A DCLK1 kinase inhibitor, its preparation method and application

Abstract

This invention discloses a compound of formula (I), or its stereoisomers, tautomers, prodrugs, pharmaceutically acceptable salts, hydrates, or solvates, wherein X is a sulfur atom or an oxygen atom; and R is optionally an aliphatic substituted amino group, aniline group, or phenolic group. This invention also discloses pharmaceutical compositions containing the compound of formula (I). The compound of formula (I) exhibits excellent DCLK1 kinase activity inhibition ability and can be used to prepare DCLK1 kinase inhibitors, drugs for the prevention and / or treatment of inflammation or cancer.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical compound synthesis technology, and in particular to a DCLK1 kinase inhibitor, its preparation method, and its application. Background Technology

[0002] Bicortin-like kinase 1 (DCLK1), also known as KIAA0369 or DCAMKL1, is a poorly studied multidomain bifunctional protein. Two tandem bicorticoid domains (DCX) at the N-terminus perform MAP functions: binding to microtubules and regulating their polymerization; the C-terminal region contains a serine / threonine kinase domain and an unstructured C-terminal tail, performing kinase-related functions.

[0003] Besides its potential function in neuronal development, DCLK1 is overexpressed in the occurrence and progression of several cancers, including gastric cancer, pancreatic cancer, colon cancer, kidney cancer, and breast cancer. Progressive research findings linking DCLK1 to inflammatory diseases (including acute lung injury and atherosclerosis) have expanded the disease applications of DCLK1. Therefore, DCLK1 is an important potential target for the treatment of inflammation or cancer, and utilizing small molecule inhibitors to suppress DCLK1 kinase activity is a wise strategy for disease treatment.

[0004] Chinese patent CN116284001A discloses a DCLK1 inhibitor, its preparation method, a pharmaceutical composition, and its application. The structure of the DCLK1 inhibitor is shown in the following formula:

[0005]

[0006] Chinese patent CN114478539A discloses a novel DCLK1 inhibitor, wherein the DCLK1 inhibitor is a compound of the following formula or a pharmaceutically acceptable salt thereof:

[0007]

[0008] Currently, several non-selective small molecule inhibitors of DCLK1 have been widely used in disease research, such as LRRK2-IN-1, XMD8-92, and XMD8-85, which have been shown to have off-target activity against DCLK1. It is worth noting that these inhibitors share a common benzopyrimidinyl-dizipine ketone active backbone, limiting further structural modifications. The discovery of DCLK1-IN-1 (a novel selective chemical probe targeting the DCLK1 kinase) has rapidly advanced the efficient analysis of DCLK1 kinase function.

[0009] Therefore, developing novel, efficient, and highly selective small molecule inhibitors of DCLK1 remains challenging. Summary of the Invention

[0010] The present invention provides a compound of formula (I), or a stereoisomer, tautomer, prodrug, pharmaceutically acceptable salt, hydrate, or solvate thereof, which has excellent DCLK1 kinase activity inhibition ability and can be used to prepare anti-inflammatory or antitumor drugs.

[0011] The technical solution of the present invention is as follows:

[0012] A compound of formula (I), or a stereoisomer, tautomer, prodrug, pharmaceutically acceptable salt, hydrate, or solvate thereof:

[0013]

[0014] In the formula, X is a sulfur atom or an oxygen atom; R is an optional aliphatic substituted amino group, aniline group or phenolic group.

[0015] "Tautomers" or "tautomer forms" refer to structural isomers with different energies that interconvert through low energy barriers. Prodrugs are substances that have weak or no activity themselves, but are converted into their corresponding biologically active forms under physiological conditions (e.g., through metabolism, solvation, or other means) after administration.

[0016] Pharmaceutically acceptable salts of compounds of formula (I) include addition salts formed by compounds of formula (I) with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, theazodisulfonic acid, acetic acid, propionic acid, lactic acid, trifluoroacetic acid, maleic acid, citric acid, fumaric acid, oxalic acid, tartaric acid, or benzoic acid.

[0017] The compound of formula (I) of the present invention, or its stereoisomers, tautomers, prodrugs, pharmaceutically acceptable salts, hydrates, and solvates, inhibit the activity of DCLK1 enzyme by binding to the DCLK1 kinase domain, and thus have good anti-inflammatory and anti-tumor activities.

[0018] Preferably, X is a sulfur atom or an oxygen atom; and R is a dimethylamino group, a tetrahydropyrrole group, a piperidinyl group, an N-methylpiperazinyl group, an N-ethylpiperazinyl group, a morpholinyl group, an N-(2-aminoethyl)morpholinyl group, an N-(3-aminopropyl)morpholinyl group, an aniline group, or a phenolic group.

[0019] The compound of formula (I) is selected from:

[0020] (4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenylcarbamate,

[0021] 3-(4-(5-chloro-4-(2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-1,1-dimethylurea

[0022] N-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)pyrrolidine-1-carboxamide,

[0023] N-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)piperidine-1-carboxamide,

[0024] N-(4-(5-chloro-4-(2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-4-methylpiperazine-1-carboxamide,

[0025] N-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-4-ethylpiperazine-1-carboxamide,

[0026] N-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)morpholine-4-carboxamide,

[0027] 1-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-3-(2-morpholinoethyl)urea,

[0028] 1-(4-(5-chloro-4-((2-isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-3-(3-morpholinopropyl)urea,

[0029] 1-(4-(5-chloro-4-(2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-3-phenylurea,

[0030] 3-(4-(5-chloro-4-(2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-1,1-dimethylthiourea,

[0031] N-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)pyrrolidine-1-thiocarboxamide,

[0032] N-(4-(5-chloro-4-(2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)piperidine-1-thiocarboxamide,

[0033] N-(4-(5-chloro-4-(2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-4-methylpiperazine-1-thiocarboxamide,

[0034] N-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-4-ethylpiperazine-1-thiocarboxamide,

[0035] N-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)morpholine-4-thiocarboxamide,

[0036] 1-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)-3-(2-morpholinoethyl)thiourea,

[0037] 1-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-3-(3-morpholinopropyl)thiourea,

[0038] 1-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)-3-phenylthiourea.

[0039] In a further preferred embodiment, X is a sulfur atom or an oxygen atom; R is an N-ethylpiperazinyl group.

[0040] The preferred compounds exhibit better DCLK1 inhibitory activity and anti-inflammatory and anti-tumor activities.

[0041] The present invention also provides a method for preparing the compound of formula (I), comprising the following steps:

[0042]

[0043] (1) Dissolve compound (A) and compound (B) in 2-methoxyethanol, heat to reflux and react, and after the reaction is completed, process to obtain compound (C);

[0044] (2) Dissolve compound (C) and ammonium chloride in an organic solvent, add iron powder, and heat the mixture under a nitrogen atmosphere to react. After the reaction is complete, process the mixture to obtain compound (D).

[0045] (3) Dissolve the compound of formula (D) in ultra-dry dichloromethane, and slowly add phenyl chloroformate or phenyl thiochloroformate dropwise in an ice-water bath. After the addition is complete, react at room temperature. After the reaction is complete, process to obtain the compound of formula (E).

[0046] (4) Dissolve the compound of formula (E) in an organic solvent, add an organic base, add RH at room temperature and heat to react, and after the reaction is completed, treat to obtain the compound of formula (I);

[0047] X is a sulfur atom or an oxygen atom; R is an optional aliphatic substituted amino group, aniline group or phenolic group.

[0048] The present invention also provides a pharmaceutical composition comprising a compound of formula (I) or a stereoisomer, tautomer, prodrug, pharmaceutically acceptable salt, hydrate, or solvate thereof; and further comprising a pharmaceutically acceptable excipient.

[0049] In the pharmaceutical composition described herein, a compound of formula (I) or its stereoisomers, tautomers, prodrugs, pharmaceutically acceptable salts, hydrates, or solvates are used as the active ingredient and are mixed with a pharmaceutically acceptable excipient to form the pharmaceutical composition. The excipient is a diluent, adjuvant, or carrier used in the pharmaceutical field.

[0050] A clinically acceptable dosage form is prepared by adding pharmaceutically acceptable excipients to a pharmaceutical composition. The dosage form may be an injection, tablet, or capsule.

[0051] Preferably, the pharmaceutical composition further includes an anti-inflammatory or antitumor drug. The compounds of the present invention, or their pharmaceutically acceptable salts or hydrates, can be used alone as anti-inflammatory or antitumor agents, or in combination with different anti-inflammatory or antitumor agents, for the treatment or prevention of inflammation or tumors.

[0052] The present invention also provides the use of compounds of formula (I) or their stereoisomers, tautomers, prodrugs, pharmaceutically acceptable salts, hydrates, and solvates in the preparation of DCLK1 kinase inhibitors.

[0053] The present invention also provides the use of the pharmaceutical composition in the preparation of DCLK1 kinase inhibitors.

[0054] The present invention also provides the use of the compound of formula (I) or its stereoisomers, tautomers, prodrugs, pharmaceutically acceptable salts, hydrates, and solvates in the preparation of medicaments for the prevention and / or treatment of inflammation or cancer.

[0055] The present invention also provides the use of the pharmaceutical composition in the preparation of medicaments for the prevention and / or treatment of inflammation or cancer.

[0056] The inflammatory diseases mentioned include sepsis, acute lung injury, arthritis, colitis, hepatitis, or chronic diseases with chronic inflammation as an important pathological pathway; the cancers mentioned include multiple myeloma, gastric cancer, lung cancer, breast cancer, esophageal cancer, colon cancer, medulloblastoma, acute myeloid leukemia, chronic leukemia, prostate cancer, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, skin cancer, ovarian cancer, glioma, thyroid cancer, or pancreatic cancer.

[0057] The chronic diseases mentioned are complications of diabetes, atherosclerosis, obesity, or hypertension.

[0058] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0059] (1) The compound of formula (I) provided by the present invention, or its stereoisomers, tautomers, prodrugs, pharmaceutically acceptable salts, hydrates, and solvates, is an effective DCLK1 kinase inhibitor that can selectively act on the DCLK1 kinase domain. Compared with the lead compound NVP-TAE684, it has a better inhibitory effect on kinase activity and can significantly inhibit the activation of the NF-κB signaling pathway in cell experiments, including reduced IKKβ phosphorylation, reduced IκB-α degradation, and reduced p65 nuclear translocation level of NF-κB.

[0060] (2) The compound of formula (I) provided by the present invention, or its stereoisomers, tautomers, prodrugs, pharmaceutically acceptable salts, hydrates, and solvates, may serve as promising lead or candidate drugs for DCLK1-related diseases. Attached Figure Description

[0061] Figure 1 The figure shows the evaluation results of the preferred compound C24 and the lead compound TAE684 in inhibiting the activation of the NF-κB signaling pathway in the example.

[0062] Figure 2 The results of the preparation of the preferred compound C24 and the lead compound TAE684 in improving the pathological damage of lung tissue in a mouse model of LPS-induced acute lung injury are shown in the following figures: (A) is a staining image of lung tissue, (B) is the wet / dry (W / D) ratio of the lung, and (C) is the total protein concentration in BALF.

[0063] Figure 3 The results of the preparation of the preferred compound C24 and the lead compound TAE684 in reducing the production of macrophages in the lungs of mice with LPS-induced acute lung injury are shown in the figure.

[0064] Figure 4 The results of the example show the reduction of inflammatory factors in mice with LPS-induced acute lung injury by the preferred compound C24 and the lead compound TAE684.

[0065] Figure 5 The results of the study on the protective effects of the preferred compound C24 and the lead compound TAE684 on septic mice are shown in the figure. Detailed Implementation

[0066] The examples and preparation methods provided below further illustrate and demonstrate the compounds of the present invention and their preparation methods. It should be understood that the scope of the following examples and preparation methods does not limit the scope of the invention in any way. The raw materials of the present invention can be obtained commercially or prepared by methods known in the art.

[0067] The structure of the compound was determined by nuclear magnetic resonance (NMR). 1 The NMR was determined by high-resolution NMR (H-NMR) and high-resolution mass spectrometry (HRMS). NMR measurements were performed using an ACF-400BRUK NMR spectrometer with deuterated chloroform (CDCl3) or deuterated dimethyl sulfoxide (DMSO-D6) as the solvent. Column chromatography was performed using 200-300 mesh silica gel.

[0068] The preparation process is as follows:

[0069]

[0070] Reaction conditions: (a) 2.5M hydrochloric acid ethanol, 2-methoxyethanol, 120℃, 6h; (b) iron powder, NH4Cl, aqueous ethanol solution (3:1, v / v), 100℃, reflux for 2h; (c) phenyl chloroformate or phenyl thiochloroformate, dried DCM, 0℃-room temperature, 1h; (d) various amine derivatives, TEA, MeCN, 0.5-2h.

[0071] Includes the following steps:

[0072] (1) Commercially available 2,5-dichloro-N-(2-(isopropylsulfonyl)phenyl)pyrimidine-4-amine (A, 1 mmol) and 2-methoxy-4-nitroaniline (B, 1.4 mmol) were added to a 25 mL round-bottom flask, dissolved in 3 mL of 2-methoxyethanol, and stirred in an oil bath. The temperature was raised to 120 °C, and 1.5 mL of hydrochloric acid-ethanol solution (2.5 M) was added dropwise. The reaction was kept under reflux for 6 hours. After the reaction was confirmed to be complete by thin-layer chromatography (TLC), the reaction apparatus was cooled to room temperature, and saturated sodium bicarbonate was added to adjust the pH to >7. The entire mixture was then transferred to a separatory funnel filled with ice water. The aqueous layer was extracted three times with 20 mL of ethyl acetate (EA), and all organic phases were collected. The organic phases were washed 2 to 3 times with saturated saline solution and collected again. The organic phases were dried over anhydrous magnesium sulfate, filtered to remove anhydrous magnesium sulfate, and the solvent ethyl acetate was removed by vacuum distillation to obtain the crude product. Add 1.5 times the amount of 200-300 mesh silica gel for column chromatography to prepare the precipitate, and then load the sample by dry method. Using petroleum ether (PE) and ethyl acetate (EA) as the mobile phase (PE:EA = 4:1), the sample is purified by silica gel column chromatography to obtain yellow intermediate C in 85% yield.

[0073] (2) Set up a reflux reaction apparatus. Take intermediate C (1 mmol) and ammonium chloride (NH4Cl, 1.5 mmol) solid and add them to a 25 mL round-bottom flask. Add 5 mL of an aqueous solution of ethanol (EtOH:H2O = 3:1) to dissolve it, forming a suspension. Finally, add iron powder (Fe, 5 mmol). Replace the air in the apparatus with nitrogen (N2) and place it in an oil bath. Heat to 100 °C and stir the reaction for 2 hours. After the reaction is complete, the reaction solvent system becomes a clear yellow-green liquid. After confirming the completeness of the reaction by thin-layer chromatography (TLC), cool the reaction apparatus to room temperature. Remove excess iron powder by diatomaceous earth filtration. Wash the filter cake repeatedly with dichloromethane (DCM) to collect as much product as possible. The collected mixture was concentrated by negative pressure distillation, dissolved again in dichloromethane, transferred to a separatory funnel, washed three times with water, and then washed three times with saturated saline solution. The organic phase was collected, dried with anhydrous magnesium sulfate, filtered to remove anhydrous magnesium sulfate, and the solvent dichloromethane was removed by negative pressure distillation to obtain a yellow-green product with a yield of 90%.

[0074] (3) Add intermediate D (1 mmol) to a 25 mL round-bottom flask, dissolve it in 10 mL of ultra-dry dichloromethane (Dry DCM), and stir in an ice-water bath (0 °C) for 5-10 minutes. Slowly add phenyl chloroformate (2 mmol) dropwise. After all the product has been added, remove the ice-water bath and gradually heat to room temperature. React for 1 hour. After the reaction is complete, use thin-layer chromatography (TLC) to detect the reaction. Add an appropriate amount of water to react with the excess phenyl chloroformate. If insoluble matter is produced, add excess dichloromethane to dissolve it. Transfer the mixture to a separatory funnel, wash it three times with water, and then wash it three times with saturated saline solution. Collect the organic phase, dry it with anhydrous magnesium sulfate, filter to remove the anhydrous magnesium sulfate, and remove the solvent dichloromethane by negative pressure distillation to obtain a yellow-green crude product. Add 1.5 times the amount of 200-300 mesh silica gel to prepare the slurry, and load it onto the sample by dry method. Using petroleum ether (PE) and ethyl acetate (EA) as the mobile phase (PE:EA = 4:1), the pale yellow product E was purified by silica gel column chromatography in a yield of 76% (i.e., X represents oxygen atoms and R represents the corresponding compound of phenolic group in general formula I).

[0075] If phenyl thiochloroformate is used for this step, the operation steps are the same, but the theoretical product E (i.e., the compound corresponding to the phenol group where X is a sulfur atom and R is a phenol group in general formula I) cannot be stored for a long time. Therefore, after monitoring the end of the reaction, the solvent is removed by negative pressure distillation, and the next step of the reaction can be carried out directly.

[0076] (4) Set up a reflux reaction apparatus. Add compound E (1 mmol) to a 25 mL round-bottom flask, dissolve it in acetonitrile (MeCN), add triethylamine (TEA, 3 mmol), and stir at room temperature. Add various amine derivatives (2 mmol, mainly dimethylamine, tetrahydropyrrole, piperidine, N-methylpiperazine, N-ethylpiperazine, morpholine, N-(2-aminoethyl)morpholine, N-(3-aminopropyl)morpholine, and aniline). Heat to 65 °C and react for 2 hours. After the reaction is complete, remove the solvent acetonitrile by vacuum distillation. Redissolve the crude product in ethyl acetate, transfer to a separatory funnel, wash three times with water, and then wash three times with saturated saline solution. Collect the organic phase, dry it with anhydrous magnesium sulfate, filter to remove anhydrous magnesium sulfate, and remove the solvent ethyl acetate by vacuum distillation to obtain a pale yellow crude product. Add 1.5 times the amount of 200-300 mesh silica gel for column chromatography to prepare slurry, and load the sample by dry method. Using dichloromethane (DCM) and methanol (MeOH) as the mobile phase (the ratio of the two was adjusted according to the specific polarity of the target product), the corresponding target compound (general formula I) was obtained by silica gel column chromatography purification.

[0077] Example 1

[0078] Preparation: (4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenylcarbamate (C10)

[0079]

[0080] Preparation method: Take the intermediate 5-chloro-N 4 -(2-isopropylsulfonyl)phenyl-N 2 1 mmol of 2-(2-methoxy-4-nitrophenyl)pyrimidine-2,4-diamine was added to a 25 mL round-bottom flask and dissolved in 10 mL of dry dichloromethane (DCM). The flask was placed in an ice-water bath (0 °C) and stirred for 5-10 minutes. 2 mmol of phenyl chloroformate was slowly added dropwise. After all the chloroformate was added, the ice-water bath was removed, and the mixture was gradually heated to room temperature and reacted for 1 hour. After the reaction was confirmed to be complete by thin-layer chromatography (TLC), an appropriate amount of water was added to react with the excess phenyl chloroformate. If insoluble matter was produced, excess dichloromethane was added to dissolve it. The mixture was transferred to a separatory funnel, washed three times with water, and then washed three times with saturated saline solution. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered to remove the anhydrous magnesium sulfate, and the solvent dichloromethane was removed by negative pressure distillation to obtain a yellow-green crude product. 1.5 times the volume of 200-300 mesh silica gel was added to prepare a slurry, which was then loaded onto the slurry using a dry method. The product was purified by silica gel column chromatography using petroleum ether (PE) and ethyl acetate (EA) as the mobile phase (PE:EA = 4:1) to obtain a pale yellow product with a yield of 76%.

[0081] Characterization data of the product: 1 H NMR (400MHz, CDCl3) δ9.58(s,1H),8.55(d,J=8.4Hz,1H),8.16(d,J=8.6Hz,2H),7.91(dd,J=7.9,1.7Hz,1H),7.64(t,1H),7.57(s,1H),7.41(t,J=7.8 Hz,3H),7.27(m,2H),7.20(dd,J=7.4,1.6Hz,2H),6.98(s,1H),6.77(dd,J= 8.7, 2.3Hz, 1H), 3.89 (s, 3H), 3.24 (p, J = 6.8Hz, 1H), 1.31 (d, J = 6.9Hz, 6H). 13C NMR (101MHz, CDCl3) δ155.33,155.07,148.72,138.35,134.54,134.35,131.16,124.74 ,124.08,123.93,123.27,119.50,111.29,103.54,55.80,55.57,36.51,15.37.ESI-MS m / z::568.2[M+H] + calcd for C 27 H 26 ClN5O5S:567.13.

[0082] Example 2

[0083] Preparation: 3-(4-(5-chloro-4-(2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-1,1-dimethylurea (C11)

[0084]

[0085] Preparation method: A reflux reaction apparatus was set up. Compound (4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenylcarbamate (C10, 1 mmol)) was added to a 25 mL round-bottom flask, dissolved in acetonitrile (MeCN), and then triethylamine (TEA, 3 mmol) was added. The mixture was stirred at room temperature, and then dimethylamine (2 mmol) was added. The temperature was raised to 65 °C and reacted for 2 hours. After the reaction was confirmed to be complete by thin-layer chromatography (TLC), the solvent acetonitrile was removed by negative pressure distillation. The crude product was redissolved in ethyl acetate and transferred to a separatory funnel. It was washed three times with water, then three times with saturated brine. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered to remove the anhydrous magnesium sulfate, and the solvent ethyl acetate was removed by vacuum distillation to obtain a pale yellow crude product. 1.5 times the volume of 200-300 mesh silica gel was added to prepare the precipitate, which was then loaded onto the column using a dry method. Using dichloromethane (DCM) and methanol (MeOH) as the mobile phase (the ratio of which was adjusted according to the specific polarity of the target product), the target product was purified by silica gel column chromatography in 79% yield.

[0086] Characterization data of the product: 1H NMR (400MHz, CDCl3) δ9.53(s,1H),8.56(d,J=8.3Hz,1H),8.13(s,1H),8.07(d,J=8.6Hz,1H),7.90(d,J=7.9Hz,1H),7.65(t,J=7.8Hz,1H),7.50(s ,1H),7.36(s,1H),7.25(t,1H),6.67(d,J=8.5Hz,1H),6.37(s,1H),3.88 (s,3H),3.24(dt,J=13.6,6.8Hz,1H),3.05(s,6H),1.31(d,J=6.8Hz,6H). 13 C NMR (101MHz, CDCl3) δ155.46,138.18,134.55,131.25,129.49,129.21,125 .80,125.07,123.51,121.79,121.70,121.20,55.82,55.67,15.37,ESI-MS m / z:519.14[M+H] + calcd for C 23 H 27 ClN6O4S:518.15.

[0087] Example 3

[0088] Preparation: N-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)pyrrolidine-1-carboxamide (C12)

[0089]

[0090] Preparation method: A reflux reaction apparatus was set up. Compound (4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenylcarbamate (C10, 1 mmol)) was added to a 25 mL round-bottom flask, dissolved in acetonitrile (MeCN), and then triethylamine (TEA, 3 mmol) was added. The mixture was stirred at room temperature, and then tetrahydropyrrole (2 mmol) was added. The temperature was raised to 65 °C and the reaction was carried out for 2 hours. After the reaction was confirmed to be complete by thin-layer chromatography (TLC), the solvent acetonitrile was removed by vacuum distillation. The crude product was redissolved in ethyl acetate and transferred to a separatory funnel. It was washed three times with water, then three times with saturated saline solution. The organic phase was collected, dried over anhydrous magnesium sulfate, and filtered to remove the anhydrous magnesium sulfate. The solvent, ethyl acetate, was removed by vacuum distillation to obtain a pale yellow crude product. 1.5 times the volume of 200-300 mesh silica gel was added to prepare a column chromatography slurry, which was then loaded onto the column using a dry method. Using dichloromethane (DCM) and methanol (MeOH) as the mobile phase (the ratio of which was adjusted according to the specific polarity of the target product), the target product was purified by silica gel column chromatography in 75% yield.

[0091] Characterization data of the product: 1 H NMR (400MHz, CDCl3) δ9.53 (s, 1H), 8.56 (d, J = 8.3Hz, 1H), 8.12 (s, 1H), 8.06 (d, J=8.4Hz,1H),7.89(d,J=7.8Hz,1H),7.65(t,J=7.7Hz,1H),7.47(d,J=21.2Hz,2 H),7.25(t,J=14.0,6.4Hz,1H),6.68(d,J=8.5Hz,1H),6.25(s,1H),3.88(s,3H) ,3.47(s,4H),3.24(dt,J=13.4,6.6Hz,1H),1.98(s,4H),1.30(d,J=6.7Hz,6H). 13 CNMR(101MHz, CDCl3)δ155.32,155.13,154.12,148.75,138.37,134.54,134.49,131.15,124.70,1 23.92,123.85,123.23,119.57,110.86,105.74,103.20,55.79,55.56,45.82,25.63,15.37.ESI-MS m / z:545.27[M+H] + calcd for C 25 H 29 ClN6O4S:544.17.

[0092] Example 4

[0093] Preparation: N-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)piperidine-1-carboxamide (C13)

[0094]

[0095] Preparation method: A reflux reaction apparatus was set up. Compound (4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenylcarbamate (C10, 1 mmol)) was added to a 25 mL round-bottom flask, dissolved in acetonitrile (MeCN), and then triethylamine (TEA, 3 mmol) was added. The mixture was stirred at room temperature, and piperidine (2 mmol) was added. The temperature was raised to 65 °C and reacted for 2 hours. After the reaction was confirmed to be complete by thin-layer chromatography (TLC), the solvent acetonitrile was removed by negative pressure distillation. The crude product was redissolved in ethyl acetate and transferred to a separatory funnel. It was washed three times with water, then three times with saturated brine. The organic phase was collected, dried over anhydrous magnesium sulfate, and filtered to remove the anhydrous magnesium sulfate. The solvent, ethyl acetate, was removed by vacuum distillation to obtain a pale yellow crude product. 1.5 times the volume of 200-300 mesh silica gel was added to prepare the precipitate, which was then loaded onto the column using a dry method. Using dichloromethane (DCM) and methanol (MeOH) as the mobile phase (the ratio of which was adjusted according to the specific polarity of the target product), the product was purified by silica gel column chromatography to obtain the corresponding target product in 76% yield.

[0096] Characterization data of the product: 1 H NMR (400MHz, CDCl3) 1 H NMR (400MHz, CDCl3) δ9.52 (s, 1H), 8.56 (d, J = 8.3Hz, 1H), 8.13 (s, 1H), 8.07 (d,J=8.5Hz,1H),7.89(d,J=7.8Hz,1H),7.64(t,J=7.7Hz,1H),7.47(s,1H), 7.33(s,1H),7.30–7.17(m,1H),6.67(d,J=8.4Hz,1H),6.51(s,1H),3.87(s, 3H), 3.46 (s, 4H), 3.24 (p, J = 6.7Hz, 1H), 1.64 (s, 6H), 1.30 (d, J = 6.8Hz, 6H). 13C NMR (101MHz, d-DMSO) δ159.15,155.80,155.35,155.11,151.99,138.69,138.57,135.30,131.34,124. 60,123.98,123.54,123.50,122.19,111.42,103.93,55.77,55.39,45.11,26.01,24.60,15.33.ESI-MS m / z:559.34[M+H] + calcd for C 26 H 31 ClN6O4S:558.18.

[0097] Example 5

[0098] Preparation: N-(4-(5-chloro-4-(2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-4-methylpiperazin-1-carboxamide (C14)

[0099]

[0100] Preparation method: A reflux reaction apparatus was set up. Compound (4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenylcarbamate (C10, 1 mmol)) was added to a 25 mL round-bottom flask, dissolved in acetonitrile (MeCN), and then triethylamine (TEA, 3 mmol) was added. While stirring at room temperature, N-methylpiperazine (2 mmol) was added, and the mixture was heated to 65 °C for 2 hours. After the reaction was confirmed to be complete by thin-layer chromatography (TLC), the solvent acetonitrile was removed by vacuum distillation. The crude product was redissolved in ethyl acetate, transferred to a separatory funnel, washed three times with water, and then washed three times with saturated saline solution. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered to remove the anhydrous magnesium sulfate, and the solvent ethyl acetate was removed by negative pressure distillation to obtain a pale yellow crude product. 1.5 times the volume of 200-300 mesh silica gel was added to prepare the precipitate, which was then loaded onto the column using a dry method. Using dichloromethane (DCM) and methanol (MeOH) as the mobile phase (the ratio of which was adjusted according to the specific polarity of the target product), the target product was purified by silica gel column chromatography in 65% yield.

[0101] Characterization data of the product: 1H NMR (400MHz, CDCl3) δ9.52 (s, 1H), 8.56 (d, J = 8.2Hz, 1H), 8.12 (d, J = 13.8Hz, 2H),7.90(d,J=7.7Hz,1H),7.65(t,J=7.5Hz,1H),7.45(s,1H),7.29(s,1H), 7.24(t,J=7.5Hz,1H),6.68(d,J=8.3Hz,1H),6.51(s,1H),3.89(s,3H),3.58 (s,4H),3.32–3.16(p,1H),2.55(s,4H),2.39(s,3H),1.31(d,J=6.6Hz,6H). 13 C NMR(101MHz,d-DMSO)δ159.13,155.81,155.41,155.13,152.02,138.68,138.21,135.30,1 31.35,124.64,124.03,123.57,111.50,104.01,55.79,55.38,54.72,43.79,15.33.ESI-MS m / z:[M+H] + 574.14, calcd for C 26 H 32 ClN7O4S:573.19.

[0102] Example 6

[0103] Preparation: N-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-4-ethylpiperazine-1-carboxamide (C15)

[0104]

[0105] Preparation method: A reflux reaction apparatus was set up. Compound (4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenylcarbamate (C10, 1 mmol)) was added to a 25 mL round-bottom flask, dissolved in acetonitrile (MeCN), and then triethylamine (TEA, 3 mmol) was added. While stirring at room temperature, N-ethylpiperazine (2 mmol) was added, and the mixture was heated to 65 °C for 2 hours. After the reaction was confirmed to be complete by thin-layer chromatography (TLC), the solvent acetonitrile was removed by vacuum distillation. The crude product was redissolved in ethyl acetate, transferred to a separatory funnel, washed three times with water, and then washed three times with saturated saline solution. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered to remove the anhydrous magnesium sulfate, and the solvent ethyl acetate was removed by vacuum distillation to obtain a pale yellow crude product. 1.5 times the volume of 200-300 mesh silica gel was added to prepare the precipitate, which was then loaded onto the column using a dry method. Using dichloromethane (DCM) and methanol (MeOH) as the mobile phase (the ratio of which was adjusted according to the specific polarity of the target product), the target product was purified by silica gel column chromatography in 67% yield.

[0106] Characterization data of the product: 1 H NMR (400MHz, CDCl3) δ9.52 (s, 1H), 8.56 (d, J = 8.3Hz, 1H), 8.18–8.03 (m, 2H), 7.9 0(d,J=7.7Hz,1H),7.65(t,J=7.6Hz,1H),7.44(s,1H),7.30(s,1H),7.27–7.22(m ,1H),6.68(d,J=8.1Hz,1H),6.52(s,1H),3.88(s,3H),3.57(s,4H),3.24(p,J=6. 5Hz,1H),2.54(s,4H),2.50(t,2H),1.31(d,J=6.7Hz,6H),1.14(t,J=7.0Hz,3H). 13 C NMR(101MHz,d-DMSO)δ159.13,155.81,155.43,152.00,138.68,138.24,135.30,131.34,124.63,1 24.01,123.57,123.52,122.41,111.50,104.01,55.79,55.38,52.62,52.02,43.99,15.33.ESI-MS m / z:588.27[M+H] + ,calcd forC 27 H 34 ClN7O4S:587.21.

[0107] Example 7

[0108] Preparation: N-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)morpholine-4-carboxamide (C16)

[0109]

[0110] Preparation method: A reflux reaction apparatus was set up. Compound (4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenylcarbamate (C10, 1 mmol)) was added to a 25 mL round-bottom flask, dissolved in acetonitrile (MeCN), and then triethylamine (TEA, 3 mmol) was added. Morpholine (2 mmol) was added while stirring at room temperature, and the mixture was heated to 65 °C for 2 hours. After the reaction was confirmed to be complete by thin-layer chromatography (TLC), the solvent acetonitrile was removed by negative pressure distillation. The crude product was redissolved in ethyl acetate and transferred to a separatory funnel. It was washed three times with water, then three times with saturated saline solution. The organic phase was collected, dried over anhydrous magnesium sulfate, and filtered to remove the anhydrous magnesium sulfate. The solvent, ethyl acetate, was removed by vacuum distillation to obtain a pale yellow crude product. 1.5 times the volume of 200-300 mesh silica gel was added to prepare the precipitate, which was then loaded onto the column using a dry method. Using dichloromethane (DCM) and methanol (MeOH) as the mobile phase (the ratio of which was adjusted according to the specific polarity of the target product), the product was purified by silica gel column chromatography to obtain the corresponding target product in 70% yield.

[0111] Characterization data of the product: 1 H NMR (400MHz, CDCl3) δ9.63(s,1H),8.53(d,J=8.3Hz,1H),8.10(s,1H),8.03(d,J=7.7Hz,1H),7.90(d,J=7.6Hz,1H),7.64(t,J=7.8Hz,1H),7.29( s,1H),7.24(s,2H),6.68(d,J=7.8Hz,1H),6.44(s,1H),3.88(s,3H),3. 76(t,4H),3.50(t,4H),3.23(p,J=6.8Hz,1H),1.31(s,3H),1.30(s,3H). 13 C NMR(101MHz,d-DMSO)δ159.10,155.80,155.67,151.98,138.67,138.07,135.30,131.34,124.5 8,124.04,123.59,123.55,122.53,111.55,104.04,66.49,55.80,55.38,44.62,15.33.ESI-MS m / z:561.2[M+H]+ calcd for C 25 H 29 ClN6O5S:560.16.

[0112] Example 8

[0113] Preparation: 1-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-3-(2-morpholinoethyl)urea (C17)

[0114]

[0115] Preparation method: A reflux reaction apparatus was set up. Compound (4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenylcarbamate (C10, 1 mmol) was added to a 25 mL round-bottom flask, dissolved in acetonitrile (MeCN), and then triethylamine (TEA, 3 mmol) was added. While stirring at room temperature, N-(2-aminoethyl)morpholine (2 mmol) was added, and the mixture was heated to 65 °C and reacted for 2 hours. After the reaction was confirmed to be complete by thin-layer chromatography (TLC), the solvent was removed by vacuum distillation. Acetonitrile was used to redissolve the crude product in ethyl acetate. The solution was transferred to a separatory funnel, washed three times with water, and then washed three times with saturated saline solution. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered to remove the anhydrous magnesium sulfate, and the solvent ethyl acetate was removed by vacuum distillation to obtain a pale yellow crude product. 1.5 times the volume of 200-300 mesh silica gel was added to prepare the precipitate, which was then loaded onto the column using a dry method. Using dichloromethane (DCM) and methanol (MeOH) as the mobile phase (the ratio of which was adjusted according to the specific polarity of the target product), the target product was purified by silica gel column chromatography in 71% yield.

[0116] Characterization data of the product: 1 H NMR (400MHz, CDCl3) δ9.55(s,1H),8.57(d,J=8.3Hz,1H),8.12(d,J=8.7Hz,2H),7. 89(d,J=7.8Hz,1H),7.63(t,J=7.7Hz,1H),7.47(d,J=20.3Hz,1H),7.42(s,1H),7.2 9–7.16(m,2H),6.73(d,J=8.4Hz,1H),5.85(s,1H),3.86(s,3H),3.73(s,4H),3.42 (d,J=3.2Hz,2H),3.31–3.18(m,1H),2.61(d,J=13.6Hz,6H),1.31(d,J=6.8Hz,6H). 13C NMR(101MHz,d-DMSO)δ159.23,155.80,155.76,155.09,152.55,138.72,138.52,135.20,131.35 ,125.38,123.94,123.49,123.42,121.74,109.52,104.33,102.08,55.73,55.39,15.33.ESI-MS m / z:604.33[M+H] + calcd for C 27 H 34 ClN7O5S:603.20.

[0117] Example 9

[0118] Preparation: 1-(4-(5-chloro-4-((2-isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-3-(3-morpholinopropyl)urea (C18)

[0119]

[0120] Preparation method: A reflux reaction apparatus was set up. Compound (4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenylcarbamate (C10, 1 mmol) was added to a 25 mL round-bottom flask, dissolved in acetonitrile (MeCN), and then triethylamine (TEA, 3 mmol) was added. While stirring at room temperature, N-(3-aminopropyl)morpholine (2 mmol) was added, and the mixture was heated to 65 °C and reacted for 2 hours. After the reaction was confirmed to be complete by thin-layer chromatography (TLC), the solvent was removed by vacuum distillation. Acetonitrile was used to redissolve the crude product in ethyl acetate. The solution was transferred to a separatory funnel, washed three times with water, and then washed three times with saturated saline solution. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered to remove the anhydrous magnesium sulfate, and the solvent ethyl acetate was removed by vacuum distillation to obtain a pale yellow crude product. 1.5 times the volume of 200-300 mesh silica gel was added to prepare the precipitate, which was then loaded onto the column using a dry method. Using dichloromethane (DCM) and methanol (MeOH) as the mobile phase (the ratio of which was adjusted according to the specific polarity of the target product), the target product was purified by silica gel column chromatography in 71% yield.

[0121] Characterization data of the product: 1H NMR (400MHz, CDCl3) δ9.55 (s, 1H), 8.57 (d, J = 8.4Hz, 1H), 8.12 (t, 2H), 7.89 (d, J =7.8Hz,1H),7.63(t,J=7.7Hz,1H),7.42(s,1H),7.25(dd,J=14.7,7.0Hz,2H),7 .17(s,1H),6.71(d,J=8.5Hz,1H),3.85(s,3H),3.65(s,4H),3.32(t,J=6.1Hz,2 H),3.24(p,J=6.8Hz,1H),2.51–2.42(m,6H),1.72(q,2H),1.31(d,J=6.8Hz,6H). 13 C NMR (101MHz, d-DMSO) δ159.23,155.78,155.09,152.53,138.71,138.54,135.21,131.35,125.33,123.93,123 .49,123.41,121.70,109.57,104.33,102.14,66.64,56.35,55.74,55.41,53.85,37.94,27.12,15.32.ESI-MS m / z:618.33[M+H] + ,calcd forC 28 H 36 ClN7O5S:617.22.

[0122] Example 10

[0123] Preparation: 1-(4-(5-chloro-4-(2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-3-phenylurea (C19)

[0124]

[0125] Preparation method: A reflux reaction apparatus was set up. Compound (4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenylcarbamate (C10, 1 mmol)) was added to a 25 mL round-bottom flask, dissolved in acetonitrile (MeCN), and then triethylamine (TEA, 3 mmol) was added. Aniline (2 mmol) was added while stirring at room temperature, and the mixture was heated to 65 °C for 2 hours. After the reaction was confirmed to be complete by thin-layer chromatography (TLC), the solvent acetonitrile was removed by negative pressure distillation. The crude product was redissolved in ethyl acetate and transferred to a separatory funnel. It was washed three times with water, then three times with saturated saline solution. The organic phase was collected, dried over anhydrous magnesium sulfate, and filtered to remove the anhydrous magnesium sulfate. The solvent, ethyl acetate, was removed by vacuum distillation to obtain a pale yellow crude product. 1.5 times the volume of 200-300 mesh silica gel was added to prepare a column chromatography slurry, which was then loaded onto the column using a dry method. Using dichloromethane (DCM) and methanol (MeOH) as the mobile phase (the ratio of which was adjusted according to the specific polarity of the target product), the target product was purified by silica gel column chromatography in 71% yield.

[0126] Characterization data of the product: 1 H NMR(400MHz,d-DMSO)δ9.56(s,1H),8.73(s,1H),8.69(s,1H),8.58(s,1H),8.4 5(s,1H),8.21(s,1H),7.81(dd,J=8.0,1.7Hz,1H),7.61(t,J=7.9Hz,1H),7.51 –7.44(m,3H),7.37(d,J=2.2Hz,1H),7.35–7.26(m,3H),7.01–6.95(m,1H),6.8 8(dd,J=8.5,2.3Hz,1H),3.76(s,3H),3.49–3.42(m,1H),1.16(d,J=6.8Hz,6H). 13 C NMR(101MHz,d-DMSO)δ159.04,155.16,153.07,140.16,138.64,137.57,135.26,131.38,129.27 ,124.04,123.64,123.54,122.39,122.29,118.68,110.13,102.56,55.81,55.35,15.32.ESI-MS m / z:567.2[M+H] + calcd for C 27 H 27 ClN6O4S:566.1.

[0127] Example 11

[0128] Preparation: 3-(4-(5-chloro-4-(2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-1,1-dimethylthiourea (C21)

[0129]

[0130] Preparation method: A reflux reaction apparatus was set up. Compound o-phenyl (4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)thiocarbamate (C20, 1 mmol) was added to a 25 mL round-bottom flask and dissolved in acetonitrile (MeCN). Triethylamine (TEA, 3 mmol) was added, and the mixture was stirred at room temperature. Dimethylamine (2 mmol) was then added, and the mixture was heated to 65 °C for 2 hours. After the reaction was confirmed to be complete by thin-layer chromatography (TLC), the solvent was removed by vacuum distillation. Acetonitrile was used to redissolve the crude product in ethyl acetate. The solution was transferred to a separatory funnel, washed three times with water, and then washed three times with saturated saline solution. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered to remove the anhydrous magnesium sulfate, and the solvent ethyl acetate was removed by vacuum distillation to obtain a pale yellow crude product. 1.5 times the volume of 200-300 mesh silica gel was added to prepare the precipitate, which was then loaded onto the column using a dry method. Using dichloromethane (DCM) and methanol (MeOH) as the mobile phase (the ratio of which was adjusted according to the specific polarity of the target product), the target product was purified by silica gel column chromatography in 69% yield.

[0131] Characterization data of the product: 1 H NMR(400MHz,d-DMSO)δ9.55(s,1H),8.99(s,1H),8.58(d,J=8.5Hz,1H),8.37(s,1H) ,8.23(s,1H),7.81(dd,J=8.0,1.6Hz,1H),7.73(ddd,J=8.7,7.3,1.7Hz,1H),7.57(d ,J=8.5Hz,1H),7.32(dd,J=15.2,1.1Hz,1H),7.08(d,J=2.2Hz,1H),6.85(dd,J=8.5 ,2.2Hz,1H),3.74(s,3H),3.44(p,J=6.8Hz,1H),3.35(s,6H),1.16(d,J=6.8Hz,6H). 13C NMR (101MHz, d-DMSO) δ186.38,163.51,160.00,155.77,143.33,142.91,140.36,136.1 0,129.63,128.99,128.52,122.55,114.71,109.55,60.78,60.14,46.11,20.08.ESI-MS m / z:535.27[M+H] + ,calcd forC 23 H 27 ClN6O3S2:534.13.

[0132] Example 12

[0133] Preparation: N-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)pyrrolidine-1-thiocarboxamide (C22)

[0134]

[0135] Preparation method: A reflux reaction apparatus was set up. Compound o-phenyl (4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)thiocarbamate (C20, 1 mmol) was added to a 25 mL round-bottom flask, dissolved in acetonitrile (MeCN), and then triethylamine (TEA, 3 mmol) was added. The mixture was stirred at room temperature, and then tetrahydropyrrole (2 mmol) was added. The temperature was raised to 65 °C and the reaction was carried out for 2 hours. After the reaction was confirmed to be complete by thin-layer chromatography (TLC), the solvent was removed by vacuum distillation. Acetonitrile was used to redissolve the crude product in ethyl acetate. The solution was transferred to a separatory funnel, washed three times with water, and then washed three times with saturated saline solution. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered to remove the anhydrous magnesium sulfate, and the solvent ethyl acetate was removed by vacuum distillation to obtain a pale yellow crude product. 1.5 times the volume of 200-300 mesh silica gel was added to prepare the precipitate, which was then loaded onto the column using a dry method. Using dichloromethane (DCM) and methanol (MeOH) as the mobile phase (the ratio of which was adjusted according to the specific polarity of the target product), the target product was purified by silica gel column chromatography in 79% yield.

[0136] Characterization data of the product: 1H NMR (400MHz, d-DMSO) δ9.63(s,1H),8.88(s,1H),8.54(s,2H),8.26(s,1H),7.82(dd,J=7.9,1.7Hz,1H),7.78–7.72(m,1H),7.53(d,J=8.5Hz,1H),7 .34(t,J=7.7Hz,1H),7.19(s,1H),6.91(dd,J=8.6,2.3Hz,1H),3.75(s,3H ),3.63(s,4H),3.45(p,J=6.8Hz,1H),1.94(s,4H),1.16(d,J=6.8Hz,6H). 13 C NMR(101MHz,DMSO-d6)δ177.82,158.14,155.53,151.05,138.38,137.91,135.65,131.4 0,124.65,124.49,124.10,123.30,117.75,109.99,104.80,56.03,55.33,15.32.ESI-MS m / z:561.2[M+H] + calcd for C 25 H 29 ClN6O3S2:560.14.

[0137] Example 13

[0138] Preparation: N-(4-(5-chloro-4-(2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)piperidine-1-thiocarboxamide (C23)

[0139]

[0140] Preparation method: A reflux reaction apparatus was set up. Compound o-phenyl (4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)thiocarbamate (C20, 1 mmol) was added to a 25 mL round-bottom flask, dissolved in acetonitrile (MeCN), and then triethylamine (TEA, 3 mmol) was added. While stirring at room temperature, piperidine (2 mmol) was added, and the mixture was heated to 65 °C and reacted for 2 hours. After the reaction was confirmed to be complete by thin-layer chromatography (TLC), the solvent acetonitrile was removed by vacuum distillation. The nitrile was redissolved in ethyl acetate, transferred to a separatory funnel, washed three times with water, and then three times with saturated saline solution. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered to remove the anhydrous magnesium sulfate, and the solvent ethyl acetate was removed by vacuum distillation to obtain a pale yellow crude product. 1.5 times the volume of 200-300 mesh silica gel was added to prepare the precipitate, which was then loaded onto the column using a dry method. The target product was purified by silica gel column chromatography using dichloromethane (DCM) and methanol (MeOH) as the mobile phase (the ratio of which was adjusted according to the specific polarity of the target product), with a yield of 75%.

[0141] Characterization data of the product: 1 H NMR (400MHz, CDCl3) δ9.52(s,1H),8.53(dd,J=8.4,1.1Hz,1H),8.22(d,J=8.5Hz,1H),8. 15(s,1H),7.90(dd,J=7.9,1.6Hz,1H),7.66(ddd,J=8.7,7.4,1.7Hz,1H),7.52(s,1H),7. 27(d,J=7.6Hz,1H),7.15(s,1H),6.83(d,J=2.3Hz,1H),6.65(dd,J=8.6,2.3Hz,1H),3.87 (s,3H),3.82(t,J=5.5Hz,4H),3.24(q,J=6.8Hz,1H),1.68(s,6H),1.30(d,J=6.8Hz,6H). 13 C NMR (101MHz, CDCl3) δ182.35,157.36,155.34,155.23,148.16,138.27,134.53,134.50,131.25,1 26.41,123.96,123.41,118.87,115.94,107.06,55.89,55.59,50.43,25.56,24.16,15.38.ESI-MS m / z:575.14[M+H] + ,calcd forC 26 H 31 ClN6O3S2:574.16.

[0142] Example 14

[0143] Preparation: N-(4-(5-chloro-4-(2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-4-methylpiperazine-1-thiocarboxamide (C24)

[0144]

[0145] Preparation method: A reflux reaction apparatus was set up. Compound o-phenyl (4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)thiocarbamate (C20, 1 mmol) was added to a 25 mL round-bottom flask, dissolved in acetonitrile (MeCN), and then triethylamine (TEA, 3 mmol) was added. While stirring at room temperature, N-methylpiperazine (2 mmol) was added, and the mixture was heated to 65 °C and reacted for 2 hours. After the reaction was confirmed to be complete by thin-layer chromatography (TLC), the solvent was removed by vacuum distillation. Acetonitrile was used as a solvent, and the crude product was redissolved in ethyl acetate. The solution was transferred to a separatory funnel, washed three times with water, and then washed three times with saturated saline solution. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered to remove the anhydrous magnesium sulfate, and the solvent ethyl acetate was removed by negative pressure distillation to obtain a pale yellow crude product. 1.5 times the volume of 200-300 mesh silica gel was added to prepare a column chromatography solution, which was then loaded onto the column using a dry method. Using dichloromethane (DCM) and methanol (MeOH) as the mobile phase (the ratio of which was adjusted according to the specific polarity of the target product), the target product was purified by silica gel column chromatography in 70% yield.

[0146] Characterization data of the product: 1 H NMR (400MHz, CDCl3) δ9.51 (s, 1H), 8.52 (dd, 1H), 8.19 (d, J = 8.6Hz, 1H), 8.11 (s, 1H) ),7.87(dd,J=8.0,1.7Hz,1H),7.69–7.60(m,2H),7.52(s,1H),7.23(dd,J=7.5,1.1 Hz,1H),6.83(d,J=2.3Hz,1H),6.67(dd,J=8.6,2.1Hz,1H),3.94(t,J=5.1Hz,4H),3 .84(s,3H),3.22(p,J=6.8Hz,1H),2.58(t,4H),2.37(s,3H),1.29(d,J=6.9Hz,6H). 13CNMR(101MHz, CDCl3)δ183.02,157.29,155.31,155.13,148.16,138.25,134.59,134.12,131.23,126.58,1 24.72,123.83,123.40,118.88,116.16,107.20,106.30,55.90,55.62,54.14,48.35,45.37,15.37.ESI-MS m / z:590.2[M+H] + ,calcd forC 26 H 32 ClN7O3S2:589.17.

[0147] Example 15

[0148] Preparation: N-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-4-ethylpiperazine-1-thiocarboxamide (C25)

[0149]

[0150] Preparation method: A reflux reaction apparatus was set up. Compound o-phenyl (4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)thiocarbamate (C20, 1 mmol) was added to a 25 mL round-bottom flask and dissolved in acetonitrile (MeCN). Triethylamine (TEA, 3 mmol) was added, and the mixture was stirred at room temperature. N-ethylpiperazine (2 mmol) was then added, and the mixture was heated to 65 °C for 2 hours. After the reaction was confirmed to be complete by thin-layer chromatography (TLC), the solvent was removed by vacuum distillation. Acetonitrile was used as a solvent, and the crude product was redissolved in ethyl acetate. The solution was transferred to a separatory funnel, washed three times with water, and then washed three times with saturated saline solution. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered to remove the anhydrous magnesium sulfate, and the solvent ethyl acetate was removed by negative pressure distillation to obtain a pale yellow crude product. 1.5 times the volume of 200-300 mesh silica gel was added to prepare a column chromatography solution, which was then loaded onto the column using a dry method. Using dichloromethane (DCM) and methanol (MeOH) as the mobile phase (the ratio of which was adjusted according to the specific polarity of the target product), the target product was purified by silica gel column chromatography in 70% yield.

[0151] Characterization data of the product: 1H NMR(400MHz, CDCl3)δ9.53(s,1H),8.53(dd,J=8.4,1.1Hz,1H),8.23(d,J=8.6Hz,1H),8.15(s,1H),7 .91(d,J=8.0Hz,1H),7.66(ddd,J=8.7,7.4,1.7Hz,1H),7.54(s,1H),7.27(d,J=7.0Hz,1H),7.25–7. 20(m,1H),6.82(d,J=2.4Hz,1H),6.65(dd,J=8.6,2.3Hz,1H),3.91(t,4H),3.87(s,3H),3.23(p,J=6 .9Hz,1H),2.55(t,J=5.1Hz,4H),2.49(q,J=7.2Hz,2H),1.30(d,J=6.9Hz,6H),1.12(t,J=7.2Hz,3H). 13 CNMR (101MHz, CDCl3) δ182.88,157.31,155.35,155.18,148.18,138.24,134.55,134.12,131.26,126. 60,124.86,123.92,123.44,118.86,116.00,107.03,55.90,55.62,52.08,48.75,15.38,11.75.ESI-MS m / z:604.13[M+H] + calcd for C 27 H 34 ClN7O3S2:603.19.

[0152] Example 16

[0153] Preparation: N-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)morpholine-4-thiocarboxamide (C26)

[0154]

[0155] Preparation method: A reflux reaction apparatus was set up. Compound o-phenyl (4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)thiocarbamate (C20, 1 mmol) was added to a 25 mL round-bottom flask, dissolved in acetonitrile (MeCN), and then triethylamine (TEA, 3 mmol) was added. The mixture was stirred at room temperature, and then morpholine (2 mmol) was added. The temperature was raised to 65 °C and reacted for 2 hours. After the reaction was confirmed to be complete by thin-layer chromatography (TLC), the solvent acetonitrile was removed by vacuum distillation. The nitrile was redissolved in ethyl acetate, the crude product was transferred to a separatory funnel, washed three times with water, and then washed three times with saturated saline solution. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered to remove the anhydrous magnesium sulfate, and the solvent ethyl acetate was removed by vacuum distillation to obtain a pale yellow crude product. 1.5 times the volume of 200-300 mesh silica gel was added to prepare the precipitate, and the sample was loaded onto the column using a dry method. Using dichloromethane (DCM) and methanol (MeOH) as the mobile phase (the ratio of which was adjusted according to the specific polarity of the target product), the target product was purified by silica gel column chromatography in 79% yield.

[0156] Characterization data of the product: 1 H NMR(400MHz, CDCl3)δ9.53(s,1H),8.53(d,J=8.4Hz,1H),8.23(d,J=8.6Hz,1H),8 .14(s,1H),7.89(dd,J=8.0,1.6Hz,1H),7.68–7.62(m,1H),7.54(s,1H),7.36(s, 1H),7.27–7.23(m,1H),6.84(d,J=2.3Hz,1H),6.67(dd,J=8.7,2.3Hz,1H),3.89– 3.81(m,7H),3.74(t,J=4.8Hz,4H),3.22(p,J=6.8Hz,1H),1.30(d,J=6.9Hz,6H). 13 C NMR (101MHz, CDCl3) δ183.47,157.30,155.35,155.16,148.20,138.28,134.52,133.92,131.26,126.80,1 24.84,123.86,123.40,118.87,116.21,107.20,106.37,66.18,55.91,55.63,49.16,29.70,15.36.ESI-MS m / z:577.3[M+H] + calcd for C 25 H 29 ClN6O4S2:576.14.

[0157] Example 17

[0158] Preparation: 1-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)-3-(2-morpholinoethyl)thiourea (C27)

[0159]

[0160] Preparation method: A reflux reaction apparatus was set up. Compound o-phenyl (4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)thiocarbamate (C20, 1 mmol) was added to a 25 mL round-bottom flask, dissolved in acetonitrile (MeCN), and then triethylamine (TEA, 3 mmol) was added. While stirring at room temperature, N-(2-aminoethyl)morpholine (2 mmol) was added, and the mixture was heated to 65 °C and reacted for 2 hours. After the reaction was confirmed to be complete by thin-layer chromatography (TLC), the mixture was distilled under negative pressure. After removing the solvent acetonitrile, the crude product was redissolved in ethyl acetate, transferred to a separatory funnel, washed three times with water, and then washed three times with saturated saline solution. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered to remove the anhydrous magnesium sulfate, and the solvent ethyl acetate was removed by negative pressure distillation to obtain a pale yellow crude product. 1.5 times the volume of 200-300 mesh silica gel was added to prepare the precipitate, and the sample was loaded by dry chromatography. Using dichloromethane (DCM) and methanol (MeOH) as the mobile phase (the ratio of which was adjusted according to the specific polarity of the target product), the target product was purified by silica gel column chromatography in 74% yield.

[0161] Characterization data of the product: 1 H NMR (400MHz, CDCl3) δ9.58 (s, 1H), 8.53 (dd, J = 8.4, 1.1Hz, 1H), 8.32 (dd, 1H), 8.15 (s ,1H),7.91(dd,J=8.0,1.5Hz,1H),7.66(ddd,J=8.7,7.4,1.6Hz,1H),7.57(s,1H),7. 29–7.24(m,1H),6.88(s,1H),6.81(dd,J=8.6,2.2Hz,1H),3.88(s,3H),3.83(s,2H), 3.67(t,4H),3.23(p,J=6.8Hz,1H),2.81(s,2H),2.67(s,4H),1.30(d,J=6.9Hz,6H). 13C NMR (101MHz, CDCl3) δ155.36,155.09,148.58,138.22,134.52,131.32,124.85,123.7 2,123.46,119.19,117.61,107.97,106.72,65.69,56.02,55.66,52.95,15.36.ESI-MS m / z:620.26[M+H] + calcd for C 27 H 34 ClN7O4S2:619.18.

[0162] Example 18

[0163] Preparation: 1-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-3-(3-morpholinopropyl)thiourea (C28)

[0164]

[0165] Preparation method: A reflux reaction apparatus was set up. Compound o-phenyl (4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)thiocarbamate (C20, 1 mmol) was added to a 25 mL round-bottom flask, dissolved in acetonitrile (MeCN), and then triethylamine (TEA, 3 mmol) was added. While stirring at room temperature, N-(3-aminopropyl)morpholine (2 mmol) was added, and the mixture was heated to 65 °C and reacted for 2 hours. After the reaction was confirmed to be complete by thin-layer chromatography (TLC), the mixture was distilled under negative pressure. After removing the solvent acetonitrile, the crude product was redissolved in ethyl acetate, transferred to a separatory funnel, washed three times with water, and then washed three times with saturated saline solution. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered to remove the anhydrous magnesium sulfate, and the solvent ethyl acetate was removed by negative pressure distillation to obtain a pale yellow crude product. 1.5 times the volume of 200-300 mesh silica gel was added to prepare the precipitate, and the sample was loaded by dry chromatography. Using dichloromethane (DCM) and methanol (MeOH) as the mobile phase (the ratio of which was adjusted according to the specific polarity of the target product), the target product was purified by silica gel column chromatography in 74% yield.

[0166] Characterization data of the product: 1H NMR (400MHz, CDCl3) δ9.58(s,1H),8.53(d,J=8.4Hz,1H),8.35(d,J=8.6Hz,1H),8.15(s,1H) ,7.91(dd,J=8.0,1.6Hz,1H),7.69–7.61(m,1H),7.56(s,1H),7.28(d,J=7.6Hz,1H),7.07(d, J=5.4Hz,1H),6.84–6.71(m,2H),3.88(s,3H),3.74(s,2H),3.39(s,4H),3.23(p,J=6.8Hz,1H ), 2.41 (t, J = 6.3Hz, 2H), 2.33 (t, J = 4.7Hz, 4H), 1.77 (p, J = 6.3Hz, 2H), 1.30 (d, J = 6.8Hz, 6H). 13 C NMR (101MHz, CDCl3) δ157.14,155.38,155.12,138.22,134.44,131.35,124.95,123.68,123.48,66.25,56.01,55.63,53.61,15.36,1.02.ESI-MS m / z:634.32[M+H] + ,calcd forC 28 H 36 ClN7O4S2:633.2.

[0167] Example 19

[0168] Preparation: 1-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)-3-phenylthiourea (C29)

[0169]

[0170] Preparation method: A reflux reaction apparatus was set up. Compound o-phenyl (4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)thiocarbamate (C20, 1 mmol) was added to a 25 mL round-bottom flask, dissolved in acetonitrile (MeCN), and then triethylamine (TEA, 3 mmol) was added. The mixture was stirred at room temperature, and aniline (2 mmol) was added. The temperature was raised to 65 °C and reacted for 2 hours. After the reaction was confirmed to be complete by thin-layer chromatography (TLC), the solvent acetonitrile was removed by vacuum distillation. The nitrile was redissolved in ethyl acetate, transferred to a separatory funnel, washed three times with water, and then washed three times with saturated saline solution. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered to remove the anhydrous magnesium sulfate, and the solvent ethyl acetate was removed by vacuum distillation to obtain a pale yellow crude product. 1.5 times the volume of 200-300 mesh silica gel was added to prepare a column chromatography slurry, which was then loaded onto the column using a dry method. Using dichloromethane (DCM) and methanol (MeOH) as the mobile phase (the ratio of which was adjusted according to the specific polarity of the target product), the target product was purified by silica gel column chromatography in 71% yield.

[0171] Characterization data of the product: 1 H NMR (400MHz, CDCl3) δ9.62 (s, 1H), 8.50 (d, J = 8.3Hz, 1H), 8.27 (d, J = 8.6Hz, 1H) ,8.15(s,1H),7.96(d,J=7.6Hz,1H),7.90(dd,J=7.9,1.6Hz,1H),7.74–7.60(m, 2H),7.41(d,J=4.3Hz,4H),7.29–7.25(m,2H),7.03(s,1H),6.84(dd,J=8.5,2.2 Hz,1H),5.29(s,1H),3.89(s,3H),3.22(p,J=6.9Hz,1H),1.30(d,J=6.8Hz,6H). 13 C NMR (101MHz, CDCl3) δ156.96,155.48,154.46,148.64,138.06,134.55,131.33,129.52,126 .97,125.18,125.03,123.87,123.68,119.30,117.75,108.24,56.04,55.70,15.37.ESI-MS m / z:583.3[M+H] + calcd for C 27 H 27 ClN6O4S:582.1. Study on the binding ability of Examples 1-19 with DCLK1

[0172] The binding kinetic measurements were performed on a FortéBio Octet Red 96 using bio-layer interferometry binding assays (BLI) to evaluate the binding ability of Examples 1-19 of the present invention to DCLK1.

[0173] Following the manufacturer's instructions, purified DCLK1 protein was biotinylated using NHS-PEG12-biotin (Thermo Fisher, 21312), and unbound NHS-PEG12-biotin was removed by passing the protein through a chromatographic column (GENEMORE, #G-MM-IGT). The biotinylated DCLK1 protein was immobilized onto a superstreptavidin (SSA) sensor (#18-5057, Octet) and electrophoresis was performed in a 200 μL buffer containing 25 mM HEPES, 150 mM NaCl, and 0.02% (v / v) Tween-20. After equilibration, association and dissociation measurements were performed using a positive control (TAE684) and a dilution (200 μM) of 18 novel compounds designed according to this invention. The binding ability of Examples 1-18 to the target protein was determined at the same drug concentration (200 μM) and compared with the lead compound TAE684 (see Table 1 for details). The optimal compound C24 was selected, which has a higher binding ability to DCLK1 than TAE684.

[0174] Table 1 shows the BLI experimental results of TAE684 at the same drug concentration (200 μM) in Implementation Case 1-19, comparing the activity levels in terms of response values ​​(nm).

[0175]

[0176]

[0177] Evaluation of the inhibitory effect of the optimal compound C24 on the NF-κB pathway

[0178] To evaluate the inhibitory effect of the optimal compound C24 on NF-κB pathway activation, Western blot (WB) was used.

[0179] Mouse mononuclear macrophage leukemia cells (RAW264.7) were pretreated with DMSO (at the same level as the treatment group), C24, or TAE684 for 0.5 h, followed by stimulation with 0.5 μg / mL LPS for 6 h. Proteins were isolated from the cells using lysis buffer or a nuclear and cytoplasmic protein extraction kit. The cells were centrifuged at 12000 rpm for 10 min at 4 °C, and the supernatant was collected. The concentration of each sample was determined using a BCA protein assay kit and normalized to the same protein concentration. After diluting the protein with 5x protein loading buffer, the protein was denatured at 100 °C for 10 min. The protein was loaded and separated into 8%–10% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes (PVDF, Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. The membranes were blocked with skim milk powder at room temperature and incubated overnight with a specific primary antibody at 4 °C on a shaker. The specific primary antibodies involved in this invention include β-actin, IκB-α, p65, and PCNA (all obtained from Cell Signaling Technology). After washing three times with TBST for 5 minutes each time, the cells were incubated with secondary antibodies at room temperature for 1 hour, followed by three more washes with TBST for 5 minutes each time. Chemiluminescence was then performed for observation. After visualization of the protein bands, the band intensity was quantitatively analyzed using density analysis on ImageJ software. The results are detailed in [link to relevant documentation]. Figure 1 .

[0180] Figure 1 The results showed that both C24 and TAE684 could reduce the activation of the NF-κB pathway to some extent, manifested as a reduction in the degradation of IκB-α and a decrease in the p65 nuclear translocation level of NF-κB. Quantitative analysis showed that C24 had a better inhibitory effect than TAE684.

[0181] Study on the protective effect of the optimal compound C24 against acute lung injury in mice.

[0182] Acute lung injury (ALI) is a type of pneumonia caused by various factors inside and outside the lungs, such as bacterial / viral pneumonia. During its development, there is an excessive inflammatory response. When pathogenic microorganisms invade the body, neutrophils and macrophages are activated to play a host defense role, producing a large number of pro-inflammatory cytokines and chemokines, causing inflammatory response and tissue damage.

[0183] The mortality rate for ALI patients remains as high as 35%-45%, and there are currently no effective treatments. Therefore, further research into effective treatments and elucidation of their underlying mechanisms are crucial for preventing and treating acute lung injury and reducing mortality. Clinical and animal experiments have confirmed that lipopolysaccharide (LPS) can activate multiple downstream pro-inflammatory signaling pathways and trigger the excessive production of inflammatory factors, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Therefore, inhibiting the release of inflammatory factors such as TNF-α and IL-6 has become an important approach to treating acute lung injury. This invention reports the protective effects of the optimal compound C24 and the lead compound TAE684 on a mouse model of LPS-induced acute lung injury and evaluates their anti-inflammatory capabilities.

[0184] Twenty-four 6-8 week old male C57BL / 6 mice were randomly divided into four groups of six each: normal control group (Con group), LPS model group (LPS group), LPS plus C24 group (LPS+C24 group), and LPS plus NVP-TAE684 group (LPS+NVP-TAE684 group). The LPS+C24 group and the LPS+NVP-TAE684 group were administered C24 or NVP-TAE684 (10 mg / kg) by gavage half an hour beforehand. The LPS group, LPS+C24 group, and LPS+NVP-TAE684 group were administered LPS (5 mg / kg) via tracheal infusion. The Con group was administered an equal volume of physiological saline via tracheal infusion. The modeling time was 6 hours.

[0185] After modeling, lung tissue from mice in each group was paraffin-embedded, sectioned, and stained with hematoxylin / eosin (H&E). The results showed that, compared with the LPS group, the LPS+C24 group and the LPS+NVP-TAE684 group exhibited significantly improved alveolar wall thickening in lung tissue, with the optimal compound C24 showing superior performance. Figure 2 (A)

[0186] After modeling, lung tissue from mice in each group was collected for wet and dry weight measurement, and the wet / dry (W / D) ratio was calculated. The results showed that both the LPS+C24 group and the LPS+NVP-TAE684 group reduced LPS-induced pulmonary edema compared to the LPS group. Figure 2 (B)

[0187] After modeling, the total protein concentration in bronchoalveolar lavage fluid (BALF) of mice in each group was quantified. The results showed that, compared with the LPS group, the LPS+C24 group and the LPS+NVP-TAE684 group significantly reduced the total protein concentration in LPS-induced BALF and weakened the degree of pulmonary microvascular barrier damage. Figure 2 Among them, the best compound C24 performed better.

[0188] After modeling, paraffin sections of lung tissue from mice in each group were subjected to immunohistochemical staining for the macrophage marker F4 / 80. The results showed that the LPS+C24 group and the LPS+NVP-TAE684 group significantly reduced the number of macrophages compared with the LPS group, and the optimal compound C24 performed better. Figure 3 ).

[0189] After modeling, mRNA was extracted from lung tissue homogenates of mice in each group. qRT-PCR detection showed that the LPS+C24 group and the LPS+NVP-TAE684 group significantly reduced the production of inflammatory factors compared to the LPS group. Figure 4 ).

[0190] Study on the protective effect of the optimal compound C24 on septic mice

[0191] Sepsis is a common, life-threatening disease caused by acute inflammation. It is a syndrome of organ dysfunction resulting from a dysregulation of the host's systemic response to infection. In sepsis, various pro-inflammatory cytokines are synthesized and released from the innate immune system as a defense mechanism against invading pathogens and infections. However, the systemic release of large amounts of pro-inflammatory cytokines during immune activation often leads to widespread inflammation, multiple organ failure, and even death.

[0192] This invention reports the protective effects of the optimal compound C24 and the lead compound TAE684 on a mouse model of sepsis, with mouse survival rate as the evaluation index.

[0193] Sepsis was induced in mice using bacterial infection. Escherichia coli strain BL21 was amplified in LB broth, and its density was measured at 600 nm using a NanoDrop 2000 spectrophotometer. The corresponding colony-forming units (CFU) were determined on LB agar plates. 0.2 mL of PBS (2 × 10⁻⁶) was added... 9 Live *E. coli* (CFU / mouse) was injected into the peritoneal cavity of 8-week-old male C57BL / 6 mice. To test the pharmacological effects of DCLK1 inhibitors in these models, 8-week-old male C57BL / 6 mice were pretreated with 20 mg / kg C24, TAE684, or a carrier (5% DMSO, 30% PEG-400, and 65% saline) via intragastric injection twice daily for three days prior to *E. coli* injection. Treatment with the compound or carrier continued twice daily until mouse death or the experiment ended on day seven. Mice survival was monitored during the experiment, every 12 hours for 7 days. Data were analyzed using logistic survival curves (n=8 per group).

[0194] Observation of the survival rate of mice with Escherichia coli-induced sepsis showed that ( Figure 5Compared with mice induced by E. coli alone, mice treated with C24 and TAE684 showed improved survival rates, with C24 showing a more significant effect.

[0195] The embodiments described above provide a detailed explanation of the technical solutions and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, and equivalent substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt thereof: ； In the formula, X is a sulfur atom or an oxygen atom; R is dimethylamino, tetrahydropyrrole, piperidinyl, N-methylpiperazinyl, N-ethylpiperazinyl, morpholinyl, N-(2-aminoethyl)morpholinyl, N-(3-aminopropyl)morpholinyl, aniline or phenolyl.

2. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The compound of formula (I) is selected from: (4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenylcarbamate, 3-(4-(5-chloro-4-(2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-1,1-dimethylurea, N-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)pyrrolidine-1-carboxamide, N-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)piperidine-1-carboxamide, N-(4-(5-chloro-4-(2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-4-methylpiperazine-1-carboxamide, N-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-4-ethylpiperazine-1-carboxamide, N-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)morpholine-4-carboxamide, 1-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-3-(2-morpholinoethyl)urea, 1-(4-(5-chloro-4-((2-isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-3-(3-morpholinopropyl)urea, 1-(4-(5-chloro-4-(2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-3-phenylurea, 3-(4-(5-chloro-4-(2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-1,1-dimethylthiourea, N-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)pyrrolidine-1-thiocarboxamide, N-(4-(5-chloro-4-(2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)piperidine-1-thiocarboxamide, N-(4-(5-chloro-4-(2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-4-methylpiperazine-1-thiocarboxamide, N-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-4-ethylpiperazine-1-thiocarboxamide, N-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)morpholine-4-thiocarboxamide, 1-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)-3-(2-morpholinoethyl)thiourea, 1-(4-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-ylamino)-3-methoxyphenyl)-3-(3-morpholinopropyl)thiourea, 1-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)-3-phenylthiourea.

3. A pharmaceutical composition, characterized in that, It includes compounds of formula (I) as described in claim 1 or 2, or pharmaceutically acceptable salts thereof; it also includes pharmaceutically acceptable excipients.

4. Use of a compound of formula (I) as described in claim 1 or 2, or a pharmaceutically acceptable salt thereof, in the preparation of a DCLK1 kinase inhibitor.

5. The use of the pharmaceutical composition as described in claim 3 in the preparation of a DCLK1 kinase inhibitor.

6. The use of a compound of formula (I) as described in claim 1 or 2, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in claim 3, in the preparation of a medicament for the prevention and / or treatment of an inflammatory disease, wherein the inflammatory disease is acute lung injury.

Images