CN116284001A

CN116284001A - DCLK1 inhibitor, preparation method, pharmaceutical composition and application

Info

Publication number: CN116284001A
Application number: CN202310044711.8A
Authority: CN
Inventors: 王淑平; 孟柳琼; 王文泽; 陈跃鹏; 叶柳; 黄诗卉; 邹毅; 朱启华; 徐云根
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2023-01-30
Filing date: 2023-01-30
Publication date: 2023-06-23

Abstract

The invention discloses a DCLK1 inhibitor, a preparation method, a pharmaceutical composition and application. The DCLK1 inhibitor has a structure as shown in formula (I), and also comprises an isomer, a pharmaceutically acceptable salt or a mixture thereof. The DCLK1 inhibitor and the pharmaceutical composition thereof have high-efficiency inhibition effect on DCLK1, can be prepared into therapeutic drugs for treating neoplastic diseases such as pancreatic cancer, and the like, can exert drug effects at molecular level, cellular level and animal level, and especially has better in vivo anti-tumor activity than positive control drugs, and the synthesis method of the compound is simple and convenient and easy.

Description

DCLK1 inhibitor, preparation method, pharmaceutical composition and application

Technical Field

The invention relates to a DCLK1 inhibitor, a preparation method, a pharmaceutical composition and application, in particular to a DCLK1 inhibitor which can be prepared into a cancer therapeutic drug, a preparation method, a pharmaceutical composition and application.

Background

Cancer, which is the most common type of malignant tumor that originates in epithelial tissue, is common in our daily lives. Because the early cancer development is too hidden, many patients have advanced stage, and metastasis and diffusion of many cancer cells can occur, so that the treatment difficulty is high and the mortality rate is extremely high. At present, the main treatment means of cancer include operation treatment, radiation therapy, chemotherapy, immunotherapy and the like, and the treatment means have long period and high price, and bring heavy economic burden and mental stress to patients and families thereof. Most cancer patients usually adopt a conservation treatment means of chemical drug treatment, although the research and development investment of the anti-cancer drugs in the world is larger and larger, on one hand, more choices are brought to the patients, and meanwhile, the defects of drug resistance, toxic and side effects and the like caused by weak selectivity when the existing anti-cancer drugs are taken for a long time are also exposed. Although the traditional chemotherapy drugs, targeted drugs and the like for treating cancers at present can effectively improve the illness state of patients, the traditional chemotherapy drugs and the targeted drugs have respective limitations. Therefore, there is a great interest in finding new therapeutic approaches, in particular drugs acting on new targets.

Bisadrenocortical hormone-like Kinase-1 (dclk1) was originally a transmembrane microtubule-associated protein Kinase found in the nervous system and has biological functions of regulating microtubule polymerization and promoting neuronal migration, and its domain has been shown to be essential 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 tract system, such as esophageal cancer, gastric cancer, colorectal cancer, pancreatic cancer, etc. Therefore, DCLK1 is considered as a new tumor marker of gastrointestinal stem cells, which also makes it a new target for cancer treatment, and no drug aiming at DCLK1 target has been successfully marketed at present. Based on the above, the research of the novel DCLK1 selective inhibitor provides a new therapeutic scheme and selection for treating various digestive tract cancers such as pancreatic cancer, and provides probe molecules for deeply researching the biological functions of DCLK 1.

Disclosure of Invention

The invention aims to: aiming at the defects of weak inhibition activity of the existing compound on DCLK1 and the like, the invention aims to provide a DCLK1 inhibitor with excellent anti-tumor activity, a preparation method, a pharmaceutical composition and application.

The technical scheme is as follows: as a first aspect to which the present invention relates, the DCLK1 inhibitor of the present invention has the structure of formula (I), which further comprises an isomer, a pharmaceutically acceptable salt or a mixture thereof,

wherein:

Y ¹ 、Y ² 、Y ³ each independently selected from CH or N;

Z ¹ 、Z ² each independently selected from O, S or NH;

R ¹ selected from H, C C1-C6 alkyl, C3-C8 cycloalkyl, CH ₂ CF ₃ Or CH (CH) ₂ CHF ₂ ；

R ² Selected from SO ₂ R ⁶ Or SOR ⁶ Wherein R is ⁶ Selecting C1-C6 alkyl, C3-C8 cycloalkyl, CF ₃ Or CH (CH) ₂ CF ₃ ；

R ³ Selected from H, F, cl, br, I, CF ₃ 、CHF ₂ 、CN、OCH ₃ 、OH or CH ₃ ；

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

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-trifluoroethyl) piperazin-1-yl or 4-isobutylpiperazin-1-yl.

Preferably, in the above structure:

Y ¹ 、Y ² 、Y ³ each independently selected from CH or N; z is Z ¹ 、Z ² Each independently selected from O or NH;

R ¹ selected from H, CH ₃ 、CH ₂ CH ₃ 、CH ₂ CHF ₂ Or CH (CH) ₂ CF ₃ ；

R ² Selected from SO ₂ R ⁶ Wherein R is ⁶ Selected from C1-C6 alkyl, CF ₃ Or CH (CH) ₂ CF ₃ ；

R ³ Selected from H, F, cl, br, CF ₃ 、OCH ₃ Or CH (CH) ₃ ；

R ⁴ Selected from CH ₃ Or CH (CH) ₂ CH ₃ ；

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-trifluoroethyl) piperazin-1-yl.

More preferably, the DCLK1 inhibitor is selected from any one of the following compounds:

the pharmaceutically acceptable salt of the DCLK1 inhibitor is a salt formed by the compound and 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.

As a second aspect of the present invention, the preparation method of the DCLK1 inhibitor is as follows:

the method comprises the following steps: when R is ¹ Is H, Y ¹ 、Y ² When CH or N are not simultaneously CH, the preparation method of the compound I-A is as follows:

specifically, the compound II is reacted with the phenyl thioether derivative III to prepare a compound IV, wherein the base is selected from triethylamine, pyridine, N-Diisopropylethylamine (DIPEA), 4-Dimethylaminopyridine (DMAP), potassium carbonate, sodium carbonate, potassium tert-butoxide, sodium tert-butoxide, cesium carbonate or sodium acetate, and preferably N, N-Diisopropylethylamine (DIPEA); the solvent is selected from N, N-Dimethylformamide (DMF), N-diethylacetamide (DMAc), N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO), isopropanol, ethanol or a mixed solvent of the above solvents, preferably isopropanol.

Compound V is prepared by reacting compound IV with m-chloroperoxybenzoic acid (m-CPBA) using a solvent selected from dichloromethane, chloroform, ethyl acetate, tetrahydrofuran, 1, 4-dioxane, DMF or a mixed solvent of any two, preferably dichloromethane, chloroform or DMF.

Preparing a compound I-A by reacting a compound V with a compound VI, wherein the acid 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 is selected from dichloromethane, chloroform, ethyl acetate, isopropanol, ethanol, tetrahydrofuran, 1, 4-dioxane, DMF or mixed solvent of any two, preferably isopropanol.

The second method is as follows: when R is ¹ When the compound is not H, the preparation method of the compound I-B is as follows:

specifically, from compounds IV and R ¹ X is reacted to prepare a compound VII, wherein the base 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 is selected from dichloromethane, chloroform, ethyl acetate, tetrahydrofuran, 1, 4-dioxane, DMF or a mixed solvent of any two, preferably tetrahydrofuran or DMF.

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

Preparing a compound I-B by reacting a compound VIII with a compound VI, wherein the acid 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 is selected from dichloromethane, chloroform, ethyl acetate, isopropanol, ethanol, tetrahydrofuran, 1, 4-dioxane, DMF or mixed solvent of any two, preferably isopropanol.

And a third method: when R is ¹ Is H, Y ¹ 、Y ² In the case of CH, the preparation method of the compound I-C is as follows：

Wherein Y is ¹ 、Y ² 、Y ³ 、Z ¹ 、Z ² 、R ¹ 、R ² 、R ³ 、R ⁴ 、R ⁵ Or R is ⁶ Is defined as before.

Specifically, compound IX is prepared from compound IV by reaction with p-toluenesulfonyl chloride (TosCl), the base used being 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 is selected from dichloromethane, chloroform, ethyl acetate, tetrahydrofuran, 1, 4-dioxane, DMF or a mixed solvent of any two, preferably tetrahydrofuran or DMF.

Compound X is prepared by reacting compound IX with m-CPBA using a solvent selected from the group consisting of dichloromethane, chloroform, ethyl acetate, tetrahydrofuran, 1, 4-dioxane, DMF or a mixture of any two, preferably dichloromethane, chloroform or DMF.

Preparation of compound XI by reaction of compound X with compound VI using a catalyst selected from Pd ₂ (dba) ₃ 、Pd(OAc) ₂ 、Pd ₂ (dppf)Cl ₂ 、Pd(PPh ₃ ) ₄ 、Pd ₂ (dppf)Cl ₂ Dichloromethane complexes or mixtures of the above catalysts, preferably Pd ₂ (dba) ₃ Or Pd (OAc) ₂ The method comprises the steps of carrying out a first treatment on the surface of the 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 is selected from toluene, chloroform, dichloromethane, acetone, acetonitrile, diethyl ether, tetrahydrofuran, 1, 4-dioxane, DMF or mixture of any two, preferablyToluene.

The compound I-C is prepared by reacting the compound with a methanol solution of X and sodium methoxide, wherein the solvent is selected from methanol, dichloromethane, chloroform, ethyl acetate, tetrahydrofuran, 1, 4-dioxane, DMF or a mixed solvent of any two, preferably methanol.

And salifying the corresponding acid with the compound I prepared by the method to obtain the pharmaceutically acceptable salt of the DCLK1 inhibitor.

As a third aspect to which the present invention relates, the pharmaceutical composition of the present invention comprises the DCLK1 inhibitor described above and a pharmaceutically acceptable carrier.

The DCLK1 inhibitor can be added with pharmaceutically acceptable carriers to prepare common medicinal preparations such as tablets, capsules, syrup, suspending agents or injection, and the preparations can be added with common medicinal auxiliary materials such as perfume, sweetener, liquid/solid filler, diluent and the like.

As a fourth aspect of the present invention, the DCLK1 inhibitor and the pharmaceutical composition thereof can be prepared as a therapeutic agent for cancer, specifically colorectal cancer, pancreatic cancer and other cancers.

The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages:

(1) The DCLK1 inhibitor and the pharmaceutical composition thereof can effectively inhibit the activity of DCLK1 at the concentration level of 1 mu M, and the inhibition rate is optimally up to 89%; can also inhibit proliferation of various tumor cells, and IC ₅₀ Values less than 10 μm;

(2) The DCLK1 inhibitor and the pharmaceutical composition thereof have wide application, can be prepared into medicines for treating tumors such as pancreatic cancer, and the like, and the antitumor activity of the animal body is obviously superior to that of a positive medicine under lower administration dosage, and the tumor inhibition rate is higher than 50%;

(3) The preparation method of the compound is simple and easy to implement and is suitable for amplification.

Drawings

FIG. 1 is tumor weight 21 days after administration of compound I-A5;

FIG. 2 is a tumor anatomy map 21 days after administration of Compound I-A5;

FIG. 3 shows the results of compound I-A5 versus tumor volume in xenograft tumor models as a function of time of administration.

Detailed Description

The technical scheme of the invention is further described below by referring to examples.

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

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

Into a 100mL three-necked flask was charged 4, 6-dichloro-1H-pyrazolo [3,4-d]Pyrimidine (II-1, 2.0g,10.58 mmol) and isopropanol (40 mL) were dissolved with stirring, 2- (isopropylthio) aniline (III-1, 1.84mL,11.64 mmol) was added, heated to 85℃and reacted for 5 hours, monitored by Thin Layer Chromatography (TLC) (Petroleum ether: ethyl acetate=3:1) to substantially complete the reaction; the reaction mixture was cooled to room temperature, filtered off with suction, and the filter cake was washed with petroleum ether (10 mL. Times.2), dried in vacuo to give 2.85g of off-white solid IV-1 in 84.2% yield. ¹ H NMR(300MHz,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.

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

To a 250mL three necked flask, IV-1 (1.0 g,3.13 mmol) and methylene chloride (75 mL) were added, stirred and dissolved, cooled to 0deg.C, and a solution of m-chloroperoxybenzoic acid (1.62 g,9.39 mmol) in methylene chloride (20 mL) was slowly added dropwise, and after about 0.5 hour, the reaction was monitored by TLC (Petroleum ether: ethyl acetate=1:1) to be substantially complete, a saturated sodium sulfite solution (30 mL) was added to quench the reaction, a methylene chloride layer was separated, extracted with methylene chloride (30 mL), the organic phase was combined, washed with a saturated sodium chloride solution (15 mL. Times.3), and dried over anhydrous sodium sulfate; suction filtration, reduced pressure distillation of the filtrate to remove the solvent, and separation and purification of the crude product by silica gel column chromatography (eluent: petroleum ether: ethyl acetate=25:1-10:1) to obtain 0.86g of off-white solid V-1, with a yield of 78.2%. ¹ H NMR(300MHz,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.

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

To a 15mL pressure-resistant bottle was added V-1 (150 mg,0.43 mmol), o-methoxyaniline (VI-1, 96. Mu.L, 0.85 mmol), p-toluenesulfonic acid monohydrate (90 mg,0.47 mmol) and isopropyl alcohol (4 mL), the mixture was heated to 110℃with stirring, the reaction was allowed to react for 8 hours, the reaction mixture was cooled to room temperature, TLC monitoring (petroleum ether: ethyl acetate=2:1) was performed to substantially complete the reaction, the solvent was distilled off under reduced pressure, the residue was added with 10mL of water, extracted with ethyl acetate (10 mL. Times.3), the organic phases were combined, washed with saturated sodium chloride solution (10 mL. Times.2), and dried over anhydrous sodium sulfate; suction filtration, reduced pressure evaporation of the filtrate to remove the solvent, purification of the crude product by silica gel column chromatography (eluent: petroleum ether: ethyl acetate=5:1-2:1) gave 80mg of brown solid I-A1 with a yield of 42.8%. ¹ H NMR(300MHz,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.

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

Using V-1 (80 mg,0.23 mmol) and 2-methoxy-4- (4-methylpiperazin-1-yl) aniline (VI-2, 50mg,0.23 mmol) as starting materials, reference was made to the preparation of compound I-A1 to give 91mg of pale yellow solid I-A2 in 74.6% yield. ¹ H NMR(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),6.59–6.51(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.

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

Using compound V-1 (150 mg,0.43 mmol) and 4-bromo-2-methoxyaniline (VI-3, 150mg,0.74 mmol) as raw materials, reference was made to the preparation method of compound I-A1 to give 120mg of pale yellow solid I-A3 in 54.4% yield. ¹ H NMR(300MHz,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.

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

Using compound V-1 (150 mg,0.43 mmol) and 2-methoxy-4-morpholinylaniline (VI-4, 89mg,0.43 mmol) as raw materials, referring to the preparation method of compound I-A1, 60mg of brown solid I-A4 was obtained, the yield was 26.9%. ¹ H NMR(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.

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

Synthesis of 2-chloro-N- (2- (isopropylsulfanyl) phenyl) -9H-purin-6-amine (IV-2)

To a 100mL three necked flask, 2, 6-dichloro-9H-purine (II-2, 1.50g,7.94 mmol) and isopropyl alcohol (35 mL) were added and dissolved with stirring, III-1 (1.33 g,7.94 mmol) was added, heating was carried out to 85℃and reflux reaction was carried out for 5 hours, TLC monitoring (Petroleum ether: ethyl acetate=3:1) showed that the reaction was substantially complete, the reaction solution was cooled to room temperature and then suction filtered, the filter cake was washed with ethyl acetate (3 mL. Times.2), and vacuum dried to obtain 2.20g of off-white solid IV-2, yield 86.7%. ¹ H NMR(300MHz,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.

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

Using compound IV-2 (79mg, 2.47 mmol) as a starting material, reference was made to the preparation of compound V-1, which gave 650mg of off-white solid V-2 in 74.8% yield. ¹ H NMR(300MHz,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.

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

V-2 (120 mg,0.34 mmol), VI-2 (83 mg,0.38 mmol), methanesulfonic acid (44. Mu.L, 0.68 mmol) and isopropyl alcohol (4 mL) were added to a 15mL pressure-resistant bottle, the reaction was heated to 110℃with stirring in a closed state for 8 hours, the reaction mixture was cooled to room temperature, TLC monitoring (dichloromethane: methanol=15:1) was carried out to substantially complete the reaction, the solvent was distilled off under reduced pressure, the residue was added with 15mL of water, extracted with ethyl acetate (10 mL. Times.3), the organic phases were combined, washed with saturated sodium chloride solution (10 mL. Times.2), and dried over anhydrous sodium sulfate; suction filtration, reduced pressure evaporation of the filtrate, and purification of the crude product by silica gel column chromatography (eluent: dichloromethane: methanol=60:1-40:1) gave 110mg of brown solid I-A5 with a yield of 60.1%. ¹ H NMR(300MHz,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,1H),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.

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

Starting from compound V-2 (180 mg,0.51 mmol) and VI-4 (117 mg,0.56 mmol), reference is made to the preparation of compound I-A5,160mg of brown solid I-A6 were obtained in 59.7% yield. ¹ H NMR(300MHz,CDCl ₃ )δ10.03(s,1H),8.89(d,J＝8.5Hz,1H),7.97(d,J＝8.7Hz,1H),7.90–7.84(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.

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

Using compound V-2 (100 mg,0.28 mmol) and VI-3 (57 mg,0.28 mmol) as raw materials, reference was made to the preparation of compound I-A5 to give 88mg of yellow solid I-A7 in 59.8% yield. ¹ H NMR(400MHz,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.

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

Using compound V-2 (100 mg,0.28 mmol) and VI-1 (32. Mu.L, 0.28 mmol) as starting materials, reference was made to the preparation of compound I-A5 to give 90mg of yellow solid I-A8 in 72.2% yield. ¹ H NMR(400MHz,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.

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

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

Into a 50mL three-necked flask was charged 2, 4-dichloro-7H-pyrrolo [2,3-d]Pyrimidine (II-3, 500mg,2.66 mmol), 2-hydroxyanisole (III-2,386. Mu.L, 3.19 mmol), potassium carbonate (730 mg,5.32 mmol) and isopropanol (25 mL), the reaction was heated to 85℃with sealed stirring under nitrogen protection for 5 hours, the reaction was cooled to room temperature, monitored by TLC (Petroleum ether: ethyl acetate=3:1) and was essentially complete, filtered off with suction, the filter cake was washed with distilled water (5 mL. Times.2) and dried in vacuo to give 560mg of pale green solid IV-3 in 72.2% yield. ¹ H NMR(300MHz,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.

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

IV-3 (3.5 g,12.0 mmol) and tetrahydrofuran (100 mL) are added into a three-necked flask of 250mL, stirred and dissolved, cooled to 0 ℃ under the protection of nitrogen, n-butyllithium (6.60 mL,13.2 mmol) is added dropwise, methyl iodide (3.73 mL,59.98 mmol) is slowly added after 15min, the reaction is closed at room temperature after 1 hour, TLC monitoring (petroleum ether: ethyl acetate=10:1) is carried out for basically complete reaction, the reaction solution is distilled off under reduced pressure, the solvent is distilled off, the residue is added with 15mL of water, extracted with ethyl acetate (10 mL×3), the organic phases are combined, washed with saturated sodium chloride solution (10 mL×2) and dried over anhydrous sodium sulfate; suction filtration, reduced pressure distillation of filtrate to remove solvent, standing and crystallization; suction filtration and vacuum drying of the filter cake gave 3.08g of tan solid VII-1 in 84.0% yield. ¹ H NMR(300MHz,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.

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

VII-1 (2.0 g,6.54 mmol) and tetrahydrofuran (20 mL) were added to a 50mL three-necked flask and stirred for dissolution, cooled to 0deg.C, a solution of m-chloroperoxybenzoic acid (2.26 g,13.08 mmol) in tetrahydrofuran (15 mL) was slowly added dropwise, the reaction was monitored by TLC to be substantially complete (Petroleum ether: ethyl acetate=5:1), quenched in saturated sodium sulfite solution (30 mL), the dichloromethane layer was separated, extracted with dichloromethane (15 mL. Times.2), and the organic phase was combined, and the reaction was quenched with saturated sodium sulfiteAnd sodium chloride solution (15 mL. Times.2), dried over anhydrous sodium sulfate; suction filtration, reduced pressure distillation of the filtrate to remove the solvent, purification of the crude product by silica gel column chromatography (eluent: petroleum ether: ethyl acetate=25:1-10:1) gives 1.65g of off-white solid VIII-1, yield 74.7%. ¹ H NMR(300MHz,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.

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

To a 25mL round bottom flask was added VIII-1 (200 mg,0.59 mmol), VI-1 (100. Mu.L, 0.89 mmol), XPhos (28 mg,0.06 mmol), cesium carbonate (579 mg,1.78 mmol) and Pd ₂ (dba) ₃ (54 mg,0.06 mmol) was added, followed by anhydrous toluene (5 mL) and heated to 110deg.C under nitrogen to react for 8 hours, TLC monitoring (petroleum ether: ethyl acetate=1:1) the reaction was essentially complete, toluene was distilled off under reduced pressure, and the crude product was purified by column chromatography over silica gel (eluent: petroleum ether: ethyl acetate=4:1-2:1) to give 88mg of off-white solid I-B1 in 35.0% yield. ¹ H NMR(400MHz,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),6.94–6.71(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.

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)

Using compound VIII-1 (200 mg,0.59 mmol) and VI-2 (131 mg,0.59 mmol) as starting materials, with reference to the preparation of compound I-B1, 210mg of yellow solid I-B2 was obtained in 67.9% yield. 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.

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)

Using compound VIII-1 (200 mg,0.59 mmol) and VI-4 (123 mg,0.59 mmol) as starting materials, reference was made to the preparation of compound I-B1 to give 120mg of brown-green solid I-B3 in 39.8% yield. 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),6.42–6.30(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.

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

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

IV-3 (500 mg,1.71 mmol) was dissolved in tetrahydrofuran (35 mL) and cooled to 0deg.C, sodium hydride (62 mg,2.57 mmol) was slowly added, p-toluenesulfonyl chloride (653 mg,3.43 mmol) was added after the reaction solution was bubble free, stirred at 0deg.C for 1 hour and then allowed to react at room temperature for 2 hours, TLC was monitored (petroleum ether: ethyl acetate=8:1) and the reaction solution was substantially complete, concentrated under reduced pressure and purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate=40:1-25:1) to give 480mg of white solid IX-1 in a yield of 62.8%. ¹ H NMR(300MHz,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.

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

IX-1 (600 mg,1.35 mmol) and DMF (15 mL) were added to a 50mL three-necked flask and dissolved with stirring, cooled to 0deg.C, the solution of m-chloroperoxybenzoic acid (813 mg,4.71 mmol) in DMF (8 mL) was slowly added dropwise, the reaction was complete as monitored by TLC (Petroleum ether: ethyl acetate=2:1) over about 0.5 hour,the reaction was quenched by the addition of saturated sodium sulfite solution (30 mL), extracted with ethyl acetate (20 mL. Times.3) and water (60 mL), the organic phases combined, washed with saturated sodium chloride solution (15 mL. Times.2), and dried over anhydrous sodium sulfate; suction filtration, reduced pressure distillation of the filtrate to remove the solvent, purification of the crude product by silica gel column chromatography (eluent: petroleum ether: ethyl acetate=25:1-10:1) to obtain 580mg of yellow solid X-1, the yield is 90.2%. ¹ H NMR(300MHz,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.

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

To a 25mL round bottom flask was added X-1 (500 mg,1.05 mmol), VI-1 (177. Mu.L, 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) was added, then anhydrous toluene (8 mL) was added and the reaction was heated to 110℃under nitrogen with sealing, and the reaction was carried out for 8 hours under TLC monitoring (petroleum ether: ethyl acetate=2:1), the toluene was distilled off under reduced pressure, and the crude product was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate=25:1-10:1) to give 160mg of yellow solid XI-1 in 27.1% yield. ¹ H NMR(300MHz,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.

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

To a 25mL round bottom flask was added intermediate XI-1 (399mg, 0.70 mmol) dissolved in anhydrous methanol (12 mL) and CH was added ₃ Methanol solution of ONa (648 μl,3.50 mmol), heated to 60deg.C and reacted for 5 hours, TLC monitoring (petroleum ether: ethyl acetate=1:1) the reaction was essentially complete, the solvent was distilled off under reduced pressure, the residue was taken up in 10mL of water, extracted with ethyl acetate (10 ml×3), the organic phases combined, washed with saturated sodium chloride solution (15 ml×2), anhydrous sulfuric acidDrying sodium; suction filtration, reduced pressure evaporation of the filtrate, and purification of the crude product by silica gel column chromatography (eluent: petroleum ether: ethyl acetate=2:1-1:1) gave 253mg of brown solid I-C1 with a yield of 88.1%. ¹ H NMR(300MHz,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.

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

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

Starting with compound X-1 (500 mg,1.05 mmol) and VI-4 (234 mg,1.15 mmol), referring to the preparation of compound XI-1, 386mg of brown solid XI-2 was obtained in 56.8% yield. ¹ H NMR(300MHz,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.

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

Using compound XI-2 (80 mg,0.12 mmol) as a starting material, referring to the preparation method of compound I-C1, 42mg of brown solid I-C2 was obtained in a yield of 68.8%. ¹ H NMR(400MHz,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,1H),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.

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)

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

To a 25mL round bottom flask was added 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) ₂ (19 mg,0.08 mmol) followed by addition of anhydrous toluene (5 mL) and heating to 110deg.C under nitrogen protection in a closed condition for 8 hours, TLC monitoring (dichloromethane: methanol=10:1) the reaction was essentially complete, toluene was distilled off under reduced pressure, and the crude product was purified by column chromatography over silica gel (eluent: dichloromethane: methanol=60:1-50:1) to give 160mg of brown solid XI-3 in 28.8% yield. ¹ H NMR(300MHz,DMSO-d ₆ )δ9.48(s,1H),8.23(d,J＝7.4Hz,1H),7.84–7.72(m,3H),7.69(d,J＝7.6Hz,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.

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)

Starting from compound XI-3 (60 mg,0.09 mmol), reference was made to the preparation method of compound I-C1, giving 40mg of brown solid I-C3 in 86.9% yield. ¹ H NMR(300MHz,CDCl ₃ )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.

Example 15: activity test of the inventive Compound for inhibiting DCLK1 at enzyme level

(1) Experimental method

Establishing a DCLK1 enzyme activity inhibition molecular screening model, and determining according to the following method: DCLK1 enzyme can catalyze the conversion of fluorescence-labeled Kinase substrate12 (supplier GL) to fluorescence-labeled products and then detect the corresponding conversion using Mobility-Shift Assay (Mobility-Shift Assay) based on microfluidic chip technology that applies the basic concept of capillary electrophoresis to microfluidic environments and detects enzymatic experiments without the addition of stopping reagents. The substrate used for the experiment is polypeptide with fluorescent label, the substrate is converted into a product under the action of enzyme in a reaction system, the charge of the substrate is correspondingly changed, and the Mobility-Shift Assay utilizes the difference of the charge of the substrate and the charge of the product, and the substrate and the product are separated by utilizing an EZReader platform and are detected respectively.

Compound powders were dissolved in 100% DMSO to prepare 10mM stock solutions. (1) 1 XKinase buffer was prepared. (2) preparation of compound concentration gradient: test compounds were tested at single concentrations of 1 μm and 200nM and diluted to 100-fold final concentration in 100% DMSO solutions in 384 well plates. The 250nL of 100-fold final concentration of compound was transferred into the destination plate using a dispenser Echo 550. (3) A2.5-fold final concentration of Kinase solution was prepared using a1 XKinase buffer. (4) Adding 10 mu L of kinase solution with 2.5 times of final concentration to each of the compound well and the positive control well; to the negative control wells, 10. Mu.L of 1 XKinase buffer was added. (5) Centrifugation at 1000rpm for 30 seconds, the reaction plate was shaken and mixed well and incubated at room temperature for 10 minutes. (6) A5/3-fold final concentration of a mixed solution of ATP and Kinase substrate was prepared using a1 XKinase buffer. (7) The reaction was initiated by adding 15. Mu.L of a 5/3-fold final concentration of the mixed solution of ATP and substrate. (8) The 384-well plate was centrifuged at 1000rpm for 30 seconds, and after shaking and mixing, incubated at room temperature for 60 minutes. (9) The kinase reaction was stopped by adding 30. Mu.L of stop detection solution, centrifuging at 1000rpm for 30 seconds, and shaking and mixing. (10) conversion was read with Caliper EZ Reader.

The calculation formula is as follows:

wherein: conversion% _sample is a Conversion reading of the sample; convertion% _min: negative control Kong Junzhi, representing conversion reading without enzyme wells; convesion% _max: positive control Kong Junzhi, represents a conversion reading without compound inhibition wells.

Fitting dose-response curve:

the log value of the concentration is taken as an X axis, the percent inhibition rate is taken as a Y axis, and a log (inhibitor) vs response-Variable slope fit quantitative effect curve of analysis software GraphPad Prism 5 is adopted, so that the percent inhibition rate of each compound on the enzyme activity is obtained.

The calculation formula is as follows:

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

(2) Experimental results

TABLE 1 inhibition of DCLK1 by target compounds at the enzyme level

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

Note that: a represents an inhibition ratio of more than 50%, and B represents an inhibition ratio of between 15% and 50%.

As can be seen from Table 1, at a concentration of 1. Mu.M, some of the compounds of the present invention had a certain inhibitory activity against DCLK1, with the inhibitory activity of compound I-A5 against DCLK1 being optimal.

Example 16: evaluation of tumor cell in vitro inhibitory Activity of Compound I-A5 Using human pancreatic cancer cell SW1990 and colon cancer cell HCT116

Materials and reagents: human colon cancer cell line HCT116 and human pancreas cancer cell line SW1990 were both purchased from the national academy of sciences cell line, wherein 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 in 5% CO ₂ Culturing in a incubator at 37 ℃; DCLK1-IN-1 and I-A5 were dissolved IN DMSO at a final concentration of 10mM/L, filtered through a 0.22. Mu.M filter, and stored IN a-20deg.C freezer. Preparation of MTT: 0.25g of MTT was weighed and dissolved in 50mL of PBS to a final concentration of 5mg/mL, filtered through a 0.22 μm filter and stored in a-20deg.C freezer.

(1) Experimental method

MTT assay cell proliferation: cells were seeded at 2000 cells/well in 96-well plates at 5% co ₂ After 24h of culture at 37 ℃, the corresponding drugs are treated for 7 days, thiazole blue dye (MTT) is added for incubation for 3-4h, the culture medium is discarded, DMSO is added for complete dissolution and uniform mixing, and cell proliferation is detected.

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

(2) Experimental results

TABLE 2 inhibitory Activity of target Compounds against HCT116 and SW1990 tumor cell lines

Note that: ++ + representing IC ₅₀ Less than 10 μm.

As can be seen from Table 2, the target compound I-A5 has a strong inhibitory activity against SW1990 and HCT 116.

Example 17: experiment for evaluating tumor inhibition effect of compound I-A5 based on xenograft tumor model of human pancreatic cancer cells SW1990

(1) Experimental method

Nude mice were inoculated subcutaneously with human pancreatic carcinoma SW1990 cells, respectively, until tumors grew to 50mm ³ Animals were then randomized into vehicle, positive drug DCLK1-IN-1, I-A5 low dose, I-A5 medium dose, and I-A5 high dose. DCLK1-IN-1 group was injected with DCLK1-IN-1 (60 mg/kg) intraperitoneally once a day; I-A5 Low dose group I-A5 (30 mg/kg) was injected intraperitoneally, once a day; the dose group in I-A5 was injected I-A5 (45 mg/kg) intraperitoneally, once daily; I-A5 high dose group I-A5 (60 mg/kg) was injected intraperitoneally, once a day. Tumor volumes were measured every two days, mice were weighed, data recorded, and animals were sacrificed on day 21;

(2) Experimental results

The results show that tumor growth was significantly inhibited in each dose group given the compound of interest I-A5 compared to the model group. The tumor weight and tumor volume were significantly reduced for each of the I-A5 dose groups compared to the positive drug and model groups (fig. 1-3). The inhibition effect of each dose group of the I-A5 on the human pancreatic cancer cell SW1990 xenograft tumor is stronger than that of a positive drug group, and the difference is obvious, so that the compound I-A5 can exert better anti-tumor effect in vivo.

In conclusion, the compound disclosed by the invention can obviously inhibit the growth of tumor cells in vitro and in vivo, and has good clinical application prospect.

Claims

1. A DCLK1 inhibitor, characterized by having a structure of formula (I), said DCLK1 inhibitor comprising an isomer, a pharmaceutically acceptable salt or a mixture thereof,

wherein:

Y ¹ 、Y ² 、Y ³ each independently selected from CH or N;

Z ¹ 、Z ² each independently selected from O, S or NH;

R ³ Selected from H, F, cl, br, I, CF ₃ 、CHF ₂ 、CN、OCH ₃ OH or CH ₃ ；

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

2. The DCLK1 inhibitor according to claim 1, wherein in the structure: r is R ¹ Selected from H, CH ₃ 、CH ₂ CH ₃ 、CH ₂ CHF ₂ Or CH (CH) ₂ CF ₃ 。

3. The DCLK1 inhibitor according to claim 1, wherein in the structure: r is R ² Selected from SO ₂ R ⁶ Wherein R is ⁶ Selected from C1-C6 alkyl, CF ₃ Or CH (CH) ₂ CF ₃ 。

4. The DCLK1 inhibitor according to claim 1, wherein in the structure: r is R ³ Selected from H, F, cl, br, CF ₃ 、OCH ₃ Or CH (CH) ₃ 。

5. The DCLK1 inhibitor according to claim 1, wherein in the structure: r is R ⁴ Selected from CH ₃ Or CH (CH) ₂ CH ₃ ，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-trifluoroethyl) piperazin-1-yl.

6. DCLK1 inhibitor according to claim 1, characterized in that it is selected from any of the following compounds:

7. the DCLK1 inhibitor according to claim 1, wherein the pharmaceutically acceptable salt is a salt of the DCLK1 inhibitor with an acid selected from the group consisting of 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.

8. A process for the preparation of a DCLK1 inhibitor according to claim 1, characterised in that it is selected from any one of the following processes:

and a third method: when R is ¹ Is H, Y ¹ 、Y ² In the case of CH, the preparation method of the compound I-C is as follows:

wherein Y is ¹ 、Y ² 、Y ³ 、Z ¹ 、Z ² 、R ¹ 、R ² 、R ³ 、R ⁴ 、R ⁵ Or R is ⁶ Is defined as in claim 1.

9. A pharmaceutical composition comprising the DCLK1 inhibitor of claim 1 and a pharmaceutically acceptable carrier.

10. Use of the DCLK1 inhibitor of claim 1 or the pharmaceutical composition of claim 8 in the preparation of a medicament for treating cancer.