DCLK1 inhibitor, preparation method, pharmaceutical composition and application

Abstract

The present invention discloses a DCLK1 inhibitor, a preparation method, a pharmaceutical composition and an application. The structure of the DCLK1 inhibitor is as shown in formula (I), and further comprises its isomers, pharmaceutically acceptable salts or mixtures thereof. The DCLK1 inhibitor and its pharmaceutical composition have a highly effective inhibitory effect on DCLK1, can be prepared as a therapeutic drug for tumor diseases such as pancreatic cancer, can exert drug efficacy at the molecular level, the cellular level and the animal level, especially the in vivo anti-tumor activity is better than that of the positive control drug, and the synthesis method of the compound is simple and easy.

Description

Technical Field

[0001] The present invention relates to a DCLK1 inhibitor, a preparation method, a pharmaceutical composition and an application, and in particular to a DCLK1 inhibitor which can be prepared as a cancer treatment drug, a preparation method, a pharmaceutical composition and an application. Background Art

[0002] Cancer refers to malignant tumors that originate from epithelial tissues. It is the most common type of malignant tumors and is common in our daily lives. Because early cancer development is too hidden, many patients are already in the late stage when they are discovered. Many cancer cells will also metastasize and spread, making treatment difficult and with a very high mortality rate. At present, the main treatments for cancer include surgery, radiotherapy, chemotherapy, immunotherapy, etc. These treatments are not only long-term, but also expensive, bringing heavy economic burdens and mental pressures to patients and their families. Most cancer patients usually take conservative treatments such as chemotherapy. Although the investment in the research and development of anticancer drugs worldwide is increasing, on the one hand, it brings more choices to patients, but also exposes the defects of existing anticancer drugs such as drug resistance after long-term use and toxic side effects caused by low selectivity. Although traditional chemotherapy drugs and targeted drugs for the treatment of cancer can effectively improve the patient's condition, they also have their own limitations. Therefore, the search for new treatments, especially drugs that act on new targets, has attracted much attention.

[0003] Doublecortin-like Kinase 1 (DCLK1) is a transmembrane microtubule-associated protein kinase originally discovered in the nervous system. It has the biological functions of regulating microtubule polymerization and promoting neuronal migration, and its domain has been shown to be indispensable for maintaining these biological functions. DCLK1 has a low level of expression in normal gastrointestinal cells, but is overexpressed in some malignant tumors of the digestive system, such as esophageal cancer, gastric cancer, colorectal cancer, and pancreatic cancer. Therefore, DCLK1 is considered to be a new gastrointestinal stem cell tumor marker, which also makes it a new target for cancer treatment. At present, no drugs targeting DCLK1 have been successfully marketed. Based on this, the research on new DCLK1 selective inhibitors provides new treatment options and options for the treatment of various digestive tract cancers such as pancreatic cancer, and also provides probe molecules for in-depth research on the biological functions of DCLK1. Summary of the invention

[0004] Purpose of the invention: In view of the shortcomings of existing compounds such as weak inhibitory activity on DCLK1, the present invention aims to provide a class of DCLK1 inhibitors with excellent anti-tumor activity, preparation methods, pharmaceutical compositions and applications.

[0005] Technical solution: As the first aspect of the present invention, the DCLK1 inhibitor of the present invention has a structure of formula (I), which also includes isomers, pharmaceutically acceptable salts or mixtures thereof,

[0006]

[0007] in:

[0008] Y 1 , Y 2 , Y 3 are independently selected from CH or N;

[0009] Z 1 , Z 2 are independently selected from O, S or NH;

[0010] R 1 is selected from H, C1-C6 alkyl, C3-C8 cycloalkyl, CH 2 CF 3 or CH 2 CHF 2 ;

[0011] R 2 Selected from SO 2 R 6 or SOR 6 , where R 6 Select C1-C6 alkyl, C3-C8 cycloalkyl, CF 3 or CH 2 CF 3 ;

[0012] R 3 Selected from H, F, Cl, Br, I, CF 3 , CHF 2 、CN、OCH 3 , OH or CH 3 ;

[0013] R 4 Selected from H, CH 3 , CH 2 CH 3 、CH(CH 3 ) 2 ;

[0014] R 5 Selected from H, F, Cl, Br, I, CF 3 , CHF 2 、CN、OCH 3 , CH 3, pyrrolidin-1-yl, piperidin-1-yl, morpholin-1-yl, piperazin-1-yl, 3,5-dimethylpiperazin-1-yl, 4-methylpiperazin-1-yl, 4-ethylpiperazin-1-yl, 4-(2,2,2-trifluoroethyl)piperazin-1-yl or 4-isobutylpiperazin-1-yl.

[0015] Preferably, in the above structure:

[0016] Y 1 , Y 2 , Y 3 are independently selected from CH or N; Z 1 , Z 2 are independently selected from O or NH;

[0017] R 1 Selected from H, CH 3 , CH 2 CH 3 , CH 2 CHF 2 or CH 2 CF 3 ;

[0018] R 2 Selected from SO 2 R 6 , where R 6 Alkyl selected from C1-C6, CF 3 or CH 2 CF 3 ;

[0019] R 3 Selected from H, F, Cl, Br, CF 3 、OCH 3 or CH 3 ;

[0020] R 4 Selected from CH 3 or CH 2 CH 3 ;

[0021] R 5 Selected from H, F, Cl, Br, CF 3 , CH 3 , pyrrolidin-1-yl, piperidin-1-yl, morpholin-1-yl, piperazin-1-yl, 4-methylpiperazin-1-yl, 4-ethylpiperazin-1-yl or 4-(2,2,2-trifluoroethyl)piperazin-1-yl.

[0022] More preferably, the above DCLK1 inhibitor is selected from any one of the following compounds:

[0023]

[0024]

[0025]

[0026]

[0027] The pharmaceutically acceptable salt of the above-mentioned DCLK1 inhibitor is a salt formed by the above-mentioned compound and an acid, wherein the acid is selected from hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid or ferulic acid.

[0028] As the second aspect of the present invention, the preparation method of the above-mentioned DCLK1 inhibitor is as follows:

[0029] Method 1: When R 1 For H, Y 1 , Y 2 When is CH or N and is not CH at the same time, the preparation method of compound IA is as follows:

[0030]

[0031] Specifically, compound IV is prepared by reacting compound II with a phenylene sulfide derivative III, and the base used is selected from triethylamine, pyridine, N,N-diisopropylethylamine (DIPEA), 4-dimethylaminopyridine (DMAP), potassium carbonate, sodium carbonate, potassium tert-butoxide, sodium tert-butoxide, cesium carbonate or sodium acetate, preferably N,N-diisopropylethylamine (DIPEA); the solvent used is selected from N,N-dimethylformamide (DMF), N,N-diethylacetamide (DMAc), N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO), isopropanol, ethanol or a mixed solvent composed of the above solvents, preferably isopropanol.

[0032] Compound V is prepared by reacting compound IV with m-chloroperbenzoic acid (m-CPBA), and the solvent used is selected from dichloromethane, chloroform, ethyl acetate, tetrahydrofuran, 1,4-dioxane, DMF or a mixed solvent of any two thereof, preferably dichloromethane, chloroform or DMF.

[0033] Compound IA is prepared by reacting compound V with compound VI, the acid used is selected from trifluoroacetic acid, trifluoromethanesulfonic acid, methanesulfonic acid, hydrochloric acid, sulfuric acid, p-toluenesulfonic acid monohydrate or a mixture of the above acids, preferably methanesulfonic acid or p-toluenesulfonic acid monohydrate; the solvent used is selected from dichloromethane, chloroform, ethyl acetate, isopropanol, ethanol, tetrahydrofuran, 1,4-dioxane, DMF or a mixed solvent of any two, preferably isopropanol.

[0034] Method 2: When R 1 When is not H, the preparation method of compound IB is as follows:

[0035]

[0036] Specifically, compound IV and R 1 X is reacted to prepare compound VII, the base used is selected from triethylamine, pyridine, DIPEA, DMAP, potassium carbonate, sodium carbonate, potassium tert-butoxide, sodium tert-butoxide, cesium carbonate or sodium acetate, preferably potassium carbonate; the solvent used is selected from dichloromethane, chloroform, ethyl acetate, tetrahydrofuran, 1,4-dioxane, DMF or a mixed solvent of any two, preferably tetrahydrofuran or DMF.

[0037] Compound VIII is prepared by reacting compound VII with m-CPBA, and the solvent used is selected from dichloromethane, chloroform, ethyl acetate, tetrahydrofuran, 1,4-dioxane, DMF or a mixed solvent of any two thereof, preferably dichloromethane, chloroform or DMF.

[0038] Compound IB is prepared by reacting compound VIII with compound VI, the acid used is selected from trifluoroacetic acid, trifluoromethanesulfonic acid, methanesulfonic acid, hydrochloric acid, sulfuric acid, p-toluenesulfonic acid monohydrate or a mixture of the above acids, preferably methanesulfonic acid or p-toluenesulfonic acid monohydrate; the solvent used is selected from dichloromethane, chloroform, ethyl acetate, isopropanol, ethanol, tetrahydrofuran, 1,4-dioxane, DMF or a mixed solvent of any two, preferably isopropanol.

[0039] Method 3: When R 1 For H, Y 1 , Y 2 When CH, the preparation method of compound IC is as follows:

[0040]

[0041]

[0042] Among them, Y 1 , Y 2 , Y 3 , Z 1 , Z 2 , R 1 , R 2 , R 3 , R 4 , R 5 or R 6 The definition of is as mentioned above.

[0043] Specifically, compound IX is prepared by reacting compound IV with p-toluenesulfonyl chloride (TosCl), and the base used is selected from triethylamine, sodium hydride, pyridine, DIPEA, DMAP, potassium carbonate, sodium carbonate, potassium tert-butoxide, sodium tert-butoxide, cesium carbonate or sodium acetate, preferably sodium hydride and potassium carbonate; the solvent used is selected from dichloromethane, chloroform, ethyl acetate, tetrahydrofuran, 1,4-dioxane, DMF or a mixed solvent of any two, preferably tetrahydrofuran or DMF.

[0044] Compound X is prepared by reacting compound IX with m-CPBA, and the solvent used is selected from dichloromethane, chloroform, ethyl acetate, tetrahydrofuran, 1,4-dioxane, DMF or a mixed solvent of any two thereof, preferably dichloromethane, chloroform or DMF.

[0045] Compound XI is prepared by reacting compound X with compound VI, wherein the catalyst used is selected from Pd 2 (dba) 3 、Pd(OAc) 2 , Pd 2 (dppf)Cl 2 、Pd(PPh 3 ) 4 , Pd 2 (dppf)Cl 2 dichloromethane complex or a mixture of the above catalysts, preferably Pd 2 (dba) 3 or Pd(OAc) 2 ; The base used is selected from triethylamine, sodium hydride, pyridine, DIPEA, DMAP, potassium carbonate, sodium carbonate, potassium tert-butoxide, sodium tert-butoxide, cesium carbonate or sodium acetate, preferably cesium carbonate; the solvent used is selected from toluene, chloroform, dichloromethane, acetone, acetonitrile, ether, tetrahydrofuran, 1,4-dioxane, DMF or a mixed solvent of any two of them, preferably toluene.

[0046] Compound IC is prepared by reacting compound X with a methanol solution of sodium methoxide, wherein the solvent used is selected from methanol, dichloromethane, chloroform, ethyl acetate, tetrahydrofuran, 1,4-dioxane, DMF or a mixed solvent of any two thereof, preferably methanol.

[0047] The corresponding acid is reacted with the compound I prepared by the above method to form a salt, thereby obtaining a pharmaceutically acceptable salt of the above DCLK1 inhibitor.

[0048] As the third aspect of the present invention, the pharmaceutical composition of the present invention comprises the above-mentioned DCLK1 inhibitor and a pharmaceutically acceptable carrier.

[0049] The above-mentioned DCLK1 inhibitors can be added with pharmaceutically acceptable carriers to prepare common pharmaceutical preparations, such as tablets, capsules, syrups, suspensions or injections. The preparations can be added with common pharmaceutical excipients such as flavors, sweeteners, liquid / solid fillers, diluents, etc.

[0050] As a fourth aspect of the present invention, the above-mentioned DCLK1 inhibitor and the pharmaceutical composition thereof can be prepared as a therapeutic drug for cancer, specifically a therapeutic drug for colorectal cancer, pancreatic cancer and other cancers.

[0051] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0052] (1) The DCLK1 inhibitor and its pharmaceutical composition can effectively inhibit DCLK1 activity at a concentration level of 1 μM, with the optimal inhibition rate reaching 89%; it can also inhibit the proliferation of various tumor cells, IC 50 Values ​​less than 10 μM;

[0053] (2) The DCLK1 inhibitor and its pharmaceutical composition are widely used and can be prepared as a therapeutic drug for pancreatic cancer and other tumors. At a lower dosage in animals, the anti-tumor activity is significantly better than that of positive drugs, and the tumor inhibition rate is higher than 50%;

[0054] (3) The preparation method of the compound is simple and easy, and suitable for scale-up. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 is the tumor weight after 21 days of administration of compound I-A5;

[0056] Figure 2 This is the anatomical image of the tumor 21 days after administration of compound I-A5;

[0057] Figure 3 The results of the effect of compound I-A5 on the tumor volume in the xenograft tumor model as a function of administration time. DETAILED DESCRIPTION

[0058] The technical solution of the present invention is further described below in conjunction with embodiments.

[0059] Example 1: N 4 -(2-(isopropylsulfonyl)phenyl)-N 6 Synthesis of -(2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine (I-A1)

[0060] Synthesis of 6-chloro-N-(2-(isopropylthio)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (IV-1)

[0061] To a 100 mL three-necked flask, add 4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (II-1, 2.0 g, 10.58 mmol) and isopropanol (40 mL), stir to dissolve, add 2-(isopropylthio)aniline (III-1, 1.84 mL, 11.64 mmol), heat to 85 °C for 5 hours, and monitor the reaction by thin layer chromatography (TLC) (petroleum ether: ethyl acetate = 3:1). The reaction is almost complete. After the reaction solution is cooled to room temperature, it is filtered, and the filter cake is washed with petroleum ether (10 mL × 2) and dried in vacuo to obtain 2.85 g of off-white solid IV-1 with a yield of 84.2%. 1 H NMR (300 MHz, DMSO-d 6 )δ13.67(s,1H),10.26(s,1H),7.62–7.54(m,1H),7.50–7.39(m,3H),7.39–7. 30(m,1H),3.59–3.44(m,1H),1.17(d,J=6.7Hz,6H).MS(ESI(+)70V)m / z[M+H] + :320.07.

[0062] Synthesis of 6-chloro-N-(2-(isopropylsulfonyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (V-1)

[0063] IV-1 (1.0 g, 3.13 mmol) and dichloromethane (75 mL) were added to a 250 mL three-necked flask, stirred to dissolve, cooled to 0 ° C, and a solution obtained by dissolving m-chloroperbenzoic acid (1.62 g, 9.39 mmol) in dichloromethane (20 mL) was slowly added dropwise. The dripping was completed in about 0.5 hours. TLC monitoring (petroleum ether: ethyl acetate = 1:1) showed that the reaction was basically complete. Saturated sodium sulfite solution (30 mL) was added to quench the reaction, and the dichloromethane layer was separated and extracted with dichloromethane (30 mL). The organic phases were combined, washed with saturated sodium chloride solution (15 mL × 3), and dried over anhydrous sodium sulfate; filtered, the filtrate was evaporated to remove the solvent under reduced pressure, and the crude product was separated and purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 25:1~10:1) to obtain 0.86 g of off-white solid V-1, with a yield of 78.2%. 1 H NMR (300 MHz, DMSO-d 6 )δ13.81(s,1H),10.32(s,1H),8.00(d,J=7.9Hz,1H),7.92–7.83(m,2H),7.65– 7.28(m,2H),3.52–3.36(m,1H),1.11(d,J=6.7Hz,6H).MS(ESI(+)70V)m / z[M+H] + :352.06.

[0064] N 4 -(2-(isopropylsulfonyl)phenyl)-N 6 Synthesis of -(2-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine (I-A1)

[0065] V-1 (150 mg, 0.43 mmol), o-anisidine (VI-1, 96 μL, 0.85 mmol), p-toluenesulfonic acid monohydrate (90 mg, 0.47 mmol) and isopropanol (4 mL) were added to a 15 mL pressure bottle, heated to 110 ° C with stirring in a closed manner, and reacted for 8 hours. The reaction solution was cooled to room temperature. TLC monitoring (petroleum ether: ethyl acetate = 2:1) showed that the reaction was basically complete. The reaction solution was evaporated under reduced pressure to remove the solvent, 10 mL of water was added to the residue, and extracted with ethyl acetate (10 mL×3). The organic phases were combined, washed with saturated sodium chloride solution (10 mL×2), and dried over anhydrous sodium sulfate; filtered, the filtrate was evaporated under reduced pressure to remove the solvent, and the crude product was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 5:1 to 2:1) to obtain 80 mg of brown solid I-A1 with a yield of 42.8%. 1 H NMR (300 MHz, CDCl 3 )δ9.80(s,1H),8.74(d,J=7.8Hz,1H),8.42(dd,J=7.7,1.5Hz,1H),7.94(s,1H),7.93–7.88(m,1H),7.79–7.60(m,2H), 7.31–7.27(m,1H),7.08–6.89(m,3H),3.92(s,3H),3.33–3.17(m,1H),1.31(d,J=6.8Hz,6H).MS(ESI(+)70V)m / z[M+H] + :439.15.

[0066] Example 2: N 4 -(2-(isopropylsulfonyl)phenyl)-N 6 Synthesis of -(2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine (I-A2)

[0067] Using V-1 (80 mg, 0.23 mmol) and 2-methoxy-4-(4-methylpiperazine-1-yl)aniline (VI-2, 50 mg, 0.23 mmol) as raw materials, referring to the preparation method of compound I-A1, 91 mg of light yellow solid I-A2 was obtained with a yield of 74.6%. 1 HNMR (300MHz, CDCl 3)δ9.73(s,1H),8.75(d,J=8.4Hz,1H),8.24(d,J=8.8Hz,1H),7.95–7.80(m,2H),7.72–7.61(m,1H),7.39(s,1H),7.25–7.21(m,1H),6.59–6.5 1(m,2H),3.88(s,3H),3.36–3.28(m,4H),3.27–3.17(m,1H),2.86–2.76(m,4H),2.50(s,3H),1.30(d,J=6.9Hz,6H).MS(ESI(+)70V)m / z[M+H] + :537.23.

[0068] Example 3: N 6 -(4-bromo-2-methoxyphenyl)-N 4 Synthesis of -(2-(isopropylsulfonyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine (I-A3)

[0069] Using compound V-1 (150 mg, 0.43 mmol) and 4-bromo-2-methoxyaniline (VI-3, 150 mg, 0.74 mmol) as raw materials, referring to the preparation method of compound I-A1, 120 mg of light yellow solid I-A3 was obtained with a yield of 54.4%. 1 H NMR (300 MHz, CDCl 3 )δ9.82(s,1H),8.67(d,J=8.4Hz,1H),8.34(d,J=8.6Hz,1H),7.96(s,1H),8.00–7.88(m,1H),7.78–7.62(m,2H),7. 31-7.22(m,1H),7.14–6.97(m,2H),3.91(s,3H),3.32–3.16(m,1H),1.31(d,J=6.7Hz,6H).MS(ESI(+)70V)m / z[M+H] + :517.06.

[0070] Example 4: N 4 -(2-(isopropylsulfonyl)phenyl)-N 6 Synthesis of -(2-methoxy-4-morpholinylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine (I-A4)

[0071] Using compound V-1 (150 mg, 0.43 mmol) and 2-methoxy-4-morpholinoaniline (VI-4, 89 mg, 0.43 mmol) as raw materials, referring to the preparation method of compound I-A1, 60 mg of brown solid I-A4 was obtained with a yield of 26.9%. 1 HNMR (300MHz, CDCl 3 )δ9.76(s,1H),8.74(d,J=7.9Hz,1H),8.19(d,J=8.7Hz,1H),8.02–7.77(m,2H),7.70–7.56(m,1H),7.47(s,1H),7.25–7.16(m,1H) ,6.66–6.37(m,2H),3.98–3.76(m,7H),3.32–3.19(m,1H),3.18–3.02(m,4H),1.29(dd,J=6.9,2.4Hz,6H).MS(ESI(+)70V)m / z[M+H] + :524.20.

[0072] Example 5: N 6 -(2-(isopropylsulfonyl)phenyl)-N 2 Synthesis of -(2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)-9H-purine-2,6-diamine (I-A5)

[0073] Synthesis of 2-chloro-N-(2-(isopropylthio)phenyl)-9H-purin-6-amine (IV-2)

[0074] To a 100 mL three-necked flask, add 2,6-dichloro-9H-purine (II-2, 1.50 g, 7.94 mmol) and isopropanol (35 mL) and stir to dissolve, add III-1 (1.33 g, 7.94 mmol), heat to 85 ° C and reflux for 5 hours. TLC monitoring (petroleum ether: ethyl acetate = 3:1) shows that the reaction is basically complete. The reaction solution is cooled to room temperature and filtered. The filter cake is washed with ethyl acetate (3 mL × 2) and vacuum dried to obtain 2.20 g of off-white solid IV-2 with a yield of 86.7%. 1 H NMR (300 MHz, DMSO-d 6 )δ9.60(s,1H),8.45(s,1H),8.09–8.02(m,1H),7.58(dd,J=7.7,1.5Hz,1H),7.47–7.36(m, 1H),7.28–7.16(m,1H),3.43–3.26(m,1H),1.19(d,J=6.7Hz,6H).MS(ESI(+)70V)m / z[M+H] + :320.07.

[0075] Synthesis of 2-chloro-N-(2-(isopropylsulfonyl)phenyl)-9H-purin-6-amine (V-2)

[0076] Using compound IV-2 (790 mg, 2.47 mmol) as raw material and referring to the preparation method of compound V-1, 650 mg of off-white solid V-2 was obtained with a yield of 74.8%. 1 H NMR (300 MHz, DMSO-d 6 )δ13.57(s,1H),10.12(s,1H),8.65(d,J=8.2Hz,1H),8.37(s,1H),8.02–7.73(m,2H), 7.46–7.33(m,1H),3.56–3.40(m,1H),1.16(d,J=6.8Hz,6H).MS(ESI(+)70V)m / z[M+H] + :352.06.

[0077] N 6 -(2-(isopropylsulfonyl)phenyl)-N 2 Synthesis of -(2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)-9H-purine-2,6-diamine (I-A5)

[0078] V-2 (120 mg, 0.34 mmol), VI-2 (83 mg, 0.38 mmol), methanesulfonic acid (44 μL, 0.68 mmol) and isopropanol (4 mL) were added to a 15 mL pressure bottle, and the mixture was heated to 110 °C with stirring in a sealed container for 8 hours. The reaction solution was cooled to room temperature. TLC monitoring (dichloromethane: methanol = 15:1) showed that the reaction was basically complete. The solvent was evaporated under reduced pressure, 15 mL of water was added to the residue, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (10 mL × 2), and dried over anhydrous sodium sulfate; the mixture was filtered, and the solvent was evaporated from the filtrate under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane: methanol = 60:1 to 40:1) to obtain 110 mg of brown solid I-A5 with a yield of 60.1%. 1 H NMR (300 MHz, CDCl 3)δ9.99(s,1H),8.87(d,J=8.4Hz,1H),8.01(d,J=8.7Hz,1H),7.90–7.83(m,1 H),7.68–7.57(m,1H),7.42(s,1H),7.23–7.14(m,1H),7.11(s,1H),6.54(d,J =2.4Hz,1H),6.45(dd,J=8.8,2.5Hz,1H),3.86(s,3H),3.38–3.17(m,5H),2. 94–2.76(m,4H),2.54(s,3H),1.29(d,J=6.8Hz,6H).MS(ESI(+)70V)m / z[M+H] + :537.23.

[0079] Example 6: N 6 -(2-(isopropylsulfonyl)phenyl)-N 2 Synthesis of -(2-methoxy-4-morpholinylphenyl)-9H-purine-2,6-diamine (I-A6)

[0080] Using compound V-2 (180 mg, 0.51 mmol) and VI-4 (117 mg, 0.56 mmol) as raw materials and referring to the preparation method of compound I-A5, 160 mg of brown solid I-A6 was obtained with a yield of 59.7%. 1 H NMR (300 MHz, CDCl 3 )δ10.03(s,1H),8.89(d,J=8.5Hz,1H),7.97(d,J=8.7Hz,1H),7.90–7.8 4(m,1H),7.67–7.58(m,1H),7.29(s,1H),7.22–7.14(m,1H),7.07(s,1H ),6.55(s,1H),6.47(d,J=8.5Hz,1H),3.89–3.83(m,7H),3.34–3.22(m,1H),3.16–3.05(m,4H),1.29(d,J=6.8Hz,6H).MS(ESI(+)70V)m / z[M+H] + :524.20.

[0081] Example 7: N 2 -(4-bromo-2-methoxyphenyl)-N 6 Synthesis of -(2-(isopropylsulfonyl)phenyl)-9H-purine-2,6-diamine (I-A7)

[0082] Using compound V-2 (100 mg, 0.28 mmol) and VI-3 (57 mg, 0.28 mmol) as raw materials and referring to the preparation method of compound I-A5, 88 mg of yellow solid I-A7 was obtained with a yield of 59.8%. 1 H NMR (400 MHz, CDCl 3 )δ9.12(s,1H),8.79(d,J=8.1.Hz,1H),8.21(d,J=7.3Hz,1H),7.57(d,J=7.5Hz,1H),7.44–7.38(m,1H),7.36(s,1H),7.16(s, 1H),7.07–7.01(m,2H),6.97(d,J=7.4Hz,1H),3.93(s,3H),3.26–3.18(m,1H),1.28(d,J=6.6Hz,6H).MS(ESI(+)70V)m / z[M+H] + :517.06.

[0083] Example 8: N 6 -(2-(isopropylsulfonyl)phenyl)-N 2 Synthesis of -(2-methoxyphenyl)-9H-purine-2,6-diamine (I-A8)

[0084] Using compound V-2 (100 mg, 0.28 mmol) and VI-1 (32 μL, 0.28 mmol) as raw materials and referring to the preparation method of compound I-A5, 90 mg of yellow solid I-A8 was obtained with a yield of 72.2%. 1 H NMR (400 MHz, CDCl 3 )δ10.09(s,1H),8.89(d,J=8.7Hz,1H),8.18(dd,J=7.8,1.6Hz,1H),7.88(dd,J=7.9,1.6Hz,1H),7.69–7.62(m,1H),7.36–7.32(m,2H),7 .23–7.17(m,1H),7.05–6.97(m,1H),6.96–6.90(m,2H),3.90(s,3H),3.35–3.22(m,1H),1.30(d,J=6.8Hz,6H).MS(ESI(+)70V)m / z[M+H] + :439.15.

[0085] Example 9: Synthesis of N-(2-methoxyphenyl)-7-methyl-4-(2-(methylsulfonyl)phenoxy)-7H-pyrrolo[2,3-d]pyrimidin-2-amine (I-B1)

[0086] Synthesis of 2-chloro-4-(2-(methylthio)phenoxy)-7H-pyrrolo[2,3-d]pyrimidine (IV-3)

[0087] To a 50 mL three-necked flask, add 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine (II-3, 500 mg, 2.66 mmol), 2-hydroxythioanisole (III-2, 386 μL, 3.19 mmol), potassium carbonate (735 mg, 5.32 mmol) and isopropanol (25 mL). Heat to 85 °C with stirring under nitrogen for 5 hours. Cool the reaction solution to room temperature. TLC monitoring (petroleum ether: ethyl acetate = 3:1) shows that the reaction is almost complete. Filter with suction, wash the filter cake with distilled water (5 mL × 2), and dry in vacuo to obtain 560 mg of light green solid IV-3 with a yield of 72.2%. 1 H NMR (300 MHz, DMSO-d 6 )δ12.46(s,1H),7.50–7.27(m,5H),6.37(s,1H),2.39(s,3H).MS(ESI(+)70V)m / z[M+H] + :292.02.

[0088] Synthesis of 2-chloro-7-methyl-4-(2-(methylthio)phenoxy)-7H-pyrrolo[2,3-d]pyrimidine (VII-1)

[0089] IV-3 (3.5 g, 12.0 mmol) and tetrahydrofuran (100 mL) were added to a 250 mL three-necked flask, stirred to dissolve, cooled to 0 ° C under nitrogen protection, n-butyl lithium (6.60 mL, 13.2 mmol) was added dropwise, and iodomethane (3.73 mL, 59.98 mmol) was slowly added after 15 min. After 1 hour, the temperature was raised to room temperature and the reaction was closed. After 5 hours, TLC monitoring (petroleum ether: ethyl acetate = 10:1) showed that the reaction was basically complete. The reaction solution was evaporated under reduced pressure to remove the solvent, 15 mL of water was added to the residue, and extracted with ethyl acetate (10 mL × 3), the organic phases were combined, washed with saturated sodium chloride solution (10 mL × 2), and dried over anhydrous sodium sulfate; filtered, the filtrate was evaporated under reduced pressure to remove the solvent, and crystallized by standing; filtered, the filter cake was dried in vacuo to obtain 3.08 g of brown-yellow solid VII-1, with a yield of 84.0%. 1 H NMR (300 MHz, CDCl 3 )δ7.38–7.32(m,1H),7.32–7.27(m,1H),7.25–7.17(m,2H),6.99(d,J=3.5Hz, 1H),6.23(d,J=3.5Hz,1H),3.82(s,3H),2.40(s,3H).MS(ESI(+)70V)m / z[M+H] +:306.04.

[0090] Synthesis of 2-chloro-7-methyl-4-(2-(methylsulfonyl)phenoxy)-7H-pyrrolo[2,3-d]pyrimidine (VIII-1)

[0091] VII-1 (2.0 g, 6.54 mmol) and tetrahydrofuran (20 mL) were added to a 50 mL three-necked flask, stirred and dissolved, cooled to 0 ° C, and a solution obtained by dissolving m-chloroperbenzoic acid (2.26 g, 13.08 mmol) in tetrahydrofuran (15 mL) was slowly added dropwise. The dripping was completed in about 0.5 hours. TLC monitoring (petroleum ether: ethyl acetate = 5:1) showed that the reaction was basically complete. Saturated sodium sulfite solution (30 mL) was added to quench the reaction, and the dichloromethane layer was separated and extracted with dichloromethane (15 mL × 2). The organic phases were combined, washed with saturated sodium chloride solution (15 mL × 2), and dried over anhydrous sodium sulfate; filtered, the filtrate was evaporated to remove the solvent under reduced pressure, and the crude product was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 25:1 to 10:1) to obtain 1.65 g of off-white solid VIII-1, with a yield of 74.7%. 1 H NMR (300 MHz, CDCl 3 )δ7.78(dd,J=7.1,1.9Hz,1H),7.50–7.33(m,2H),7.37–7.28(m,1H),7.08(dd,J=7.1, 1.9Hz,1H),6.41(d,J=7.6Hz,1H),3.89(s,3H),3.19(s,3H).MS(ESI(+)70V)m / z[M+H] + :338.03.

[0092] Synthesis of N-(2-methoxyphenyl)-7-methyl-4-(2-(methylsulfonyl)phenoxy)-7H-pyrrolo[2,3-d]pyrimidin-2-amine (I-B1)

[0093] VIII-1 (200 mg, 0.59 mmol), VI-1 (100 μL, 0.89 mmol), XPhos (28 mg, 0.06 mmol), cesium carbonate (579 mg, 1.78 mmol) and Pd were added to a 25 mL round bottom flask. 2 (dba) 3 (54 mg, 0.06 mmol), followed by the addition of anhydrous toluene (5 mL), and the mixture was heated to 110 ° C under nitrogen protection for 8 hours. The reaction was basically completed under TLC monitoring (petroleum ether: ethyl acetate = 1:1). The reaction solution was evaporated under reduced pressure to remove toluene, and the crude product was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 4:1 to 2:1) to obtain 88 mg of off-white solid I-B1. The yield was 35.0%.1 H NMR (400 MHz, CDCl 3 )δ8.26(d,J=8.1Hz,1H),8.14(d,J=7.5Hz,1H),7.75–7.67(m,1H),7.58(s,1H),7.50–7.42(m,2H),6.9 4–6.71(m,4H),6.38(d,J=3.5Hz,1H),3.87(s,3H),3.78(s,3H),3.20(s,3H).MS(ESI(+)70V)m / z[M+H] + :425.12.

[0094] Example 10: Synthesis of N-(2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)-7-methyl-4-(2-(methylsulfonyl)phenoxy)-7H-pyrrolo[2,3-d]pyrimidin-2-amine (I-B2)

[0095] Using compound VIII-1 (200 mg, 0.59 mmol) and VI-2 (131 mg, 0.59 mmol) as raw materials and referring to the preparation method of compound I-B1, 210 mg of yellow solid I-B2 was obtained with a yield of 67.9%. 1 HNMR (400MHz, CDCl 3 )δ8.23–8.06(m,2H),7.73–7.64(m,1H),7.45(d,J=7.9Hz,2H),7.32(s,1H),6.80(d,J=3.6Hz,1H),6.51(d,J=2.5Hz,1H),6.41– 6.28(m,2H),3.84(s,3H),3.75(s,3H),3.20(s,3H),3.17–3.11(m,4H),2.65–2.58(m,4H),2.37(s,3H).MS(ESI(+)70V)m / z[M+H] + :523.20.

[0096] Example 11: Synthesis of N-(2-methoxy-4-morpholinylphenyl)-7-methyl-4-(2-(methylsulfonyl)phenoxy)-7H-pyrrolo[2,3-d]pyrimidin-2-amine (I-B3)

[0097] Using compound VIII-1 (200 mg, 0.59 mmol) and VI-4 (123 mg, 0.59 mmol) as raw materials and referring to the preparation method of compound I-B1, 120 mg of brown-green solid I-B3 was obtained with a yield of 39.8%. 1 HNMR (300MHz, CDCl 3)δ8.21–8.08(m,2H),7.74–7.64(m,1H),7.51–7.40(m,2H),7.35(s,1H),6.81(d,J=3.6Hz,1H),6.56–6.47(m,1H),6.4 2–6.30(m,2H),3.91–3.86(m,4H),3.85(s,3H),3.76(s,3H),3.21(s,3H),3.13–3.04(m,4H).MS(ESI(+)70V)m / z[M+H] + :510.17.

[0098] Example 12: Synthesis of N-(2-methoxyphenyl)-4-(2-(methylsulfonyl)phenoxy)-7H-pyrrolo[2,3-d]pyrimidin-2-amine (I-C1)

[0099] Synthesis of 2-chloro-4-(2-(methylthio)phenoxy)-7-p-methylbenzenesulfonyl-7H-pyrrolo[2,3-d]pyrimidine (IX-1)

[0100] IV-3 (500 mg, 1.71 mmol) was added to a 100 mL three-necked flask and dissolved in tetrahydrofuran (35 mL). The mixture was cooled to 0 ° C. Sodium hydride (62 mg, 2.57 mmol) was slowly added. After the reaction solution was free of bubbles, p-toluenesulfonyl chloride (653 mg, 3.43 mmol) was added. After stirring at 0 ° C for 1 hour, the mixture was moved to room temperature for 2 hours. The reaction was basically completed under TLC monitoring (petroleum ether: ethyl acetate = 8:1). The reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 40:1-25:1) to obtain 480 mg of white solid IX-1 with a yield of 62.8%. 1 H NMR (300 MHz, CDCl 3 )δ8.15–8.08(m,2H),7.59(d,J=4.0Hz,1H),7.37–7.31(m,3H),7.30–7.28(m,1H),7.25–7.20(m, 1H),7.16–7.10(m,1H),6.48(d,J=3.9Hz,1H),2.42(s,3H),2.38(s,3H).MS(ESI(+)70V)m / z[M+H] + :446.03.

[0101] Synthesis of 2-chloro-4-(2-(methylsulfonyl)phenoxy)-7-p-methylbenzenesulfonyl-7H-pyrrolo[2,3-d]pyrimidine (X-1)

[0102] IX-1 (600 mg, 1.35 mmol) and DMF (15 mL) were added to a 50 mL three-necked flask, stirred and dissolved, cooled to 0 ° C, and a solution obtained by dissolving m-chloroperbenzoic acid (813 mg, 4.71 mmol) in DMF (8 mL) was slowly added dropwise. The addition was completed in about 0.5 hours. TLC monitoring (petroleum ether: ethyl acetate = 2:1) showed that the reaction was complete. Saturated sodium sulfite solution (30 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (20 mL × 3) and water (60 mL). The organic phases were combined, washed with saturated sodium chloride solution (15 mL × 2), and dried over anhydrous sodium sulfate; filtered, the filtrate was evaporated to remove the solvent under reduced pressure, and the crude product was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 25:1-10:1) to obtain 580 mg of yellow solid X-1 with a yield of 90.2%. 1 H NMR (300 MHz, CDCl 3 )δ8.13(d,J=7.4Hz,1H),7.85–7.71(m,3H),7.60(d,J=7.4Hz,1H),7.51–7.39(m,2H),7 .33–7.28(m,2H),7.17–7.05(m,1H),3.20(s,3H),2.39(s,3H).MS(ESI(+)70V)m / z[M+H] + :478.02.

[0103] Synthesis of N-(2-methoxyphenyl)-4-(2-(methylsulfonyl)phenoxy)-7-p-methylbenzenesulfonyl-7H-pyrrolo[2,3-d]pyrimidin-2-amine (XI-1)

[0104] A 25 mL round bottom flask was charged with X-1 (500 mg, 1.05 mmol), VI-1 (177 μL, 1.57 mmol), XPhos (50 mg, 0.11 mmol), cesium carbonate (1.02 g, 3.14 mmol) and Pd 2 (dba) 3 (96 mg, 0.11 mmol), followed by the addition of anhydrous toluene (8 mL), and the reaction mixture was sealed and heated to 110 ° C under nitrogen protection for 8 hours. The reaction was basically completed under TLC monitoring (petroleum ether: ethyl acetate = 2:1). The reaction solution was evaporated under reduced pressure to remove toluene, and the crude product was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 25:1-10:1) to obtain 160 mg of yellow solid XI-1. The yield was 27.1%. 1 H NMR (300 MHz, CDCl 3)δ8.36(s,1H),8.15–7.95(m,4H),7.74–7.66(m,1H),7.61–7.56(m,1H),7.51–7.37(m,3H),7.23(s,1H), 7.01–6.81(m,3H),6.50(d,J=3.9Hz,1H),3.87(s,3H),3.18(s,3H),2.36(s,3H).MS(ESI(+)70V)m / z[M+H] + :565.11.

[0105] Synthesis of N-(2-methoxyphenyl)-4-(2-(methylsulfonyl)phenoxy)-7H-pyrrolo[2,3-d]pyrimidin-2-amine (I-C1)

[0106] In a 25 mL round-bottom flask, intermediate XI-1 (395 mg, 0.70 mmol) was dissolved in anhydrous methanol (12 mL). CH 3 A methanol solution of ONa (648 μL, 3.50 mmol) was heated to 60°C for 5 hours. The reaction was basically completed under TLC monitoring (petroleum ether: ethyl acetate = 1:1). The solvent was evaporated under reduced pressure, 10 mL of water was added to the residue, and the mixture was extracted with ethyl acetate (10 mL×3). The organic phases were combined, washed with saturated sodium chloride solution (15 mL×2), and dried over anhydrous sodium sulfate; the filtrate was filtered, and the solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 2:1 to 1:1) to obtain 253 mg of brown solid I-C1 with a yield of 88.1%. 1 H NMR (300 MHz, CDCl 3 )δ9.19(s,1H),8.15(d,J=7.5Hz,1H),8.03(d,J=8.0Hz,1H),7.78–7.68(m,1H),7.59(s,1H),7.53–7.44(m,2H ),6.93–6.80(m,3H),6.78–6.67(m,1H),6.47–6.39(m,1H),3.86(s,3H),3.23(s,3H).MS(ESI(+)70V)m / z[M+H] + :411.10.

[0107] Example 13: Synthesis of N-(2-methoxy-4-morpholinylphenyl)-4-(2-(methylsulfonyl)phenoxy)-7H-pyrrolo[2,3-d]pyrimidin-2-amine (I-C2)

[0108] Synthesis of N-(2-methoxy-4-morpholinylphenyl)-4-(2-(methylsulfonyl)phenoxy)-7-p-methylbenzenesulfonyl-7H-pyrrolo[2,3-d]pyrimidin-2-amine (XI-2)

[0109] Using compound X-1 (500 mg, 1.05 mmol) and VI-4 (234 mg, 1.15 mmol) as raw materials, referring to the preparation method of compound XI-1, 386 mg of brown solid XI-2 was obtained, and the yield was 56.8%. 1 H NMR (300 MHz, DMSO-d 6 )δ8.31(s,1H),7.91(d,J=8.0Hz,2H),7.82(s,1H),7.71–7.53(m,1H),7.43(d,J=4.0Hz,1H),7.38–7.31(m,4H),7.26–7.22(m,2H),6.64(d, J=2.5Hz,1H),6.49(d,J=4.0Hz,1H),3.78(s,3H),3.77–3.73(m,4H),3.23(s,3H),3.15–3.07(m,4H),2.34(s,3H).MS(ESI(+)70V)m / z[M+H] + :650.17.

[0110] Synthesis of N-(2-methoxy-4-morpholinylphenyl)-4-(2-(methylsulfonyl)phenoxy)-7H-pyrrolo[2,3-d]pyrimidin-2-amine (I-C2)

[0111] Using compound XI-2 (80 mg, 0.12 mmol) as raw material and referring to the preparation method of compound I-C1, 42 mg of brown solid I-C2 was obtained with a yield of 68.8%. 1 H NMR (400 MHz, DMSO-d 6 )δ11.61(s,1H),7.98(dd,J=7.9,1.7Hz,1H),7.87–7.80(m,1H),7.70(d,J=8.8Hz,1H),7.65–7.52(m,2H),7.37(s,1H),7.12–7.06(m,1 H),6.59(d,J=2.6Hz,1H),6.41–6.26(m,2H),3.78(s,3H),3.76–3.70(m,4H),3.33(s,3H),3.09–3.00(m,4H).MS(ESI(+)70V)m / z[M+H] + :496.16.

[0112] Example 14: Synthesis of N-(2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)-4-(2-(methylsulfonyl)phenoxy)-7H-pyrrolo[2,3-d]pyrimidin-2-amine (I-C3)

[0113] Synthesis of N-(2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)-4-(2-(methylsulfonyl)phenoxy)-7-p-methylbenzenesulfonyl-7H-pyrrolo[2,3-d]pyrimidin-2-amine (XI-3)

[0114] Add X-1 (400 mg, 0.84 mmol), VI-2 (185 mg, 0.84 mmol), XPhos (40 mg, 0.08 mmol), cesium carbonate (818 mg, 2.51 mmol) and Pd(OAc) to a 25 mL round bottom flask. 2 (19 mg, 0.08 mmol), followed by the addition of anhydrous toluene (5 mL), and the reaction mixture was sealed and heated to 110 ° C under nitrogen protection for 8 hours. The reaction was basically completed under TLC monitoring (dichloromethane: methanol = 10: 1). The reaction solution was evaporated under reduced pressure to remove toluene, and the crude product was purified by silica gel column chromatography (eluent: dichloromethane: methanol = 60: 1-50: 1) to obtain 160 mg of brown solid XI-3, with a yield of 28.8%. 1 H NMR (300 MHz, DMSO-d 6 )δ9.48(s,1H),8.23(d,J=7.4Hz,1H),7.84–7.72(m,3H),7.69(d,J=7.6H z,1H),7.51–7.36(m,2H),7.33–7.28(m,2H),7.14–7.08(m,2H),6.46(dd ,J=7.5,1.5Hz,1H),6.33(d,J=1.6Hz,1H),3.84(s,3H),3.32–3.21(m,7H ),2.70–2.62(m,4H),2.41(s,3H),2.32(s,3H).MS(ESI(+)70V)m / z[M+H] + :663.20.

[0115] Synthesis of N-(2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)-4-(2-(methylsulfonyl)phenoxy)-7H-pyrrolo[2,3-d]pyrimidin-2-amine (I-C3)

[0116] Using compound XI-3 (60 mg, 0.09 mmol) as raw material and referring to the preparation method of compound I-C1, 40 mg of brown solid I-C3 was obtained with a yield of 86.9%. 1 H NMR (300 MHz, CDCl3 )δ10.01(s,1H),8.12(d,J=7.9Hz,1H),7.91(d,J=8.8Hz,1H),7.77–7.63(m,1H),7.51–7.39(m,2H),7.18(s,1H),6.69(s,1H),6.51(d, J=2.5Hz,1H),6.37–6.27(m,2H),3.82(s,3H),3.22(s,3H),3.19–3.10(m,4H),2.67–2.59(m,4H),2.38(s,3H).MS(ESI(+)70V)m / z[M+H] + :509.19.

[0117] Example 15: Experiment on the inhibition of DCLK1 activity by the compounds of the present invention at the enzyme level

[0118] (1) Experimental methods

[0119] A molecular screening model for DCLK1 enzyme activity inhibition was established, and the assay was performed as follows: DCLK1 enzyme can catalyze the conversion of fluorescently labeled Kinase substrate 12 (supplier GL) into fluorescently labeled products, and then the corresponding conversion rate is detected using a mobility detection technology (Mobility-Shift Assay) based on microfluidic chip technology. This technology applies the basic concept of capillary electrophoresis to a microfluidic environment and detects enzymatic experiments without adding a stop reagent. The substrate used in the experiment is a fluorescently labeled polypeptide. Under the action of the enzyme in the reaction system, the substrate is converted into a product, and the charge it carries also changes accordingly. Mobility-Shift Assay uses the difference in charge between the substrate and the product, and uses the EZReader platform to separate the two and detect them separately.

[0120] Dissolve the compound powder in 100% DMSO to prepare a 10mM stock solution. (1) Prepare 1×Kinase buffer. (2) Preparation of compound concentration gradient: The test compound is tested at a single concentration of 1μM and 200nM, and diluted to a 100-fold final concentration of 100% DMSO solution in a 384-well plate. Use a dispenser Echo550 to transfer 250nL of the 100-fold final concentration of the compound to the target plate. (3) Use 1×Kinase buffer to prepare a kinase solution at 2.5 times the final concentration. (4) Add 10μL of 2.5-fold final concentration of the kinase solution to the compound well and the positive control well respectively; add 10μL of 1×Kinase buffer to the negative control well. (5) Centrifuge at 1000rpm for 30 seconds, shake the reaction plate to mix, and incubate at room temperature for 10 minutes. (6) Use 1×Kinase buffer to prepare a mixed solution of ATP and Kinase substrate at 5 / 3 times the final concentration. (7) Add 15 μL of a mixed solution of ATP and substrate at 5 / 3 times the final concentration to start the reaction. (8) Centrifuge the 384-well plate at 1000 rpm for 30 seconds, shake and mix, and incubate at room temperature for 60 minutes. (9) Add 30 μL of stop detection solution to stop the kinase reaction, centrifuge at 1000 rpm for 30 seconds, shake and mix. (10) Read the conversion rate using Caliper EZ Reader.

[0121] Calculation formula:

[0122]

[0123] Where: Conversion%_sample is the conversion rate reading of the sample; Conversion%_min: the mean value of the negative control wells, representing the conversion rate reading of the wells without enzyme activity; Conversion%_max: the mean value of the positive control wells, representing the conversion rate reading of the wells without compound inhibition.

[0124] Fitting dose-effect curve:

[0125] The log value of the concentration was used as the X-axis and the percentage inhibition rate was used as the Y-axis. The log (inhibitor) vs response-Variable slope of the analysis software GraphPad Prism 5 was used to fit the dose-effect curve to obtain the percentage inhibition rate of each compound on the enzyme activity.

[0126] The calculation formula is as follows:

[0127] Y=Bottom+(Top-Bottom) / (1+10^((LogIC 50 -X)*Hill Slope))

[0128] (2) Experimental results

[0129] Table 1. Inhibitory activity of target compounds against DCLK1 at the enzyme level

[0130] Cpd. DCLK1(1000nM) I-A2 A I-A4 B I-A5 A，89％ I-A6 B I-A8 B

[0131] Note: A means the inhibition rate is greater than 50%, and B means the inhibition rate is between 15% and 50%.

[0132] As can be seen from Table 1, at a concentration of 1 μM, some compounds of the present invention have certain inhibitory activity against DCLK1, among which compound I-A5 has the best inhibitory activity against DCLK1.

[0133] Example 16: Evaluation of the in vitro tumor cell inhibitory activity of compound I-A5 using human pancreatic cancer cells SW1990 and colon cancer cells HCT116

[0134] Materials and reagents: Human colon cancer cell line HCT116 and human pancreatic cancer cell line SW1990 were purchased from the Cell Bank of the Chinese Academy of Sciences. HCT116 cells were cultured in McCoy'5A medium containing 10% fetal bovine serum (FBS), and SW1990 cells were cultured in L-15 medium containing 10% fetal bovine serum (FBS). Both cells were cultured in 5% CO 2 , cultured in a 37°C constant temperature incubator; DCLK1-IN-1 and I-A5 were dissolved in DMSO to a final concentration of 10 mM / L, filtered with a 0.22 μM filter membrane, and stored in a -20°C refrigerator. Preparation of MTT: weigh 0.25 g of MTT and dissolve it in 50 mL of PBS to a final concentration of 5 mg / mL, filtered with a 0.22 μM filter membrane, and stored in a -20°C refrigerator.

[0135] (1) Experimental methods

[0136] MTT assay for cell proliferation: cells were seeded at a density of 2000 cells / well in a 96-well plate and incubated in 5% CO 2 After culturing at 37°C for 24 h, the cells were treated with corresponding drugs for 7 days, and thiazolyl blue dye (MTT) was added for incubation for 3-4 h. The culture medium was discarded, DMSO was added to fully dissolve and mix, and cell proliferation was detected.

[0137] Cell growth inhibition rate = (1-absorbance value of experimental group / absorbance value of control group) × 100%.

[0138] (2) Experimental results

[0139] Table 2. Inhibitory activity of target compounds against HCT116 and SW1990 tumor cell lines

[0140]

[0141] Note: +++ indicates IC 50 Less than 10μM.

[0142] As shown in Table 2, the target compound I-A5 has strong inhibitory activity against SW1990 and HCT116.

[0143] Example 17: Experimental study on evaluating the anti-tumor effect of compound I-A5 based on a xenograft tumor model of human pancreatic cancer cells SW1990

[0144] (1) Experimental methods

[0145] Nude mice were subcutaneously inoculated with human pancreatic cancer SW1990 cells and the tumors were grown to 50 mm. 3 Afterwards, the animals were randomly divided into a vehicle group, a positive drug DCLK1-IN-1 group, an I-A5 low-dose group, an I-A5 medium-dose group, and an I-A5 high-dose group. The DCLK1-IN-1 group was injected with DCLK1-IN-1 (60 mg / kg) by intraperitoneal injection once a day; the I-A5 low-dose group was injected with I-A5 (30 mg / kg) by intraperitoneal injection once a day; the I-A5 medium-dose group was injected with I-A5 (45 mg / kg) by intraperitoneal injection once a day; the I-A5 high-dose group was injected with I-A5 (60 mg / kg) by intraperitoneal injection once a day. The volume of the transplanted tumor was measured every two days, the weight of the mice was weighed, the data was recorded, and the animals were killed on the 21st day;

[0146] (2) Experimental results

[0147] The results showed that compared with the model group, the tumor growth of each dose group of the target compound I-A5 was significantly inhibited. Compared with the positive drug group and the model group, the tumor weight and tumor volume of each dose group of I-A5 were significantly reduced ( Figure 1 to Figure 3 ). The inhibitory effects of each dose group of I-A5 on human pancreatic cancer cell SW1990 xenograft tumors were stronger than those of the positive drug group, and the differences were significant, indicating that compound I-A5 can exert a better anti-tumor effect in vivo.

[0148] In summary, the compounds disclosed in the present invention can significantly inhibit the growth of tumor cells in vivo and in vitro, and have good prospects for clinical application.

Claims

1. A DCLK1 inhibitor, It is characterized in that Select any of the following compounds:

2. A pharmaceutically acceptable salt of the DCLK1 inhibitor according to claim 1, It is characterized in that It is a salt formed by the DCLK1 inhibitor and an acid, wherein the acid is selected from hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid or ferulic acid.

3. A pharmaceutical composition, It is characterized in that Comprising the DCLK1 inhibitor according to claim 1 and a pharmaceutically acceptable carrier.

4. Use of the DCLK1 inhibitor according to claim 1 or the pharmaceutical composition according to claim 3 in the preparation of a cancer therapeutic drug.

5. The use according to claim 4, It is characterized in that The medicine is a medicine for treating colorectal cancer and pancreatic cancer.

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