4-(2-(methylamino) thiazole-5-yl) pyrimidine-2-amine derivative and application thereof

Abstract

The invention discloses a 4-(2-(methylamino) thiazole-5-yl) pyrimidine-2-amine derivative and application thereof, the structural formula of the 4-(2-(methylamino) thiazole-5-yl) pyrimidine-2-amine derivative is shown in the specification, in the formula, R1 is hydrogen or methyl; r2 is a substituted benzoyl group, a substituted phenylacetyl group, and a substituted or unsubstituted benzene propionyl group; and R3 is a substituted phenyl group, a 5-nitrofuran-2-yl group or a 1H-imidazole-2-yl group. As a novel CDK9 inhibitor, the ESCC inhibitor fills the blank of special medicines for ESCC; pharmaceutical salts and compositions can be prepared, and development of oral or injection preparations is facilitated; the compound is suitable for esophageal cancer single-drug treatment or is combined with other targeting / immune / chemotherapeutic drugs to improve the curative effect and reduce the drug resistance risk; meanwhile, other CDK9 related tumors such as lung cancer and breast cancer can be expanded.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry technology, specifically relating to a 4-(2-(methylamino)thiazo-5-yl)pyrimidine-2-amine derivative and its applications. Background Technology

[0002] Epidemiological data shows that in 2020, there were approximately 604,000 new cases of esophageal cancer and 544,000 deaths worldwide, ranking eighth in incidence and sixth in mortality among all cancers globally. China is a high-incidence area for esophageal cancer, accounting for more than half of the global cases, with approximately 85.79% of these being ESCC subtypes. It is predicted that by 2040, the number of new cases and deaths from esophageal cancer worldwide will climb to 987,700 and 914,300 respectively, representing increases of 63.5% and 68%; while China is expected to see as many as 530,000 new cases and 525,000 deaths, making the disease control situation extremely serious.

[0003] Cyclin-dependent kinase 9 (CDK9), a key factor regulating gene transcription elongation, plays a crucial role in tumorigenesis and development. CDK9 forms a positive transcription elongation factor b (P-TEFb) complex with cyclin T, driving gene transcription, including various short-lived oncoproteins such as MYC and MCL-1, by catalyzing the phosphorylation of the C-terminal domain (CTD) of RNA polymerase II. Furthermore, CDK9 directly or indirectly participates in the DNA damage response (DDR) process. Recent studies have shown that CDK9 is significantly overexpressed in both EAC and ESCC tissues and cell lines of esophageal cancer, and CDK9 inhibitors (alone or in combination) have demonstrated good antitumor activity in esophageal cancer models. These findings collectively indicate that CDK9 is a highly promising molecular target for esophageal cancer treatment, and the development of novel small-molecule inhibitors based on this target has significant clinical implications. However, novel CDK9 inhibitors targeting esophageal cancer are rarely reported, and there are currently no marketed CDK9 inhibitors for esophageal cancer treatment.

[0004] Existing CDK9 inhibitors, such as CDKI-73, have shown some activity in esophageal cancer models, but their in vitro IC50 is limited. 50 The activity levels were relatively high (4.58 μM for KYSE510 and 19.33 μM for KYSE150), resulting in limited in vivo tumor-suppressive efficacy, and the selectivity needs further optimization. Most reported thiazolium-pyrimidine compounds target other kinases such as Aurora or CDK4 / 6, lacking structural optimization and comprehensive validation targeting CDK9, especially ESCC. To address these issues, novel CDK9 inhibitors with higher activity and selectivity are needed to meet the clinical treatment needs of esophageal cancer. Summary of the Invention

[0005] The purpose of this invention is to overcome the deficiencies of the prior art and provide a 4-(2-(methylamino)thiazo-5-yl)pyrimidine-2-amine derivative.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned 4-(2-(methylamino)thiazol-5-yl)pyrimidine-2-amine derivative.

[0007] Another object of the present invention is to provide the use of the above-mentioned 4-(2-(methylamino)thiazo-5-yl)pyrimidine-2-amine derivative.

[0008] The technical solution of the present invention is as follows:

[0009] A 4-(2-(methylamino)thiazo-5-yl)pyrimidine-2-amine derivative, the structural formula of which is or ,in,

[0010] R1 is hydrogen or methyl;

[0011] R2 is a substituted benzoyl group, a substituted phenylacetyl group, or a substituted or unsubstituted phenylpropionyl group;

[0012] R3 is a substituted phenyl, 5-nitrofuran-2-yl, or 1H-imidazol-2-yl.

[0013] In a preferred embodiment of the present invention, its structural formula is selected from one of the following: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , .

[0014] The use of the above-mentioned 4-(2-(methylamino)thiazo-5-yl)pyrimidine-2-amine derivative in the preparation of CDK9-related tumor therapeutic drug compositions.

[0015] In a preferred embodiment of the present invention, the structural formula of the 4-(2-(methylamino)thiazo-5-yl)pyrimidine-2-amine derivative is selected from... , and .

[0016] More preferably, the structural formula of the 4-(2-(methylamino)thiazolyl-5-yl)pyrimidine-2-amine derivative is as follows: .

[0017] In a preferred embodiment of the present invention, the CDK9-related tumor is esophageal cancer.

[0018] A CDK9-related tumor treatment drug composition, characterized in that its active ingredient includes the 4-(2-(methylamino)thiazol-5-yl)pyrimidine-2-amine derivative described above.

[0019] In a preferred embodiment of the present invention, the structural formula of the 4-(2-(methylamino)thiazo-5-yl)pyrimidine-2-amine derivative is selected from... , and .

[0020] More preferably, the structural formula of the 4-(2-(methylamino)thiazolyl-5-yl)pyrimidine-2-amine derivative is as follows: .

[0021] In a preferred embodiment of the present invention, the CDK9-related tumor is esophageal cancer.

[0022] The beneficial effects of this invention are:

[0023] 1. This invention has excellent anti-proliferative activity: it shows significant inhibitory effects on various esophageal squamous cell carcinoma (ESCC) cell lines, which is superior to the positive control CDKI-73; it can achieve high-efficiency inhibition at low concentrations, and the long-term proliferation inhibition effect is further confirmed by colony formation experiments.

[0024] 2. This invention has good selectivity: it has low toxicity to normal esophageal epithelial cells and a high selectivity index; CDK9 knockdown experiments show that the activity of the compound is highly dependent on the target site, avoiding non-specific toxicity.

[0025] 3. The mechanism of action of this invention is comprehensive: it regulates the transcriptional elongation process in a concentration-dependent manner, downregulates RNA Pol II CTDp-Ser2 phosphorylation, MCL-1 and c-Myc protein and mRNA expression levels, and upregulates Cleaved PARP; at the same time, it induces G2 / M phase arrest and apoptosis, and effectively blocks tumor cell migration and invasion through scratch assay and MMP2 expression inhibition.

[0026] 4. This invention has strong kinase inhibitory ability: it has a highly efficient inhibitory effect on the CDK9 / Cyclin T1 complex; thermodynamic migration experiments directly confirm the stable binding of the compound to the target protein and improve the thermal stability of the complex.

[0027] 5. This invention has significant in vivo antitumor effects: In a nude mouse xenograft model of esophageal cancer, continuous administration of moderate doses significantly inhibited the growth of tumor volume and weight; there was no weight loss or pathological changes in major organs; the expression of proliferation markers Ki67 and Nur77 in tumor tissue was downregulated, and the mechanism was consistent with that in vitro.

[0028] 6. The synthesis process of this invention is efficient and simple: the multi-step synthetic route starting from commercially available raw materials has a high overall yield, mild reaction conditions (room temperature to reflux, no need for precious metal catalysts), intermediates are easy to monitor and purify, NMR / HRMS data fully support structural confirmation, and it is convenient for laboratory and industrial-scale production.

[0029] 7. This invention has broad clinical application potential: as a novel CDK9 inhibitor, it fills the gap in dedicated drugs for ESCC; it can be prepared into pharmaceutical salts and compositions, facilitating the development of oral or injectable formulations; it is suitable for monotherapy of esophageal cancer, or in combination with other targeted / immunotherapy / chemotherapy drugs to improve efficacy and reduce the risk of drug resistance; it can also be extended to other CDK9-related tumors such as lung cancer and breast cancer. Attached Figure Description

[0030] Figure 1 This is a Western blot analysis diagram showing the effect of the representative compound on the expression level of KYSE150-related proteins in Example 11 of the present invention.

[0031] Figure 2 This is a graph showing the results of the kinase activity experiment of the representative compound in Example 12 of the present invention.

[0032] Figure 3 The figure shows the results of the thermodynamic migration experiment of 12B and CDK9 / Cyclin T1 in Example 13 of the present invention.

[0033] Figure 4 This is a diagram showing the results of the cell colony formation and CDK9 knockdown experiments in Example 14 of this invention.

[0034] Figure 5 This is a graph showing the evaluation results of the effects of compound 12B in inducing cell cycle arrest and apoptosis in esophageal cancer cells in Example 15 of this invention.

[0035] Figure 6 This is a scratch test result of the evaluation of the effect of compound 12B on the migration of esophageal cancer cells in Example 16 of the present invention.

[0036] Figure 7 This is a Western blot analysis result of the effect of compound 12B in Example 16 of the present invention on the expression of MMP2, a migration marker protein of esophageal cancer cells.

[0037] Figure 8 This is a graph showing the results of Western blot analysis of the intervention effect of compound 12B on the expression of related proteins in ESCC cells in Example 17 of the present invention.

[0038] Figure 9 This is a diagram showing the results of in vivo experiments using compound 12B in a nude mouse xenograft tumor model in Example 18 of this invention. Detailed Implementation

[0039] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0040] The preparation method of the 4-(2-(methylamino)thiazo-5-yl)pyrimidine-2-amine derivative provided by the present invention is as follows:

[0041] (1) Commercially available N-methylthiourea was reacted to give the corresponding 2-methylamino-substituted thiazolidinone intermediates (1,3).

[0042] (2) The 2-methylamino-substituted thiazolidinone intermediate (1,3) reacts with N,N-dimethylformamide dimethyl acetal (DMF-DMA) under nitrogen protection to form the corresponding enone intermediate (4,5).

[0043] (3) The enone intermediate (4,5) and the guanidine nitrate salts (6a-6b) of the synchronously synthesized m-aminoacetanilide or p-aminoacetanilide are ring-closed under nitrogen protection, 104-106 °C, and alkaline conditions to form the corresponding 2-aminopyrimidine rings, which are the key intermediates of the reaction (7a-7b, 8a-8b).

[0044] (4) The key intermediate (7a-7b, 8a-8b) is deacetylated under acidic conditions to obtain the naked amino group, and then the key intermediate (9a-9b, 10a-10b) is obtained.

[0045] (5) The key intermediate (9a-9b, 10a-10b) is reacted with different types of carboxylic acids or acyl chlorides by condensation reaction to obtain target compounds 11a-11p. The amino group reacts with the corresponding aldehyde group to obtain target compounds 12a-12h and 12A-12G.

[0046] The specific reaction route is shown below:

[0047]

[0048] Example 1: Preparation of intermediate 1-(4-methyl-2-(methylamino)thiazolyl)ethane-1-one (1)

[0049] In a dry 100 mL reaction flask, methanol (30 mL), N-methylthiourea (5.0 g, 0.055 mol), and pyridine (4.4 g, 0.11 mol) were added sequentially at room temperature. Then, 3-chloro-2,4-pentanedione (7.4 g, 0.055 mol) was added dropwise with stirring. After the addition was complete, the reaction was allowed to proceed at room temperature for 4 h. After 2 h, the reaction was stopped by TLC monitoring. The reaction solution was filtered, the filter cake was collected, and dried to obtain a white solid product—intermediate 1:1-(4-methyl-2-(methylamino)thiazolyl-5-yl)ethane-1-one 5.2 g (yield: 88%).

[0050] Example 2: Preparation of intermediate (E)-N,N-dimethyl-N'-(methylaminomethylthio)formamide (2)

[0051] In a dry 100 mL reaction flask, N-methylthiourea (5.0 g, 0.055 mol) was dissolved in 30 mL of methanol at room temperature. N,N-dimethylformamide dimethyl acetal (7.5 mL, 0.055 mol) was added with stirring. The reaction was allowed to proceed overnight at room temperature. The reaction was stopped when TLC monitoring showed the reaction was complete. The product was evaporated to dryness to give 6.1 g of a white solid product – intermediate 2: (E)-N,N-dimethyl-N'-(methylaminomethylthio)formamide (yield: 75%).

[0052] Example 3: Synthesis of intermediate 1-(2-(methylamino)thiazol-5-yl)ethane-1-one (3) Preparation

[0053] In a dry 100 mL reaction flask, intermediate 2 (5 g, 0.034 mol) was dissolved in 30 mL of acetonitrile. Chloroacetone (2 mL, 0.034 mol) was added with stirring, and the mixture was refluxed for 4 h. The reaction was stopped by TLC monitoring. After cooling, the reaction solution was concentrated, neutralized with saturated sodium bicarbonate solution, filtered, and the filter cake was washed twice with water and dried to give a nearly white solid product - intermediate 3: 4.7 g of 1-(2-(methylamino)thiazolyl-5-yl)ethane-1-one (yield: 88%).

[0054] Example 4: Preparation of intermediate (E)-3-(dimethylamino)-1-(4-methyl-2-(methylamino)thiazolyl-5-yl)prop-2-en-1-one (4)

[0055] In a dry 100 mL reaction flask, intermediate 1 (2.0 g, 0.012 mol) and N,N-dimethylformamide dimethyl acetal (5 mL, 0.036 mol) were added sequentially, and the mixture was refluxed for 12 h. The reaction was stopped by TLC monitoring after it was complete. The reaction solution was cooled and concentrated under reduced pressure to obtain a pale yellow solid. An appropriate amount of ethyl acetate was added, and the mixture was filtered. The filter cake was washed twice with ethyl acetate and dried to obtain an orange-yellow solid intermediate 4: (E)-3-(dimethylamino)-1-(4-methyl-2-(methylamino)thiazolyl-5-yl)prop-2-en-1-one 1.7 g (yield: 70%).

[0056] Example 5: Preparation of intermediate (E)-3-(dimethylamino)-1-(2-(methylamino)thiazolyl-5-yl)prop-2-en-1-one (5)

[0057] In a dry 100 mL reaction flask, 1-(2-(methylamino)thiazol-5-yl)ethane-1-one (intermediate 3.2 g, 0.013 mol) and N,N-dimethylformamide dimethyl acetal (3 mL, 0.013 mol) were added sequentially, and the mixture was refluxed for 12 h. The reaction was stopped after TLC monitoring showed the reaction was complete. The reaction solution was cooled and concentrated under reduced pressure to obtain a pale yellow solid. An appropriate amount of ethyl acetate was added, and the mixture was filtered through a funnel. The filter cake was washed twice with ethyl acetate and dried to obtain an orange-yellow solid intermediate, intermediate 5: (E)-3-(dimethylamino)-1-(2-(methylamino)thiazol-5-yl)prop-2-en-1-one 1.5 g (yield: 70%).

[0058] Example 6: Preparation of intermediate N-(3-guanidinophenyl)acetamide nitrate (6a)

[0059] In a dry 100 mL reaction flask, 75% ethanol, m-aminoacetanilide (2.0 g, 0.013 mol), and cyanamide (1.64 g, 0.039 mol) were added sequentially. Concentrated hydrochloric acid (0.63 mL, 0.020 mol) was slowly added dropwise while stirring. After the addition was complete, the temperature was raised to reflux temperature and the reaction continued for 24 h. The reaction was stopped when TLC monitoring showed it was complete. After cooling, the reaction solution was concentrated under reduced pressure to remove the ethanol. An aqueous solution of ammonium nitrate was then added until a solid appeared, at which point the addition was stopped. The reaction solution was continued to react for 1 h in an ice-water mixture at 0 °C with stirring. A large amount of nearly white solid precipitated visibly. The solid was filtered, the filter cake was washed twice with water, and dried to obtain 3.3 g of nearly white solid intermediate 6a: N-(3-guanidinylphenyl)acetamide nitrate (yield: 88%).

[0060] Replacing m-aminoacetanilide with p-aminoacetanilide in the above experimental steps can prepare the intermediate N-(4-guanidinylphenyl)acetamide nitrate (6b).

[0061] Example 7: Preparation of N-(3-((4-(4-methyl-2-(methylamino)thiazolyl-5-yl)pyrimidin-2-yl)amino)phenyl)acetamide (7a)

[0062] In a dry 100 mL reaction flask, intermediate 4 (2 g, 0.009 mol), intermediate 6a (4.83 g, 0.018 mol), and sodium hydroxide (0.72 g, 0.018 mol) were dissolved in 30 mL of 75% ethanol. The reaction was carried out at 105 °C for 48 h. The reaction was stopped when TLC monitoring showed that the reaction was complete. After the reaction solution was cooled to room temperature, it was filtered, the filter cake was washed twice with ethyl acetate, and dried to obtain a pale yellow solid product - intermediate 7a: 1.8 g of N-(3-((4-(4-methyl-2-(methylamino)thiazolyl-5-yl)pyrimidin-2-yl)amino)phenyl)acetamide (yield: 69%).

[0063] Similarly, in the above experimental steps:

[0064] 1) When intermediate 6a is replaced with 6b, intermediate N-(4-((4-(4-methyl-2-(methylamino)thiazolyl-5-yl)pyrimidin-2-yl)amino)phenyl)acetamide (7b) can be prepared (yield: 83%).

[0065] 2) When intermediate 4 is replaced with 5, intermediate N-(3-((4-(2-(methylamino)thiazolyl-5-yl)pyrimidin-2-yl)amino)phenyl)acetamide (8a) can be prepared (yield: 77%).

[0066] 3) When intermediate 4 is replaced with 5 and 6a is replaced with 6b, intermediate N-(4-((4-(2-(methylamino)thiazolyl-5-yl)pyrimidin-2-yl)amino)phenyl)acetamide (8b) can be prepared (yield: 88%).

[0067] Example 8: Intermediate N 1 Preparation of -(4-(4-methyl-2-(methylamino)thiazo-5-yl)pyrimidin-2-yl)phenyl-1,3-diamine (9a)

[0068] In a dry 100 mL reaction flask, intermediate 7a (2 g), 10 mL of methanol, and 10 mL of water were added sequentially. While stirring, 10 mL of 1M dilute hydrochloric acid was added dropwise. After the addition was complete, the mixture was heated to reflux temperature in an oil bath and refluxed for 4 hours. The reaction was stopped when TLC monitoring showed the reaction was complete. The reaction solution was cooled to room temperature, and saturated sodium bicarbonate was slowly added dropwise until the pH of the entire system became weakly alkaline. A pale yellow solid was observed to precipitate continuously. The mixture was then filtered through a funnel, the filter cake was washed twice with water, and dried to obtain the pale yellow solid product – intermediate 9a:N. 1 -(4-(4-methyl-2-(methylamino)thiazolyl-5-yl)pyrimidin-2-yl)phenyl-1,3-diamine 1.4 g.

[0069] Similarly, in the above experimental steps: when intermediate 7a is replaced with 7b, 8a, or 8b, the corresponding intermediate N can be prepared respectively. 1 -(4-(4-methyl-2-(methylamino)thiazo-5-yl)pyrimidin-2-yl)phenyl-1,4-diamine (9b), N 1 -(4-(2-(methylamino)thiazo-5-yl)pyrimidin-2-yl)phenyl-1,3-diamine (10a) and N 1 -(4-(2-(methylamino)thiazolyl-5-yl)pyrimidin-2-yl)phenyl-1,4-diamine (10b).

[0070] Example 9: Preparation of target products 11a-11p

[0071] In a dry 50 mL reaction flask, DMF (10 mL), various carboxylic acids or acyl chlorides (0.11 mol / 0.15 mol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) (0.2 mol) were added sequentially at room temperature or in an ice bath. Then, intermediate 9a or 9b (0.1 mmol) and N,N-diisopropylethylamine (DIEA) (0.2 mmol) were added as a catalyst while stirring. The reaction was allowed to proceed overnight at room temperature. TLC monitoring showed the reaction was complete. The reaction was stopped, and the reaction solution was extracted three times with EA, washed with saturated sodium chloride solution, and the organic phases were combined. After drying in anhydrous MgSO4, the mixture was filtered to prepare slag, and then separated by silica gel column chromatography (V... DCM :V MeOH =20:1), to obtain the corresponding target product 11a-11p (yield: 40%-81%).

[0072] Target products 11a-11p are respectively: 2-methyl-N-(3-((4-(4-methyl-2-(methylamino)thiazol-5-yl)pyrimidin-2-yl)amino)phenyl)benzamide (11a), 2-fluoro-N-(3-((4-(4-methyl-2-(methylamino)thiazol-5-yl)pyrimidin-2-yl)amino)phenyl)benzamide (11b), 2-fluoro-N-(3-((4-(4-methyl-2-(methylamino)thiazol-5-yl)amino)phenyl)benzamide (11b), 2-fluoro-N-(3-((4-(4-methyl-2-(methylamino)thiazol-5-yl)amino)phenyl)benzamide (11b), 2-fluoro-N-(3-((4-(4-methyl-2-(methylamino)thiazol-5-yl)amino)phenyl)benzamide (11b). 4-(3-((4-(4-methyl-2-(methylamino)thiazolyl)pyrimidin-2-yl)amino)phenyl)benzamide (11c), 4-fluoro-N-(3-((4-(4-methyl-2-(methylamino)thiazolyl)pyrimidin-2-yl)amino)phenyl)benzamide (11d), N-(3-((4-(4-methyl-2-(methylamino)thiazolyl)pyrimidin-2-yl)amino)phenyl)-4-nitrobenzamide (11e), 33-bromo-4-methoxy-N-(3-((4-(4-) - Methyl-2-(methylamino)thiazolyl-5-yl)pyrimidin-2-yl)amino)phenyl)benzamide (11f), N-(3-((4-(4-methyl-2-(methylamino)thiazolyl-5-yl)pyrimidin-2-yl)amino)phenyl)-2-(p-tolyl)acetamide (11g), 3-(4-methoxyphenyl)-N-(3-((4-(4-methyl-2-(methylamino)thiazolyl-5-yl)pyrimidin-2-yl)amino)phenyl)propionamide (11h), 3-(4-fluorophenyl)-N-(3-( (4-(4-methyl-2-(methylamino)thiazolyl-5-yl)pyrimidin-2-yl)amino)phenyl)propionamide (11i), N-(3-((4-(4-methyl-2-(methylamino)thiazolyl-5-yl)pyrimidin-2-yl)amino)phenyl)-3-phenylpropionamide (11j), 2-fluoro-N-(4-(4-methyl-2-(methylamino)thiazolyl-5-yl)pyrimidin-2-yl)amino)phenyl)benzamide (11k), 4-fluoro-N-(4-(4-methyl-2-(methylamino)thiazolyl-5-yl)pyrimidin-2-yl)amino)phenyl)benzamide (11l), N-(4-((4-(4-methyl-2-) (methylamino)thiazol-5-yl)pyrimidin-2-yl)amino)phenyl)-4-nitrobenzamide (11m), N-(4-((4-(4-methyl-2-(methylamino)thiazol-5-yl)pyrimidin-2-yl)amino)phenyl)2-(p-tolyl)acetamide (11n), 2-(3-methoxyphenyl)-N-(4-(4-(-4-methyl-2-(methylamino)thiazol-5-yl)pyrimidin-2-yl)amino)phenyl)acetamide (11o), 3-(4-bromophenyl)-N-(4-(4-(-4-methyl-2-(methylamino)thiazol-5-yl)pyrimidin-2-yl)amino)phenyl)propionamide (11p).

[0073] Example 10: Preparation of target products 12a-12h and 12A-12G

[0074] In a dry 50 mL reaction flask, intermediates 9a, 9b, 10a, or 10b (0.1 mmol) were added to a round-bottom flask, dissolved in an appropriate amount of anhydrous ethanol, followed by the addition of various aldehydes (0.1 mmol), and then acetic acid (0.1 mmol) was slowly added dropwise. The mixture was refluxed under N2 protection and the reaction was monitored after 4 h. The reaction was stopped after TLC monitoring indicated completion. The reaction solution was directly filtered and washed 2-3 times with ethyl acetate, then dried to obtain the corresponding target products 12a-12h and 12A-12G (yields: 44%-67%).

[0075] The target products 12a-12h and 12A-12G are respectively: (E)-5-(2-((3-(((2,3-dimethoxybenzyl)amino)phenyl)amino)pyrimidin-4-yl)-N,4-dimethylthiazol-2-amine (12a) and (E)-5-(2-((3-((3-bromo-4-methoxybenzyl)amino)phenyl)amino)pyrimidin-4-yl)-N,4-dimethylthiazol-2-amine (12a) and (E)-5-(2-((3-((3-bromo-4-methoxybenzyl)amino)phenyl)amino)pyrimidin-4-yl)-N,4-dimethylthiazol-2-amine (12a). (E)-N,4-dimethylthiazol-2-amine (12b), (E)-2,5-difluoro-4-((3-((4-(4-methyl-2-(methylamino)thiazol-5-yl)pyrimidin-2-yl)amino)phenyl)imino)methyl)phenol (12c), (E)-4-((3-((4-(4-methyl-2-(methylamino)thiazol-5-yl)pyrimidin-2-yl)amino)phenyl)imino)methyl)phenol (12d), (E)-N-4-dimethyl-5-(2-((3-(((5-nitrofuran-2-yl)methylene)amino)phenyl)amino)pyrimidin-4-yl)thiazol-2-amine (12e), (E)-5-(2-((3-(((1H-imidazol-2-yl)methylene)amino)phenyl)amino)pyrimidin-4-yl) -N,4-Dimethylthiazol-2-amine (12f), (E)-N-4-dimethyl-5-(2-((4-(((5-nitrofuran-2-yl)methylene)amino)phenyl)amino)pyrimidin-4-yl)thiazol-2-amine (12g), (E)-5-(2-((4-(((1H-imidazol-2-yl)methylene)amino)phenyl)amino)pyrimidin-4-yl)-N,4-dimethylthiazol-2-amine (12h), (E)-5-(2-((3-(((2,3-dimethoxybenzyl)amino)phenyl)amino)pyrimidin-4-yl)-N-methylthiazol-2-amine (12A), (E)-5-(2-((3-(((3-bromo-4-methoxybenzyl)amino)phenyl) (12B)-(E)-2,5-difluoro-4-((3-((4-(2-(methylamino)thiazol-5-yl)pyrimidin-2-yl)amino)phenyl)imino)methyl)phenol (12C)-4-((3-((4-(4-2-(methylamino)thiazol-5-yl)pyrimidin-2-yl)amino)phenyl)imino)methyl)phenol (12C) (12D) phenol, (E)-N-methyl-5-(2-((3-(((5-nitrofuran-2-yl)methylene)amino)phenyl)aminopyrimidine-4-yl)thiazole-2-amine (12E), (E)-N-4-dimethyl-5-(2-((4-((((5-nitrofuran-2-yl)methylene)amino)phenyl)amino)pyrimidine-4-yl)thiazole-2-amine (12F), (E)-5-(2-((4-(((1H-imidazol-2-yl)methylene)amino)phenyl)amino)pyrimidine-4-yl)-N-methylthiazole-2-amine (12G).

[0076] Table 1. Structure and proton NMR spectrum of the compounds described in this invention 1 ¹H NMR and high-resolution mass spectrometry (HRMS)

[0077] compound structural <![CDATA[ 1 H NMR and HRMS<!-- 9 --> ]]> 11a <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 10.24(s, 1H), 9.44 (s, 1H), 8.33 (d,J = 5.4 Hz, 1H), 8.06 – 8.01 (m,2H), 7.62 – 7.57 (m, 1H), 7.45(d, J = 7.4 Hz, 1H), 7.41 – 7.37(m, 1H), 7.31 (d, J = 7.3 Hz,3H), 7.23 (d, J = 8.1 Hz, 1H),6.88 (d, J = 5.5 Hz, 1H), 2.82(s, 3H), 2.48 (s, 3H), 2.40 (s,3H). 13 C NMR (151 MHz, DMSO-d6) δ169.86, 168.17, 160.02, 159.15,158.03, 152.88, 141.29, 139.71,137.89, 135.63, 130.92, 129.96,128.81, 127.67, 126.05, 118.17,115.25, 113.95, 111.73, 107.39,31.25, 19.80, 19.13. HRMS (ESI,m / z): calcd for C 23 H 23 N6OS [M + H] + 431.1576, found 431.1644.]]> 11b <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 10.35(s, 1H), 9.48 (s, 1H), 8.33 (d,J = 5.4 Hz, 1H), 8.03 (s, 2H),7.66 (d, J = 7.4 Hz, 1H), 7.59(t, J = 8.1 Hz, 2H), 7.37 – 7.31(m, 3H), 7.24 (t, J = 8.1 Hz,1H), 6.89 (d, J = 5.4 Hz, 1H),2.83 (s, 3H), 2.49 (s, 3H). 13 CNMR (151 MHz, DMSO-d6) δ 169.92,163.10, 160.19, 159.98, 159.16,158.54, 158.01, 152.91, 141.37,139.31, 130.36, 128.94, 124.98,118.16, 116.65, 116.51, 115.47,113.91, 111.65, 107.44, 31.25,19.11. HRMS (ESI, m / z): calcdfor C 22 H 20 FN6OS [M + H] + 435.1325,found 445.1393. <!-- 10 -->]]> 11c <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 10.15(s, 1H), 9.45 (s, 1H), 8.34 (d,J = 5.4 Hz, 1H), 8.10 (t, J =2.1 Hz, 1H), 8.01 (d, J = 4.8Hz, 1H), 7.78 (s, 1H), 7.75 (d,J = 4.2 Hz, 1H), 7.57 – 7.54 (m,1H), 7.42 – 7.40 (m, 2H), 7.32 –7.29 (m, 1H), 7.24 (t, J = 8.0Hz, 1H), 6.89 (d, J = 5.4 Hz,1H), 2.78 (s, 3H), 2.48 (s, 3H),2.41 (s, 3H). 13 C NMR (151 MHz,DMSO-d6) δ 165.97, 160.03,159.14, 158.05(*2), 152.90,141.23, 139.57, 138.08, 135.55,132.48, 128.70, 128.64, 125.29,118.25, 115.39, 114.85, 112.67,107.36(*2), 31.22, 21.44,19.11.HRMS (ESI, m / z): calcd forC 23 H 23 N6OS [M + H] + 431.1576, found431.1646.]]> 11d <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 10.22(s, 1H), 9.70 (s, 1H), 8.50 (d,J = 5.2 Hz, 1H), 8.17 (s, 1H),8.04 (d, J = 6.5 Hz, 1H), 7.73(s, 1H), 7.58 (d, J = 8.1 Hz,1H), 7.38 (t, J = 8.5 Hz, 3H),7.27 (dd, J = 12.6, 4.5 Hz, 2H),7.12 (d, J = 5.3 Hz, 1H), 3.56(s, 3H), 2.67 (s, 3H). 13 C NMR(151 MHz, DMSO-d6) δ 169.53,164.82, 163.59, 162.99, 160.17,159.76, 159.09, 148.49, 141.02,139.54, 131.97, 130.90, 128.81,126.09, 116.13, 115.98, 115.68,115.03, 112.71, 108.84, 38.23,18.80. HRMS (ESI, m / z): calcdfor C 22 H 20 FN6OS [M + H] + 435.1325,found 435.2029. <!-- 11 -->]]> 11e <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 10.54(s, 1H), 9.51 (s, 1H), 8.37 (d,J = 8.3 Hz, 2H), 8.34 (d, J =5.4 Hz, 1H), 8.19 (d, J = 8.3Hz, 2H), 8.12 (s, 1H), 8.02 (q,J = 4.9 Hz, 1H), 7.60 (d, J =8.1 Hz, 1H), 7.33 (d, J = 8.0Hz, 1H), 7.27 (t, J = 8.0 Hz,1H), 6.90 (d, J = 5.4 Hz, 1H),2.78 (s, 3H), 2.48 (s, 3H). 13 CNMR (151 MHz, DMSO-d6) δ 169.93,164.26, 159.98, 159.15, 158.04,152.96, 149.58, 141.37, 141.20,139.07, 129.70(*2), 128.90,123.99(*2), 118.21, 115.80,114.82, 112.53, 107.43, 31.23,19.11. HRMS (ESI, m / z): calcdfor C 22 H 20 N7O3S [M + H] + 462.1270,found 462.1339.]]> 11f <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 10.17(s, 1H), 9.57 (s, 1H), 8.36 (d,J = 5.5 Hz, 1H), 8.24 (s, 1H),8.09 (s, 1H), 8.04 – 8.01 (m,1H), 7.53 (d, J = 8.0 Hz, 1H),7.32 (d, J = 8.1 Hz, 1H), 7.28 –7.22 (m, 3H), 6.93 (d, J = 5.5Hz, 1H), 3.95 (s, 3H), 2.83 (s,3H), 2.50 (s, 3H). 13 C NMR (151MHz, DMSO-d6) δ 169.84, 163.91,159.54, 158.88, 158.34, 157.61,140.90, 139.51, 132.74, 129.62,128.81, 128.70, 118.19, 115.64,115.16, 112.85, 112.60(*2),110.79, 107.37, 57.08, 31.48,18.43. HRMS (ESI, m / z): calcdfor C 23 H 22 BrN6O2S [M + H] + 525.0630, found 525.2871. <!-- 12 -->]]> 11g <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 10.08(s, 1H), 9.61 (s, 1H), 8.45 (d,J = 5.3 Hz, 1H), 7.92 (d, J =2.1 Hz, 1H), 7.50 (d, J = 9.3Hz, 1H), 7.26 (dd, J = 7.7, 2.5Hz, 1H), 7.22 (s, 1H), 7.17 –7.15 (m, 3H), 7.12 (d, J = 7.9Hz, 2H), 7.04 (d, J = 5.4 Hz,1H), 3.56 (s, 2H), 2.61 (s, 3H),2.28 (s, 3H), 2.27 (s, 3H). 13 CNMR (151 MHz, DMSO-d6) δ 172.04,169.64, 159.99, 159.59, 158.73,148.43, 140.99, 136.34, 135.88,133.61, 131.54, 129.83, 129.46,129.44, 129.26, 125.39, 114.97,113.60, 111.15, 108.85, 43.32,34.76, 21.16, 18.77. HRMS (ESI,m / z): calcd for C 24 H 25 N6OS [M + H] + 445.1732, found 445.1801.]]> 11h <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 9.87(s, 1H), 9.45 (s, 1H), 8.38 (s,1H), 8.08 (q, J = 4.7 Hz, 1H),7.86 (s, 1H), 7.59 – 7.55 (m,1H), 7.29 (s, 1H), 7.23 (dd, J =8.3, 4.6 Hz, 3H), 6.94 (d, J =5.5 Hz, 1H), 6.92 – 6.89 (m,2H), 3.77 (s, 3H), 2.91 (s, 3H),2.90 (d, J = 4.5 Hz, 2H), 2.64(d, J = 7.9 Hz, 2H), 2.53 (s,3H); 13 C NMR (151 MHz, DMSO-d6) δ170.82, 169.85, 159.66,157.99(*3),141.27,139.35,133.57,129.66(*3),128.86,114.70,114.19(*2),113.45,110.89,107.38(*2), 55.43,38.72, 30.52, 30.29, 19.10. HRMS(ESI, m / z): calcd for C 25 H 27 N6O2S[M + H] + 474.1838, found474.1863. <!-- 13 -->]]> 11i <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 9.84(s, 1H), 9.40 (s, 1H), 8.32 (d,J = 5.4 Hz, 1H), 8.05 (d, J =4.8 Hz, 1H), 7.80 (d, J = 2.1Hz, 1H), 7.52 (d, J = 6.9 Hz,1H), 7.30 – 7.26 (m, 2H), 7.23(dd, J = 7.4, 2.1 Hz, 1H), 7.17(t, J = 8.0 Hz, 1H), 7.11 (t, J= 8.9 Hz, 2H), 6.88 (d, J = 5.5Hz, 1H), 2.90 (d, J = 7.7 Hz,2H), 2.86 (s, 3H), 2.61 (d, J =7.5 Hz, 2H), 2.47 (s, 3H); 13 CNMR (151 MHz, DMSO-d6) δ 170.61,160.36, 159.98, 157.98(*2),141.28, 139.64, 130.52(*2),130.47, 128.87, 118.15, 115.50,115.36(*2), 114.74, 110.90,107.39(*2), 38.41, 30.58, 30.48,19.09. HRMS (ESI, m / z): calcdfor C 24 H 23 FN6OS [M + H] + 463.1638,found 463.1653. <!-- 14 -->]]> 11j <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 9.84(s, 1H), 9.42 (s, 1H), 8.32 (d,J = 5.4 Hz, 1H), 8.07 (s, 1H),7.82 (s, 1H), 7.52 (d, J = 8.1Hz, 1H), 7.27 (dt, J = 17.9, 8.5Hz, 5H), 7.18 (q, J = 8.3, 7.8Hz, 2H), 6.89 (d, J = 5.4 Hz,1H), 2.92 (d, J = 7.9 Hz, 2H),2.86 (s, 3H), 2.63 (t, J = 7.8Hz, 2H), 2.48 (s, 3H). 13 C NMR(151 MHz, DMSO-d6) δ 170.72,169.83, 159.96, 159.15, 157.95(*2), 141.74, 141.27, 139.68,128.88, 128.79(*2), 128.72,126.40, 118.15, 114.72, 113.33,110.90, 107.39(*2), 38.37,31.36, 31.26, 19.07. HRMS (ESI,m / z): calcd for C 24 H 24 N6OS [M + H] + 445.1732, found 445.1801.]]> 11k <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 10.35(s, 1H), 9.69 (s, 1H), 8.50 (d,J = 5.2 Hz, 1H), 7.80 (s, 1H),7.70 (s, 1H), 7.67 (d, J = 6.1Hz, 2H), 7.56 (q, J = 6.2 Hz,1H), 7.43 (dd, J = 18.1, 8.5 Hz,2H), 7.35 – 7.31 (m, 2H), 7.12(d, J = 5.3 Hz, 1H), 3.50 (s,3H), 2.67 (s, 3H). 13 C NMR (151MHz, DMSO-d6) δ 166.27, 162.77,160.16, 160.01, 157.31, 148.37,136.91, 133.43, 130.36, 129.78,126.61, 125.67, 125.56, 124.97,120.52, 119.63, 116.65, 116.52,116.50, 108.54, 36.81, 18.75.HRMS (ESI, m / z): calcd forC 22 H 20 FN6OS [M + H] + 435.1325,found 445.1396. <!-- 15 -->]]> 11l <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 10.20(s, 1H), 9.66 (s, 1H), 8.50 (d,J = 5.2 Hz, 1H), 8.06 – 8.04 (m,1H), 7.79 (d, J = 6.0 Hz, 3H),7.71 (d, J = 8.6 Hz, 1H), 7.41 –7.34 (m, 4H), 7.11 (d, J = 5.3Hz, 1H), 3.56 (s, 3H), 2.67 (s,3H). 13 C NMR (151 MHz, DMSO-d6) δ169.67, 164.46, 163.61, 160.02,159.73, 148.38, 136.88, 133.48,131.94, 131.02, 130.96, 130.77,130.71, 121.23, 119.48, 116.17,116.02, 115.80, 115.66, 108.49,38.22, 18.80. HRMS (ESI, m / z):calcd for C 22 H 20 FN6OS [M + H] + 435.1325, found 435.1213.]]> 11m <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 10.71(s, 1H), 9.48 (s, 1H), 8.37 –8.34 (m, 3H), 8.27 (d, J = 8.8Hz, 2H), 8.20 (d, J = 4.8 Hz,1H), 7.77 (d, J = 7.7 Hz, 4H),6.90 (d, J = 5.5 Hz, 1H), 2.87(s, 3H), 2.48 (s, 3H). 13 C NMR(151 MHz, DMSO-d6) δ 169.62,163.83, 159.87, 159.04, 158.10,152.73, 149.47, 141.14, 137.50,132.87, 129.72(*2), 123.92(*2),121.39, 119.22, 118.31(*2),114.34, 107.11, 31.11, 19.14.HRMS (ESI, m / z): calcd forC 22 H 20 N7O3S [M + H] + 462.1270,found 445.1338. <!-- 16 -->]]> 11n <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 10.03(s, 1H), 9.37 (s, 1H), 8.30 (d,J = 5.4 Hz, 1H), 8.09 – 8.01 (m,1H), 7.67 (d, J = 8.7 Hz, 2H),7.49 (d, J = 8.6 Hz, 2H), 7.22(d, J = 7.8 Hz, 2H), 7.13 (d, J= 7.6 Hz, 2H), 6.87 (d, J = 5.4Hz, 1H), 3.55 (s, 2H), 2.85 (s,3H), 2.46 (s, 3H), 2.28 (s, 3H). 13 C NMR (151 MHz, DMSO-d6) δ169.26, 159.88, 158.99,158.10,152.65, 136.60, 135.96, 133.59,133.50, 129.39(*4), 129.32,119.88(*2), 119.49(*2), 118.43,106.94, 43.36, 31.13, 21.12,19.10. HRMS (ESI, m / z): calcdfor C 24 H 25 N6OS [M + H] + 445.1732,found 445.1797.]]> 11o <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 10.04(s, 1H), 9.37 (s, 1H), 8.31 (d,J = 5.4 Hz, 1H), 8.04 (d, J =4.8 Hz, 1H), 7.69 – 7.67 (m,2H), 7.51 – 7.49 (m, 2H), 7.24(d, J = 8.2 Hz, 1H), 6.92 – 6.91(m, 2H), 6.88 (d, J = 5.5 Hz,1H), 6.84 – 6.82 (m, 1H), 3.75(s, 2H), 3.58 (s, 3H), 2.86 (s,3H), 2.47 (s, 3H). 13 C NMR (151MHz, DMSO-d6)δ 169.61,168.95,159.88,159.66,158.98,158.15(*2),152.69,138.08, 136.65, 133.43,129.77, 121.76(*2), 119.90,119.49, 115.34(*2), 112.32,106.96,55.44, 43.79, 31.13,19.11. HRMS (ESI, m / z): calcdfor C 24 H 25 N6O2S [M + H] + 461.1681,found 461.1750. <!-- 17 -->]]> 11p <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 9.99(s, 1H), 9.59 (s, 1H), 8.46 (d,J = 5.3 Hz, 1H), 7.94 (d, J =2.1 Hz, 1H), 7.47 – 7.45 (m,3H), 7.26 – 7.23 (m, 4H), 7.17(d, J = 8.1 Hz, 1H), 7.05 (d, J= 5.3 Hz, 1H), 3.65 (s, 3H),3.07 (t, J = 7.5 Hz, 2H), 2.66(t, J = 7.7 Hz, 2H), 2.61 (s,3H). 13 C NMR (151 MHz, DMSO-d6) δ172.73, 170.57, 160.13, 159.44,158.87, 148.26, 141.23, 140.73,139.73, 131.59, 131.55, 131.27,131.07, 128.85, 125.33, 119.50,114.89, 113.57, 111.15, 108.80,38.04, 35.61, 29.61, 18.79. HRMS(ESI, m / z): calcd for C 24 H 24 BrN6OS[M + H] + 524.0837, found524.1188.]]> 12a <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 9.57(s, 1H), 8.82 (s, 1H), 8.36 (d,J = 5.4 Hz, 1H), 7.98 (q, J =4.7 Hz, 1H), 7.83 (s, 1H), 7.64– 7.62 (m, 1H), 7.31 (t, J = 8.0Hz, 1H), 7.24 (dd, J = 8.2, 1.6Hz, 1H), 7.19 (t, J = 7.9 Hz,1H), 6.92 (d, J = 5.4 Hz, 1H),6.83 (dd, J = 8.3, 2.4 Hz, 1H),3.88 (s, 3H), 3.84 (s, 3H), 2.73(s, 3H), 2.48 (s, 3H). 13 C NMR(151 MHz, DMSO-d6) δ 170.01,159.92, 159.02, 158.11, 155.85,153.19, 153.01, 152.82, 150.08,142.03, 129.65, 129.55, 124.81,118.60, 118.36, 116.79, 116.17,114.29, 111.29, 107.35, 61.95,56.36, 31.18, 19.12. HRMS (ESI,m / z): calcd for C 24 H 25 N6OS [M + H] + 461.1681, found 461.1751. <!-- 18 -->]]> 12b <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 9.56(s, 1H), 8.83(s, 1H), 8.41(dd, J= 5.6Hz, 1H), 7.90 (dd, J = 4.7Hz, 2H), 7.78 (s, 1H), 7.66 (dd,1H), 7.52 (q, J = 8.1 Hz, 1H),7.30 (dd, J = 8.3, 2.5 Hz, 2H),7.19 (d, J = 8.1 Hz, 1H), 6.92(m, J = 5.6 Hz, 1H), 6.63 (dd, J= 8.3, 2.5 Hz, 1H), 3.85 (s,3H), 2.83 (s, 3H), 2.48 (s,3H). 13 CNMR(151MHz,DMSO-d6)δ169.55,164.24,159.63,160.15,158.43,157.66,157.20,142.51,140.83,136.44,129.65,129.61,129.55,120.52 116.17, 114.29,112.95,112.40,108.66,107.31,55.36,31.35,19.11. HRMS (ESI, m / z):calcd for C 23 H 22 BrN6OS [M + H] + 510.4260, found 510.4289.]]> 12c <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 13.07(s, 1H), 9.52 (s, 1H), 8.43 (s,1H), 8.25 (q, J = 6.0 Hz, 1H),8.22 (m, 1H), 8.10 (s, 1H), 7.65(d, J = 7.4, 2.1 Hz, 2H), 7.32(dd, J = 8.0 Hz, 1H), 7.19 (d, J= 6.0 Hz, 1H), 6.88 (dd, J =8.1, 2.1 Hz, 2H), 2.84 (s, 3H),2.49 (s, 3H). 13 C NMR (151 MHz,DMSO-d6) δ 180.60, 171.84,160.16, 158.92, 157.65, 157.40,151.65, 151.47, 149.15, 145.37,144.42, 142.16, 141.60, 129.48,129.10, 124.56, 117.00, 113.50,111.15, 107.03, 31.40, 19.15.HRMS (ESI, m / z): calcd forC 22 H 18 F2N6OS [M + H] + 453.1234,found 453.1250. <!-- 19 -->]]> 12d <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 9.66(s, 1H), 8.56 (s, 1H), 8.52 (q,J = 6.0 Hz, 1H), 8.18 (dd, J =6.3 Hz, 1H), 8.22 (s, 1H), 7.79(s, 2H), 7.60 (t, J = 7.8, 2.2Hz, 1H), 7.43 (q, J = 6.0 Hz,2H), 7.19 (d, J = 7.8 Hz, 2H),6.88 (dd, J = 8.1, 2.2 Hz, 2H),2.84 (s, 3H),2.48 (s, 3H). 13 C NMR(151 MHz, DMSO-d6) δ 169.87,162.15, 160.16, 157.92, 157.77,157.60, 153.68, 151.50, 150.11,144.39, 142.40, 141.51, 141.33,129.65, 129.27, 123.15, 116.01,114.44, 110.24, 107.09, 31.56,19.02. HRMS (ESI, m / z): calcdfor C 22 H 21 N6OS [M + H] + 417.1419,found 417.1465.]]> 12e <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 9.61(s, 1H), 8.63 (s, 1H), 8.37 (d,J = 5.4 Hz, 1H), 8.02 (d, J =4.9 Hz, 1H), 7.95 (s, 1H), 7.84(d, J = 3.9 Hz, 1H), 7.70 – 7.65(m, 1H), 7.45 (d, J = 3.8 Hz,1H), 7.35 (t, J = 8.0 Hz, 1H),6.95 (dd, J = 12.5, 7.1 Hz, 2H),2.81 (s, 3H), 2.48 (s, 3H). 13 CNMR (151 MHz, DMSO-d6) δ 169.94,159.85, 159.06, 158.11, 153.27,153.05, 152.84, 150.78, 147.91,142.15, 129.73, 118.42, 118.29,114.65, 114.19, 112.15, 107.49(*2), 31.24, 19.12. HRMS (ESI, m / z): calcd for C 20 H 18 N7O3S [M + H] + 436.4620, found 436.1182. <!-- 20 -->]]> 12f <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 9.60(s, 1H), 8.52 (s, 1H), 8.36 (d,J = 5.4 Hz, 1H), 8.11 (q, J =4.9 Hz, 1H), 7.95 (s, 1H), 7.56(d, J = 9.3 Hz, 1H), 7.31 (q, J= 5.9, 3.9 Hz, 4H), 6.94 (d, J =5.4 Hz, 1H), 6.88 (d, J = 9.5Hz, 1H), 2.78 (s, 3H), 2.48 (s,3H). 13 C NMR (151 MHz, DMSO-d6) δ181.73, 169.84, 159.86, 158.91,158.18, 152.95, 151.71, 150.58(*2), 142.14, 129.63(*2), 118.40,117.05, 114.70, 110.90, 107.27,31.08, 19.08. HRMS (ESI, m / z):calcd for C 19 H 19 N8S [M + H] + 391.1375, found 391.1443.]]> 12g <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 9.75(s, 1H), 8.69 (s, 1H), 8.38 (d,J = 5.4 Hz, 1H), 7.89 (d, J =8.9 Hz, 2H), 7.83 (d, J = 3.9Hz, 1H), 7.44 (d, J = 8.9 Hz,2H), 7.37 (d, J = 3.9 Hz, 1H),6.96 (d, J = 5.6 Hz, 2H), 2.90(s, 3H), 2.51 (s, 3H). 13 C NMR(151 MHz, DMSO-d6) δ 169.74,159.58, 158.95, 158.02, 154.03,152.61, 144.85, 143.11, 141.28,122.81(*2), 119.65(*2), 118.14,117.32, 114.95(*2), 107.69,31.41, 18.77. HRMS (ESI, m / z):calcd for C 20 H 18 N7O3S [M + H] + 436.4620, found 436.1183. <!-- 21 -->]]> 12h <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 9.58(s, 1H), 8.82 (s, 1H), 8.36 (d,J = 5.4 Hz, 1H), 7.98 (q, J =4.9 Hz, 1H), 7.93 (s, 1H), 7.56(d, J = 9.3 Hz, 1H), 7.31 (t, J= 8.0 Hz, 2H), 7.24 (dd, J =8.2, 1.6 Hz, 2H), 6.94 (d, J =5.4 Hz, 1H), 6.88 (d, J = 9.5Hz, 1H), 2.73 (s, 3H), 2.48 (s,3H). 13 C NMR (151 MHz, DMSO-d6) δ170.73, 169.55, 159.86, 158.91,155.18, 152.95, 151.71, 150.58,148.48,142.14, 129.63(*2),118.40, 117.05, 114.79, 110.90,108.11, 31.18, 19.12. HRMS (ESI,m / z): calcd for C 19 H 18 N8S [M + H] + 391.1375, found 391.1443.]]> 12A <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 9.60(s, 1H), 8.83 (s, 1H), 8.36 (d,J = 5.8 Hz, 1H), 8.00 (t, J =4.7 Hz, 1H), 7.81 (s, 1H), 7.62(d, 1H), 7.30 (t, J = 8.0 Hz,1H), 7.24 (dd, J = 8.5, 1.6 Hz,1H), 7.21 (t, J = 7.9 Hz, 1H),6.90 (q, J = 5.8 Hz, 1H), 6.85(m, J = 8.5, 2.4 Hz, 1H), 3.88(s, 3H), 3.86 (s, 3H), 2.71 (s,3H). 13 C NMR (151 MHz, DMSO-d6) δ169.55, 159.90, 159.41, 157.85,157.81, 153.19, 153.00, 152.82,150.08, 141.03, 129.63, 129.55,125.62, 118.61, 118.43, 116.66(*2) , 115.19, 114.28, 111.34,107.35, 55.30, 31.19,. HRMS(ESI, m / z): calcd for C 23 H 21 N6OS[M + H] + 447.1525, found447.1560. <!-- 22 -->]]> 12B <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 9.55(s, 1H), 8.83(s, 1H), 8.40(d, J= 5.6Hz, 1H), 7.90 (q, J = 4.7Hz, 2H), 7.78 (s, 1H), 7.66 (dd,1H), 7.52 (q, J = 8.0 Hz, 1H),7.31 (dd, J = 8.5, 2.6 Hz, 2H),7.19 (d, J = 8.0 Hz, 1H), 6.92(d, J = 5.6 Hz, 1H), 6.63 (dd, J= 8.5, 2.6 Hz, 1H), 3.85 (s,3H), 2.83 (s, 3H). 13 C NMR(151MHz,DMSO-d6)δ170.01,163.21,160.93,160.15,158.43,157.66(*2),142.51,140.83,136.44,129.55(*2),129.65, 120.52 116.17, 114.29,112.40(*2), 107.31(*2), 55.36,31.30. HRMS (ESI, m / z): calcdfor C 22 H 20 BrN6OS [M + H] + 496.0524,found 496.0602.]]> 12C <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 13.15(s, 1H), 9.62 (s, 1H), 8.48 (s,1H), 8.33 (d, J = 5.3 Hz, 1H),8.18 (d, J = 4.8 Hz, 1H), 8.06(s, 1H), 7.91 (s, 1H), 7.62 (dd,J = 8.0, 2.2 Hz, 1H), 7.32 (t, J= 8.0 Hz, 2H), 7.19 (d, J = 5.3Hz, 2H), 6.88 (dd, J = 8.1, 2.1Hz, 1H), 2.84 (s, 3H). 13 C NMR(151 MHz, DMSO-d6) δ 181.74,172.87, 160.16, 158.92, 157.64,157.47, 151.67, 150.54, 149.14,145.35, 143.44, 142.14, 141.63,129.66, 129.11, 124.54, 117.01,114.46, 111.19, 106.03, 31.39.HRMS (ESI, m / z): calcd forC 21 H 17 F2N6OS [M + H] + 439.1074,found 439.1112. <!-- 23 -->]]> 12D <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 9.68(s, 1H), 8.55 (s, 1H), 8.50 (d,J = 6.0 Hz, 1H), 8.22 (t, J =6.3 Hz, 1H), 8.22 (s, 1H), 7.79(s, 1H),7.70 – 7.63 (m, 1H) 7.60(t, J = 8.0, 2.1 Hz, 2H), 7.43(dd, J = 6.0 Hz, 2H), 7.19 (q, J= 8.4 Hz, 2H), 6.88 (d, J = 8.0,2.1 Hz, 2H), 2.83 (s, 3H). 13 C NMR(151 MHz, DMSO-d6) δ 169.85,162.70, 162.16, 158.66, 158.49,157.64, 155.37, 155.72, 150.02,143.25, 143.44, 141.60, 141.37,129.53, 128.25, 124.15, 124.53,115.47, 110.24, 108.60, 31.22.HRMS (ESI, m / z): calcd forC 21 H 19 N6OS [M + H] + 403.1263, found403.1290.]]> 12E <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 9.63(s, 1H), 8.62 (s, 1H), 8.33 (d,J = 5.3 Hz, 1H), 8.17 (q, J =4.7 Hz, 1H), 8.05 (s, 1H), 7.92(t, J = 2.1 Hz, 1H), 7.83 (d, J= 3.9 Hz, 1H), 7.67 (dd, J =8.1, 2.1 Hz, 1H), 7.45 (d, J =3.9 Hz, 1H), 7.35 (t, J = 8.0Hz, 1H), 7.19 (d, J = 5.3 Hz,1H), 6.96 (dd, J = 8.0, 2.6 Hz,1H), 2.85 (s, 3H). 13 C NMR (151MHz, DMSO-d6) δ 172.84, 160.10,158.97, 157.63, 153.22, 152.83,150.72, 147.90, 143.45, 142.14,129.80, 124.45, 118.51, 118.26,114.66, 114.20, 112.17, 106.17,31.40. HRMS (ESI, m / z): calcdfor C 19 H 16 N7O3S [M + H] + 422.0957,found 422.1011. <!-- 24 -->]]> 12F <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 9.61(s, 1H), 8.47 (s, 1H), 8.33 (d,J = 5.3 Hz, 1H), 8.17 (q, J =4.8 Hz, 1H), 8.06 (s, 1H), 7.90(t, J = 2.1 Hz, 1H), 7.61 (dd, J= 8.0, 2.1 Hz, 1H), 7.32 (t, J =8.0 Hz, 2H), 7.19 (d, J = 5.3Hz, 1H), 6.88 (dd, J = 8.0, 2.1Hz, 2H), 2.83 (s, 3H). 13 C NMR(151 MHz, DMSO-d6) δ 181.74,172.87, 160.16, 158.92, 157.66,151.67, 150.54, 149.13, 145.35,143.44, 142.13, 129.67, 124.54,117.02, 114.48, 111.18, 106.03,105.26, 31.39. HRMS (ESI, m / z):calcd for C 19 H 16 N7O3S [M + H] + 422.0957, found 422.1013.]]> 12G <![CDATA[ 1 H NMR (600 MHz, DMSO-d6) δ 9.48(s, 1H), 8.60 (s, 1H), 8.42 (d,J = 5.4 Hz, 1H), 7.80 (q, J =4.9 Hz, 1H), 7.92 (s, 1H), 7.38(d, J = 9.3 Hz, 1H), 7.22 (t, J= 8.0 Hz, 2H), 7.18 (dd, J =8.2, 1.6 Hz, 2H), 6.94 (d, J =5.4 Hz, 1H), 6.88 (d, J = 9.5Hz, 1H), 2.83 (s, 3H). 13 C NMR(151 MHz, DMSO-d6) δ 169.89,169.55, 160.05, 157.84, 155.13,152.90, 150.51, 150.38, 148.48,142.14, 128.99, 126.51,117.33,117.06, 114.79, 110.90, 108.11,31.18. HRMS (ESI, m / z): calcdfor C 18 H 16 N8S [M + H] + 377.1219,found 377.1230.]]>

[0078] Example 10: MTT assay to evaluate the anti-esophageal cancer cell activity of the patented compound

[0079] Cell line culture: Human esophageal cancer cells (KYSE510, KYSE150) were cultured using RPMI-1640 medium.

[0080] Preparation of compound solution: Dissolve the compound in DMSO to prepare a 10 mM stock solution for subsequent experiments. Store the stock solution in a -20°C freezer.

[0081] The MTT assay was used to test the inhibitory effects of 10 μM and 1 μM compounds on the proliferation of KYSE510 cells: Logarithmic-phase cells were seeded into 96-well plates and cultured overnight at 37 °C. The test compound (final concentration 10 μM or 1 μM) was added to each well, with three replicates per group. A proportionally diluted DMSO solution was used as a control. After 72 h of further culture, 0.5 mg / mL MTT was added, and the cells were cultured at 37 °C for 3–4 h. The supernatant was discarded, and 150 μL of DMSO was added to each well. The plates were shaken for 1 min to fully dissolve the formazan. The absorbance at 492 nm was then measured using a microplate reader. The experimental data are expressed as follows: The calculated inhibition rates are shown in Table 2. Most compounds (except 11d, 11g, 11h, 11i, 11j, and 11p) exhibited strong inhibitory activity against esophageal cancer KYSE510 cells, with inhibition rates greater than 60% at a test concentration of 10 μM. Compounds 12a, 12e, 12B, 12D, and 12E showed the best inhibitory activity, with inhibition rates greater than 60% at a test concentration of 1 μM (significantly better than the positive control compound CDKI-73).

[0082] Inhibitory effects of compounds on different tumor cells (IC50) 50 Evaluation: The MTT assay was used to test the inhibitory effect (inhibition rate) of the compound on KYSE510 and KYSE150 at different concentrations, and the IC50 of the compound in different cell lines was calculated using Graphpad Prism. 50The experimental results are shown in Table 3. Consistent with the MTT assay results of the 10 μM and 1 μM compounds, the tested compounds showed significantly better inhibitory effects on KYSE510 and KYSE150 than the positive control compound CDKI-73. Furthermore, the tested compounds generally exhibited better inhibitory effects on esophageal cancer cells KYSE510 and KYSE150 than on esophageal epithelial cells HET-1A.

[0083] Table 2. Inhibitory effects of 10 μM and 1 μM compounds on the proliferation of esophageal cancer KYSE510 cells.

[0084]

[0085] Table 3. Inhibitory effects of some active compounds and positive control CDKI-73 on human esophageal cancer (KYSE510 and KYSE150) and esophageal epithelial cells HET-1A (IC50, 20 ... 50 (µM)

[0086]

[0087] Example 11: Effects of representative compounds on the expression of proteins such as RNA PO II CTD p-Ser2, MCL-1, and Cleaved PARP

[0088] CDK9 inhibitors can block the phosphorylation (p-Ser2) of the C-terminal domain of RNA polymerase II by CDK9 / Cyclin T1, thereby inhibiting the expression of the anti-apoptotic oncoprotein MCL-1 and inducing apoptosis (PARP cleavage). Therefore, in this example, KYSE150 cells were treated with 1 μM of the active compound for 24 h, and the expression of RNA Pol II CTD p-Ser2, MCL-1, and Cleaved PARP proteins was measured to evaluate the inhibitory effect of this series of compounds on CDK9. Figure 1 The results showed that 12B, 12a, and 12C significantly downregulated p-Ser2 and MCL-1, and upregulated Cleaved PARP.

[0089] Example 12: Experiment on the activity of CDK9 / Cyclin T1 kinase, a representative compound

[0090] The effects of compounds 12B, 12a, and 12C on CDK9 / Cyclin T1 kinase activity were determined by Eurofins DiscoverX using the Human CMGC Kinase Enzymatic LANCE Assay Kit (Kinase Profiler, Eurofins, Celle L'Evescault, France). The results are as follows: Figure 2As shown in the figure. Experimental results indicate that 12B, 12a, and 12C exhibit superior inhibitory effects on CDK9 / Cyclin T1 kinase activity compared to the positive compound CDKI-73. 12B can inhibit CDK9 / Cyclin T1 kinase activity in a concentration-dependent manner, with an IC50 value of [missing information]. 50 The values ​​are 43.2 nM respectively.

[0091] Example 13: Thermodynamic migration experiment of 12B and CDK9 / Cyclin T1

[0092] After treating KYSE150 cells with 1 µM 12B or DMSO for 3 h, the cells were washed with PBS, collected into EP tubes using a scraper, centrifuged at 300 g for 3 min, the supernatant was discarded, and lysis buffer of 1.5 mL 250 mM NaCl was added. The cells were lysed on ice for 2 h, centrifuged at 12000 g for 10 min, and the supernatant was aliquoted into 200 µL PCR tubes. Different temperature programs were set in the PCR instrument, and the cells were incubated for 3 min, then placed at room temperature for 3 min, centrifuged at 12000 g for 10 min, and the supernatant was added to loading buffer. The samples were then boiled for subsequent protein immunoblotting experiments. The experimental results are shown below. Figure 3 As shown in the figure, the experimental results indicate that all proteins degrade with increasing temperature, but the stability of CDK9 protein and its complex Cyclin T1 significantly increased after incubation with the addition of small molecules. This result suggests that 12B can directly bind to CDK9 / Cyclin T1 and improve protein stability.

[0093] Example 14: Evaluation of the significant inhibitory effect of compound 12B on esophageal cancer cell proliferation

[0094] The MTT assay was used to determine the inhibitory effects of different concentrations of 12B (30, 10, 3.333, 1.111, 0.370, 0.123, 0.041, 0.014, 0.004 µM) on the proliferation of five human esophageal cancer cell lines (KYSE510_shCDK9-2, KYSE150, KYSE510, KYSE140, EC-9706, ECA-109) and one human normal esophageal epithelial cell line (Het-1A). The calculated IC50 results are shown in Table 4. Compound 12B exhibits excellent inhibitory effects on different esophageal cancer cell lines, with IC50 values ​​of [missing data]. 50 The concentration was between 0.2387 and 0.8497 μM (significantly better than the positive control CDKI-73), while the inhibitory effect on normal esophageal cell lines was greatly reduced.

[0095] Table 4.12. Inhibitory effects of 2B and CDKI-73 on Het-1A in different esophageal cancer cells and normal esophageal epithelial cells (IC50).50 )

[0096]

[0097] Furthermore, the ability of compound 12B to inhibit the proliferation of esophageal cancer cells was further determined by a cell colony formation assay. 1000–2000 log-phase KYSE510 shCDK9-2, KYSE150, and KYSE510 cells were seeded in 12-well plates and cultured at 37°C for 24 h. After two weeks of culture, medium containing different concentrations (10, 30, and 90 nM) of 12B was added. The medium was then discarded, and the cells were washed three times with 1×PBS, fixed with methanol at room temperature for 15 min, discarded, washed three times with 1×PBS, and air-dried. The cells were then stained with 0.5% crystal violet solution (diluted with PBS) at room temperature for 10–15 min, the staining solution was removed, and the cells were washed with ultrapure water until no background color was visible. Colony counts were observed under a microscope, and photographs were taken. The experimental results are shown below. Figure 4 As shown. Compound 12B significantly inhibited colony formation of KYSE150 and KYSE510 cells in vitro, and 90 nM 12B achieved the same inhibitory effect as 1 μM CDKI-73. Figure 4 A). Furthermore, CDK9 knockdown experiments demonstrated that the inhibitory effect of 12B on esophageal cancer cell proliferation was CDK9-dependent. CDK9 knockout significantly reduced the inhibitory effect of 12B on KYSE510 cell colony formation. Figure 4 B), while significantly increasing IC 50 value( Figure 4 C).

[0098] Example 15: Evaluation of the effects of compound 12B on esophageal cancer cell cycle arrest and apoptosis.

[0099] The effects of compound 12B on cell cycle progression were detected by flow cytometry. Log-phase tumor cells (KYSE150) were seeded in 6-well plates and cultured overnight at 37°C. After 8 h of further culture with different concentrations (0.3, 1, 3 μM) of 12B, the culture medium was discarded. Cells were rinsed with pre-chilled 1×PBS, digested with trypsin, and collected into pre-chilled 1.5 mL EP tubes. After centrifugation at 1000 rpm for 5 min, the supernatant was removed. Cells were washed twice with pre-chilled PBS, centrifuged again, and the supernatant was removed. The tube walls were gently tapped to prevent cell clumping. Cells were fixed with 70% ethanol at 4°C for at least 4 h, centrifuged at 1000 rpm for 5 min, and the supernatant was removed. Cells were washed twice with pre-chilled 1×PBS, centrifuged again, and the supernatant was removed. The tube walls were gently tapped to prevent cell clumping. 500 μL of a working concentration of 1 μg / mL LDAPI solution (2 mg / mL DAPI stock solution was prepared with ultrapure water and diluted with 1×PBS to the working concentration before use) or a PI solution containing RNase was added to gently resuspend the cells. Cells were stained at room temperature in the dark for 20 min, and cell cycle distribution was detected by flow cytometry. Experimental results are as follows Figure 5 As shown in Figures A and B, after KYSE150 cells were treated with compound 12B at 0.3 μM and 1 μM for 8 h, 41.30% and 62.31% of the cells were arrested in the G2 / M phase, respectively.

[0100] The effect of compound 12B on cell apoptosis was investigated by flow cytometry. Log-phase KYSE150 cells were seeded in 12-well plates and cultured overnight at 37°C. After treatment with medium containing 0.3-1 μM 12B for 24 h, the medium was discarded, and the cells were washed with pre-chilled 1×PBS. After trypsin digestion, the cells were collected into pre-chilled 1.5 mL EP tubes, centrifuged at 1000 rpm for 5 min, and the supernatant was removed. The cells were washed twice with pre-chilled PBS, centrifuged again, and the supernatant was removed. The tube walls were gently tapped to prevent cell clumping. The cells were stained with Annexin V-FITC apoptosis detection kit (Yeasen) at room temperature in the dark for 20 min, and the apoptosis status was detected by flow cytometry. Figure 5 The results showed that after treatment with 12B at concentrations of 0.3 μM and 1.0 μM, the percentage of total apoptotic cells increased from 5.06% (control) to 11.10% (0.3 μM) and 11.35% (1.0 μM), respectively. The 1.0 μM 12B (11.35%) was more effective at inducing apoptosis than the 3.0 μM lead compound CDKI-73 (8.19%).

[0101] Following apoptosis, the expression of related proteins undergoes significant changes. CDK9, playing a crucial role in transcription, regulates downstream short-lived apoptotic proteins such as MCL-1, Bcl-2, XIAP, and Myc. MCL-1, an anti-apoptotic protein of the Bcl-2 family, plays a vital regulatory role in cell growth and differentiation, inhibiting apoptosis by suppressing cytochrome C release. The Myc gene, a classic oncogene, encodes the MYC protein, which participates in regulating cell proliferation, differentiation, and apoptosis. This study investigated the effects of compound 12B on the expression of apoptosis marker proteins MCL-1 and C-Myc using qRT-PCR. The concentration of extracted total RNA was measured, and a portion was diluted to 200 ng / µL. A reverse transcription system was prepared, incubated at 42 ℃ for 2 min, and 10 µL of Hifair was added and mixed thoroughly. The PCR program was then set to incubate at 25 ℃ for 5 min – 42 ℃ for 30 min – 85 ℃ for 5 min. The cDNA obtained from reverse transcription was diluted in 380 µL of sterile ddH2O and used as a template for qPCR. qPCR primers were designed online, and the forward and reverse primers were reconstituted to a concentration of 100 µM. 10 µL of each primer and 80 µL of sterile ddH2O were mixed to prepare the primer mix. qPCR was performed in 96-well plates, with each well containing 4 µL of diluted cDNA template, 5 µL of SYBR Green mix, and 1 µL of primer mix. The results were then analyzed. This experiment used a kit from Yeason, and the results are as follows: Figure 5 As shown in D, the mRNA expression levels of the short-lived proteins Mcl-1 and C-Myc, which are regulated by CDK9, were downregulated after 12B treatment in a dose-dependent manner.

[0102] Example 16: Evaluation of the intervention effect of compound 12B on the migration of esophageal cancer cells

[0103] ESCC is a highly aggressive cancer characterized by its high metastatic potential. CDK9 has also been identified as a target gene for inhibiting tumor cell migration. KYSE150 cells were seeded in 6-well plates and incubated until the cell density reached approximately 95%. Artificial wounds were created by streaking the densely packed cells using a 10 μL pipette tip (ensuring the wounds were as straight as possible and each wound was roughly the same size and shape). DMSO, different concentrations (3 μM, 1 μM, 0.3 μM, 0.1 μM) of compound 12B, and the positive control compound CDKI-73 (1 μM) were added to the 6-well plates. The plates were incubated at 37°C in a 5% CO2 incubator, and the streaks were photographed using an electron microscope at 0 h, 12 h, 24 h, 48 h, and 72 h. The experimental results are as follows: Figure 6As shown, after 72 h, the control cells treated with DMSO showed the greatest degree of scratch healing. Compared with the DMSO-treated group, 12B treatment significantly inhibited and slowed cell migration, and the inhibitory effect was more pronounced with increasing 12B concentration. At the same concentration, 12B had a stronger ability to inhibit KYSE150 cell migration than the positive control compound CDKI-73.

[0104] In addition, KYSE510 cells were treated with DMSO, different concentrations (3 μM, 1 μM, 0.3 μM, 0.1 μM) of compound 12B, and the positive compound CDKI-73 (1 μM) for 24 h before Western blot analysis. The results are as follows: Figure 7 As shown, 12B concentration-dependently inhibited the expression level of MMP2 (a marker protein of cell migration activity). At a treatment concentration of 1 μM, the inhibitory effect of 12B was superior to that of the positive compound CDKI-73.

[0105] Example 17: Evaluation of the intervention effect of compound 12B on the regulation of CDK9 transcriptional elongation in ESCC cells

[0106] CDK9 is a cyclin-dependent kinase that plays a crucial role in cellular transcription. CDK9 plays a vital role in global transcriptional regulation, particularly in regulating the transcription of MCL-1 and C-Myc genes; overactivation of CDK9 primarily leads to the overexpression of MCL-1 and C-Myc genes. C-PARP plays a key role in apoptosis; it is a small fragment formed by the specific enzymatic cleavage of PARP and serves as a marker of apoptosis, its presence indicating the occurrence of apoptosis. KYSE510 and KYSE150 cells were treated for 24 h with DMSO, different concentrations (3 μM, 1 μM, 0.3 μM, 0.1 μM) of compound 12B, and the positive compound CDKI-73 (1 μM), respectively. Western blot analysis was then performed to analyze the expression levels of the aforementioned related proteins in each cell type. The experimental results are as follows: Figure 8 As shown, 12B treatment for 24 h inhibited the phosphorylation of RNA Pol IICTD Ser2 in KYSE150 and KYSE510 cells, thereby blocking Pol II activation; this inhibition was concentration-dependent. Furthermore, the inhibitory effect of 1 μM CDKI-73 was weaker than that of 12B at the same concentration. After treatment with 12B, ESCC cells showed reduced expression of MCL-1 and C-Myc by inhibiting CDK9. 12B can inhibit ESCC cell proliferation by inducing apoptosis; therefore, the expression level of C-PARP in ESCC cells increased after 12B treatment.

[0107] Example 18: Compound 12B can inhibit the growth of esophageal cancer KYSE150 cell xenografts in nude mice.

[0108] Ten female BALB / c nude mice aged 4-6 weeks were selected and housed in the SPF-grade animal facility of the Experimental Animal Center of Xiamen University. After several days of acclimatization, KYSE150 cells in the logarithmic growth phase were prepared into a cell suspension (3×10⁻⁶ cells / mL). 6 Cells / nude mice were injected subcutaneously into the right anterior axillary region of nude mice. The tumor volume reached approximately 100 mm. 3 Later (tumor volume = length × width × width / 2 mm) 3 Mice were randomly divided into two groups of five mice each, with the aim of maintaining a similar average tumor volume across the groups. A control group and a drug-treated group (20 mg / kg) were established. Mice in the control group received a 200 μL injection of a blank solvent intraperitoneally at a fixed time each day for 14 consecutive days. Mice in the drug-treated group received a 200 μL injection of the drug solution intraperitoneally at a fixed time each day for 14 consecutive days. Starting from the first day of drug administration, the tumor size (length and width) of each mouse was measured and recorded daily using calipers, and the weight of each mouse was also measured. On the 14th day of drug administration, all mice were sacrificed, and subcutaneous tumors were dissected, weighed, and photographed for comparison. Heart, liver, spleen, lung, and kidney samples from each group were subjected to Hematologic & Escherichia coli (H&E) staining to assess the toxicity of the compound. Tumor tissue samples were divided into two parts: one part for H&E staining and immunohistochemistry, and the other part for preparing protein lysis buffer samples for Western blotting analysis. Experimental results are as follows: Figure 7 As shown.

[0109] Tumor inhibition rate (TGI) calculation = (1 - (T - T0) / (V - V0) × 100%)

[0110] T: Mean tumor volume on day 14 in the treatment group; T0: Mean tumor volume on day 1 in the treatment group;

[0111] V: Mean volume of tumors in the control group on day 14; V0: Mean volume of tumors in the control group on day 1.

[0112] Drug solvent ratio: 10% DMSO + 5% Tween 80 + 85% D5W, where D5W represents 5% glucose aqueous solution.

[0113] Figure 9 Image A shows images of KYSE150 xenograft tumors in the control group and the drug-treated group after the experiment.

[0114] Figure 9 The results showed that 12B significantly inhibited the weight of KKYSE150 xenograft tumors;

[0115] Figure 9 The results showed that 12B significantly inhibited the growth of KYSE150 cell xenograft tumors during drug administration;

[0116] Figure 9 The D-value shows that compound 12B did not cause a significant reduction in body weight in nude mice during administration;

[0117] Figure 9 The middle section (E) shows the H&E staining of major organs in mice of the control and treatment groups;

[0118] Figure 9 The middle part (F) shows the expression of Ki67 and Nur77 in tumor tissues of the control group and those treated with compound 12B;

[0119] Figure 9 The image shows the expression of related proteins in xenogeneic tumor suppression in KYSE150 cells after 12B administration.

[0120] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A 4-(2-(methylamino)thiazo-5-yl)pyrimidine-2-amine derivative, characterized in that: Its structural formula is or , Wherein, R1 is hydrogen or methyl; R2 is a substituted benzoyl group, a substituted phenylacetyl group, or a substituted or unsubstituted phenylpropionyl group; R3 is a substituted phenyl, 5-nitrofuran-2-yl, or 1H-imidazol-2-yl.

2. The 4-(2-(methylamino)thiazo-5-yl)pyrimidine-2-amine derivative as described in claim 1, characterized in that: Its structural formula is selected from one of the following: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , .

3. Use of the 4-(2-(methylamino)thiazo-5-yl)pyrimidine-2-amine derivative of claim 1 or 2 in the preparation of a CDK9-related tumor therapeutic composition.

4. The use as described in claim 3, characterized in that: The structural formula of the 4-(2-(methylamino)thiazo-5-yl)pyrimidine-2-amine derivative is selected from... , and .

5. The use as described in claim 4, characterized in that: The structural formula of the 4-(2-(methylamino)thiazo-5-yl)pyrimidine-2-amine derivative is as follows: .

6. The use as described in any one of claims 3 to 5, characterized in that: The CDK9-related tumors mentioned are esophageal cancer.

7. A CDK9-related tumor therapeutic drug composition, characterized in that: Its active ingredient includes the 4-(2-(methylamino)thiazo-5-yl)pyrimidine-2-amine derivative as described in claim 1 or 2.

8. The CDK9-related tumor therapeutic composition as described in claim 7, characterized in that: The structural formula of the 4-(2-(methylamino)thiazo-5-yl)pyrimidine-2-amine derivative is selected from... , and .

9. The CDK9-related tumor therapeutic composition as described in claim 8, characterized in that: The structural formula of the 4-(2-(methylamino)thiazo-5-yl)pyrimidine-2-amine derivative is as follows: .

10. A CDK9-related tumor therapeutic composition according to any one of claims 7 to 9, characterized in that: The CDK9-related tumors mentioned are esophageal cancer.

Images