceptors or participate in similar signal transduction pathways, and they are mainly involved in non-adaptive immune functions (including neutrophil activation, natural killer cell recognition and osteoclast activity). Integrins are cell surface receptors that play a key role in the activation of both leukocyte adhesion and innate and acquired immunity.

[0006] Ligand binding results in the activation of both immunoreceptors and integrins, which results in the activation of Src family kinases and phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic surface of receptor-associated transmembrane aptamers. Syk binds to the phosphorylated ITAM motifs of the aptamers, resulting in the activation of Syk and subsequent phosphorylation and activation of downstream signal transduction pathways.

[0007] Syk is crucial for B-cell activation by B-cell receptor (BCR) signal transduction. Once Syk is bound to phosphorylated BCR, it is activated, thus causing early signal transduction events after BCR activation. B-cell signal transduction by BCR can lead to a wide range of biological outputs, which in turn depends on the developmental stage of B-cells. The intensity and duration of BCR signals must be accurately regulated. Abnormal BCR-mediated signal transduction can cause B-cell activation disorders and / or the formation of pathogenic autoantibodies, thereby leading to a variety of autoimmune diseases and / or inflammatory diseases. Mice lacking Syk show impaired B-cell maturation, reduced immunoglobulin production, harm to T-cell-independent immune response, and a significant reduction in the sustained calcium signal for BCR stimulation.

[0008] A large amount of evidence supports the role of B-cells and the human immune system in the pathogenesis of autoimmune and / or inflammatory diseases. The development of protein-based treatments (such as Rituxan) for reducing B-cells represents a method for treating a variety of autoimmune and inflammatory diseases. It is known that autoantibodies and their resulting immune complexes play a pathogenic role in autoimmune and / or inflammatory diseases. The pathogenic response to these antibodies depends on signal transduction through Fc receptors, which in turn depends on Syk. Due to the role of Syk in B-cell activation and FcR-dependent signal transduction, inhibitors of Syk can be used as inhibitors of B-cell-mediated pathogenic activity (including autoantibody production). Therefore, the inhibition of the activity of the Syk enzyme in cells is proposed to treat autoimmune diseases through its effect on the production of autoantibodies.

[0009] Syk also plays an important role in FCεRI-mediated mast cell degranulation and eosinophil activation. Therefore, Syk is involved in allergic diseases (including asthma). Syk binds to the phosphorylated γ chain of FCεRI (through its SH2 domain), and it is critical for downstream signal transduction. Mast cells lacking Syk show a lack of degranulation, arachidonic acid and cytokine secretion. This also suggests pharmacological agents that inhibit Syk activity in mast cells. In animal models of asthma, treatment with Syk antisense oligonucleotides inhibits antigen-induced infiltration of eosinophils and neutrophils. Syk-deficient eosinophils also show impaired activation in response to FCεRI stimulation. Therefore, small molecule inhibitors of Syk will be used to treat allergy-induced inflammatory diseases (including asthma).

[0010] Syk is also expressed in mast cells and monocytes and has been shown to be important for the function of these cells. For example, Syk deficiency in mice is associated with impaired IgE-mediated mast cell activity (which is a significant reduction in the release of TNF-α and other inflammatory cytokines). Syk kinase inhibitors have also been shown to inhibit mast cell degranulation in cell tests.

[0011] VEGFR2, also known as KDR or Flk-1, was identified as a receptor for VEGF and VEGFC and is an early marker of endothelial cell progenitors, with expression restricted to endothelial cells in vivo. VEGFR2 has been shown to be a major signal transducer for angiogenesis and the development of pathological conditions such as cancer and diabetic retinopathy. Studies have shown that anti-VEGF can have an inhibitory effect on the expression and activation of proinflammatory factors, thereby reducing ocular surface inflammation. VEGFR2 transduces the main signal for angiogenesis through its powerful tyrosine kinase activity. However, unlike other representative tyrosine kinase receptors, VEGFR2 does not use the Ras pathway as the main downstream signaling, but instead uses the phospholipase C protein kinase C pathway to express mitogen-activated protein (MAP) kinase activation and DNA synthesis. Therefore, inhibiting VEGFR2 activity and its downstream signaling is an important target for the treatment of diseases involving angiogenesis and inflammation.

[0012] In diseases such as tumors, angiogenesis is an important step in the development of the disease. If angiogenesis can be inhibited, (Folkman, 2002, The role of angiogenesis in tumor growth and metastasis, Semin Oncol. 29 (6 Suppl 16): 15-8; Witmer et al., 2003), vascular endothelial growth factor and angiogenesis in eye diseases, Prog Retin Eye Res. 22: 1-29. Vascular endothelial growth factor is a secretory angiogenic mitogen, Science, 246: 1306-1309; vascular endothelial growth factor is a key factor in inducing angiogenesis. Ferrara et al., 2003, Biology of VEGF and its receptors, Nature Medicine 9: 9-22). The VEGFR2 protein molecule consists of three parts: the extracellular region, the transmembrane region and the cytoplasmic region. VEGF can bind to the extracellular region of VEGFR2. This binding can induce the phosphorylation of the tyrosine kinase group in the cytoplasmic region of VEGFR2, thereby initiating intracellular signal transduction, causing proliferation, migration and angiogenesis of vascular endothelial cells, and preventing the correction of vascular endothelial cells. In view of the key role of VEGFR2 in promoting the signal transduction of angiogenesis, inhibiting the binding of VEGF to VEGF2 can achieve the effect of preventing angiogenesis and inhibiting inflammation. Abnormal angiogenesis is related to the pathology of many diseases, including cancer, age-related macular degeneration (AMD), proliferative diabetic retinopathy (PDR) and ocular surface inflammation (DED). Thrombospondin-1 (TSP-1) regulates VEGFR-2 signal transduction through CD36 in microvascular endothelial cells. VEGF-A initiates angiogenesis by binding to VEGFR-2 and inducing its autophosphorylation, and promotes Syk phosphorylation. Activation of Syk further phosphorylates the y1175 residue of VEGFR-2 and mediates endothelial cell migration. In the absence of VEGF-A, the TSP-1-CD36 complex promotes Fyn activation, returning the angiogenic switch to the "off" position.

[0013] Therefore, inhibition of Syk and VEGFR-2 activity can be used to treat allergic diseases, autoimmune diseases and inflammatory diseases, including but not limited to dry eye and allergic conjunctivitis, retinal inflammatory diseases, age-related macular degeneration (AMD), proliferative diabetic retinopathy (PDR) and retinopathy of prematurity (ROP), cancer, rheumatoid arthritis, glomerulonephritis, multiple vasculitides, idiopathic thrombocytopenic purpura (ITP), myasthenia gravis, allergic rhinitis, chronic obstructive pulmonary disease (COPD), adult respiratory distress syndrome (ARDs) and asthma, etc. Summary of the invention

[0014] The present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,

[0015]

[0016] in,

[0017] R1 and R2 are each independently selected from H and pyrazolyl, and R1 and R2 are not pyrazolyl or H at the same time;

[0018] R3 and R4 are each independently selected from H, F, Cl, Br, I, OH, NH2, CN, C 1-3 Alkyl and C 1-3 Alkoxy, the C 1-3 Alkyl and C 1-3 The alkoxy group is optionally substituted with 1, 2 or 3 halogens;

[0019] T1 is selected from CH and N;

[0020] D1 is selected from -O-, -C(R5)(R6)-, -N(R7)- and R5 and R6 are each independently selected from H, F, Cl, Br, I, OH and C 1-3 Alkyl, the C 1-3 The alkyl group is optionally substituted with 1, 2 or 3 halogens;

[0021] Alternatively, R5 and R6 together with the carbon atom to which they are commonly attached form an oxetane group;

[0022] R7 is selected from H, and C 1-3 Alkyl, the C 1-3 The alkyl group is optionally substituted with 1, 2 or 3 halogens;

[0023] R8 is selected from H and -C(=O)-C 1-3 alkyl;

[0024] n is selected from 1 and 2.

[0025] In some embodiments of the present invention, the above R1 is selected from H, Other variables are as defined in the present invention.

[0026] In some embodiments of the present invention, the above R2 is selected from H, Other variables are as defined in the present invention.

[0027] In some embodiments of the present invention, the above R3 is selected from H, F, Cl, Br, I, OH, NH2, CN, CH3 and CH3O, and other variables are as defined in the present invention.

[0028] In some embodiments of the present invention, the above R4 is selected from H, F, Cl, Br, I, OH, NH2, CN, CH3 and CH3O, and other variables are as defined in the present invention.

[0029] In some embodiments of the present invention, the above R5 is selected from H, F, Cl, Br, I, OH, NH2 and CH3, and other variables are as defined in the present invention.

[0030] In some embodiments of the present invention, the above R5 is selected from H, F, Cl, Br, I, OH and CH3, and other variables are as defined in the present invention.

[0031] In some embodiments of the present invention, the above R6 is selected from H, F, Cl, Br, I, OH, NH2 and CH3, and other variables are as defined in the present invention.

[0032] In some embodiments of the present invention, the above R6 is selected from H, F, Cl, Br, I, OH and CH3, and other variables are as defined in the present invention.

[0033] In some embodiments of the present invention, the above R5 and R6 together with the carbon atom to which they are commonly connected form Other variables are as defined in the present invention.

[0034] In some embodiments of the present invention, the above R7 is selected from H, CH3 and Other variables are as defined in the present invention.

[0035] In some embodiments of the present invention, the above R8 is selected from H and -C(=O)-CH3, and other variables are as defined in the present invention.

[0036] In some embodiments of the present invention, the above Selected from Wherein, R5, R6, R7 and R8 are as defined in the present invention, and other variables are as defined in the present invention.

[0037] In some embodiments of the present invention, the above Selected from

[0038] Other variables are as defined in the present invention.

[0039] Some other solutions of the present invention are obtained by any combination of the above variables.

[0040] In some embodiments of the present invention, the above compound or a pharmaceutically acceptable salt thereof is selected from:

[0041]

[0042] wherein R3, R4, T1, D1 and n are as defined in the present invention.

[0043] The present invention also provides a compound represented by the following formula or a pharmaceutically acceptable salt thereof:

[0044]

[0045] The present invention also provides the use of the above compound or a pharmaceutically acceptable salt thereof in the preparation of drugs related to spleen tyrosine kinase (Syk) and vascular endothelial growth factor 2 (VEGFR2) dual inhibitors.

[0046] In some embodiments of the present invention, the above-mentioned application is characterized in that the spleen tyrosine kinase (Syk) and vascular endothelial growth factor 2 (VEGFR2) dual-target inhibitor related drugs are used to treat allergic diseases, autoimmune diseases and inflammatory diseases, such as dry eye and allergic conjunctivitis, retinal inflammatory diseases, age-related macular degeneration (AMD), proliferative diabetic retinopathy (PDR) and retinopathy of prematurity (ROP), cancer, rheumatoid arthritis, glomerulonephritis, multiple vasculitides, idiopathic thrombocytopenic purpura (ITP), myasthenia gravis, allergic rhinitis, chronic obstructive pulmonary disease (COPD), adult respiratory distress syndrome (ARDs) and asthma.

[0047] Technical Effects

[0048] As a dual inhibitor of spleen tyrosine kinase (Syk) and vascular endothelial growth factor 2 (VEGFR2), the compound of the present invention exhibits good dual inhibitory activity of Syk and VEGFR2, shows very good target specificity, has no other deviation from the target selectivity problem, has no obvious inhibitory effect on the five major subtypes of CYP450 enzymes (CYP1A2, 2C9, 2C19, 2D6, 3A4), and exhibits statistically significant efficacy in the scopolamine-induced mouse dry eye model. In addition, the compound of the present invention has a high eye-to-blood ratio and is suitable for ocular administration, and has great development prospects.

[0049] Definition and Description

[0050] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered to be uncertain or unclear in the absence of a special definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient.

[0051] The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0052] The term "pharmaceutically acceptable salt" refers to salts of compounds of the invention, prepared from compounds of the invention having specific substituents with relatively nontoxic acids or bases. When the compounds of the invention contain relatively acidic functional groups, base addition salts can be obtained by contacting such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts. When the compounds of the invention contain relatively basic functional groups, acid addition salts can be obtained by contacting such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid, and salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain basic and acidic functional groups, and thus can be converted into any base or acid addition salt.

[0053] Pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid radicals or bases. Generally, the preparation method of such salts is: in water or an organic solvent or a mixture of the two, these compounds in free acid or base form are reacted with a stoichiometric amount of an appropriate base or acid to prepare.

[0054] The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl. All of these isomers and their mixtures are included within the scope of the present invention.

[0055] Unless otherwise indicated, the term "enantiomer" or "optical isomer" refers to stereoisomers that are mirror images of one another.

[0056] Unless otherwise indicated, the term "cis-trans isomers" or "geometric isomers" arises from the inability of a ring to rotate freely about double bonds or single bonds of ring carbon atoms.

[0057] Unless otherwise indicated, the term "diastereomer" refers to stereoisomers that have two or more chiral centers and that are not mirror images of each other.

[0058] Unless otherwise indicated, "(+)" indicates dextrorotatory, "(-)" indicates levorotatory, and "(±)" indicates racemic.

[0059] Unless otherwise specified, the key is a solid wedge. and dotted wedge key To indicate the absolute configuration of a stereocenter, use a straight solid bond. and straight dashed key To indicate the relative configuration of a stereocenter, use a wavy line Indicates a wedge-shaped solid key or dotted wedge key Or use a wavy line Represents a straight solid bond or straight dashed key

[0060] Unless otherwise indicated, the terms "enriched in one isomer", "isomerically enriched", "enriched in one enantiomer" or "enantiomerically enriched" mean that the content of one isomer or enantiomer is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

[0061] Unless otherwise indicated, the term "isomer excess" or "enantiomeric excess" refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the content of the other isomer or enantiomer is 10%, the isomer or enantiomeric excess (ee value) is 80%.

[0062] Optically active (R)- and (S)-isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereoisomers are separated by conventional methods known in the art, and then the pure enantiomer is recovered. In addition, the separation of enantiomers and diastereomers is usually accomplished by using chromatography, which uses a chiral stationary phase and is optionally combined with a chemical derivatization method (for example, a carbamate is generated from an amine). The compounds of the present invention may contain non-natural proportions of atomic isotopes on one or more atoms that constitute the compound. For example, the compound can be labeled with a radioactive isotope, such as tritium ( 3 H), iodine-125( 125 I) or C-14( 14 C). For another example, deuterium can be used to replace hydrogen to form a deuterated drug. The bond between deuterium and carbon is stronger than the bond between ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs have the advantages of reducing toxic side effects, increasing drug stability, enhancing efficacy, and extending the biological half-life of drugs. All isotopic composition changes of the compounds of the present invention, whether radioactive or not, are included in the scope of the present invention.

[0063] The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include a variant of deuterium and hydrogen, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is oxygen (i.e., =O), it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that it may be substituted or not substituted, and unless otherwise specified, the type and number of the substituents may be arbitrary on the basis of chemical achievable.

[0064] When any variable (e.g., R) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 0-2 Rs, the group may be optionally substituted with up to two Rs, and each occurrence of R is an independent choice. In addition, combinations of substituents and / or variants thereof are permitted only if such combinations result in stable compounds.

[0065] When the number of a linking group is 0, such as -(CRR)0-, it means that the linking group is a single bond.

[0066] When the number of a substituent is 0, it means that the substituent does not exist, for example, -A-(R)0 means that the structure is actually -A.

[0067] When a substituent is vacant, it means that the substituent does not exist. For example, when X in AX is vacant, it means that the structure is actually A.

[0068] When one of the variables is selected from a single bond, it means that the two groups it connects are directly connected. For example, when L in ALZ represents a single bond, it means that the structure is actually AZ.

[0069] When a substituent has bonds that cross-link to two or more atoms in a ring, the substituent may be bonded to any atom in the ring, e.g. Indicates that the substituent R can be substituted at any position on the cyclohexyl group or cyclohexadiene. When the listed substituents do not specify which atom is connected to the substituted group, the substituent can be bonded through any atom thereof. For example, a pyridyl substituent can be connected to the substituted group through any carbon atom on the pyridine ring.

[0070] When the listed linking group does not indicate its linking direction, its linking direction is arbitrary, for example, The connecting group L is -MW-, in which case -MW- can connect ring A and ring B in the same direction as the reading order from left to right to form You can also connect ring A and ring B in the opposite direction of the reading order from left to right to form Combinations of linkers, substituents, and / or variations thereof are permissible only if such combinations result in stable compounds.

[0071] Unless otherwise specified, when a group has one or more connectable sites, any one or more sites of the group can be connected to other groups through chemical bonds. When the chemical bond connection mode is non-positional and there are H atoms at the connectable sites, when the chemical bonds are connected, the number of H atoms at the site will decrease with the number of connected chemical bonds to become a group with the corresponding valence. The chemical bond connecting the site to other groups can be a straight solid bond. Straight dotted key or wavy line For example, the straight solid bond in -OCH3 indicates that it is connected to other groups through the oxygen atom in the group; The straight dashed bond in the group indicates that the two ends of the nitrogen atom in the group are connected to other groups; The wavy line in the phenyl group indicates that it is connected to other groups through the carbon atoms at positions 1 and 2 in the phenyl group; It means that any connectable site on the piperidine group can be connected to other groups through one chemical bond, including at least These four connection methods, even if the H atom is drawn on -N-, Still includes For groups connected in this way, when one chemical bond is connected, the H at that site will be reduced by one and become a corresponding monovalent piperidine group.

[0072]

[0043] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

[0073] Unless otherwise specified, the term “C 1-3 "Alkyl" is used to refer to a straight or branched chain saturated hydrocarbon group consisting of 1 to 3 carbon atoms. 1-3 Alkyl groups include C 1-2 and C 2-3 Alkyl, etc.; it may be monovalent (such as methyl), divalent (such as methylene) or polyvalent (such as methine). 1-3 Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.

[0074] Unless otherwise specified, the term “C 1-3 "Alkoxy" refers to those alkyl groups containing 1 to 3 carbon atoms which are attached to the rest of the molecule via an oxygen atom. 1-3 Alkoxy includes C 1-2 , C 2-3 , C3 and C2 alkoxy, etc. 1-3 Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), and the like.

[0075] Unless otherwise specified, C n-n+m or C n -C n+m Any specific case including n to n+m carbons, such as C 1-12 Including C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 , and C 12 , also includes any range from n to n+m, such as C 1-12 Including C 1-3 , C 1-6 , C 1-9 , C 3-6 , C 3-9 , C 3-12 , C 6-9 , C 6-12 , and C9-12 Etc.; similarly, n-membered to n+m-membered means that the number of atoms in the ring is n to n+m, for example, 3-12-membered ring includes 3-membered ring, 4-membered ring, 5-membered ring, 6-membered ring, 7-membered ring, 8-membered ring, 9-membered ring, 10-membered ring, 11-membered ring, and 12-membered ring, and also includes any range from n to n+m, for example, 3-12-membered ring includes 3-6-membered ring, 3-9-membered ring, 5-6-membered ring, 5-7-membered ring, 6-7-membered ring, 6-8-membered ring, and 6-10-membered ring, etc.

[0076] Unless otherwise specified, when a compound contains a double bond structure, such as a carbon-carbon double bond, a carbon-nitrogen double bond, and a nitrogen-nitrogen double bond, and each atom on the double bond is connected to two different substituents (in a double bond containing a nitrogen atom, a lone pair of electrons on the nitrogen atom is regarded as a substituent connected to it), if the atoms on the double bond in the compound and their substituents are separated by , it represents the (Z) isomer, (E) isomer or a mixture of both isomers of the compound.

[0077] The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the embodiments of the present invention.

[0078] The structure of the compound of the present invention can be confirmed by conventional methods known to those skilled in the art. If the present invention relates to the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, single crystal X-ray diffraction (SXRD) is used to collect diffraction intensity data of the cultured single crystal using a Bruker D8 venture diffractometer, the light source is CuKα radiation, and the scanning mode is: / ω scanning, after collecting relevant data, the direct method (Shelxs97) is further used to analyze the crystal structure to confirm the absolute configuration.

[0079] The solvent used in the present invention is commercially available.

[0080] The present invention uses the following abbreviations: Syk represents tyrosine kinase; VEGFR2 represents vascular endothelial growth factor receptor 2; VEGF represents vascular endothelial growth factor; CD36 represents thrombospondin receptor; DTT represents 1,4-dimercaptothreitol; ATP represents adenosine 5'-triphosphate; MgCl2 represents magnesium chloride; MnCl2 represents manganese chloride; EDTA represents ethylenediaminetetraacetic acid; HEPES Buffer stands for 4-hydroxyethylpiperazineethanesulfonic acid buffer; HTRF stands for high-throughput drug screening; DMSO stands for dimethyl sulfoxide; DIEA stands for N,N-diisopropylethylamine; CDI stands for N,N-carbonyldiimidazole; Pd(dppf)Cl2 stands for 1,1-bis(diphenylphosphinoferrocene)palladium dichloride; NaOH stands for sodium hydroxide; psi stands for pressure unit; KOH stands for potassium hydroxide; BOC stands for tert-butyloxycarbonyl, which is an amine protecting group; HLM stands for human liver microsomes; NADPH stands for reduced nicotinamide adenine dinucleotide phosphate; SEM stands for 2-(trimethylsilyl)ethoxymethyl; and room temperature stands for 15 to 30 degrees Celsius.

[0081] Compounds are named according to the conventional nomenclature in the art or using The software names were used, and commercially available compounds were named using the supplier's catalog names. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 Tear secretion on the 7th and 12th day after modeling. Note: PRE(D-1) means the day before modeling, D7 means the 7th day, and D12 means the 12th day; p means significant difference.

[0083] Figure 2 Corneal fluorescence staining scores on the 7th and 12th days after modeling. Note: PRE (D-1) means the day before modeling, D7 means the 7th day, and D12 means the 12th day; p means significant difference. DETAILED DESCRIPTION

[0084] The present invention is described in detail below by way of examples, but it is not intended to impose any adverse limitations on the present invention. The present invention has been described in detail herein, and specific embodiments thereof are also disclosed therein. It will be apparent to those skilled in the art that various changes and modifications may be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0085] Example 1: Preparation of Compound 1

[0086]

[0087] Step 1: Synthesis of 1-2 To a solution of 4-bromo-2-fluoro-1-nitrobenzene (5 g, 22.73 mmol) in acetonitrile (100 ml), imidazole (1.55 g, 22.73 mmol) and DIEA (2.94 g, 22.73 mmol) were added. The reaction solution was stirred at 90 degrees Celsius for 16 hours. The reaction solution was concentrated, water (100 ml) was added, and it was extracted twice with ethyl acetate (50 ml). The organic phases were combined, dried, filtered, and concentrated to obtain compound 1-2.

[0088] 1 H NMR (400MHz, DMSO-d6) δ = 8.12 (d, J = 8.5Hz, 1H), 8.04 (d, J = 2.0Hz, 1H), 7.98-7.93 (m, 2H), 7.45 (s, 1H), 7.09 (s, 1H).

[0089] Step 2: Synthesis of 1-3 Add stannous chloride dihydrate (20.2 g, 89.52 mmol) to a solution of compound 1-2 (4.00 g, 14.92 mmol) in ethanol (240 ml). Stir at 90 degrees Celsius for 3 hours. The reaction solution was concentrated under reduced pressure, ethyl acetate (300 ml) was added, and the solution was neutralized with saturated sodium bicarbonate solution to pH = 9 to precipitate a white solid. The precipitated solid was filtered out. Water (100 ml) was added to the filtrate, and the mixture was extracted twice with ethyl acetate (100 ml). The organic phases were combined, washed with saturated brine (200 ml), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 1-3.

[0090] 1 H NMR (400MHz, DMSO-d6) δ = 7.79 (s, 1H), 7.34 (s, 1H), 7.30 (dd, J = 2.4, 8.7Hz, 1H), 7.24 (d, J = 2.3Hz, 1H), 7.11 (s, 1H), 6.83 (d, J = 8.5Hz, 1H), 5.18 (s, 2H).

[0091] Step 3: 1-4 Synthesis

[0092] Under nitrogen protection, CDI (749.18 mg, 4.62 mmol) was added to dichlorobenzene (20 ml) of compound 1-3 (1.1 g, 4.62 mmol). The mixture was stirred at 190 degrees Celsius for 2 hours. The reaction solution was cooled to room temperature and filtered, and the filter cake was dried to obtain compound 1-4.

[0093] 1H NMR (400MHz, DMSO-d6) δ = 11.95 (s, 1H), 8.61 (d, J = 1.0Hz, 1H), 8.45 (d, J = 2.0Hz, 1H), 7.62-7.54 (m, 2H), 7.34-7.25 (m, 1H).

[0094] Step 4: Synthesis of 1-5 Add compound 1-4 (300 mg, 1.14 mmol) and dimethylaniline (621.65 mg, 5.13 mmol) to phosphorus oxychloride (5 ml). Stir at 110 degrees Celsius for 1.5 hours. The reaction solution is concentrated, and the pH is adjusted to 9 with saturated sodium bicarbonate aqueous solution. Solids precipitate, and the precipitated solids are filtered and the filter cake is dried to obtain compound 1-5.

[0095] 1 H NMR (400MHz, DMSO-d6) δ = 9.01 (s, 1H), 8.80 (d, J = 2.0Hz, 1H), 7.95 (d, J = 8.8Hz, 1H), 7.91 (s, 1H), 7.84 (dd, J = 2.0, 8.8Hz, 1H).

[0096] Step 5: Synthesis of 1-6 To a solution of compound 1-5 (270.0 mg, 955.68 μmol) in isopropanol (3 ml), 4-morpholineaniline (170.33 mg, 955.68 μmol) and DIEA (148.21 mg, 1.15 mmol) were added. The reaction mixture was stirred at 100 degrees Celsius for 32 hours. The reaction mixture was filtered and the filter cake was dried to obtain compound 1-6.

[0097] 1 H NMR (400MHz, DMSO-d6) δ=9.65(s,1H),8.74(d,J=1.0Hz,1H),8.48(s,1H),7.99(d,J=9 .0Hz,2H),7.59-7.55(m,2H),7.08-6.91(m,3H),3.78-3.72(m,4H),3.10-3.05(m,4H).

[0098] Step 6: 1-8 synthesis

[0099] Compound 1-6 (100 mg, 235.69 μmol), compound 1-7 (83.19 mg, 282.83 μmol), potassium carbonate (97.72 mg, 707.07 μmol) and Pd(dppf)Cl2 (17.25 mg, 23.57 μmol) were added to 1,4-dioxane (4 ml) and water (1 ml). The reaction solution was stirred at 80 degrees Celsius for 4 hours under nitrogen protection. Water (15 ml) was added to the reaction solution and extracted twice with ethyl acetate (15 ml). The organic phases were combined, washed twice with saturated brine (20 ml), dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 1-8.

[0100] Step 7: 1 Synthesis

[0101] Trifluoroacetic acid (1.54 g, 13.51 mmol) was added to a solution of compound 1-8 (110.0 mg, 215.02 μmol) in dichloromethane (1 ml). Stir at 25 degrees Celsius for 1 hour. The reaction solution was adjusted to pH 8 with aqueous ammonia, water (10 ml) was added, and extracted twice with dichloromethane (10 ml). The combined organic phases were washed twice with saturated brine (10 ml), dried over anhydrous sodium sulfate, filtered, and concentrated. 3 ml of acetone was added to the crude product, heated to 60 degrees Celsius and stirred for 0.5 hours, then cooled to room temperature, filtered, and dried in vacuo to obtain compound 1.

[0102] 1 H NMR(400MHz, DMSO-d6)δ:8.81(s,1H),8.43(s,1H),8.23(s,2H),7.92(d,J=8.8Hz,2H),7.80(s,1H),7.77-7.7 2(m,1H),7.71-7.63(m,1H),7.07(d,J=8.8Hz,2H),3.79-3.76(m,4H),3.16(br.s.,4H);MS(ESI)m / z:412[M+H] + .

[0103] Example 2: Preparation of Compound 2

[0104]

[0105] Step 1: 2-2 Synthesis

[0106] Compound 1-6 (140 mg, 329.96 μmol), compound 2-1 (116.47 mg, 395.95 μmol), potassium carbonate (45.60 mg, 329.96 μmol) and Pd(dppf)Cl2 (24.14 mg, 33 μmol) were added to 1,4-dioxane (4 ml) and water (1 ml). The reaction solution was stirred at 80 degrees Celsius for 4 hours under nitrogen protection. Water (15 ml) was added to the reaction solution and extracted twice with ethyl acetate (15 ml). The organic phases were combined, washed twice with saturated brine (20 ml), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 2-2. MS (ESI) m / z: 512 [M+H] + .

[0107] Step 2: 2 Synthesis

[0108] Trifluoroacetic acid (23.10 g, 202.57 mmol) was added to a solution of compound 2-2 (150.0 mg, 293.22 μmol) in dichloromethane (1 ml). Stir at 25 degrees Celsius for 2 hours. The reaction solution was adjusted to pH 8 with aqueous ammonia, water (10 ml) was added, and extracted twice with dichloromethane (20 ml). The combined organic phases were washed twice with saturated brine (10 ml), dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by high performance liquid chromatography (chromatographic column: 3-Phenomenex LunaC1875×30mm×3μm; mobile phase: [water (0.1% trifluoroacetic acid)-acetonitrile]; B (acetonitrile)%: 45%-75%, 7min) to obtain compound 2 (trifluoroacetate).

[0109] 1 H NMR(400MHz,DMSO-d6)δ:9.76(br.s.,1H),8.81(s,1H),8.54(s,1H),8.06-7.90(m,3H),7.81-7.63( m,3H),7.01(d,J=8.3Hz,2H),6.92(s,1H),3.77(br.s.,4H),3.12(br.s.,2H); MS(ESI)m / z:412[M+H] + .

[0110] Example 3: Preparation of Compound 3

[0111]

[0112] Step 1: Synthesis of 3-2 To a solution of 4-bromo-1-fluoro-2-nitrobenzene (10 g, 45.45 mmol) in acetonitrile (200 ml), imidazole (3.09 g, 45.45 mmol) and DIEA (5.87 g, 45.45 mmol) were added. The reaction solution was stirred at 90 degrees Celsius for 16 hours. The reaction solution was concentrated, water (100 ml) was added, and it was extracted twice with ethyl acetate (50 ml). The organic phases were combined, washed once with saturated brine (50 ml), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 3-2.

[0113] 1 H NMR (400MHz, DMSO-d6) δ = 8.19-8.09 (m, 1H), 7.86 (dd, J = 2.3, 8.5Hz, 1H), 7.67-7.56 (m, 1H), 7.39-7.34 (m, 1H), 7.23 (s, 1H), 7.05 (t, J = 1.3Hz, 1H).

[0114] Step 2: Synthesis of 3-3 Add stannous chloride dihydrate (36.87 g, 163.38 mmol) to a solution of compound 3-2 (7.30 g, 27.23 mmol) in ethanol (350 ml). Stir at 80 degrees Celsius for 3 hours. The reaction solution was concentrated under reduced pressure, ethyl acetate (300 ml) was added, and the solution was neutralized with saturated sodium bicarbonate solution to pH = 9 to precipitate a white solid. The precipitated solid was filtered off. Water (100 ml) was added to the filtrate, and the mixture was extracted twice with ethyl acetate (100 ml). The organic phases were combined, washed with saturated brine (300 ml), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 3-3.

[0115] Step 3: 3-4 Synthesis

[0116] Under nitrogen protection, CDI (2.23 g, 13.78 mmol) was added to dichlorobenzene (40 ml) of compound 3-3 (1.64 g, 6.89 mmol). The mixture was stirred at 190 degrees Celsius for 2 hours. The reaction solution was cooled to room temperature and filtered, and the filter cake was dried to obtain compound 3-4.

[0117] 1 H NMR (400MHz, DMSO-d6) δ = 8.54 (d, J = 1.0Hz, 1H), 8.08 (d, J = 8.5Hz, 1H), 7.60 (d, J=1.3Hz, 1H), 7.52 (d, J=2.0Hz, 1H), 7.47 (dd, J=2.1, 8.7Hz, 1H), 7.06 (s, 1H).

[0118] Step 4: Synthesis of 3-5 Compound 3-4 (800 mg, 3.03 mmol) and dimethylaniline (1.66 g, 13.70 mmol) were added to phosphorus oxychloride (10 ml). Stirred at 110 degrees Celsius for 1.5 hours. The reaction solution was concentrated, and the pH was adjusted to 9 with saturated sodium bicarbonate aqueous solution to precipitate solids. The precipitated solids were filtered, and the filter cake was dried and purified by column chromatography (silica gel, petroleum ether / ethyl acetate = 100 / 1 to 2:1) to obtain compound 3-5.

[0119] 1 H NMR (400MHz, DMSO-d6) δ = 8.98 (s, 1H), 8.41 (d, J = 8.8Hz, 1H), 8.25 (d, J = 1.8Hz, 1H), 7.99 (dd, J = 2.0, 8.8Hz, 1H), 7.92 (s, 1H).

[0120] Step 5: Synthesis of 3-6 To compound 3-5 (230.0 mg, 814.10 μmol) in isopropanol (5 ml) were added 4-morpholineaniline (174.12 mg, 976.92 μmol) and DIEA (126.26 mg, 976.92 μmol). The reaction solution was stirred at 100 degrees Celsius for 16 hours. The reaction solution was filtered and the filter cake was dried to obtain compound 3-6.

[0121] 1 H NMR (400MHz, DMSO-d6) δ=9.72(s,1H),8.68(d,J=1.3Hz,1H),8.13(d,J=8.8Hz,1H),8.00(d,J=9.0Hz,2H),7.80(d,J=2.3 Hz, 1H), 7.71 (d, J = 1.3Hz, 1H), 7.52 (dd, J = 2.1, 8.7Hz, 1H), 6.95 (d, J = 9.0Hz, 2H), 3.79-3.70 (m, 4H), 3.12-3.00 (m, 4H).

[0122] Step 6: 3 Synthesis Compound 3-6 (90 mg, 212.12 μmol), compound 1-7 (74.87 mg, 254.54 μmol), potassium carbonate (87.95 mg, 636.36 μmol) and Pd(dppf)Cl2 (15.52 mg, 21.21 μmol) were added to 1,4-dioxane (4 ml) and water (1 ml). The reaction solution was stirred at 80 degrees Celsius for 4 hours under nitrogen protection. Water (15 ml) was added to the reaction solution and extracted twice with ethyl acetate (15 ml). The organic phases were combined, washed twice with saturated brine (20 ml), dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by HPLC (chromatographic column: 3-Phenomenex Luna C18 75×30 mm×3 μm; mobile phase: [water (0.1% trifluoroacetic acid)-acetonitrile]; B (acetonitrile) %: 45%-75%, 7 min) to obtain compound 3 (trifluoroacetate).

[0123] 1 H NMR(400MHz,DMSO-d6)δ:8.74(s,1H),8.25-8.12(m,3H),8.01-7.87(m,3H),7.77(s,1H),7.70(d,J =8.3Hz,1H),7.08(d,J=8.3Hz,2H),3.79(d,J=4.3Hz,4H),3.17(br.s.,4H); MS(ESI)m / z:412[M+H] + .

[0124] Example 4: Preparation of Compound 4

[0125]

[0126] Compound 3-6 (100 mg, 235.69 μmol), compound 2-1 (83.19 mg, 282.83 μmol), potassium carbonate (97.72 mg, 707.07 μmol) and Pd(dppf)Cl2 (17.25 mg, 23.57 μmol) were added to 1,4-dioxane (4 ml) and water (1 ml). The reaction solution was stirred at 90 degrees Celsius for 4 hours under nitrogen protection. Water (20 ml) was added to the reaction solution and extracted twice with ethyl acetate (20 ml). The organic phases were combined, washed twice with saturated brine (20 ml), dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by HPLC (chromatographic column: 3-Phenomenex Luna C18 75×30 mm×3 μm; mobile phase: [water (0.1% trifluoroacetic acid)-acetonitrile]; B (acetonitrile) %: 45%-75%, 7 min) to obtain compound 4 (trifluoroacetate).

[0127] 1 H NMR(400MHz, DMSO-d6)δ:8.76(s,1H),8.24(d,J=8.5Hz,1H),8.17(s,1H),7.95(d,J=8.0Hz,2H),7.88(dd,J=1.6,8.4Hz,1H) ,7.82-7.73(m,2H),7.10(d,J=8.8Hz,2H),6.85(d,J=2.3Hz,1H),3.79(br.s.,4H),3.22-3.13(m,4H); MS(ESI)m / z:412[M+H] + .

[0128] Example 5: Preparation of Compound 5

[0129]

[0130] Step 1: Synthesis of 5-3 To a solution of p-fluoronitrobenzene (2 g, 14.17 mmol) in DMSO (20 ml), potassium carbonate (5.88 g, 42.52 mmol) and tert-butyl piperazine-1-carboxylate (3.96 g, 21.26 mmol) were added. The reaction solution was stirred at 80 degrees Celsius for 16 hours. The reaction solution was concentrated, water (100 ml) was added, and it was extracted three times with ethyl acetate (50 ml). The organic phases were combined, dried, filtered, and concentrated to obtain compound 5-3.

[0131] 1 H NMR (400MHz, DMSO-d6) δ = 8.06 (d, J = 8.8 Hz, 2H), 7.01 (d, J = 9.6 Hz, 2H), 3.47 (s, 8H), 1.42 (s, 9H).

[0132] Step 2: 5-4 Synthesis

[0133] Compound 5-3 (4.2 g, 13.67 mmol) was added in small portions to a dioxane hydrochloride solution (4M, 59.81 ml) and stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure to obtain compound 5-4 (crude hydrochloride), which was directly used in the next step.

[0134] 1 H NMR (400MHz, DMSO-d6) δ = 9.45 (br, 1H), 8.11 (d, J = 9.6Hz, 2H), 7.10 (d, J = 9.6Hz, 2H), 3.67-3.69 (m, 4H), 3.20-3.22 (m, 4H).

[0135] Step 3: 5-5 synthesis

[0136] Under nitrogen protection, sodium acetate (4.04 g, 49.22 mmol) was added to a solution of compound 5-4 (3.4 g, hydrochloride) in ethanol (40 ml), and oxetane (1.77 g, 14.61 mmol) and zinc chloride (4.47 g, 32.81 mmol) were added after stirring at room temperature for 1 hour, and sodium cyanoborohydride (3.09 g, 49.22 mmol) was added after stirring at room temperature for 2 hours. Stir at 40 degrees Celsius for 16 hours. Water (150 ml) was added to the reaction solution, and extracted three times with ethyl acetate (100 ml). The organic phases were combined, washed twice with saturated brine (80 ml), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 5-5.

[0137] 1 H NMR (400MHz, DMSO-d6) δ = 8.05 (d, J = 9.6Hz, 2H), 7.03 (d, J = 9.2Hz, 2H), 4.45-4.57 (m, 5H), 3.47 (br, 4H), 2.38-2.22 (br, 4H).

[0138] Step 4: 5-6 Synthesis

[0139] Under nitrogen protection, palladium carbon (0.5 g, 2.25 mmol) was added to a solution of compound 5-5 (4.2 g, 15.95 mmol) in methanol (150 ml), and the mixture was replaced with a hydrogen balloon three times. The reaction solution was stirred at room temperature for 16 hours under hydrogen conditions. The reaction solution was filtered, and the filtrate was concentrated and dried to obtain compound 5-6.

[0140] MS (ESI) m / z: 234 [M+H] + .

[0141] Step 5: Synthesis of 5-7 Compound 5-6 (991 mg, 4.25 mmol) and DIEA (686 mg, 5.31 mmol) were added to a solution of compound 1-5 (1.0 g, 3.54 mmol) in isopropanol (10 ml). The reaction solution was stirred at 100 degrees Celsius for 16 hours. After the reaction solution cooled to room temperature, it was filtered and the filter cake was dried to obtain compound 5-7.

[0142] Step 6: Synthesis of 5-9 Compound 5-7 (200 mg, 417 μmol), compound 5-8 (135.3 mg, 417 μmol), potassium carbonate (173 mg, 1.25 mmol) and Pd(dppf)Cl2 (30.53 mg, 41.72 μmol) were added to 1,4-dioxane (4 ml) and water (1 ml). The reaction solution was stirred at 100 degrees Celsius for 16 hours under nitrogen protection. Water (30 ml) was added to the reaction solution and extracted three times with ethyl acetate (15 ml). The organic phases were combined, washed twice with saturated brine (20 ml), dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 5-9.

[0143] MS (ESI) m / z: 597 [M+H] + .

[0144] Step 7:5 Synthesis Add tetrabutylammonium fluoride (153 mg, 586.5 μmol) and ethylenediamine (70.5 mg, 1.17 mmol) to a solution of compound 5-9 (350 mg, 586.5 μmol) in tetrahydrofuran (10 ml). The reaction solution was stirred at 75 degrees Celsius for 16 hours. The reaction solution was adjusted to pH 8 with sodium hydroxide solution, water (10 ml) was added, and extracted twice with dichloromethane (10 ml). The combined organic phases were washed twice with saturated brine (10 ml), dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was separated by silica gel column chromatography (petroleum ether: ethyl acetate = 10% to 100%) to obtain compound 5.

[0145] 1 H NMR (400MHz, DMSO-d6) δ = 9.44 (s, 1H), 8.74 (d, J = 1.0Hz, 1H), 8.39 (d, J = 1.6Hz, 1H), 8.22 (s, 2H), 8.00 (d, J = 9.0Hz, 3H), 7.75-7 .68(m,2H),7.63(d,J=8.5Hz,1H),6.96(d,J=9.0Hz,2H),4.61-4.52(m,2H),4.48(t,J=6.1Hz,3H),3.20-3.03(m,4H),2.42(br t,J=4.7Hz,4H); MS(ESI)m / z:467[M+H] + .

[0146] Example 6: Preparation of Compound 6

[0147]

[0148] Step 1: Synthesis of 6-2 Compound 6-1 (338.55 mg, 1.77 mmol) and DIEA (1.14 g, 8.85 mmol) were added to a solution of compound 1-5 (0.5 g, 1.77 mmol) in dimethyl sulfoxide (7 ml). The reaction solution was stirred at 120 degrees Celsius for 16 hours. After the reaction solution cooled to room temperature, 50 ml of water was added, and solids precipitated. The solids were filtered, and the filter cake was slurried with 10 ml of isopropanol, filtered, and dried to obtain compound 6-2.

[0149] Step 2: Synthesis of 6-3 Compound 6-2 (400 mg, 914.63 mmol), compound 5-8 (1.19 g, 3.66 mmol), potassium carbonate (379.23 mg, 2.74 mmol) and Pd(dppf)Cl2 (66.92 mg, 91.46 μmol) were added to 1,4-dioxane (12 ml) and water (3 ml). The reaction solution was stirred at 100 degrees Celsius for 16 hours under nitrogen protection. Water (100 ml) was added to the reaction solution and extracted three times with ethyl acetate (150 ml). The organic phases were combined, washed twice with saturated brine (100 ml), dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was separated by silica gel column chromatography (dichloromethane / methanol, methanol: 0% to 10%) to obtain compound 6-3.

[0150] MS (ESI) m / z: 555 [M+H] + .

[0151] Step 3: 6 Synthesis

[0152] Compound 6-3 (280 mg, 504.72 μmol) was added to dioxane hydrochloride (4M, 5 ml). The reaction solution was stirred at 25 degrees Celsius for 16 hours. The reaction solution was adjusted to pH 8 with 2M sodium hydroxide aqueous solution, water (10 ml) was added, and it was extracted twice with dichloromethane (10 ml). The combined organic phases were washed twice with saturated brine (10 ml), dried over anhydrous sodium sulfate, filtered and concentrated. 3 ml of acetone was added to the crude product, stirred for 0.5 hours, filtered, and the filter cake was dried to obtain compound 6. 1H NMR (400MHz, DMSO-d6) δ = 10.80 (br s, 1H), 8.99 (s, 1H), 8.56 (s, 1H), 8.29 (s, 2H), 8.03 (s, 1H), 7.94 (br s,2H),7.87-7.81(m,1H),7.79-7.68(m,1H),7.11(br d,J=8.9Hz,2H),5.52(s,1H),3.84(br d,J=11.3Hz,3H),3.51(br d,J=11.0Hz,4H),2.83(br d,J=4.4Hz,4H); MS(ESI)m / z:425[M+H] + .

[0153] Example 7: Preparation of Compound 7

[0154]

[0155] Step 1: Synthesis of 7-2 Compound 7-1 (362.85 mg, 1.77 mmol) and DIEA (1.14 g, 8.85 mmol) were added to a solution of compound 1-5 (0.5 g, 1.77 mmol) in dimethyl sulfoxide (7 ml). The reaction solution was stirred at 120 degrees Celsius for 16 hours. After the reaction solution was cooled to room temperature, 50 ml of water was added, and solids precipitated. The solids were filtered, and the filter cake was slurried with 10 ml of isopropanol, filtered, and dried to obtain compound 7-2.

[0156] Step 2: Synthesis of 7-3 Compound 7-2 (123 mg, 272.51 μmol), compound 5-8 (441.87 mg, 1.36 mmol), potassium carbonate (112.99 mg, 817.53 μmol) and Pd(dppf)Cl2 (19.94 mg, 27.25 μmol) were added to 1,4-dioxane (4 ml) and water (1 ml). The reaction solution was stirred at 100 degrees Celsius for 16 hours under nitrogen protection. Water (50 ml) was added to the reaction solution and extracted three times with ethyl acetate (100 ml). The organic phases were combined, washed twice with saturated brine (50 ml), dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was separated by silica gel column chromatography (dichloromethane: methanol = 0% to 10%) to obtain compound 7-3. MS (ESI) m / z: 569 [M + H] + .

[0157] Step 3: 7 Synthesis

[0158] Compound 7-3 (127 mg, 223.28 μmol) was added to dioxane hydrochloride (4M, 5 ml). The reaction solution was stirred at 25 degrees Celsius for 16 hours. The reaction solution was adjusted to pH 8 with 2M sodium hydroxide aqueous solution, water (10 ml) was added, and it was extracted twice with dichloromethane (10 ml). The combined organic phases were washed twice with saturated brine (10 ml), dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was separated by silica gel column chromatography (dichloromethane / methanol, methanol: 0% to 20%) to obtain compound 7.

[0159] 1 H NMR(400MHz,DMSO-d6)δ=11.20(br s,1H),9.17(d,J=1.1Hz,1H),8.68(s,1H),8.37(s,2H),8.19(s,1H),7.96-7.68(m,2H),7.46(s,1H),7.33(s,1H),7.20(s,1H),6.96(br d,J=8.8Hz,2H),3.96-3.74(m,2H),3.74-3.60(m,1H),3.56-3.34(m,5H),3.31-3.03(m,2H),2.81(d,J=4.8Hz,3H); MS(ESI)m / z:439[M+H] + .

[0160] Example 8: Preparation of Compound 8

[0161]

[0162] Step 1: Synthesis of 8-2 To a solution of compound 5-1 (1 g, 7.09 mmol) in DMSO (20 ml), potassium carbonate (2.94 g, 21.26 mmol) and compound 8-1 (1.06 g, 9.21 mmol) were added. The reaction solution was stirred at 100 degrees Celsius for 16 hours. The reaction solution was concentrated, water (50 ml) was added, and it was extracted three times with ethyl acetate (50 ml). The organic phases were combined, dried, filtered, and concentrated to obtain compound 8-2. MS (ESI) m / z: 237 [M+H] + .

[0163] Step 2: 8-3 Synthesis

[0164] Under nitrogen protection, ammonium chloride (1.19 g, 22.22 mmol) and iron powder (2.48 g, 44.44 mmol) were added to a solution of compound 8-2 (1.5 g, 15.95 mmol) in ethanol (8 ml) and water (2 ml). The reaction solution was stirred at 100 degrees Celsius for 16 hours. The reaction solution was filtered, water (50 ml) was added to the filtrate, and extracted three times with ethyl acetate (50 ml). The organic phases were combined, dried, filtered, concentrated and dried to obtain compound 8-3. MS (ESI) m / z: 207 [M+H] + .

[0165] Step 3: Synthesis of 8-4 Compound 8-3 (401.63 mg, 1.95 mmol) and DIEA (1.14 g, 8.85 mmol) were added to a solution of compound 1-5 (0.5 g, 1.77 mmol) in dimethyl sulfoxide (7 ml). The reaction solution was stirred at 120 degrees Celsius for 16 hours. After the reaction solution cooled to room temperature, 50 ml of water was added, and solids precipitated. The solids were filtered, and the filter cake was slurried with 10 ml of isopropanol, filtered, and dried to obtain compound 8-4.

[0166] Step 4: Synthesis of 8-5 Compound 8-4 (340 mg, 751.64 μmol), compound 5-8 (731.26 mg, 2.25 mmol), potassium carbonate (311.65 mg, 2.25 mmol) and Pd(dppf)Cl2 (55 mg, 75.16 μmol) were added to 1,4-dioxane (4 ml) and water (1 ml). The reaction solution was stirred at 100 degrees Celsius for 16 hours under nitrogen protection. Water (50 ml) was added to the reaction solution and extracted three times with dichloromethane (100 ml). The organic phases were combined, washed twice with saturated brine (50 ml), dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was separated by silica gel column chromatography (dichloromethane / tetrahydrofuran, tetrahydrofuran: 0% to 100%) to obtain compound 8-5. MS (ESI) m / z: 570 [M+H] + .

[0167] Step 5: 8 Synthesis

[0168] Compound 8-5 (350 mg, 614.28 μmol) was added to dioxane hydrochloride (4M, 5 ml). The reaction solution was stirred at 25 degrees Celsius for 16 hours. The reaction solution was adjusted to pH 8 with 2M sodium hydroxide aqueous solution, water (10 ml) was added, and it was extracted twice with dichloromethane (10 ml). The combined organic phases were washed twice with saturated brine (10 ml), dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was separated by silica gel column chromatography (dichloromethane / methanol, methanol: 0% to 20%) to obtain compound 8.

[0169] 1H NMR (400MHz, DMSO-d6)δ=12.71-12.02(m,1H),10.29(br s,1H),8.91(s,1H),8.53(s,1H),8.37(br d,J=7.5Hz,2H),8.29(s,2H),7.93(s,1H),7.84(br d,J=8.4Hz,3H),7.80(s,1H),7.77(s,1H),3.24-2.97(m,1H),2.67(s,1H),1.80(br s,3H),1.49-0.33(m,6H); MS(ESI)m / z:440[M+H] + .

[0170] Example 9: Preparation of Compound 9

[0171]

[0172] Step 1: 9-2 Synthesis

[0173] A solution of compound 9-1 (45 g, 189.62 mmol) in methanol (500 ml) and water (200 ml) was cooled to 0 degrees Celsius, and sodium periodate (44.61 g, 208.58 mmol) was added. The reaction solution was stirred at 20 degrees Celsius for 16 hours. The reaction solution was filtered, and the filter cake was washed three times with ethyl acetate (50 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 9-2.

[0174] Step 2: 9-3 Synthesis

[0175] Under nitrogen protection, trifluoroacetamide (17.85 g), rhodium acetate (523.43 mg), magnesium oxide (12.73 g), diacetoxy iodobenzene (38.15 g) and potassium carbonate (54.56 g) were added to a solution of compound 9-2 (20 g, 78.95 mmol) in dichloromethane (300 ml) . The reaction solution was stirred at 20 degrees Celsius for 16 hours. The reaction solution was filtered and the filter cake was washed three times with dichloromethane (100 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product was separated and purified by silica gel column chromatography (petroleum ether / ethyl acetate, ethyl acetate: 0% to 50%) to obtain compound 9-3.

[0176] Step 3: 9-4 Synthesis

[0177] Compound 9-3 (16.0 g) was added to a hydrobromic acid (45%, w / v) acetic acid solution (60 ml). The reaction solution was stirred at 20 degrees Celsius for 10 hours. The reaction solution was adjusted to pH 8 with a 2M sodium hydroxide aqueous solution, water (100 ml) was added, and the mixture was extracted three times with dichloromethane (150 ml). The combined organic phases were washed twice with saturated brine (100 ml), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 9-4.

[0178] Step 4: 9-5 Synthesis

[0179] To a solution of compound 5-1 (1 g, 7.09 mmol) in DMSO (10 ml), potassium carbonate (2.94 g, 21.26 mmol) and compound 9-4 (1.14 g, 8.50 mmol) were added. The reaction solution was stirred at 100 degrees Celsius for 16 hours. The reaction solution was concentrated, water (50 ml) was added, and extracted three times with ethyl acetate (50 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 9-5. MS (ESI) m / z: 256 [M+H] + .

[0180] Step 5: 9-6 synthesis

[0181] Under nitrogen protection, ammonium chloride (1.05 g, 19.59 mmol) and iron powder (1.09 g, 19.59 mmol) were added to a solution of compound 9-5 (1.0 g, 3.92 mmol) in ethanol (20 ml) and water (5 ml). The reaction solution was stirred at 90 degrees Celsius for 1 hour. The reaction solution was filtered, water (50 ml) was added to the filtrate, and extracted three times with ethyl acetate (50 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 9-6. MS (ESI) m / z: 226 [M+H] + .

[0182] Step 6: Synthesis of 9-7 Compound 9-6 (0.3 g, 1.33 mmol) and DIEA (516.25 mg, 3.99 mmol) were added to a solution of compound 1-5 (376.18 mg, 1.33 mmol) in dimethyl sulfoxide (10 ml). The reaction solution was stirred at 120 degrees Celsius for 16 hours. After the reaction solution cooled to room temperature, 50 ml of water was added, and solids precipitated. The solids were filtered, and the filter cake was slurried with 10 ml of isopropanol, filtered, and dried to obtain compound 9-7.

[0183] Step 7: 9 Synthesis Compound 9-7 (200 mg, 424.29 μmol), compound 9-8 (164.66 mg, 848.58 μmol), potassium carbonate (175.92 mg, 1.27 mmol) and Pd(dppf)Cl2 (31.05 mg, 42.43 μmol) were added to 1,4-dioxane (8 ml) and water (2 ml). The reaction solution was stirred at 100 degrees Celsius for 16 hours under nitrogen protection. Water (50 ml) was added to the reaction solution and extracted three times with dichloromethane (100 ml). The organic phases were combined, washed twice with saturated brine (50 ml), dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by HPLC (chromatographic column: 3-Phenomenex Luna C18 75×30 mm×3 μm; mobile phase: [water (0.1% trifluoroacetic acid)-acetonitrile]; B (acetonitrile) %: 45%-75%, 7 min) to obtain compound 9 (trifluoroacetate).

[0184] 1 H NMR (400MHz, DMSO-d6): δ = 10.75 (br s, 1H), 9.08 (s, 1H), 8.62 (s, 1H), 8.32 (s, 2H), 8.14 (s, 1H), 7.97 (br d,J=7.5Hz,2H),7.83-7.89(m,1H),7.76-7.81(m,1H),7.18(brd,J=9.0Hz,2H),4.28(br d,J=14.6Hz,4H),4.08(br d,J=13.6Hz,2H),3.76(br t,J=10.8Hz,2H),3.59-3.67(m,2H); MS(ESI)m / z:459[M+H] + .

[0185] Example 10: Preparation of Compound 10

[0186]

[0187] Step 1: 10-1 Synthesis

[0188] Triethylamine (817.81 μL) and acetyl chloride (209.65 μL) were added to a solution of compound 9-5 (0.5 g, 1.96 mmol) in dichloromethane (10 ml). The reaction solution was stirred at 25 degrees Celsius for 2 hours. Water (50 ml) was added to the reaction solution, and it was extracted three times with dichloromethane (100 ml). The organic phases were combined, washed twice with saturated brine (50 ml), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 10-1.

[0189] Step 5: 10-2 Synthesis

[0190] Under nitrogen protection, ammonium chloride (449.76 mg) and iron powder (469.55 mg) were added to a solution of compound 10-1 (0.5 g, 1.68 mmol) in ethanol (20 ml) and water (5 ml). The reaction solution was stirred at 90 degrees Celsius for 1 hour. The reaction solution was filtered, water (50 ml) was added to the filtrate, and extracted three times with ethyl acetate (50 ml). The organic phases were combined, dried, filtered, concentrated and dried to obtain compound 10-2. MS (ESI) m / z: 268 [M+H] + .

[0191] Step 6: Synthesis of 10-3 Compound 10-2 (0.2 g) and DIEA (390.91 μL) were added to a solution of compound 1-5 (211.35 mg) in dimethyl sulfoxide (10 mL). The reaction solution was stirred at 120 degrees Celsius for 16 hours. After the reaction solution cooled to room temperature, 50 mL of water was added, and solids precipitated. The solids were filtered, and the filter cake was slurried with 10 mL of isopropanol, filtered, and dried to obtain compound 10-3.

[0192] Step 7: Synthesis of 10 Add compound 10-3 (200 mg), compound 9-8 (151.18 mg), potassium carbonate (161.52 mg) and Pd(dppf)Cl2 (28.50 mg) to 1,4-dioxane (8 ml) and water (2 ml). Under nitrogen protection, the reaction solution was stirred at 100 degrees Celsius for 16 hours. Water (50 ml) was added to the reaction solution and extracted three times with dichloromethane (100 ml). The organic phases were combined, washed twice with saturated brine (50 ml), dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product was separated and purified by silica gel column chromatography (dichloromethane / methanol, methanol: 0% to 10%) to obtain compound 10.

[0193] 1 H NMR (400MHz, DMSO-d6): δ=13.01(br s,1H),9.55(s,1H),8.76(s,1H),8.42(s,1H),8.33(br s,1H),8.09(br d,J=9.0Hz,2H),7.72-7.78(m,2H),7.64(d,J=8.5Hz,1H),7.07(br d,J=9.0Hz,2H),3.87-3.95(m,2H),3.59-3.68(m,3H),3.41(br d,J=11.3Hz,2H),3.31(br s,2H),1.99(s,3H); MS(ESI)m / z:501[M+H] + .

[0194] Example 11: Preparation of Compound 11

[0195]

[0196] Step 1: 11-2 Synthesis

[0197] Under nitrogen protection, compound 1-5 (300 mg, 1.06 mmol) and compound 11-1 (225.85 mg, 1.27 mmol) were mixed and stirred at 120 degrees Celsius for 16 hours. After the reaction solution cooled to room temperature, 10 ml of dichloromethane and 2 ml of methanol were added, stirred for 15 minutes, and filtered. The filter cake was collected and vacuum dried to obtain compound 11-2. MS (ESI) m / z: 423, 425 [M+H] + .

[0198] Step 2: Synthesis of 11 Add compound 11-2 (100 mg), compound 9-8 (236 mg), potassium carbonate (97.75 mg) and Pd(dppf)Cl2 (17.29 mg) to 1,4-dioxane (4 ml) and water (1 ml). Under nitrogen protection, the reaction solution was stirred at 100 degrees Celsius for 16 hours. Water (20 ml) was added to the reaction solution and extracted three times with dichloromethane (30 ml). The organic phases were combined, washed twice with saturated brine (30 ml), dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was separated and purified by silica gel column chromatography (dichloromethane / methanol, methanol: 0% to 10%) to obtain compound 11.

[0199] 1 H NMR (400MHz, DMSO-d6): δ = 10.61 (br s, 1H), 9.05 (s, 1H), 8.60 (s, 1H), 8.32 (s, 2H), 8.12 (s, 1H), 8.00 (br d,J=8.1Hz,2H),7.84-7.91(m,1H),7.76-7.82(m,1H),7.33(brd,J=8.4Hz,2H),5.75(s,1H),3.96(br d,J=10.5Hz,2H),3.45(td,J=10.7,3.6Hz,2H),2.73-2.84(m,1H),1.64-1.78(m,4H); MS(ESI)m / z:411[M+H] + .

[0200] Example 12: Preparation of Compound 12

[0201]

[0202] Step 1: Synthesis of 12-2 To a solution of compound 5-1 (0.5 g) in DMSO (20 ml), potassium carbonate (1.47 g) and compound 12-1 (585.89 mg) were added. The reaction solution was stirred at 100 degrees Celsius for 16 hours. The reaction solution was concentrated, water (50 ml) was added, and extracted three times with ethyl acetate (50 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 12-2. MS (ESI) m / z: 249 [M+H] + .

[0203] Step 2: 12-3 Synthesis

[0204] Under nitrogen protection, ammonium chloride (603.26 mg) and iron powder (1.26 g) were added to a solution of compound 12-2 (0.8 g) in ethanol (8 ml) and water (2 ml). The reaction solution was stirred at 100 degrees Celsius for 16 hours. The reaction solution was filtered, water (50 ml) was added to the filtrate, and extracted three times with ethyl acetate (50 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 12-3. MS (ESI) m / z: 219 [M+H] + .

[0205] Step 3: Synthesis of 12-4 Compound 12-3 (154.53 mg) and DIEA (123.30 μL) were added to a solution of compound 1-5 (200 mg) in isopropanol (2 mL). The reaction solution was stirred at 120 degrees Celsius for 16 hours. After the reaction solution was cooled to room temperature, water (50 mL) was added and extracted three times with ethyl acetate (50 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was separated and purified by silica gel column chromatography (dichloromethane / tetrahydrofuran, tetrahydrofuran: 0% to 20%) to obtain compound 12-4. MS (ESI) m / z: 464, 466 [M+H] + .

[0206] Step 4: Synthesis of 12-5 Compound 12-4 (126.54 mg), compound 5-8 (441.87 mg), potassium carbonate (112.99 mg) and Pd(dppf)Cl2 (19.94 mg) were added to 1,4-dioxane (8 ml) and water (2 ml). The reaction solution was stirred at 100 degrees Celsius for 16 hours under nitrogen protection. Water (50 ml) was added to the reaction solution and extracted three times with dichloromethane (100 ml). The organic phases were combined, washed twice with saturated brine (50 ml), dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was separated by silica gel column chromatography (dichloromethane / tetrahydrofuran, tetrahydrofuran: 0% to 100%) to obtain compound 12-5. MS (ESI) m / z: 582 [M+H] + .

[0207] Step 5: 12 Synthesis

[0208] Compound 12-5 (50 mg) was added to dioxane hydrochloride (4M, 5 ml). The reaction solution was stirred at 25 degrees Celsius for 16 hours. The reaction solution was adjusted to pH 8 with 2M sodium hydroxide aqueous solution, water (10 ml) was added, and it was extracted twice with dichloromethane (10 ml). The combined organic phases were washed twice with saturated brine (10 ml), dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 12.

[0209] 1 H NMR (400MHz, DMSO-d6) δ = 10.14 (br, 1H), 8.84-8.86 (m, 1H), 8.51 (s, 1H), 8.42 (d, J = 8.9Hz, 2H), 8.27 (s, 1H), 7.87-7.62 (m, 3H),7.33-7.10(m,2H),6.67(s,1H),5.75(s,1H),3.98-3.91(m,4H),3.71(s,4H),0.98-0.68(m,4H); MS(ESI)m / z:452[M+H] + .

[0210] Biological test data:

[0211] Experimental Example 1: In vitro test of the inhibitory effect of compounds on Syk kinase (enzyme experiment)

[0212] Purpose

[0213] The interaction between substrate and enzyme was detected by HTRF, and the IC 50 The value was used as an indicator to evaluate the inhibitory effect of the compound on Syk kinase.

[0214] Experimental Materials

[0215] Syk kinase (Invitrogen, PV3857)

[0216] DTT (Sigma #43815)

[0217] ATP (Sigma#A7699)

[0218] MgCl2(Sigma#63020)

[0219] MnCl2(Sigma#M1787)

[0220] EDTA (Invitrogen #15575-020)

[0221] HEPES Buffer(Invitrogen#15630-080)

[0222] KinEASE TM TK(Cisbio#62TK0PEC,20000tests)

[0223] Low volume,384-well,white polystyrene plate(Greiner#784075)

[0224] 384 Well Microplates(Greiner#781946)

[0225] Centrifuge (Eppendorf #5810R)

[0226] Pipette (Eppendorf)

[0227] Pipette (Greiner)

[0228] Pipette (Eppendorf)

[0229] Mutidorp pipette

[0230] POD 810 Plate Assembler fully automated microplate pretreatment system

[0231] Envision Reader

[0232] Experimental steps and methods:

[0233] a) Compound dilution and plate making

[0234] 1) The compound powder is weighed and dissolved in a certain amount of DMSO with an initial concentration of 10 mM.

[0235] 2) The compound concentration was diluted to 0.74 mM, and plated using POD18, 135 nL per well, the starting concentration of the compound was 10 μM, 11 concentration points, and 3-fold decreasing gradient dilution.

[0236] b) Enzyme and substrate reaction stage

[0237] 1) Prepare the experimental buffer solution by diluting the 5× HTRF Buffer in the kit to 1× and adding the specified amount of DTT and MgCl2 solution for later use.

[0238] 2) Prepare SYK enzyme reaction solution with 1×HTRF Buffer so that the final reaction concentration of SYK kinase is 0.0156 ng / μL.

[0239] 3) Prepare TK-Substrate-biotin / ATP mixed solution so that the final substrate concentration is controlled at 0.2 μM and the ATP concentration is controlled at 2 μM.

[0240] 4) Use a Mutidorp pipette to add samples, add 5 μL of SYK enzyme solution and TK-Substrate-biotin / ATP mixture to each well, and incubate at 23 °C for 1 hour.

[0241] c) Detection stage:

[0242] 1) Add 13.33 mL of EDTA solution to the test buffer of the kit, add the specified amount of Eu-labeled antibody and XL-665 to prepare the test solution.

[0243] 2) Use a Mutidorp pipette to add 10 μL of the test solution to each well and incubate at 23°C for 1 hour to terminate the reaction of the enzyme and substrate mixture.

[0244] 3) After centrifugation, read the value on Envision.

[0245] d) Data analysis: Use XL-Fit to analyze the data and calculate the IC of the compound 50 value.

[0246] Experimental Results

[0247] The experimental results are shown in Table 1.

[0248] Experimental Example 2: In vitro test of the inhibitory effect of compounds on VEGFR2 kinase (enzyme experiment)

[0249] Purpose

[0250] The VEGFR2 Kinase Assay Kit is intended for use MAX was used as a detection reagent to measure VEGFR2 kinase activity, with the IC 50 The values ​​are used as indicators for compound screening and analysis.

[0251] Experimental methods and procedures

[0252] ADP-GLO TM The kinase assay is a luminescent kinase assay that measures the formation of adenosine diphosphate by kinase; ADP is converted to ATP by UltraGlo TM Luciferase converts the luminescent signal into a light signal. The luminescent signal is positively correlated with the amount of ADP and the kinase activity. This assay is well suited for determining the activity of compounds, making it ideal for primary screening and kinase selectivity analysis. ADP-Glo TMKinase assays can be used to monitor the activity of nearly any ADP-generating enzyme (i.e., kinase or ATPase):

[0253] 1) Dilute enzyme, substrate, ATP and inhibitor in kinase buffer;

[0254] 2) 384-well plate: 1 μL inhibitor or (5% dimethyl sulfoxide solution), 2 μL KDR enzyme, 2 μL substrate / ATP mixture;

[0255] 3) Incubate at room temperature for 60 minutes;

[0256] 4) Add 5 μL ADP-GLO TM Reagents;

[0257] 5) Incubate at room temperature for 40 minutes;

[0258] 6) Add 10 μL of kinase detection reagent;

[0259] 7) Incubate at room temperature for 30 minutes;

[0260] 8) Record luminescence (integration time 0.5-1 sec). Data are displayed as relative light units (rlu), which directly correlates to the amount of ATP produced. Correlation between the percentage of ATP converted to ADP per kinase amount and the corresponding signal-to-background ratio.

[0261] 9) Data analysis: (a) Titration of KDR enzyme with 50 μM ATP to show the luminescent signal generated by KDR enzyme; (b) Staurosporine dose response with 1.5 ng KDR to determine inhibitor IC 50 value.

[0262] Experimental results: The results are shown in Table 1:

[0263] Table 1 Experimental data of the compounds of the present invention inhibiting Syk enzyme and VEFGR2 enzyme

[0264] Compound No. <![CDATA[Inhibiting Syk enzyme IC 50 (nM)]]> <![CDATA[Inhibiting VEFGR2 enzyme IC 50 (nM)]]> Compound 1 15 23 Compound 2 125 31 Compound 5 17 19 Compound 6 50 44 Compound 7 34 42 Compound 8 30 31 Compound 9 23 22 Compound 10 11 41 Compound 11 34 56 Compound 12 315 88

[0265] The results show that: The results show that the compounds of the present invention have significant inhibitory effects on spleen tyrosine kinase (Syk) and vascular endothelial growth factor 2 (VEGFR2).

[0266] Experimental Example 3: Activity inhibition test of compounds on sixty-seven non-target kinases (enzyme test)

[0267] Purpose

[0268] In order to investigate the specificity of the compound's inhibition of the target, this study tested the activity and selectivity of the test compound 1 on the non-target kinases of the relevant pathways on the Eurofins kinaseProfilerTM platform, using IC 50 The values ​​are used as indicators for compound screening and analysis.

[0269] Experimental methods and procedures

[0270] The detection of compound 1 on each selected kinase used the Eurofins standard Kinase ProfilerTM experimental process, which followed the relevant standard operating procedures. The inhibitory activity IC values ​​of the tested compound 1 on 67 kinases including Abl(h), B-Raf(h), BTK(h) and JAK(h) were analyzed. 50 value.

[0271] Experimental results: The experimental results are shown in Table 2:

[0272] Table 2 IC inhibitory activity of compound 1 on sixty-seven non-target kinases 50

[0273]

[0274]

[0275] The results show that the compounds of the present invention exhibit very good targeting specificity and have no other deviations from the target selectivity issues.

[0276] Experimental Example 4: Evaluation of Compounds' Inhibition of Cytochrome P450 Enzymes in Vitro

[0277] Purpose

[0278] In order to investigate the activity of the compounds on human liver microsomal CYP450 enzymes, this study used LC-MS / MS to determine the inhibitory effect of compound 1 on the five major subtypes of human hepatocyte CYP450 enzymes (CYP1A2, 2C9, 2C19, 2D6, and 3A4). 50 The values ​​are used as indicators for compound screening and analysis.

[0279] Experimental methods and procedures

[0280] 1) Prepare test compound and standard inhibitor working solutions (100×);

[0281] 2) Take the microsomes out of the -80°C freezer, thaw on ice, label them with the date, and return them to the freezer immediately after use;

[0282] 3) Add 20 μl of substrate solution to the corresponding wells;

[0283] 4) Add 20 μl of phosphate buffered saline to the blank wells;

[0284] 5) Add 2 μl of test compound and positive control working solution to the corresponding wells;

[0285] 6) Add 2 μl of solvent to the wells without inhibitor and the blank wells;

[0286] 7) Prepare human liver microsome working solution;

[0287] 8) Add 158 μl of HLM working solution to all wells of the incubation plate;

[0288] 9) Preheat the plate in a 37°C water bath for about 10 minutes;

[0289] 10) preparing NADPH cofactor solution;

[0290] 11) Add 20 μl of NADPH cofactor to all incubation wells;

[0291] 12) Incubate all CYPs in a 37°C water bath for 10 minutes;

[0292] 13) At the time point, the reaction was terminated by adding 400 μL of cold solution (200 ng / mL Tolbutamide in acetonitrile and 200 ng / mL Labetalol in acetonitrile);

[0293] 14) The samples were centrifuged at 4000 rpm for 20 min to precipitate proteins;

[0294] 15) Transfer 200 μl of supernatant into 100 μl of HPLC water and shake for 10 minutes;

[0295] 16) Start LC / MS / MS analysis.

[0296] Experimental Results

[0297] The experimental results are shown in Table 3:

[0298]

[0299] The results showed that the compounds of the present invention had no significant inhibitory effect on the five major subtypes of CYP450 enzymes (CYP1A2, 2C9, 2C19, 2D6, and 3A4).

[0300] Experimental Example 5: Pharmacokinetic evaluation of ocular administration in mice

[0301] Purpose

[0302] In order to investigate the eye-to-blood ratio of the in vivo exposure after ocular administration of the compound, this study used the LC-MS / MS method to determine the exposure of compound 1 in the cornea and plasma after ocular administration in mice, and used the drug concentration at the same time point as an indicator for compound screening and analysis.

[0303] Experimental methods and procedures

[0304] C57BL / 6 mice, weighing 18-20 g, were randomly divided into 4 groups after acclimation for 3-5 days, with three mice in each group, and the drug was administered by eye drops, 5 μL in each eye.

[0305] The test animals (C57BL / 6 mice) were not fasted before administration, and were allowed to drink water freely before, during and after the experiment.

[0306] After eye drops, blood samples were collected from the saphenous vein at 1 hour and 3 hours, and after anticoagulation with commercial EDTA-K2, the samples were transferred to a centrifuge for centrifugation. After blood samples were collected from the saphenous vein, the corneas were collected under isoflurane anesthesia. At each time point, the plasma and corneas of mice in the same group were mixed together for testing.

[0307] Treatment of plasma and cornea:

[0308] Plasma: Transfer to 4℃, 3200g, 10min and centrifuge to separate plasma within 30min, and take 20μL plasma from each mouse. The plasma of mice in the same group at each time point is mixed together for testing.

[0309] Cornea: Remove the cornea, rinse it twice with pre-cooled saline, and gently dry it with a paper towel. Then put it into a 1.5 mL centrifuge tube, use an analytical balance, weigh it by peeling method, and finally weigh it. At each time point, the corneas of mice in the same group were mixed together for testing.

[0310] Corneal homogenization method: 1 g cornea was added with 9 mL of pre-cooled methanol: 15 mM phosphate buffer solution = 1:2, and an ultrasonic cell disruptor was used (amplitude rod No. 2, amplitude 50%, working 5 s, stopping 5 s, ultrasonic 3 min).

[0311] Experimental Results

[0312] The experimental results are shown in Table 4:

[0313] Table 4 Plasma and corneal drug concentrations and eye-to-blood ratios of compound 1 in mice after eye drops

[0314]

[0315] The results show that the compound of the present invention has a high eye-to-blood ratio and is suitable for ocular administration.

[0316] Example 6: Efficacy test of dry eye model induced by subcutaneous injection of scopolamine in mice

[0317] Purpose

[0318] Dry eye, also known as keratoconjunctivitis sicca, is a common ophthalmic disease, mostly caused by ocular surface damage or tear film instability caused by abnormal tear quality or quantity or dynamics. Dry eye is often accompanied by symptoms such as itchy eyes, dryness, and foreign body sensation in the eyes, which have a serious impact on patients' vision and quality of life. This study aims to use a mouse dry eye model induced by subcutaneous injection of scopolamine, administer the vehicle and compound 1 to mice by eye drops, evaluate the improvement of compound 1 on the dry eye model, and explore the effective dose of the test article.

[0319] Experimental methods and procedures

[0320] Twelve animals were selected from 20 female C57BL / 6J mice and divided into two groups according to the tear test results of each animal before the experiment, with 6 animals in each group.

[0321] On the 1st to 12th day of the experiment, dry eye was induced in mice by subcutaneous injection of scopolamine hydrobromide solution, 4 times / day, 0.1 mL / mouse / time, with an interval of 3±0.5h between two injections.

[0322] On the 1st to 12th day of the experiment, the animal was given a vehicle and 0.5% compound 1 (i.e., 5 mg / mL) by eye drops, 4 times / day, 3 μL / eye / time, with an interval of 3±0.5h between two administrations. Note that the modeling agent must be given before the eye drops (except for the examinations on the 7th and 12th days).

[0323] Before modeling, tear tests and corneal fluorescence staining scores were performed on all experimental animals on days 7 and 12. Tear tests on days 7 and 12 were performed 30 minutes after the second administration, and corneal fluorescence staining scores were performed 30 minutes after the third administration.

[0324] On the 13th day of the experiment, blood samples were collected from the first three animals in Group 2 1 hour after administration, and then the animals were euthanized, and their eyeballs were collected and their corneas were collected; blood samples were collected from the last three animals in Group 2 3 hours after administration, and then the animals were euthanized, and their eyeballs were collected and their corneas were collected.

[0325] The day of modeling was designated as the first day of the experiment.

[0326] Drug delivery preparation

[0327] 1) Weigh the required amount of test sample into a suitable container.

[0328] 2) Add the required volume of 25% (w / v) 2-hydroxypropyl-β-cyclodextrin and 0.2% (v / v) castor oil sterile injection aqueous solution to the above container, continue stirring until visually homogeneous, and if necessary, heat and / or sonicate in a 37±2°C water bath. Measure and record the pH value, and if necessary, adjust the pH value to between 7.0 and 7.5 with appropriate concentrations of hydrochloric acid or sodium hydroxide.

[0329] 3) The prepared test sample is filtered into sterile containers using a 0.22 μm GV filter membrane under a laminar flow hood and then packaged for use.

[0330] The prepared drug preparation should be stored in a refrigerator at 2°C to 8°C away from light and used within 2 days. At least 30 minutes before administration, the prepared drug preparation should be taken out of the refrigerator and equilibrated to room temperature.

[0331] Modeling preparations

[0332] 1) Weigh the required amount of modeling agent into a suitable container.

[0333] 2) Add the required volume of physiological saline to the above container and vortex until the modeling agent is visually dissolved.

[0334] 3) Under a laminar flow hood, filter the preparation using a 0.22 μm GV filter membrane into a sterile container.

[0335] The prepared drug preparation should be stored in a refrigerator at 2-8°C away from light and used within 7 days. At least 30 minutes before administration, take the prepared drug preparation out of the refrigerator and equilibrate to room temperature.

[0336] Experimental Results

[0337] Tear secretion test

[0338] 1) Weigh about 1 g of phenol red powder into a suitable container;

[0339] 2) Add 20-30 mL of normal saline and mix continuously until the solution is saturated;

[0340] 3) Filter the saturated solution through filter paper into another clean container;

[0341] 4) Place the cotton thread into the filtered reagent and soak for 5 to 10 minutes;

[0342] 5) Take out the soaked cotton thread, dry it and set it aside. Try to use it up within five days.

[0343] Before the formal modeling of experimental animals, adaptive tear tests were performed once a day for at least 2 days.

[0344] Tear secretion was tested in both eyes of all experimental animals before modeling, at least 30 minutes after the second administration on day 7 and day 12. The left eye of each animal was measured first, and after the tear secretion test of the left eye of all experimental animals was completed, the right eye of each animal was measured again.

[0345] During the test, use tweezers to insert the phenol red cotton thread into the eyelid below the outer canthus of the mouse eye as much as possible at one time, start timing for 30 seconds immediately, remove the cotton thread, and use a vernier caliper to measure the length of the tear trace. If the cotton thread falls off during the test, wait for the animal to be still for more than 10 minutes before taking the second measurement. The tear secretion test results on the 7th and 12th days after modeling are shown in Figure 1 .

[0346] Corneal fluorescein staining

[0347] Corneal fluorescence staining scores were performed on all experimental animals before modeling, at least 30 minutes after the third administration on day 7 and day 12. Each animal was scored first on the left eye and then on the right eye.

[0348] First, approximately 1 μL of 1% sodium fluorescein was dripped onto the cornea of ​​the eye to be tested using a pipette, the upper and lower eyelids of the eye to be tested were closed several times, the cornea was rinsed several times with normal saline, and then corneal fluorescence staining of the eye to be tested was scored using a handheld slit lamp.

[0349] The scoring criteria are as follows: the animal cornea is divided into 5 areas, namely the upper, lower, nasal, temporal and central areas. Each area is scored from 0 to 3 points. Each area is scored, and the score of a single eye is the sum of the scores of the five areas. 0 points, no staining; 1 point, slight staining, punctate staining and the number is less than 5; 2 points, moderate staining, punctate staining but no plaque staining; 3 points, severe staining, obvious fluorescent plaques. The average value of the total score of each group of eyes was used for comparison between groups. The corneal fluorescent staining scoring results on the 7th and 12th days after modeling are shown in the figure. Figure 2 .

[0350] The results showed that the compound of the present invention exhibited statistically significant efficacy in the scopolamine-induced dry eye model in mice.

Claims

1. A method for preparing a compound of formula 1, the method comprising the following steps: Step 1: Add imidazole and DIEA to an acetonitrile solution of the compound of formula 1-1, stir the reaction solution at 90 degrees Celsius for 16 hours, concentrate the reaction solution, add water, extract twice with ethyl acetate, combine the organic phases, dry, filter and concentrate to obtain a compound of formula 1-2; Step 2: Add stannous chloride dihydrate to an ethanol solution of the compound of formula 1-2, stir at 90 degrees Celsius for 3 hours, concentrate the reaction solution under reduced pressure, add ethyl acetate, neutralize with saturated sodium bicarbonate solution to pH = 9 to precipitate a white solid, filter out the precipitated solid, add water to the filtrate, extract twice with ethyl acetate, combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, filter and concentrate to obtain a compound of formula 1-3; Step 3: Under nitrogen protection, CDI is added to the dichlorobenzene solution of the compound of formula 1-3, and the mixture is stirred at 190 degrees Celsius for 2 hours. The reaction solution is cooled to room temperature and then filtered. The filter cake is dried to obtain the compound of formula 1-4; Step 4: Add the compound of formula 1-4 and dimethylaniline to phosphorus oxychloride, stir at 110 degrees Celsius for 1.5 hours, concentrate the reaction solution, adjust the pH to 9 with saturated sodium bicarbonate aqueous solution to precipitate solid, filter the precipitated solid, and dry the filter cake to obtain the compound of formula 1-5; Step 5: Add 4-morpholineaniline and DIEA to the isopropanol solution of the compound of formula 1-5, stir the reaction solution at 100 degrees Celsius for 32 hours, filter the reaction solution, and dry the filter cake to obtain the compound of formula 1-6; Step 6: Add the compound of formula 1-6, the compound of formula 1-7, potassium carbonate and Pd(dppf)Cl2 to 1,4-dioxane and water, then, under nitrogen protection, stir the reaction solution at 80 degrees Celsius for 4 hours, add water to the reaction solution, extract twice with ethyl acetate, combine the organic phases, wash twice with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to obtain the compound of formula 1-8; Step 7: Add trifluoroacetic acid to a dichloromethane solution of the compound of formula 1-8, stir at 25 degrees Celsius for 1 hour, adjust the pH of the reaction solution to 8 with aqueous ammonia, add water, extract twice with dichloromethane, combine the organic phases, wash twice with saturated brine, dry over anhydrous sodium sulfate, filter and concentrate, add acetone to the crude product, heat to 60 degrees Celsius, stir for 0.5 hours, then cool to room temperature, filter and dry in vacuo to obtain a compound of formula 1.

2. A method for preparing a compound of formula 2, the method comprising the following steps: Step 1: Add a compound of formula 1-6, a compound of formula 2-1, potassium carbonate and Pd(dppf)Cl2 to 1,4-dioxane and water, then, under nitrogen protection, stir the reaction solution at 80 degrees Celsius for 4 hours, add water to the reaction solution, extract twice with ethyl acetate, combine the organic phases, wash twice with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to obtain a compound of formula 2-2; Step 2: Add trifluoroacetic acid to a dichloromethane solution of the compound of formula 2-2, stir at 25 degrees Celsius for 2 hours, adjust the pH of the reaction solution to 8 with aqueous ammonia, add water, extract twice with dichloromethane, combine the organic phases, wash twice with saturated brine, dry over anhydrous sodium sulfate, filter and concentrate, and separate and purify the crude product by high performance liquid chromatography to obtain a compound of formula 2.

3. A method for preparing a compound of formula 3, the method comprising the following steps: Step 1: Add imidazole and DIEA to an acetonitrile solution of the compound of formula 3-1, stir the reaction solution at 90 degrees Celsius for 16 hours, concentrate the reaction solution, add water, extract twice with ethyl acetate, combine the organic phases, wash once with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to obtain a compound of formula 3-2; Step 2: Add stannous chloride dihydrate to an ethanol solution of the compound of formula 3-2, stir at 80 degrees Celsius for 3 hours, concentrate the reaction solution under reduced pressure, add ethyl acetate, neutralize with saturated sodium bicarbonate solution to pH = 9 to precipitate a white solid, filter out the precipitated solid, add water to the filtrate, extract twice with ethyl acetate, combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, filter and concentrate to obtain a compound of formula 3-3; Step 3: Under nitrogen protection, CDI is added to the dichlorobenzene solution of the compound of formula 3-3, and the mixture is stirred at 190 degrees Celsius for 2 hours. The reaction solution is cooled to room temperature and filtered, and the filter cake is dried to obtain a compound of formula 3-4; Step 4: Add the compound of formula 3-4 and dimethylaniline to phosphorus oxychloride, stir at 110 degrees Celsius for 1.5 hours, concentrate the reaction solution, adjust the pH to 9 with saturated sodium bicarbonate aqueous solution to precipitate solid, filter the precipitated solid, dry the filter cake, and purify by column chromatography to obtain a compound of formula 3-5; Step 5: Add 4-morpholineaniline and DIEA to an isopropanol solution of the compound of formula 3-5, stir the reaction solution at 100 degrees Celsius for 16 hours, filter the reaction solution, and dry the filter cake to obtain a compound of formula 3-6; Step 6: Add the compound of formula 3-6, the compound of formula 1-7, potassium carbonate and Pd(dppf)Cl2 to 1,4-dioxane and water, then stir the reaction solution at 80 degrees Celsius for 4 hours under nitrogen protection, add water to the reaction solution, extract twice with ethyl acetate, combine the organic phases, wash twice with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to obtain a crude product; the crude product is separated and purified by high performance liquid chromatography to obtain a compound of formula 3.

4. A method for preparing a compound of formula 4, the method comprising: Add the compound of formula 3-6, the compound of formula 2-1, potassium carbonate and Pd(dppf)Cl2 to 1,4-dioxane and water, then stir the reaction solution at 90 degrees Celsius for 4 hours under nitrogen protection, add water to the reaction solution, extract twice with ethyl acetate, combine the organic phases, wash twice with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to obtain a crude product; the crude product is separated and purified by high performance liquid chromatography to obtain a compound of formula 4.

5. A method for preparing a compound of formula 5, the method comprising the following steps: Step 1: Add potassium carbonate and a compound of formula 5-2 to a DMSO solution of a compound of formula 5-1, stir the reaction solution at 80 degrees Celsius for 16 hours, concentrate the reaction solution, add water, extract with ethyl acetate three times, combine the organic phases, dry, filter, and concentrate to obtain a compound of formula 5-3; Step 2: Add a small amount of the compound of formula 5-3 to a dioxane hydrochloride solution in batches, stir at room temperature for 16 hours, and then concentrate the reaction solution under reduced pressure to obtain a compound of formula 5-4; Step 3: Under nitrogen protection, sodium acetate is added to the ethanol solution of the compound of formula 5-4, and after stirring at room temperature for 1 hour, oxetanone and zinc chloride are added, and after stirring at room temperature for 2 hours, sodium cyanoborohydride is added, and stirred at 40 degrees Celsius for 16 hours, and then water is added to the reaction solution, extracted three times with ethyl acetate, and then the organic phases are combined, washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a compound of formula 5-5; Step 4: under nitrogen protection, palladium carbon is added to a methanol solution of the compound of formula 5-5, and the mixture is replaced with a hydrogen balloon three times, and then the reaction solution is stirred at room temperature for 16 hours under hydrogen conditions, and then the reaction solution is filtered, and the filtrate is concentrated and dried to obtain a compound of formula 5-6; Step 5: Add the compound of formula 5-6 and DIEA to the isopropanol solution of the compound of formula 1-5, stir the reaction solution at 100 degrees Celsius for 16 hours, then cool the reaction solution to room temperature, filter, and dry the filter cake to obtain the compound of formula 5-7; Step 6: Add the compound of formula 5-7, the compound of formula 5-8, potassium carbonate and Pd(dppf)Cl2 to 1,4-dioxane and water, then, under nitrogen protection, stir the reaction solution at 100 degrees Celsius for 16 hours, add water to the reaction solution, extract three times with ethyl acetate, combine the organic phases, wash twice with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to obtain a compound of formula 5-9; Step 7: Add tetrabutylammonium fluoride and ethylenediamine to a tetrahydrofuran solution of the compound of formula 5-9, stir the reaction solution at 75 degrees Celsius for 16 hours, then adjust the pH of the reaction solution to 8 with sodium hydroxide solution, add water, extract twice with dichloromethane, then combine the organic phases, wash twice with saturated brine, dry over anhydrous sodium sulfate, filter and concentrate to obtain a crude product; the crude product is separated by silica gel column chromatography to obtain a compound of formula 5.

6. A method for preparing a compound of formula 6, the method comprising the following steps: Step 1: Add the compound of formula 6-1 and DIEA to a dimethyl sulfoxide solution of the compound of formula 1-5, stir the reaction solution at 120 degrees Celsius for 16 hours, then cool the reaction solution to room temperature, add water, precipitate solids, filter, slurry the filter cake with isopropanol, filter and dry to obtain a compound of formula 6-2; Step 2: Add the compound of formula 6-2, the compound of formula 5-8, potassium carbonate and Pd(dppf)Cl2 to 1,4-dioxane and water, then, under nitrogen protection, stir the reaction solution at 100 degrees Celsius for 16 hours, add water to the reaction solution, extract three times with ethyl acetate, combine the organic phases, wash twice with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to obtain a crude product; the crude product is separated by silica gel column chromatography to obtain a compound of formula 6-3; Step 3: Add the compound of formula 6-3 to dioxane hydrochloride, stir the reaction solution at 25 degrees Celsius for 16 hours, then adjust the pH of the reaction solution to 8 with sodium hydroxide aqueous solution, add water, extract twice with dichloromethane, then combine the organic phases, wash twice with saturated brine, dry over anhydrous sodium sulfate, filter and concentrate to obtain a crude product; add acetone to the crude product, stir for 0.5 hours, filter, and dry the filter cake to obtain a compound of formula 6.

7. A method for preparing a compound of formula 7, the method comprising the following steps: Step 1: Add the compound of formula 7-1 and DIEA to a dimethyl sulfoxide solution of the compound of formula 1-5, stir the reaction solution at 120 degrees Celsius for 16 hours, then cool the reaction solution to room temperature, add water, precipitate solids, filter, slurry the filter cake with isopropanol, filter and dry to obtain a compound of formula 7-2; Step 2: Add the compound of formula 7-2, the compound of formula 5-8, potassium carbonate and Pd(dppf)Cl2 to 1,4-dioxane and water, then, under nitrogen protection, stir the reaction solution at 100 degrees Celsius for 16 hours, add water to the reaction solution, extract three times with ethyl acetate, combine the organic phases, wash twice with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to obtain a crude product; the crude product is separated by silica gel column chromatography to obtain a compound of formula 7-3; Step 3: Add the compound of formula 7-3 to dioxane hydrochloride, stir the reaction solution at 25 degrees Celsius for 16 hours, then adjust the pH of the reaction solution to 8 with aqueous sodium hydroxide solution, add water, extract twice with dichloromethane, then combine the organic phases, wash twice with saturated brine, dry over anhydrous sodium sulfate, filter and concentrate to obtain a crude product; the crude product is separated by silica gel column chromatography to obtain a compound of formula 7.

8. A method for preparing a compound of formula 8, the method comprising the following steps: Step 1: Add potassium carbonate and a compound of formula 8-1 to a DMSO solution of a compound of formula 5-1, stir the reaction solution at 100 degrees Celsius for 16 hours, concentrate the reaction solution, add water, extract with ethyl acetate three times, combine the organic phases, dry, filter, and concentrate to obtain a compound of formula 8-2; Step 2: Under nitrogen protection, ammonium chloride and iron powder are added to the ethanol and aqueous solution of the compound of formula 8-2, and the reaction solution is stirred at 100 degrees Celsius for 16 hours, then the reaction solution is filtered, water is added to the filtrate, and extracted three times with ethyl acetate, and then the organic phases are combined, dried, filtered, and concentrated to obtain a compound of formula 8-3; Step 3: Add the compound of formula 8-3 and DIEA to the dimethyl sulfoxide solution of the compound of formula 1-5, stir the reaction solution at 120 degrees Celsius for 16 hours, then cool the reaction solution to room temperature, add water, precipitate solid, filter, slurry the filter cake with isopropanol, filter and dry to obtain the compound of formula 8-4; Step 4: Add the compound of formula 8-4, the compound of formula 5-8, potassium carbonate and Pd(dppf)Cl2 to 1,4-dioxane and water, then, under nitrogen protection, stir the reaction solution at 100 degrees Celsius for 16 hours, add water to the reaction solution, extract three times with dichloromethane, combine the organic phases, wash twice with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to obtain a crude product; the crude product is separated by silica gel column chromatography to obtain a compound of formula 8-5; Step 5: Add the compound of formula 8-5 to dioxane hydrochloride, stir the reaction solution at 25 degrees Celsius for 16 hours, then adjust the pH of the reaction solution to 8 with sodium hydroxide aqueous solution, add water, extract twice with dichloromethane, then combine the organic phases, wash twice with saturated brine, dry over anhydrous sodium sulfate, filter and concentrate to obtain a crude product; the crude product is separated by silica gel column chromatography to obtain a compound of formula 8.

9. A method for preparing a compound of formula 9, the method comprising the following steps: Step 1: Cool an aqueous solution of the compound of formula 9-1 to 0 degrees Celsius, add sodium periodate, and then stir the reaction solution at 20 degrees Celsius for 16 hours, filter the reaction solution, wash the filter cake three times with ethyl acetate, and then combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate to obtain a compound of formula 9-2; Step 2: Under nitrogen protection, trifluoroacetamide, rhodium acetate, magnesium oxide, diacetoxyiodobenzene and potassium carbonate are added to a dichloromethane solution of the compound of formula 9-2, and the reaction solution is stirred at 20 degrees Celsius for 16 hours, and then the reaction solution is filtered, the filter cake is washed three times with dichloromethane, the organic phases are combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product; the crude product is separated and purified by silica gel column chromatography to obtain a compound of formula 9-3; Step 3: Add the compound of formula 9-3 to a hydrobromic acid and acetic acid solution, stir the reaction solution at 20 degrees Celsius for 10 hours, then adjust the pH of the reaction solution to 8 with an aqueous sodium hydroxide solution, add water, extract three times with dichloromethane, then combine the organic phases, wash twice with saturated brine, dry over anhydrous sodium sulfate, filter and concentrate to obtain a compound of formula 9-4; Step 4: Add potassium carbonate and a compound of formula 9-4 to a DMSO solution of the compound of formula 5-1, stir the reaction solution at 100 degrees Celsius for 16 hours, concentrate the reaction solution, add water, extract with ethyl acetate three times, combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate to obtain a compound of formula 9-5; Step 5: Under nitrogen protection, ammonium chloride and iron powder are added to the ethanol and aqueous solution of the compound of formula 9-5, and the reaction solution is stirred at 90 degrees Celsius for 1 hour, then the reaction solution is filtered, water is added to the filtrate, and extracted three times with ethyl acetate, and then the organic phases are combined, dried, filtered, and concentrated to obtain a compound of formula 9-6; Step 6: Add the compound of formula 9-6 and DIEA to the dimethyl sulfoxide solution of the compound of formula 1-5, stir the reaction solution at 120 degrees Celsius for 16 hours, then cool the reaction solution to room temperature, add water, precipitate solid, filter, slurry the filter cake with isopropanol, filter and dry to obtain the compound of formula 9-7; Step 7: Add a compound of formula 9-7, a compound of formula 9-8, potassium carbonate and Pd(dppf)Cl2 to 1,4-dioxane and water, then stir the reaction solution at 100 degrees Celsius for 16 hours under nitrogen protection, add water to the reaction solution, extract three times with dichloromethane, combine the organic phases, wash twice with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to obtain a crude product; the crude product is separated and purified by high performance liquid chromatography to obtain a compound of formula 9.

10. A method for preparing a compound of formula 10, the method comprising the following steps: Step 1: Add triethylamine and acetyl chloride to a dichloromethane solution of the compound of formula 9-5, stir the reaction solution at 25 degrees Celsius for 2 hours, then add water to the reaction solution, extract three times with dichloromethane, combine the organic phases, wash twice with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to obtain a compound of formula 10-1; Step 2: Under nitrogen protection, ammonium chloride and iron powder are added to the ethanol and aqueous solution of the compound of formula 10-1, and the reaction solution is stirred at 90 degrees Celsius for 1 hour, then the reaction solution is filtered, water is added to the filtrate, and extracted with ethyl acetate three times, and then the organic phases are combined, dried, filtered, and concentrated to obtain the compound of formula 10-2; Step 3: Add the compound of formula 10-2 and DIEA to the dimethyl sulfoxide solution of the compound of formula 1-5, stir the reaction solution at 120 degrees Celsius for 16 hours, then cool the reaction solution to room temperature, add water, precipitate solid, filter, slurry the filter cake with isopropanol, filter and dry to obtain the compound of formula 10-3; Step 4: Add a compound of formula 10-3, a compound of formula 9-8, potassium carbonate and Pd(dppf)Cl2 to 1,4-dioxane and water, then stir the reaction solution at 100 degrees Celsius for 16 hours under nitrogen protection, add water to the reaction solution, extract three times with dichloromethane, combine the organic phases, wash twice with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to obtain a crude product; the crude product is separated and purified by silica gel column chromatography to obtain a compound of formula 10.

11. A method for preparing a compound of formula 11, the method comprising the following steps: Step 1: Under nitrogen protection, the compound of formula 1-5 and the compound of formula 11-1 are mixed and stirred at 120 degrees Celsius for 16 hours, and then the reaction solution is cooled to room temperature, and dichloromethane and methanol are added. After stirring for 15 minutes, the filter cake is collected and vacuum dried to obtain a compound of formula 11-2; Step 2: Add a compound of formula 11-2, a compound of formula 9-8, potassium carbonate and Pd(dppf)Cl2 to 1,4-dioxane and water, then stir the reaction solution at 100 degrees Celsius for 16 hours under nitrogen protection, add water to the reaction solution, extract three times with dichloromethane, combine the organic phases, wash twice with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to obtain a crude product; the crude product is separated and purified by silica gel column chromatography to obtain a compound of formula 11.

12. A method for preparing a compound of formula 12, the method comprising the following steps: Step 1: Add potassium carbonate and a compound of formula 12-1 to a DMSO solution of a compound of formula 5-1, stir the reaction solution at 100 degrees Celsius for 16 hours, concentrate the reaction solution, add water, extract with ethyl acetate three times, combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate to obtain a compound of formula 12-2; Step 2: Under nitrogen protection, ammonium chloride and iron powder are added to the ethanol and aqueous solution of the compound of formula 12-2, and the reaction solution is stirred at 100 degrees Celsius for 16 hours, then the reaction solution is filtered, water is added to the filtrate, and extracted three times with ethyl acetate, and then the organic phases are combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a compound of formula 12-3; Step 3: Add the compound of formula 12-3 and DIEA to the isopropanol solution of the compound of formula 1-5, stir the reaction solution at 120 degrees Celsius for 16 hours, then cool the reaction solution to room temperature, add water, extract with ethyl acetate three times, then combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate to obtain a crude product; the crude product is separated and purified by silica gel column chromatography to obtain a compound of formula 12-4; Step 4: Add the compound of formula 12-4, the compound of formula 5-8, potassium carbonate and Pd(dppf)Cl2 to 1,4-dioxane and water, then, under nitrogen protection, stir the reaction solution at 100 degrees Celsius for 16 hours, add water to the reaction solution, extract three times with dichloromethane, combine the organic phases, wash twice with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to obtain a crude product; the crude product is separated by silica gel column chromatography to obtain a compound of formula 12-5; Step 5: Add the compound of formula 12-5 to dioxane hydrochloride, stir the reaction solution at 25 degrees Celsius for 16 hours, then adjust the pH of the reaction solution to 8 with aqueous sodium hydroxide solution, add water, extract twice with dichloromethane, then combine the organic phases, wash twice with saturated brine, dry over anhydrous sodium sulfate, filter and concentrate to obtain the compound of formula 12.

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