Tetrahydrobenzothiazolyl-containing 2, 4-diaminopyrimidine compound and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a 2, 4-diaminopyrimidine compound containing tetrahydrobenzothiazolyl and application of the 2, 4-diaminopyrimidine compound. The invention provides a tetrahydrobenzothiazolyl-containing 2, 4-diaminopyrimidine compound with a structure as shown in a formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof, and an application of the tetrahydrobenzothiazolyl-containing 2, 4-diaminopyrimidine compound. The tetrahydrobenzothiazolyl-containing 2, 4-diaminopyrimidine compound disclosed by the invention has obvious inhibitory activity on cyclin-dependent kinase 2 (CDK2), has an obvious treatment effect on diseases related to the cyclin-dependent kinase 2, such as tumor diseases such as human colon cancer, and has no obvious side effect; the compound can be used for preparing new target candidate drugs for human colon cancer and other diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a tetrahydrobenzothiazolyl-containing 2,4-diaminopyrimidine compound and application thereof. Background Art

[0002] Colon cancer is one of the most common malignancies worldwide, accounting for 10% of new cancer cases and 9.4% of cancer deaths worldwide. The development and progression of colon cancer is a complex biological process involving multiple genes, proteins, and signaling pathways. Currently, the early diagnosis rate for colon cancer remains low, and the five-year survival rate for advanced cases is less than 40%, posing a serious threat to public health and increasing the economic burden. Therefore, in-depth research into the molecular mechanisms of colon cancer pathogenesis, drug resistance, recurrence, and metastasis, as well as the active search for new anti-tumor molecular targets, is of great significance for drug development and disease treatment.

[0003] Cyclin-dependent kinases (CDKs), a key class of cell cycle or transcriptional regulatory proteases, are closely associated with tumorigenesis and treatment. Existing studies have shown that regulating cyclin-dependent kinases can block the tumor cell cycle, control proliferation, and promote apoptosis, thereby achieving the goal of tumor treatment. In recent years, with in-depth research into cell cycle regulation mechanisms, particularly the revelation of the central role of cyclin-dependent kinases (CDKs) in cell cycle regulation, CDKs have become a key area of ​​research and development for novel anticancer drugs. Studies have shown that CDK2 is a potential target for reversing resistance to CDK4 / 6 inhibitors and has been listed as one of the most promising targets for cancer therapy, garnering considerable attention. Currently, there are very few highly active and selective CDK2 inhibitors, and most inhibitors have poor metabolic stability, providing extremely limited information for the development of similar CDK2 inhibitors. The development of highly active, non-toxic, and selective CDK2 inhibitors through rational drug design remains a key challenge in this emerging area of ​​colorectal cancer treatment.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a tetrahydrobenzothiazolyl-containing 2,4-diaminopyrimidine compound and its application. The tetrahydrobenzothiazolyl-containing 2,4-diaminopyrimidine compound has significant inhibitory activity against cyclin-dependent kinase 2 (CDK2) and can be used to prepare new target candidate drugs for diseases such as human colon cancer.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a tetrahydrobenzothiazolyl-containing 2,4-diaminopyrimidine compound having a chemical structure as shown in the general formula (I):

[0008]

[0009] In the general formula (I),

[0010] R 1 is a methyl, hydrogen or fluorine atom;

[0011] R 2 It is a sulfonamide group, a methylsulfonyl group, a hydrogen atom, an N-methylpiperazinyl group or an N-ethylpiperazinyl group.

[0012] Preferably, it has any of the following structures:

[0013]

[0014]

[0015] The tetrahydrobenzothiazolyl 2,4-diaminopyrimidine compounds provided by the present invention have significant inhibitory activity against cyclin-dependent kinase 2 (CDK2) type, have significant therapeutic effects on CDK2-related diseases such as in vitro models of colon cancer, and can be used to prepare new target candidate drugs for pulmonary arterial hypertension.

[0016] In a second aspect, the present invention provides pharmaceutically acceptable salts or stereoisomers of the tetrahydrobenzothiazolyl-containing 2,4-diaminopyrimidine compound.

[0017] In a third aspect, the present invention provides a cyclin-dependent kinase 2 activity inhibitor, the main active ingredient of which is the tetrahydrobenzothiazolyl-containing 2,4-diaminopyrimidine compound or a pharmaceutically acceptable salt or stereoisomer thereof.

[0018] In a fourth aspect, the present invention provides the use of the tetrahydrobenzothiazolyl-containing 2,4-diaminopyrimidine compound, or a pharmaceutically acceptable salt or stereoisomer thereof, or the cyclin-dependent kinase 2 activity inhibitor in the preparation of a drug for treating cyclin-dependent kinase 2-related diseases.

[0019] Preferably, in the application, the tetrahydrobenzothiazolyl-containing 2,4-diaminopyrimidine compound has the structure shown below:

[0020]

[0021] In a fifth aspect, the present invention provides an anti-tumor drug, the main active ingredient of which is the tetrahydrobenzothiazolyl-containing 2,4-diaminopyrimidine compound or a pharmaceutically acceptable salt or stereoisomer thereof.

[0022] Preferably, the anti-tumor drug is used to treat human colon cancer and has good anti-colon cancer activity.

[0023] In a more specific embodiment, the present invention uses the CCK8 method standard operating procedure to determine the tumor inhibitory activity of tetrahydrobenzothiazolyl 2,4-diaminopyrimidine compounds. The results show that the compound has strong inhibitory activity against human colon cancer cells HCT116 and is superior to the control drug, and has good prospects for development as an anti-colon cancer drug.

[0024] Beneficial effects:

[0025] The present invention provides a tetrahydrobenzothiazolyl-containing 2,4-diaminopyrimidine compound having a structure as represented by formula (I), or a pharmaceutically acceptable salt or stereoisomer thereof, and uses thereof. The tetrahydrobenzothiazolyl-containing 2,4-diaminopyrimidine compound of the present invention has significant inhibitory activity against cyclin-dependent kinase 2 (CDK2), and has significant therapeutic effects on cyclin-dependent kinase 2-related diseases, such as human colon cancer and other tumor diseases, without significant side effects. The compound can be used to prepare new target drug candidates for diseases such as human colon cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0027] Figure 1 These are the experimental results of Example 10 of the present invention. Flow cytometry analysis of the effect of compound M-01 on the cell cycle of HCT116 cells after treatment with compound M-01 for 24 hours was performed, wherein Figure A shows the control group; Figure B shows the M-01, 0.65 μM treatment group; Figure C shows the M-01, 1.30 μM treatment group; and Figure D shows the M-01, 2.60 μM treatment group. DETAILED DESCRIPTION

[0028] The technical problem to be solved by the present invention is to provide a tetrahydrobenzothiazolyl-containing 2,4-diaminopyrimidine compound, which has a significant inhibitory effect on the proliferation of human colon tumor cells. In vitro, it can arrest the cell cycle of human colon tumor cells (HCT-116) at the G2 / M phase and exhibit dose-dependent resistance, showing good anti-colon cancer activity.

[0029] The compound provided by the present invention has a chemical structure as shown in the general formula (I):

[0030]

[0031] Among them, R 1 is a methyl, hydrogen or fluorine atom; R 2 It is a sulfonamide group, a methylsulfonyl group, a hydrogen atom, an N-methylpiperazinyl group or an N-ethylpiperazinyl group.

[0032] The preparation process of the compound provided by the present invention is as follows:

[0033]

[0034] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

[0035] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the exact ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed herein.

[0036] In the description of this specification, the reference terms "one embodiment", "some embodiments", "specific implementation methods", or "some specific implementation methods" and the like mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiments of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0037] In the examples provided herein, if specific techniques or conditions are not specified, the experiments were performed according to those described in literature in the field or according to the product instructions. Reagents or instruments used without manufacturer's indication are conventional products available through regular channels.

[0038] Example 1

[0039] This example provides compound M-01, whose structural formula and name are as follows:

[0040]

[0041] 4-(5-methyl-4-((4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)amino)pyrimidin-2-yl)amiro)benzenesulfonamide.

[0042] This example provides a method for synthesizing compound M-01, and the specific process is as follows:

[0043] Synthesis of Intermediate L2: Compound L1 (2,4-dichloro-5-methylpyrimidine) (1490 mg, 10 mmol) was added to a 100 ml reaction flask and dissolved in 40 mL of N,N-dimethylformamide (DMF). Sodium hydroxide (440 mg, 11 mmol) and 4,5,6,7-tetrahydrobenzothiazol-2-amine (1700 mg, 11 mmol) were slowly added with stirring at -5°C. After completion of the addition, the reaction was monitored by thin-layer chromatography (TLC). After completion, a large amount of water was added to the reaction system to terminate the reaction. The mixture was extracted twice with ethyl acetate, and the organic layer was collected and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation under reduced pressure, dried, dissolved in methanol, and recrystallized to obtain 1960 mg of a white solid in a yield of 73.0%.

[0044] 1 H NMR (400MHz, DMSO-d6) δ1.79(s,4H),2.18(s,3H),2.55(s,2H),2.64(s,2H),8.13(s,1H),11.23(s,1H).

[0045] Synthesis of compound M-01: Compound L2 (134.5 mg, 0.5 mmol) and 4-aminobenzenesulfonamide (88 mg, 0.51 mmol) were dissolved in 15 mL of ethylene glycol ethyl ether and stirred. 0.5 ml of concentrated hydrochloric acid was slowly added dropwise to reflux. After the reaction was completed by TCL detection, the reaction was quenched with saturated sodium chloride solution, extracted with ethyl acetate, and the organic layer was collected and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation under reduced pressure and recrystallized from methanol to obtain 130 mg of a white solid with a yield of 62.6%.

[0046] 1H NMR(400MHz,DMSO-d6)δ:1.81(s,4H,2×CH2),2.16(s,3H,CH3),2.59(s,2H,CH2),2.68(s,2H,CH2),7.16(s,2H,N H2),7.71(d,J=8.8Hz,2H,ArH),7.96(d,J=8.8Hz,2H,ArH),8.04(s,1H,ArH),9.34(s,1H,NH),10.55(s,1H,NH). 13 C NMR(100MHz,DMSO-d6)δ:157.32,156.13,144.34,136.13,126.70,118.61,23.42,23.03,22.75,13.68.HRMSm / z(ESI)calcd for C 18 H 20 N6O2S2[M+H] + 417.1167, found 417.1160.

[0047] Example 2

[0048] This example provides compound M-02, whose structural formula and name are as follows:

[0049]

[0050] N-(4-(Methylsulfonyl)phenyl)-N4-(4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)pyrimidine-2,4-diamine 2 -(4-(methylsulfonyl)phenyl)-N 4 This example provides a method for synthesizing compound M-02. Compared to the method for synthesizing compound M-01 in Example 1, the only difference is that the raw material in step 1 is 2,4-dichloropyrimidine, and the raw material in step 2 is 4-(methylsulfonyl)aniline.

[0051] This example obtained 45 mg of a white solid with a yield of 46.5%.

[0052] 1H NMR(400MHz,DMSO-d6)δ:1.79(s,4H,2×CH2),2.57(s,2H,CH2),2.67(s,2H,CH2),3.16(s,3H,CH3),6.57(d,J=5.6Hz,1H,A rH),7.81(d,J=9.2Hz,2H,ArH),8.08(d,J=8.8Hz,2H,ArH),8.20(d,J=5.6Hz,1H,ArH),9.68(s,1H,NH),11.34(s,1H,NH). 13 C NMR(100MHz,DMSO-d6)δ:158.84,157.91,156.81,155.83,145.66,144.58,132.72,12 8.14,121.52,119.20,100.64,44.43,26.44,23.41,23.07,22.75.HRMSm / z(ESI)calcd forC 18 H 19 N5O2S2[M+H] + 402.1058,found 402.1052.

[0053] Example 3

[0054] This example provides compound M-03, whose structural formula and name are as follows:

[0055]

[0056] 4-(4-((4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)amino)pyrimidin-2-yl)amino)benzenesulfonamide.

[0057] This example provides a method for synthesizing compound M-03. Compared with the method for synthesizing compound M-01 in Example 1, the only difference is that the raw material in step 1 is 2,4-dichloropyrimidine, and the raw material in step 2 is 4-aminobenzenesulfonamide.

[0058] This example obtained 60 mg of a white solid with a yield of 55.5%.

[0059] 1H NMR(400MHz,DMSO-d6)δ:1.80(s,4H,2×CH2),2.57(s,2H,CH2),2.67(s,2H,CH2),6.55(d,J=5.2Hz,1H,ArH),7.18(s,2H,N H2),7.73(d,J=8.8Hz,2H,ArH),7.98(d,J=8.8Hz,2H,ArH),8.19(d,J=5.6Hz,1H,ArH),9.55(s,1H,NH),11.31(s,1H,NH). 13 C NMR(100MHz,DMSO-d6)δ:158.97,157.89,156.86,155.86,144.57,143.98,136.58 ,126.70,121.47,119.05,100.29,26.45,23.41,23.08,22.76.HRMSm / z(ESI)calcd for C 17 H 18 N6O2S2[M+H] + 403.1011, found 403.1007.

[0060] Example 4

[0061] This example provides compound M-04, whose structural formula and name are as follows:

[0062]

[0063] 5-Methyl-N 2 -(4-(methylsulfonyl)phenyl)-N4 - (4,5,6,7-Tetrahydrobenzo[d]thiazol-2-yl)pyrimidine-2,4-diamine

[0064] 5-methyl-N 2 -(4-(methlsulfonyl)phenyl)-N 4 -(4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)pyrimidine-2,4-diamine.

[0065] This example provides a method for synthesizing compound M-04. Compared with the method for synthesizing compound M-01 in Example 1, the only difference is that the raw material in step 1 is 2,4-dichloro-5-methylpyrimidine, and the raw material in step 2 is 4-(methylsulfonyl)aniline.

[0066] This example obtained 51 mg of a white solid with a yield of 59.2%.

[0067] 1 H NMR(400MHz,DMSO-d6)δ:1.81(s,4H,2×CH2),2.16(s,3H,CH3),2.59(s,2H,CH2),2.67(s,2H,CH2),3. 15(s,3H,CH3),7.79(d,J=8.4Hz,2H,ArH),8.05–8.07(m,3H,ArH),9.48(s,1H,NH),10.58(s,1H,NH). 13 C NMR(100MHz,DMSO-d6)δ:157.16,156.05,145.99,132.22,128.15,118.76,44.46,23.40,23.01,22.75,13.70.HRMSm / z(ESI)calcdfor C 19 H 21 N5O2S2[M+H] + 416.1215, found 416.1205.

[0068] Example 5

[0069] This example provides compound M-05, whose structural formula and name are as follows:

[0070]

[0071] 5-Fluoro-N 2 -(4-(methylsulfonyl)phenyl)-N 4 -(4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)pyrimidine-2,4-diamine

[0072] 5-fluoro-N2-(4-(methylsulfonyl)phenyl)-N 4 -(4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)pyrimidine-2,4-diamine.

[0073] This example provides a method for synthesizing compound M-05. Compared with the method for synthesizing compound M-01 in Example 1, the only difference is that the raw material in step 1 is 2,4-dichloro-5-fluoropyrimidine, and the raw material in step 2 is 4-(methylsulfonyl)aniline.

[0074] This example obtained 50 mg of a white solid with a yield of 55%.

[0075] 1H NMR(400MHz,DMSO-d6)δ:1.81(s,4H,2×CH2),2.56(s,2H,CH2),2.65(s,2H,CH2),3.17(s,3H,CH3),7.81(d, J=8.8Hz,2H,ArH),8.04(d,J=8.8Hz,2H,ArH),8.23(d,J=2.8Hz,1H,ArH),9.65(s,1H,NH),11.96(s,1H,NH). 13 C NMR(100MHz,DMSO-d6)δ:154.59,145.83,140.91,132.49,128.19,118.70,44.45,26.76,25.11,23.20,22.76,22.66.HRMS(ESI)m / z:calcd for C 18 H 18 FN5O2S2[M+H] + 420.0964,found 420.0957.

[0076] Example 6

[0077] This example provides compound M-06, whose structural formula and name are as follows:

[0078]

[0079] 5-Methyl-N 2 -(4-(4-methylpiperazin-1-yl)phenyl)-N 4 -(4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)pyrimidine-2,4-diamine

[0080] 5-methyl-N 2 -(4-(4-methylpiperazin-1-yl)phenyl)-N 4 -(4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)pyrimidine-2,4-diamine.

[0081] This example provides a method for synthesizing compound M-06. Compared with the method for synthesizing compound M-01 in Example 1, the only difference is that the raw material in step 1 is 2,4-dichloro-5-methylpyrimidine, and the raw material in step 2 is 4-(4-methylpiperazin-1-yl)aniline.

[0082] This example obtained 58 mg of a white solid with a yield of 65.8%.

[0083] 1H NMR(400MHz,DMSO-d6)δ:1.79(s,4H,2×CH2),2.09(s,3H,CH3),2.23(s,3H,CH3),2.45–2.49(m,4H,2×CH2),2.56(s,2H,CH2),2.62(s,2H,C H2),3.04–3.08(m,4H,2×CH2),6.87(d,J=9.0Hz,2H,ArH),7.50(d,J=8.9Hz,2H,ArH),7.90(s,1H,ArH),8.65(s,1H,NH),10.34(s,1H,NH). 13 CNMR(100MHz,DMSO-d6)δ:158.52,156.52,146.77,132.98,122.42,116.09,55.07,49.24,46.08,29.40,23.43,23.06,22.69,13.48.HRMSm / z(ESI)calcd for C 23 H 29 N7S[M+H] + 436.2278,found436.2275.

[0084] Example 7

[0085] This example provides compound M-07, whose structural formula and name are as follows:

[0086]

[0087] N 2 -(4-(4-ethylpiperazin-1-yl)phenyl)-5-methyl-N 4 -(4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)pyrimidine-2,4-diamine

[0088] N 2 -(4-(4-ethylpiperazin-1-yl)phenyl)-5-methyl-N 4 (4,5,,6,,7-tetrahydrobenzo[d]thiazol-2-yl)pyrimidine-2,4-diamine.

[0089] This example provides a method for synthesizing compound M-07. Compared with the method for synthesizing compound M-01 in Example 1, the only difference is that the raw material in step 1 is 2,4-dichloro-5-methylpyrimidine, and the raw material in step 2 is 4-(4-ethylpiperazin-1-yl)aniline.

[0090] This example obtained 62 mg of a white solid with a yield of 68.5%.

[0091] 1 H NMR(400MHz, DMSO-d6)δ:1.03(t,J=7.2Hz,3H,CH3),1.79(s,4H,2×CH2),2.10(s,3H,CH3),2.37(q,J=7.1Hz,2H,CH2),2.50(s,4H,2×CH2),2.57(s,2H,CH2 ),2.62(s,2H,CH2),2.99–3.13(m,4H,2×CH2),6.86(d,J=9.0Hz,2H,ArH),7. 51(d,J=8.8Hz,2H,ArH),7.90(s,1H,ArH),8.66(s,1H,NH),10.40(s,1H,NH). 13 C NMR(100MHz,DMSO-d6)δ:158.52,156.52,132.98,122.42,116.04,52.78,52.02,49.34,26.22,23.43,23.06,22.69,13.48,12.28.HRMSm / z(ESI)calcd for C 24 H 31 N7S[M+H] + 450.2434,found450.2438.

[0092] Example 8

[0093] In this example, the CCK8 method was used to determine the anti-tumor activities of compounds M-01 to M-07 provided in Examples 1 to 7.

[0094] The assay includes the following steps:

[0095] Trypsinized cells were plated at 3 × 10 4Cells were seeded at a density of 1 μg / mL in a 96-well plate, with 0.1 mL added to each well. The compound was dissolved in DMSO to prepare a 1 M stock solution. Culture medium was then used to prepare final concentrations of 10, 20, 40, 80, 160, and 320 μM in the drug-addition medium. A commercial 5-fluorouracil (5-FU) solution at the same concentration was used as a control. After 24 hours of culture in the 96-well plate, the supernatant was aspirated and replaced with the drug-addition medium, which was then cultured for another 24 hours. The culture medium was discarded, and 100 μL of 1% CCK-8 culture medium was added to each well and incubated at room temperature for several minutes. The OD value of the control at 450 nm was measured on a microplate reader to a value of approximately 1.0. Three replicates were performed for each concentration. The inhibition rate (%) = (1–T–B / C–B) × 100%, where T, C, and B represent the absorbance values ​​of the experimental well, blank control well, and background, respectively. The half-maximal inhibitory concentration (IC) was calculated using the formula Xm-i(Σp-0.5). 50 .

[0096] The bioassay results are shown in Table 1:

[0097] Table 1 Antitumor activity data of compounds M01 to M07

[0098] project <![CDATA[InvitrocytotoxicityIC 50 (μM),HCT116]]> M-01 1.29±0.14 M-02 3.63±0.66 M-03 2.19±0.11 M-04 3.25±0.41 M-05 5.42±0.43 M-06 1.64±0.05 M-07 0.85±0.13

[0099] Experimental results show that this series of compounds has significant inhibitory activity against human colon cancer cells (HCT-116).

[0100] Example 9

[0101] In this example, the cyclin-dependent kinase 2 (CDK2) enzyme inhibitory activities of compounds M-01 to M-07 provided in Examples 1 to 7 were determined.

[0102] The experimental protocol for in vitro CDK2 enzyme inhibition activity test is as follows:

[0103] 1) Drug preparation: Prepared with DMSO to 10mM L -1 The mother solution was dissolved by ultrasonic acceleration, and then gradient diluted with DMSO and kinase buffer to ensure that the final concentration of DMSO was less than 1%.

[0104] 2) Kinase reaction buffer composition: 40 mM Tris, pH 7.4, 10 mM MgCl2, 0.1 mg / ml BSA, 1 mM DTT, 10 μM ATP.

[0105] 3) Add 1 μl of drug to each well of a white ELISA plate, then add 10 μl of various CDK enzymes and mix well.

[0106] 4) Add 5 μl of Histone H1 substrate and 34 μl of assay buffer to the above reaction wells, mix well, and incubate at 30°C for 40 min.

[0107] 5) Add 50 μl of ATP detection solution and react at room temperature for 5 minutes. Immediately measure the chemiluminescence signal on a microplate reader. The enzyme activity is inversely proportional to the chemiluminescence value. Substitute the value into the following formula to calculate the percentage activity: % activity = {(Lu drug - Lu background) / (Lu enzyme - Lu background)} × 100%. Use Graphpad Prism 5 software to calculate the IC value of the compound. 50 value.

[0108] Table 2 CDK2 enzyme inhibitory activity data of compounds M01-M07

[0109] project <![CDATA[IC 50 (nM),CDK2]]> M-01 4.7±0.3 M-02 93%@1.0μM M-03 87%@1.0μM M-04 33.4±2.4 M-05 90%@1.0μM M-06 36%@1.0μM M-07 30%@1.0μM Positive drugs 29.4±0.95

[0110] The positive control in the above table is the compound 5-methyl-N- 2 -(4-(methylsulfonyl)phenyl)-N 4 -(6,7-tetrahydro-4H-pyrano[4,3-d]thiazol-2-yl)pyrimidine-2,4-diamine, whose structural formula is:

[0111]

[0112] As shown in Table 2, the compound M-01 of the present invention has a strong inhibitory effect on CDK2 protein kinase, and the in vitro enzymatic inhibition activity (IC 50 ) at 4.7 nM, with good in vitro activity, significantly better than the control.

[0113] Example 10

[0114] This example studies the effect of the compound M-01 provided in Example 1 on the cell cycle of the human colon cancer cell line HCT116.

[0115] The experimental plan is as follows:

[0116] 1) Cell Culture: Human colon cancer cell line HCT116 was cultured in DMEM medium containing 10% fetal bovine serum. Human colon cancer cell line HCT116 was cultured in DMEM:F12 (1:1) medium (containing 100 IU / ml penicillin and 100 IU / ml streptomycin) at 37°C in a humidified atmosphere of 5% CO2, with the medium changed every two days.

[0117] 2) Cell seeding: Cells in logarithmic phase growth were digested with 0.25% trypsin and seeded in 96-well plates at a concentration of 4,000 cells / well for HCT116 and 8,000 cells / well for PC-3. Experimental procedures could be started after 24 hours when cells were growing well.

[0118] 3) Drug design: Drugs at concentrations of 0, 0.65 μM, 1.30 μM, and 2.60 μM were designed to treat the human colon cancer cell line HCT116.

[0119] 4) Drug treatment: Discard the original culture medium in the culture plate, add new drug-containing culture medium according to the concentration designed in the previous step to treat the cells, and place the cell plate in a 37°C, 5% CO2 constant temperature incubator for further culturing for 24 hours.

[0120] 5) Cell collection: Collect the supernatant in a 15 ml centrifuge tube, wash once with PBS and combine the liquid, digest the cells with trypsin containing EDTA and collect them in the same centrifuge tube, and centrifuge at 800 rpm for 5 minutes.

[0121] 6) Wash cells: After centrifugation, discard the supernatant, add an appropriate amount of PBS to gently suspend the cells, and centrifuge at 800 rpm for 5 minutes. Repeat this operation twice.

[0122] 7) Fix cells: After centrifugation, discard the supernatant, resuspend the cells in 1 ml of PBS, quickly add 3 ml of -20°C pre-cooled anhydrous ethanol, cover tightly, and place in a 4°C refrigerator to fix overnight.

[0123] 8) Cell washing: Centrifuge the fixed cells at 1200 rpm for 5 min, discard the supernatant, then resuspend and wash with an appropriate amount of PBS and centrifuge at 1200 rpm for 5 min.

[0124] 9) Cycle detection: Discard the supernatant, add 300 μL PBS, gently suspend the cells and transfer them to a flow cytometer. Add 10 μL PI and 1.25 μL RNase in a dark place, mix thoroughly and incubate in a 37°C incubator for 30 min. Detect the cycle distribution of each cell on a flow cytometer.

[0125] 10) Apoptosis Detection: Take 50,000-100,000 resuspended cells and centrifuge at 1000 rpm for 5 min. Aspirate the supernatant with a pipette and gently resuspend the cells in 200 μL Binding Buffer. Then add 5 μL Annexin V-PE and 5 μL 7-AAD staining solution and mix well. Incubate at room temperature (20-25°C) in the dark for 10-20 min, and then analyze cell apoptosis by flow cytometry.

[0126] The experimental results are shown in Figure 1 .

[0127] This example further explored the effect of compound M-01 on the HCT-116 cell cycle. Flow cytometric analysis revealed that, in HCT116 cells, as the concentration of M-01 increased, the proportion of cells in the G2 / M phase of the treated group increased after 24 hours compared to the untreated group, while the proportions of cells in the G0 / G1 and S phases decreased. Comparison of the distribution of G2 / M phase cells between the blank control group and each drug-treated group revealed significant differences between the HCT-116 drug-treated and blank controls, indicating that compound M-01 can induce G2 / M cycle arrest in HCT116 colon cancer cells and warrants further study.

[0128] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A 2,4-diaminopyrimidine compound containing a tetrahydrobenzothiazolyl group, characterized in that: It has a chemical structure as shown in the general formula (I): In the general formula (I), R 1 is a methyl, hydrogen or fluorine atom; R 2 It is a sulfonamide group, a methylsulfonyl group, a hydrogen atom, an N-methylpiperazinyl group or an N-ethylpiperazinyl group.

2. The tetrahydrobenzothiazolyl-containing 2,4-diaminopyrimidine compound according to claim 1, characterized in that: It has any of the following structures:

3. A pharmaceutically acceptable salt or stereoisomer of the tetrahydrobenzothiazolyl-containing 2,4-diaminopyrimidine compound according to claim 1 or 2.

4. A cyclin-dependent kinase 2 activity inhibitor, characterized in that The main active ingredient is the tetrahydrobenzothiazolyl-containing 2,4-diaminopyrimidine compound according to claim 1 or 2, or the salt or stereoisomer according to claim 3.

5. Use of the tetrahydrobenzothiazolyl-containing 2,4-diaminopyrimidine compound according to claim 1 or 2, or a pharmaceutically acceptable salt or stereoisomer thereof, or the cyclin-dependent kinase 2 activity inhibitor according to claim 4 in the preparation of a medicament for treating cyclin-dependent kinase 2-related diseases.

6. The application according to claim 5, characterized in that: The tetrahydrobenzothiazolyl-containing 2,4-diaminopyrimidine compound has the structure shown below:

7. An antitumor drug, characterized in that: The main active ingredient is the tetrahydrobenzothiazolyl-containing 2,4-diaminopyrimidine compound according to claim 1 or 2, or the salt or stereoisomer according to claim 3.

8. The anti-tumor drug according to claim 7, characterized in that: The anti-tumor drug is used for treating colon cancer.

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