A pyridopyrimidine compound containing a morpholine structure, and a preparation method and application thereof

Abstract

The application discloses a pyridopyrimidine compound containing a morpholine structure and a preparation method and application thereof, and belongs to the technical field of medicines. The pyridopyrimidine compound containing the morpholine structure has a strong ability of inhibiting PI3K alpha kinase, and thus can be used as an active ingredient for preparing a therapeutic drug for diseases caused by abnormal activation of PI3K alpha kinase, and shows potential application value in preparation of a drug for treating and / or preventing proliferative diseases and cancers. The application provides important technical support for development of an anticancer drug with high efficiency and better selectivity.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a pyridopyrimidine compound containing a morpholine structure, its preparation method, and its application. Background Technology

[0002] In terms of cancer types, lung cancer, breast cancer in women, and colorectal cancer are the three cancers with the highest number of newly diagnosed cases worldwide. Data reveals the serious threat that cancer poses to human health, especially breast cancer and lung cancer, the most common cancers in both women and men. Breast cancer has a particularly significant impact on women's health globally, accounting for one-quarter of newly diagnosed cancer cases and one-sixth of cancer deaths among women worldwide. To address the challenge of breast cancer, improving its prevention and treatment capabilities has become a hot topic for governments and health organizations worldwide. Developing new drugs for breast cancer using molecular targeted therapy strategies has become a current focus. Currently, clinically applied personalized targeted therapies mainly target the abnormal activation of the PI3K signaling pathway to treat breast cancer. Through clear molecular targets, target detection technologies, and marketed targeted drugs, clinical efficacy has been significantly improved. Phosphatidylinositol 3-kinase is a family of enzymes with serine / threonine (Ser / Thr) kinase activity, widely involved in regulating key biological processes such as cell growth, differentiation, proliferation, and endocytosis. Based on their sequence homology, structural characteristics, and substrate specificity, PI3Ks are classified into three main classes: Type I, Type II, and Type III, with Type I PI3Ks being the most extensively studied. Type I PI3Ks can be further divided into Class IA and Class IB: Class IA includes PI3Kα, PI3Kβ, and PI3Kδ, while Class IB only contains PI3Kγ. Class IA PI3Ks are heterodimers composed of the catalytic subunit p110α, p110β, or p110δ and the regulatory subunit p85, while Class IB PI3Ks are composed of the catalytic subunit p110γ and the regulatory subunit p101 or p87.

[0003] The activation mechanisms of PI3K are complex and diverse. Class IA PI3Ks primarily transmit signals through receptor tyrosine kinases (RTKs). When insulin or other growth factors bind to RTKs, autophosphorylation of tyrosine residues in the receptor's intracellular domain is triggered, thereby activating PI3K. In contrast, PI3Kγ activation mainly depends on the interaction between GN-β (guanine nucleotide-binding protein-β) and GN-γ (guanine nucleotide-binding protein-γ) in G-protein-coupled receptors. Furthermore, PI3K activation can also be achieved through Ras proteins with GTPase activity. Insulin can also indirectly activate PI3K through insulin receptor substrate 1 (IR1). Simultaneously, cytokines can also mediate PI3K activation through Janus kinase 1 (JK1). These diverse activation mechanisms make PI3K play a crucial role in cellular signaling networks.

[0004] Studies have shown that the PI3K-AKT-mTOR signaling pathway is a key intracellular signaling network that plays a central role in multiple aspects of human life activities. Abnormal activation of this pathway is closely related to the occurrence, development, and treatment resistance of various tumors. The PI3K-AKT-mTOR pathway prevents cell death by promoting cell survival and inhibiting apoptosis, which is particularly important in tumor development. Tumors may utilize the PI3K-AKT-mTOR pathway to suppress the immune system's attack, achieving immune evasion. Sustained activation of the PI3K-AKT-mTOR pathway may lead to tumor resistance to chemotherapy and targeted therapies, and it interacts with other signaling pathways such as MAPK and JAK, which can further promote tumor development. Abnormal activation of the PI3K-AKT-mTOR pathway is associated with various tumor types, including breast cancer, colorectal cancer, lung cancer, prostate cancer, and ovarian cancer. Due to the crucial role of the PI3K-AKT-mTOR pathway in tumor development, it has become an important target for cancer treatment. Currently marketed drugs have various problems, and further improvements in efficacy are still needed. Summary of the Invention

[0005] To address the above problems, this invention provides a pyridopyrimidine compound containing a morpholine structure, its preparation method, and its application.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] One of the technical solutions of this invention is to provide a pyridopyrimidine compound containing a morpholine structure, the structural formula of which is shown in Formula I:

[0008]

[0009] In Equation I, when R1 is When R2 is selected

[0010] When R1 is When R2 is selected

[0011] The names of the pyridine-pyrimidine compounds containing the morpholine structure in Formula I of this invention are as follows:

[0012] [1]N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)hexanoamide;

[0013] [2]N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)propionamide;

[0014] [3]N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)isobutyramide;

[0015] [4]N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)heptamide;

[0016] [5]N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-2-methoxyacetamide;

[0017] [6]N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)furan-2-carboxamide;

[0018] [7]N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)thiophene-2-carboxamide;

[0019] [8]N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)tetrahydro-2H-pyran-4-carboxamide;

[0020] [9]N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)cyclopropaneformamide;

[0021]

[10] N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)cyclobutanecarboxamide;

[0022]

[11] N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)cyclopentanecarboxamide;

[0023]

[12] N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)cyclohexanecarboxamide;

[0024]

[13] N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-4-methylbenzamide;

[0025]

[14] N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-3-methylbenzamide;

[0026]

[15] N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-4-ethylbenzamide;

[0027]

[16] N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-4-propylbenzamide;

[0028]

[17] 4-(tert-butyl)-N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)benzamide;

[0029]

[18] N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-3-phenylpropionamide;

[0030]

[19] 3-Chloro-N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)benzamide;

[0031]

[20] N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-3-fluorobenzamide;

[0032]

[21] N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-3-(trifluoromethyl)benzamide;

[0033]

[22] N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-4-(trifluoromethyl)benzamide;

[0034]

[23] N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-2-methoxybenzamide;

[0035]

[24] N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-3-methoxybenzamide;

[0036]

[25] N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-4-methoxybenzamide;

[0037]

[26] N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)propionamide;

[0038]

[27] N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)isobutyramide;

[0039]

[28] N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)hexanoamide;

[0040]

[29] N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)heptamide;

[0041]

[30] N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-2-methoxyacetamide;

[0042]

[31] N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)furan-2-carboxamide;

[0043]

[32] N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)thiophene-2-carboxamide;

[0044]

[33] N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyridino[3,4-d]pyrimidin-2-yl)phenyl)cyclopropaneformamide;

[0045]

[34] N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyridino[3,4-d]pyrimidin-2-yl)phenyl)cyclobutanecarboxamide;

[0046]

[35] N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyridino[3,4-d]pyrimidin-2-yl)phenyl)cyclopentanecarboxamide;

[0047]

[36] N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyridino[3,4-d]pyrimidin-2-yl)phenyl)cyclohexanecarboxamide;

[0048]

[37] N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyridino[3,4-d]pyrimidin-2-yl)phenyl)-4-methylbenzamide;

[0049]

[38] N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-4-ethylbenzamide;

[0050]

[39] 4-(tert-butyl)-N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyridino[3,4-d]pyrimidin-2-yl)phenyl)benzamide;

[0051]

[40] N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-3-phenylpropionamide;

[0052]

[41] N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-3-fluorobenzamide;

[0053]

[42] N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-2-methoxybenzamide;

[0054]

[43] N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyridino[3,4-d]pyrimidin-2-yl)phenyl)-3-methoxybenzamide.

[0055] The second technical solution of this invention: provides a method for preparing the above-mentioned pyridopyrimidine compounds containing morpholine structures, wherein R1 is... The preparation route for the compound represented by Formula I is as follows:

[0056]

[0057] In the above routes, compound 2 was prepared by the Miyaura borylation reaction; compound 4 was prepared by the nucleophilic substitution reaction; compound 5 was prepared by the de-Boc reaction; compound 6-a was prepared by the aromatic nucleophilic substitution reaction; compound 6-a and compound 2 were combined by the Suzuki coupling reaction to prepare compound 7-a; and compound 8-a was prepared by the amidation reaction.

[0058] When R1 is The preparation route for the compound represented by Formula I is as follows:

[0059]

[0060] In the above routes, compound 2 was prepared by the Miyaura borylation reaction; compound 4 was prepared by the nucleophilic substitution reaction; compound 5 was prepared by the de-Boc reaction; compound 6-b was prepared by the aromatic nucleophilic substitution reaction; compound 6-b and compound 2 were combined by the Suzuki coupling reaction to prepare compound 7-b; and compound 8-b was prepared by the amidation reaction.

[0061] The third technical solution of the present invention is to provide a pharmaceutically acceptable salt of the above-mentioned pyridopyrimidine compounds containing a morpholine structure.

[0062] Fourth technical solution of the present invention: a pharmaceutical preparation, wherein the active ingredient is a pyridopyrimidine compound containing a morpholine structure or a pharmaceutically acceptable salt of a pyridopyrimidine compound containing a morpholine structure.

[0063] Optionally, the dosage form of the pharmaceutical preparation includes, but is not limited to, injections, tablets, capsules, aerosols, suppositories, films, pellets, topical ointments, or encapsulated tablets.

[0064] Fifth technical solution of the present invention: the use of the above-mentioned pyridopyrimidine compound containing a morpholine structure in the preparation of a drug for treating and / or preventing cancer.

[0065] The sixth technical solution of the present invention: the use of the above-mentioned pyridopyrimidine compound containing a morpholine structure in the preparation of a medicament for treating and / or preventing proliferative diseases.

[0066] The seventh technical solution of the present invention: the use of a pharmaceutically acceptable salt of the above-mentioned pyridopyrimidine compound containing a morpholine structure in the preparation of a medicament for treating and / or preventing cancer.

[0067] The eighth technical solution of the present invention: the use of a pharmaceutically acceptable salt of the above-mentioned pyridopyrimidine compound containing a morpholine structure in the preparation of a medicament for treating and / or preventing proliferative diseases.

[0068] The ninth technical solution of the present invention: the application of the above-mentioned pharmaceutical preparation in the preparation of a drug for treating and / or preventing cancer.

[0069] The tenth technical solution of the present invention: the application of the above-mentioned pharmaceutical preparation in the preparation of a drug for treating and / or preventing tumor growth.

[0070] The cancers mentioned above include breast cancer, lung cancer, liver cancer, or colorectal cancer.

[0071] The beneficial technical effects of the present invention are as follows:

[0072] The pyridopyrimidine compounds containing morpholine structures provided by this invention possess strong inhibitory capabilities against PI3Kα kinase. Therefore, they can be used as active ingredients in the preparation of therapeutic drugs targeting diseases caused by abnormal activation of PI3Kα kinase, and show potential application value in the preparation of drugs for the treatment and / or prevention of proliferative diseases and cancer. This invention provides important technical support for the development of highly effective and more selective anticancer drugs. Attached Figure Description

[0073] Figure 1 The diagram shows the molecular docking results of the pyridine-pyrimidine compound containing a morpholine structure prepared in Example 26 with PI3Kα.

[0074] Figure 2 The effect of pyridine-pyrimidine compounds containing morpholine structures prepared in Example 26 at different concentrations on the hemolysis rate of erythrocytes.

[0075] Figure 3 The results show the analysis of total protein, urea, glucose, creatinine, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, and creatine kinase in the blood of KM mice in each group after drug administration.

[0076] Figure 4 The changes in body weight of KM mice during drug administration (a) and the organ index of KM mice after drug administration (b).

[0077] Figure 5 H&E staining images of the five internal organs of KM mice in each group after drug administration (scale bar is 100 μm).

[0078] Figure 6 Time-dependent effects of morpholine-containing pyridopyrimidine compounds prepared in Example 26 at different concentrations on MCF-7.

[0079] Figure 7 The figure shows the effect of pyrrolopyrimidine compounds containing morpholine structures prepared in Experiment Example 26 on the morphology of cancer cells.

[0080] Figure 8 The effect of morpholine-containing pyrrolopyrimidine compounds prepared for flow cytometry analysis on cancer cell apoptosis is shown in Figure 26.

[0081] Figure 9 The effect of morpholine-containing pyrrolopyrimidine compounds prepared for flow cytometry analysis on the cell cycle of cancer cells is shown in Figure 26.

[0082] Figure 10 The figure shows the effect of pyrrolopyrimidine compounds containing morpholine structures prepared in Experiment Example 26 on cancer cell proliferation.

[0083] Figure 11 The figure shows the effect of morpholine-containing pyrrolopyrimidine compounds prepared in Example 26 on the migration ability of MCF-7 cells, as detected by the cell scratch assay.

[0084] Figure 12 The graph shows the effect of pyrrolopyrimidine compounds containing morpholine structures prepared in Example 26 on the mitochondrial membrane potential of cancer cells.

[0085] Figure 13The effect of morpholine-containing pyrrolopyrimidine compounds prepared in Example 26 on reactive oxygen species content in MCF-7 cells, as detected by the DCFH-DA probe. Detailed Implementation

[0086] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0087] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0088] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0089] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0090] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0091] In the embodiments of the present invention, the proton and carbon NMR spectra of the compounds were determined using a Bruker ARX-400, and the mass spectra were determined using an Agilent 1100 LC / MSD; all reagents used were analytical grade or chemically pure.

[0092] The synthetic routes for the pyridopyrimidine compounds containing morpholine structures in Examples 1-25 of this invention are shown in Route 1:

[0093] Route 1:

[0094]

[0095] The synthetic routes for the pyridopyrimidine compounds containing morpholine structures in Examples 26-43 of this invention are shown in Route 2:

[0096] Route 2:

[0097]

[0098] Example 1

[0099] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)hexanoamide:

[0100] Step 1: Preparation of 2-amino-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)benzonitrile (Compound 2)

[0101]

[0102] Compound 1 (3.0 g, 15.23 mmol), bis(pinacol)diboron (5.03 g, 19.8 mmol), potassium acetate (4.48 g, 45.69 mmol), and Pd(dppf)Cl2 catalyst (557.4 mg, 0.76 mmol) were mixed in 40 mL of 1,4-dioxane solvent. The mixture was stirred at 80 °C for 16 h under nitrogen protection. After the reaction was complete, the solvent was removed by concentration, and the mixture was purified by silica gel column chromatography using a petroleum ether to ethyl acetate ratio of 5:1. Compound 2 (3.0 g, 12.3 mmol) was finally obtained.

[0103] Step 2: Preparation of tert-butyl 2-chloro-4-morpholino-5,8-dihydropyrido[3,4-d]pyrimidine-7(6H)-carboxylic acid (compound 4):

[0104]

[0105] Compound 3, morpholine, and N,N-diisopropylethylamine were dissolved in N,N-dimethylformamide in a molar ratio of 1:2:2. The solution was reacted at 60°C for 4 hours. After the reaction was complete, ice water was added and the mixture was shaken. A large amount of yellow precipitate appeared, which was then filtered. The filter cake was placed in an oven at 50°C overnight. The filter cake was then washed with dichloromethane. The washings were evaporated to dryness at 40°C. Finally, recrystallization was performed using a solvent of petroleum ether to ethyl acetate in a volume ratio of 13:1, resulting in a pale yellow precipitate. This precipitate was then filtered, and the filter cake was dried to obtain compound 4.

[0106] Step 3: Preparation of 4-(2-chloro-5,6,7,8-tetrahydropyridino[3,4-d]pyrimidin-4-yl)morpholine (compound 5)

[0107]

[0108] Compound 4 (8 g, equivalent to 22.6 mmol) was added to a pre-cooled solution of dichloromethane (30 mL) containing HCl / 1,4-dioxane (60 mL) under ice bath conditions. This pre-cooling step was intended to control the reaction rate and prevent excessive exothermic reaction. The reaction mixture was then stirred overnight at room temperature to ensure complete reaction. After this step, thin-layer chromatography (TLC) confirmed the complete consumption of the starting material. Next, the reaction mixture was concentrated to remove the solvent, and the resulting solid was dissolved in a mixture of methanol and dichloromethane (10:1 ratio, total volume 200 mL). An appropriate amount of sodium bicarbonate (NaHCO3) was then added to this solution, and the mixture was stirred at a constant temperature for 3 hours to promote the completion of the reaction and remove byproducts. After the reaction was complete, insoluble matter was removed by filtration, and the filtrate was concentrated to give compound 5.

[0109] Step 4: Preparation of 4-(2-chloro-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl)morpholine (compound 6):

[0110]

[0111] Compound 5 (3.0 g, 11.82 mmol), 3-iodopyridine (3.6 g, 17.7 mmol), Ruphos (551.5 mg, 1.182 mmol), Cs₂CO₃ (7.7 g, 23.64 mmol), and Pd₂(dba)₃ catalyst (541 mg, 0.591 mmol) were mixed. This mixture was then placed in 120 mL of 1,4-dioxane solvent. The reaction mixture was purged for 1 minute under nitrogen protection to remove air from the container, ensuring an anaerobic environment and preventing possible side reactions. This purging step was then repeated in another 120 mL of 1,4-dioxane to further ensure an anaerobic reaction environment. The reaction mixture was then heated at 100 °C for 18 hours to promote the reaction. After the reaction was complete, the mixture was concentrated to remove the solvent, yielding a solid residue. This residue was then purified by silica gel column chromatography to separate the target product (compound 6) and byproducts. The eluent is a mixed solvent system of dichloromethane / methanol at a volume ratio of 60:1 to optimize the elution efficiency of the target product.

[0112] Step 5: Preparation of 2-amino-5-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)benzonitrile (7):

[0113]

[0114] Compound 6 (1.0 g, 3 mmol), compound 2 (810.6 mg, 3.3 mmol), sodium carbonate (960.4 mg, 9.1 mmol), and Pd(PPh3)4 (176.3 mg, 0.15 mmol) were weighed. These compounds were added to 15 mL of an acetonitrile / water mixture. The reaction mixture was then purged under nitrogen for 1 minute to remove air from the container, ensuring the reaction proceeded under anaerobic conditions. This step was crucial for preventing oxidative side reactions and improving reaction selectivity. The reaction mixture was then heated at 80°C for 16 hours to promote complete reaction. After the reaction was complete, the mixture was concentrated by vacuum distillation or rotary evaporation to remove the solvent, yielding the residual solid product. Purification was then performed by silica gel column chromatography using a hexane-dichloromethane / methanol eluent system at a volume ratio of 60:1 to optimize the elution efficiency and purity of the target product. Finally, compound 7 was successfully isolated.

[0115] Step Six: Preparation of N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)hexanoamide:

[0116]

[0117] Compound 7 (70 mg, 0.17 mmol) was dissolved in 1 mL of pyridine solvent to form a homogeneous solution. This solution was cooled in an ice bath for 2 minutes to lower the reaction temperature and control the reaction rate. Subsequently, 3 to 5 drops of hexanoyl chloride reagent were added dropwise to the cooled solution. This step required careful handling to ensure that the acyl chloride was uniformly dispersed and reacted fully with compound 7. After the reaction was complete, the mixture was concentrated to remove excess solvent. Next, pre-chromatographic elution purification was performed using a dichloromethane / methanol mixed solvent system at a volume ratio of 20:1. This step aimed to utilize the difference in solubility of the compound in different solvents to separate the target product and other byproducts by column chromatography.

[0118] 1H NMR (400MHz, CDCl3) δ8.66-8.58(m,2H),8.55(d,J=8.8Hz,1H),8.12(d,J=4.4H z,1H),7.79(s,1H),7.33-7.20(m,2H),4.44(s,2H),3.91-3.84(m,4H),3.60-3 .54(m,6H),2.87(t,J=5.4Hz,2H),2.49(t,J=7.6Hz,2H),1.77(td,J=8.5,5.7H z,2H),1.39(tq,J=8.1,4.3Hz,4H),1.27(d,J=10.0Hz,1H),0.97-0.86(m,3H). 13 C NMR (150MHz, CDCl3-d) δ171.85,164.76,162.04,158.84,145.57,141.88,140.09,137.18,133.83,131.95,123.99,121.59,120.63,116. 65,114.11,101.51,66.89(2C),52.73,48.38(2C),45.29,38.04,31.43,26.87,25.13,22.52,14.05.HRMS(ESI)(m / z):[M+H]+:512.2765.

[0119] Example 2

[0120] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)propionamide:

[0121] Following the method of Example 1, in step six, hexanoyl chloride is replaced with an equimolar amount of propionyl chloride.

[0122] 1 H NMR (400MHz, CDCl3) δ8.66-8.59(m,2H),8.42(s,1H),8.15-8.09(m,1H),7.74(s,1H),7.36-7.25(m,2H),4.45(s,2H),3.87(t,J=4 .6Hz,4H),3.57(q,J=5.0Hz,6H),2.88(t,J=5.3Hz,2H),2.53(q,J=7.5Hz,2H),1.35-1.22(m,4H).HRMS(ESI)(m / z):[M+H]+:calcd for C 26 H 27N7O2:470.2304, found:470.2288.

[0123] Example 3

[0124] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)isobutyramide:

[0125] Following the method of Example 1, in step six, hexanoyl chloride is replaced with an equimolar amount of isobutyryl chloride.

[0126] 1 H NMR (400MHz, DMSO) δ8.64-8.54(m,2H),8.43(d,J=2.9Hz,1H),8.01(d,J=4.5Hz, 1H),7.71(d,J=8.5Hz,1H),7.49-7.41(m,1H),7.26(dd,J=8.5,4.5Hz,1H),4.44( s,2H),3.76(t,J=4.6Hz,4H),3.56(d,J=5.0Hz,5H),2.86(d,J=5.3Hz,2H),2.77- 2.68(m,1H),1.23(s,3H),1.15(d,J=6.8Hz,5H).HRMS(ESI)(m / z):[M+H]+:calcd forC 27 H 29 N7O2:484.2461, found:484.2413.

[0127] Example 4

[0128] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)heptamide:

[0129] Following the method of Example 1, in step six, hexanoyl chloride is replaced with an equimolar amount of heptanyl chloride.

[0130] 1H NMR (400MHz, CDCl3) δ8.67-8.58(m,2H),8.55(d,J=8.8Hz,1H),8.43(d,J=2.8Hz,1H),7.84(s,1H),7.33-7.21(m,2H),4.45(s,2H),3.91-3.8 5(m,4H),3.57(t,J=4.7Hz,6H),2.88(t,J=5.4Hz,2H),2.49(t,J=7.6Hz,2H),1.78(p,J=7.5Hz,2H),1.48-1.24(m,7H),0.91(q,J=4.9Hz,3H). 13 C NMR (150MHz, CDCl3-d) δ171.85,164.76,162.04,158.84,145.57,141.88,140.09,137.18,133.83,131.95,123.99,121.59,120.63,11 6.65,114.11,101.51,66.89(2C),52.73,48.38(2C),45.29,38.04,31.43,26.87,25.13,22.52,14.05.HRMS(ESI)(m / z):[M+H]+:calcd for C 30 H 35 N7O2:526.2930, found:526.2941.

[0131] Example 5

[0132] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-2-methoxyacetamide:

[0133] Following the method of Example 1, in step six, hexanoyl chloride is replaced with an equimolar amount of 2-methoxyacetyl chloride.

[0134] 1 H NMR (400MHz, CDCl3) δ9.08(s,1H),8.68-8.60(m,2H),8.59(d,J=8.8Hz,1H),8.42(d,J=2.8Hz,1H),8.12(d,J=4.5Hz ,1H),7.36-7.25(m,2H),4.45(s,2H),4.10(s,2H),3.91-3.84(m,4H),3.57(d,J=4.6Hz,9H),2.88(t,J=5.3Hz,2H). 13C NMR (150MHz, CDCl3-d) δ168.51,164.97,162.28,159.03,146.01,145.79,141.19,140.32,134.00,132.32,124.20,121.81,120.65 ,116.47,114.36,102.32,72.32,67.10(2C),59.99,52.95,48.58(2C)(d,J=3.3Hz),45.51,27.09.HRMS(ESI)(m / z):[M+H]+:calcd for C 26 H 27 N7O3:486.2254, found:486.2232.

[0135] Example 6

[0136] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)furan-2-carboxamide:

[0137] Following the method of Example 1, in step six, hexanoyl chloride is replaced with an equimolar amount of furan-2-formyl chloride.

[0138] 1 H NMR (400MHz, CDCl3) δ8.66-8.17(m,1H),7.87(d,J=6.4Hz,1H),7.34-7.27(m,1H) ,4.30(s,1H),3.52(dd,J=76.1,5.6Hz,4H),3.18(s,16H),2.73(d,J=6.6Hz,1H). 13 CNMR(150MHz, CDCl3-d)δ164.97,162.30,159.01,156.23,147.19,145.71(d,J=16.1Hz),141.63,140.46,134.42,134.09 ,132.32,124.16,121.74,120.61,117.05,116.64,114.36,113.30,102.08,67.11(2C),52.96,48.60(2C),45.53,27.11.

[0139] Example 7

[0140] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)thiophene-2-carboxamide:

[0141] Following the method of Example 1, in step six, hexanoyl chloride is replaced with an equimolar amount of thiophene-2-formyl chloride.

[0142] 1 H NMR (400MHz, CDCl3) δ8.71-8.65(m,3H),8.46-8.38(m,2H),8.13(dd,J=4.5,1.4Hz,1H),7.74(dd,J=3.8,1.1Hz,1H),7.65(dd,J=5.0,1 .1Hz,1H),7.37-7.25(m,2H),7.19(dd,J=5.0,3.8Hz,1H),4.47(s,2H),3.92-3.85(m,4H),3.59(q,J=4.4Hz,6H),2.89(t,J=5.4Hz,2H). 13 C NMR (150MHz, CDCl3-d) δ164.99,162.24,160.07,159.02,145.83,141.92,138.58,134.41,134.19,132.77,132.13,129.55,128.59,124 .29,121.94,120.65,116.83,114.37,101.96,67.12(2C),52.93,48.60(2C)(d,J=2.7Hz),45.50,27.09.HRMS(ESI)(m / z):[M+H]+:calcd for C 28 H 25 N7O2S:524.1869,found:524.1872.

[0143] Example 8

[0144] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)tetrahydro-2H-pyran-4-carboxamide:

[0145] Following the method of Example 1, in step six, hexanoyl chloride is replaced with an equimolar amount of tetrahydro-2H-pyran-4-formyl chloride.

[0146] 1H NMR (400MHz, CDCl3) δ8.68-8.60(m,2H),8.55(d,J=8.7Hz,1H),8.43(d,J=2.8Hz,1H),8. 19-8.10(m,1H),7.81(s,1H),7.36-7.23(m,2H),4.46(s,2H),4.09(dt,J=11.5,3.5Hz,2 H),3.91-3.84(m,4H),3.62-3.52(m,7H),3.52-3.49(m,2H),3.48-3.41(m,1H),2.88(t, J=5.4Hz,2H),2.63(ddt,J=13.4,8.6,4.8Hz,1H),2.04-1.95(m,1H),1.94-1.79(m,1H). 13 CNMR(150MHz, CDCl3-d)δ173.07,164.97,162.22,158.97,146.14,145.82,141.85,134.07,132.12,124.29,121.95,120.98,11 6.84,114.37,102.00,67.38,67.09,52.91,48.58(d,J=2.9Hz),45.47,43.65,29.36,27.07.HRMS(ESI)(m / z):[M+H]+:calcdfor C 29 H 31 N7O3:526.2567, found:526.2547.

[0147] Example 9

[0148] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)cyclopropaneformamide:

[0149] Following the method of Example 1, in step six, hexanoyl chloride is replaced with an equimolar amount of cyclopropaneformyl chloride.

[0150] 1H NMR (400MHz, CDCl3) δ8.68-8.50(m,4H),8.12(d,J=4.4Hz,1H),7.98(s,1H),7.30(ddd,J=11.9,7.1,3.1Hz,2H),4.45(s,2H),3.87(t,J=4.6 Hz, 4H), 3.57 (q, J = 4.9Hz, 6H), 2.87 (t, J = 5.4Hz, 2H), 1.66 (tt, J = 8.1, 4.5Hz, 1H), 1.16 (dt, J = 7.0, 3.5Hz, 2H), 0.97 (dq, J = 7.5, 4.1Hz, 2H). 13 C NMR (150MHz, CDCl3-d) δ172.66,164.98,162.25,159.09,146.04,145.79,142.23,140.31,137.42,134.02(d,J=8.0Hz),132.20,124.20 ,121.80,120.77,116.96,114.29,101.44,67.11(2C),52.95,48.60(2C),45.52,27.08,16.57,9.38(C).HRMS(ESI)(m / z):[M+H]+:calcd for C 27 H 27 N8O3:482.2304, found:482.2299.

[0151] Example 10

[0152] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)cyclobutane formamide:

[0153] Following the method of Example 1, in step six, hexanoyl chloride is replaced with an equimolar amount of cyclobutaneformyl chloride.

[0154] 1H NMR (400MHz, DMSO) δ10.10(s,1H),8.63-8.59(m,1H),8.43(d,J=3.0Hz,1H),8.01(d,J=4.5Hz,1 H),7.74(d,J=8.6Hz,1H),7.49-7.41(m,1H),7.26(dd,J=8.5,4.5Hz,1H),4.44(s,2H),3.76(t, J=4.6Hz,4H),3.56(t,J=4.8Hz,6H),2.86(t,J=5.2Hz,2H),2.32-2.18(m,2H),2.21-2.13(m,2H ),2.06-1.92(m,1H),1.86(td,J=9.5,4.7Hz,1H),1.23(s,2H).HRMS(ESI)(m / z):[M+H]+:calcd for C 28 H 29 N7O2:496.2461, found:496.2480.

[0155] Example 11

[0156] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)cyclopentaneformamide:

[0157] Following the method of Example 1, in step six, hexanoyl chloride is replaced with an equimolar amount of cyclopentaneformyl chloride.

[0158] 1 H NMR (400MHz, CDCl3) δ8.69-8.58(m,2H),8.55(d,J=8.8Hz,1H),8.42(d,J=2.9Hz,1H),8.11(d,J=4.4Hz,1H),7.80(s,1H),7.32-7.19(m,2H),4.44( s,2H),3.90-3.83(m,4H),3.56(dt,J=6.1,3.2Hz,5H),2.90-2.72(m,3H), 2.48(s,1H),2.09-1.75(m,5H),1.75-1.55(m,2H),1.24(d,J=4.7Hz,1H). 13C NMR(150MHz, CDCl3-d)δ175.27,164.96,162.18,159.08,145.90(d,J=14.2 Hz),142.30,139.84,136.97,134.03,132.12,124.32,121.98,120.76,116 .90,114.27,101.69,67.10(2C),52.88,48.59(2C),47.44(d,J=4.5Hz),45 .44,30.80(2C),27.05,26.31,26.21(2C).HRMS(ESI)(m / z):[M+H]+:calcd forC 29 H 31 N7O2:510.2617, found:510.2622.

[0159] Example 12

[0160] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)cyclohexaneformamide:

[0161] Following the method of Example 1, in step six, hexanoyl chloride is replaced with an equimolar amount of cyclohexaneformyl chloride.

[0162] 1 H NMR (400MHz, CDCl3) δ8.69-8.58(m,2H),8.55(d,J=8.8Hz,1H),8.43(d,J=2.9Hz,1H),8.11(d ,J=4.5Hz,1H),7.81(s,1H),4.44(s,2H),3.90-3.83(m,4H),3.57(q,J=4.8Hz,6H),2.86(t,J= 5.3Hz,2H),2.03(dd,J=13.1,3.6Hz,2H),1.87(dt,J=12.1,3.3Hz,2H),1.82-1.68(m,1H),1.6 3(dd,J=13.4,6.0Hz,1H),1.55(dd,J=12.2,8.9Hz,1H),1.49-1.33(m,2H),1.36-1.18(m,4H).

[0163] Example 13

[0164] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-4-methylbenzamide:

[0165] Following the method of Example 1, in step six, hexanoyl chloride is replaced with an equimolar amount of 4-methylbenzoyl chloride.

[0166] 1 H NMR (400MHz, CDCl3) δ8.79-8.72(m,1H),8.69(s,1H),8.72-8.64(m,1H),8.52(s,1H),8.43(s,1H),8.13(d,J=4.4Hz,1H),7.90 -7.83(m,2H),7.38-7.27(m,4H),4.48(s,2H),3.89(t,J=4.6Hz,4H),3.59(q,J=5.3Hz,6H),2.89(t,J=5.1Hz,2H),2.46(s,3H). 13 CNMR(150MHz, CDCl3-d)δ165.67,164.98,162.19,159.11,146.03,145.87,143.94,142.37,134.21(d,J=12.0Hz),132.16,131.11,130.17(2C),1 27.62(2C),124.37,122.07,120.69,116.98,114.30,102.00,67.12(2C) ,52.90,48.62(2C),45.47,27.06,21.97.HRMS(ESI)(m / z):[M+H]+:calcd for C 31 H 29 N7O2:532.2461, found:532.2473.

[0167] Example 14

[0168] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-3-methylbenzamide:

[0169] Following the method of Example 1, in step six, hexanoyl chloride is replaced with an equimolar amount of 3-methylbenzoyl chloride.

[0170] 1H NMR (400MHz, DMSO) δ8.69(s,2H),8.45(s,1H),7.86(s,2H),7.48(s,3H),5.77(d,J=5.5Hz ,4H),3.78(s,4H),3.59(s,6H),2.53(s,5H),2.43(s,2H).HRMS(ESI)(m / z):[M+H]+:calcd for C 31 H 29 N7O2:532.2461, found:532.2471.

[0171] Example 15

[0172] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-4-ethylbenzamide:

[0173] Following the method of Example 1, in step six, hexanoyl chloride is replaced with an equimolar amount of 4-ethylbenzoyl chloride.

[0174] 1 H NMR (400MHz, CDCl3) δ8.75(d,J=9.6Hz,1H),8.71-8.64(m,2H),8.53(s,1H),8.13(d,J=4.5Hz,1H),7.92-7.86(m,2H),7.37(d,J=8.1Hz,2H),7.37 -7.25(m,2H),4.47(s,2H),3.88(t,J=4.6Hz,4H),3.59(dt,J=6.3,3.2Hz ,6H),2.89(t,J=5.4Hz,2H),2.75(q,J=7.6Hz,2H),1.29(t,J=7.6Hz,4H). 13 C NMR (150MHz, CDCl3-d) δ165.70,164.97,162.26,159.07,150.10,145.81,142 .36,140.20,137.32,134.26,134.15,132.15,131.34,128.99(2C),127.72(2C ),124.23,121.85,120.70,116.97,114.33,102.02,67.11(2C),52.94,48.55( 2C),45.50,29.24,27.09,15.59(d,J=2.0Hz).HRMS(ESI)(m / z):[M+H]+:calcd for C 32 H 31N7O2:546.2617, found:546.2651.

[0175] Example 16

[0176] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-4-propylbenzamide:

[0177] Following the method of Example 1, in step six, hexanoyl chloride is replaced with an equimolar amount of 4-propylbenzoyl chloride.

[0178] 1 H NMR (400MHz, CDCl3) δ8.79-8.72(m,1H),8.71-8.64(m,2H),8.53(s,1H),8.45-8. 40(m,1H),8.13(d,J=4.5Hz,1H),7.92-7.85(m,2H),7.40-7.31(m,3H),7.31(dd,J =8.5,4.5Hz,1H),4.48(s,2H),3.89(t,J=4.7Hz,4H),3.59(q,J=5.2Hz,6H),2.89 (t,J=5.3Hz,2H),2.69(t,J=7.6Hz,2H),1.76-1.63(m,2H),0.97(t,J=7.3Hz,3H). 13 C NMR (150MHz, CDCl3-d) δ165.73,164.98,162.24,159.08,148.60,145.83,142. 37,134.26,134.15,132.15,131.36,129.58(2C),127.63(2C),124.27,123.91 ,121.90,120.70,120.59,116.98,114.32,102.04,102.02,67.11(2C),52.92, 48.60(2C),45.49,38.29,27.08,24.60,14.09.HRMS(ESI)(m / z):[M+H]+:calcd for C 33 H 33 N7O2: 560.2774, found: 560.2769.

[0179] Example 17

[0180] Preparation of 4-(tert-butyl)-N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)benzamide:

[0181] Following the method of Example 1, in step six, hexanoyl chloride is replaced with an equimolar amount of 4-(tert-butyl)benzoyl chloride.

[0182] 1 H NMR (400MHz, CDCl3) δ8.71-8.65(m,2H),8.53(s,1H),8.43(d,J=2.8Hz,1H),8.16-8.10(m,1H),7.94-7.87(m,2H),7.60-7.53(m,2 H),7.39-7.27(m,2H),4.48(s,2H),3.89(t,J=4.6Hz,4H),3.59(q,J=5.1Hz,6H),3.49(s,1H),2.89(t,J=5.4Hz,2H),1.37(s,9H). 13 CNMR(150MHz, CDCl3-d)δ165.68,164.98,162.24,159.08,156.91,145.83,142.37,140.11,137.22,134.26,134.15,132.14,131.09,127.47(2 C),126.47(2C),124.26,121.90,120.66,116.96,114.32,102.01,67.1 1(2C),52.93,48.59(2C)(d,J=3.6Hz),45.49,35.48,31.46(3C),27.08.

[0183] Example 18

[0184] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-3-phenylpropionamide:

[0185] Following the method of Example 1, in step six, hexanoyl chloride is replaced with an equimolar amount of 3-phenylpropionyl chloride.

[0186] 1H NMR(400MHz, CDCl3)δ8.61(dd,J=6.5,2.1Hz,2H),8.53(d,J=9.5Hz,1H),8.43(s,1H),8.13(s,1H),7.66(s,1H),7.36-7.19(m,7H) ,4.45(s,2H),3.91-3.84(m,4H),3.57(q,J=4.6Hz,6H),3.10(t,J=7.6Hz,2H),2.91-2.77(m,4H).HRMS(ESI)(m / z):[M+H]+:calcd for C 32 H 32 N7O2:546.2617, found:546.2635.

[0187] Example 19

[0188] Preparation of 3-chloro-N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)benzamide:

[0189] Following the method of Example 1, in step six, hexanoyl chloride is replaced with an equimolar amount of 3-chloro-benzoyl chloride.

[0190] 1 H NMR (400MHz, CDCl3) δ8.74-8.64(m,3H),8.48(s,1H),8.43(d,J=2.9Hz,1H),8.13(dd,J=4.4,1.5Hz,1H),7.98(t,J=1.9Hz,1H),7.80(dt,J=7.8,1.4Hz ,1H),7.63-7.56(m,1H),7.49(t,J=7.9Hz,1H),7.32-7.19(m,2H),4.46(s, 2H), 3.88 (dd, J=5.5, 3.7Hz, 4H), 3.62-3.54 (m, 6H), 2.88 (t, J=5.3Hz, 2H). 13 C NMR (150MHz, CDCl3-d) δ164.99,164.44,162.30,158.94,146.03,145.80,141.75,135.88,135.75,134.84,134.18,133.18,132.22, 130.75,128.33(d,J=2.6Hz),125.23,124.24,121.86,121.06,116.83,114.45,102.51,67.11(2C),52.95,48.61(2C),45.52,27.11

[0191] Example 20

[0192] Preparation method of N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-3-fluorobenzamide:

[0193] Following the method of Example 1, in step six, hexanoyl chloride is replaced with an equimolar amount of 3-fluorobenzoyl chloride.

[0194] 1 H NMR (400MHz, CDCl3) δ8.70(s,3H),8.50(s,1H),8.16-8.10(m,1H),7.75-7.66(m,2H),7.53(td,J=8.0,5.4Hz,1H) ,7.37-7.20(m,3H),4.47(s,2H),3.89(t,J=4.6Hz,4H),3.59(t,J=4.9Hz,6H),3.49(s,1H),2.89(t,J=5.3Hz,2H).

[0195] Example 21

[0196] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-3-(trifluoromethyl)benzamide:

[0197] Following the method of Example 1, in step six, hexanoyl chloride is replaced with an equimolar amount of 3-(trifluoromethyl)benzoyl chloride.

[0198] 1 H NMR (400MHz, CDCl3) δ8.71(d,J=7.5Hz,3H),8.50(s,1H),8.28(s,1H),8.11(d,J=8.1Hz,1H),7.89(d,J=7.8Hz,1H),7.71(t ,J=8.0Hz,1H),4.47(s,2H),3.89(t,J=4.7Hz,4H),3.62-3.56(m,6H),2.89(s,2H),1.26(s,1H),0.88(s,1H),0.07(s,1H). 13C NMR (150MHz, CDCl3-d) δ165.00,164.38,162.35,158.90,141.60,135.03,134.89,134.18,132.23,130.18(d,J=17.0Hz),125. 26(q,J=3.9Hz),121.22,116.82,114.50,102.74,67.11(2C),52.99,48.61(2C),45.54,27.14.HRMS(ESI)(m / z):[M+H]+:calcd forC 31 H 26 F3N7O2:586.2178,found:586.2192.

[0199] Example 22

[0200] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-4-(trifluoromethyl)benzamide:

[0201] Following the method of Example 1, in step six, hexanoyl chloride is replaced with an equimolar amount of 4-(trifluoromethyl)benzoyl chloride.

[0202] 1 H NMR (400MHz, DMSO) δ11.00(s,1H),8.73-8.63(m,2H),8.44(d,J=3.0Hz,1H),8.22(d,J=8.1Hz,2H),8.00(dd,J=12.9,6.6Hz,3H),7.78(d,J=8.5Hz ,1H),7.46(ddd,J=8.7,3.3,1.3Hz,1H),7.27(dd,J=8.5,4.5Hz,1H),4.4 6(s,2H),3.81-3.74(m,4H),3.58(q,J=5.2Hz,6H),2.88(t,J=5.3Hz,2H). 13C NMR(150MHz,DMSO-d6)δ165.03,164.35,162.31,157.89,145.78,141.81,1 39.63,137.66,137.20,136.15,132.91(d,J=3.5Hz),132.28,129.26(2C),1 27.10(2C),126.15(q,J=3.8Hz),124.13,121.51,117.22,114.60,109.50,6 6.53(2C),52.41,48.17(2C),44.55,26.56.HRMS(ESI)(m / z):[M+H]+:calcd forC 31 H 26 F3N7O2:586.2178,found:586.2188.

[0203] Example 23

[0204] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-2-methoxybenzamide:

[0205] Following the method of Example 1, in step six, hexanoyl chloride is replaced with an equimolar amount of 2-methoxybenzoyl chloride.

[0206] 1 H NMR(400MHz, CDCl3)δ11.02(s,1H),8.70-8.61(m,2H),8.44(s,1H),8.33(d,J =7.8Hz,1H),8.15-8.10(m,1H),7.57(t,J=7.8Hz,1H),7.40-7.28(m,2H),7.16 (t,J=7.6Hz,1H),7.10(d,J=8.4Hz,1H),4.48(s,2H),4.19(s,3H),3.89(t,J=4 .6Hz, 4H), 3.80 (d, J = 5.7Hz, 1H), 3.59 (q, J = 6.0Hz, 6H), 2.89 (t, J = 5.3Hz, 2H). 13C NMR (150MHz, CDCl3-d) δ164.97,164.05,162.23,159.25,157.98,145.80,14 3.37,140.33,137.48,134.56,134.07,133.69,133.06,132.45,124.17,121 .91,121.74,120.88(d,J=5.8Hz),117.47,114.20,111.92,101.83,67.13(2 C),56.44,52.95,48.61(2C),45.53,27.08.HRMS(ESI)(m / z):[M+H]+:calcd for C 31 H 29 N7O3:548.2410, found:548.2455.

[0207] Example 24

[0208] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-3-methoxybenzamide:

[0209] Following the method of Example 1, in step six, hexanoyl chloride is replaced with an equimolar amount of 3-methoxybenzoyl chloride.

[0210] 1 H NMR (400MHz, CDCl3) δ8.74(d,J=9.5Hz,1H),8.70(s,2H),8.54(s,1H),8.43(d,J=2.8Hz,1H),8.12(d,J=4.5Hz,1H),7.55-7.41(m, 3H),7.41-7.29(m,2H),7.19-7.12(m,1H),4.48(s,2H),3.90(d,J=6.0Hz,7H),3.60(dt,J=9.1,5.1Hz,6H),2.89(t,J=5.3Hz,2H). 13CNMR(150MHz,CDCl3-d)δ165.62,164.98,162.27,160.55,159.03,146.02 ,145.80,142.15,140.22,134.15,132.17,130.53,124.24,121.86,120.78 ,119.52,119.16,116.91,114.37,112.83,102.23,67.11(2C),55.88,52. 94,48.59(2C)(d,J=3.4Hz),45.51,27.10.HRMS(ESI)(m / z):[M+H]+:calcd for C 31 H 29 N7O 33 :548.2410,found:548.2414.

[0211] Example 25

[0212] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyridin-3-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-4-methoxybenzamide:

[0213] Following the method of Example 1, in step six, hexanoyl chloride is replaced with an equimolar amount of 4-methoxybenzoyl chloride.

[0214] 1 H NMR (400MHz, CDCl3) δ9.08-8.54(m,3H),8.41-8.10(m,4H),8.03-7.76(m,2H),5.89(d,J=2.1Hz,2H),4.75(s,2H),4. 00(d,J=2.1Hz,3H),3.90(d,J=5.1Hz,4H),3.84(s,2H),3.72(s,3H),3.11-2.77(m,2H),1.18(dd,J=7.9,6.0Hz,2H). 13 C NMR(150MHz,DMSO-d6)δ165.12,163.98,162.64,161.13,157.83,147.07,142.38,135.06,132.44,131.90,130.11,127.80,127.05 ,126.59,125.57,117.12,114.01(d,J=5.9Hz),108.99,66.22,55.70,51.19,47.91,43.38,25.72.HRMS(ESI)(m / z):[M+H]+:calcd for C31 H 29 N7O3:548.2410, found:548.2416.

[0215] Example 26

[0216] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)propionamide:

[0217] Following the method of Example 1, in step four, 3-iodopyridine is replaced with an equimolar amount of 2-bromopyrazine, and in step six, hexanoyl chloride is replaced with an equimolar amount of propionyl chloride.

[0218] 1 H NMR (400MHz, CDCl3) δ8.63(d,J=8.8Hz,2H),8.56(d,J=8.7Hz,1H),8.23(s,1H),8.16-8.09(m,1H),7.91(d,J=2.6Hz,1H),7.76(s,1H),4.72(s ,2H),3.92(t,J=5.3Hz,2H),3.86(t,J=4.6Hz,4H),3.55(t,J=4.6Hz,4H),2.82(t,J=5.3Hz,2H),2.53(q,J=7.5Hz,2H),1.30(t,J=7.5Hz,3H). 13 C NMR (150MHz, CDCl3-d) δ172.56,165.10,162.01,159.05,154.23,142.24,142.10,134.13,134.09,133.61,132.18,130.94,120 .76,116.85,114.67,101.66,67.09(2C),50.13,48.63(2C),41.20,31.35,30.06,26.60,9.67.HRMS(ESI)(m / z):[M+H]+:calcd for C 25 H 26 N8O2:471.2257, found:471.2284.

[0219] Example 27

[0220] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)isobutyramide:

[0221] Following the method of Example 26, simply replace the isopropionyl chloride in step six with an equimolar amount of isobutyryl chloride.

[0222] 1 H NMR (400MHz, CDCl3) δ8.67-8.60(m,2H),8.56(d,J=8.7Hz,1H),8.24(d,J=1.5Hz,1H),8.12(dd,J=2.6,1.5Hz,1H),7.91(d,J=2.6Hz,1H),7.79(s,1H ),4.72(s,2H),3.92(t,J=5.3Hz,2H),3.89-3.83(m,4H),3.55(t,J=4.6Hz ,4H),2.82(t,J=5.4Hz,2H),2.66(p,J=6.9Hz,1H),1.32(d,J=6.9Hz,6H). 13 C NMR (150MHz, CDCl3-d) δ175.82,165.10,162.01,159.06,154.24,142.26,142.18,134.14,134.07,133.57,132.12,130.91,120 .79,116.85,114.67,101.80,67.09(2C),50.14,48.63(2C),41.20,37.35,26.60,19.81,0.35.HRMS(ESI)(m / z):[M+H]+:calcd for C 26 H 28 N8O2: 485.2413, found: 485.2422

[0223] Example 28

[0224] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)hexanoamide:

[0225] Following the method of Example 26, simply replace the isopropionyl chloride in step six with an equimolar amount of hexanoyl chloride.

[0226] 1H NMR (400MHz, CDCl3) δ8.64(d,J=9.1Hz,2H),8.57(d,J=8.7Hz,1H),8.25(s,1 H),8.13(d,J=2.6Hz,1H),7.72(s,1H),4.74(s,2H),3.93(t,J=5.3Hz,2H),3 .90-3.83(m,4H),3.56(t,J=4.6Hz,4H),2.83(t,J=5.4Hz,2H),2.49(t,J=7. 6Hz,2H),1.84-1.72(m,3H),1.40(dq,J=7.3,3.5Hz,4H),0.98-0.90(m,3H). 13 C NMR (150MHz, CDCl3-d) δ172.04,165.10,162.01,159.06,154.24,142.25,142.10,134.11(d,J=9.5Hz),133.60,132.18,130.93,120.79, 116.86,114.67,101.67,67.09(2C),50.14,48.63(2C),41.20,38.28,31.64,26.60,25.35,22.74,14.27.HRMS(ESI)(m / z):[M+H]+:calcd for C 28 H 32 N8O2: 513.2726, found: 513.2719

[0227] Example 29

[0228] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)heptamide:

[0229] Following the method of Example 26, simply replace the isopropionyl chloride in step six with an equimolar amount of heptanyl chloride.

[0230] 1H NMR (400MHz, CDCl3) δ8.67-8.60 (m, 2H), 8.56 (d, J = 8.7Hz, 1H), 8.25 (s, 1H), 8.15-8. 10(m,1H),7.92(d,J=2.6Hz,1H),7.73(s,1H),4.73(s,2H),3.93(t,J=5.3Hz,2H),3.9 0-3.83(m,4H),3.56(t,J=4.6Hz,4H),2.83(t,J=5.4Hz,2H),2.49(t,J=7.6Hz,2H),1 .78(p,J=7.5Hz,2H), 1.38(dtd,J=26.7,7.3,2.6Hz,6H), 0.91(td,J=5.7,2.8Hz,3H). 13 C NMR (150MHz, CDCl3-d) δ172.04,165.10,162.02,159.06,154.23,142.24,142.10,134.14,134.08,133.62,132.18,130.95,12 0.79,116.86,114.67,101.67,67.09(2C),50.14,48.64(2C),41.20,38.33,31.59,30.06,29.19,26.60,25.63,22.84,14.39.

[0231] Example 30

[0232] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-2-methoxyacetamide:

[0233] Following the method of Example 26, simply replace the isopropionyl chloride in step six with an equimolar amount of 2-methoxyacetyl chloride.

[0234] 1 H NMR (400MHz, CDCl3) δ9.08(s,1H),8.69-8.62(m,2H),8.59(d,J=8.7Hz,1H),8.24(s,1H),8.12(dd,J=2.6,1.4Hz,1H),7.91(d,J= 2.6Hz,1H),4.74(s,2H),4.10(s,2H),3.93(t,J=5.4Hz,2H),3.86(t,J=4.6Hz,4H),3.57(d,J=6.6Hz,7H),2.83(t,J=5.4Hz,2H). 13C NMR (150MHz, CDCl3-d) δ168.52,165.09,162.03,159.03,154.23,142.24,141.21,134.54,134.02,133.61,132.34,130.94, 120.66,116.48,114.71,102.32,72.33,67.08(2C),60.00,50.14,48.63(2C),41.20,26.61.HRMS(ESI)(m / z):[M+H]+:calcd forC 25 H 26 N8O3:486.2206, found:487.2200.

[0235] Example 31

[0236] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)furan-2-carboxamide:

[0237] Following the method of Example 26, simply replace the isopropionyl chloride in step six with an equimolar amount of furan-2-formyl chloride.

[0238] 1 H NMR (400MHz, DMSO) δ8.68-8.63(m,1H),8.45(s,1H),8.14(d,J=2.4Hz,1H),8.03(s,1H),7.89(d,J=2.5Hz,1H),7.77(d,J=8.6Hz,1H), 7.41(d,J=3.5Hz,1H),6.76(d,J=3.3Hz,1H),5.75(s,2H),4.75(s,2H),3.88(s,2H),3.76(s,4H),3.56(s,4H),2.84(d,J=5.7Hz,2H). 13 C NMR (150MHz, CDCl3-d) δ165.10,162.03,159.04,156.24,154.24,150.06,147.20,145.67,141.67,136.44,133.60,132.35,130.9 3,120.62,117.07,116.65,114.71,113.31,102.09,67.09(2C),50.14,48.64(2C),41.20,26.61.HRMS(ESI)(m / z):[M+H]+:calcd for C 27 H 34N8O3:509.2050, found:509.2051.

[0239] Example 32

[0240] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)thiophene-2-carboxamide:

[0241] Following the method of Example 26, simply replace the isopropionyl chloride in step six with an equimolar amount of thiophene-2-formyl chloride.

[0242] 1 H NMR(400MHz, CDCl3)δ8.69(dd,J=5.7,1.6Hz,3H),8.39(s,1H),8.25(d,J=1.5Hz ,1H),8.13(dd,J=2.6,1.5Hz,1H),7.92(d,J=2.6Hz,1H),7.74(dd,J=3.8,1.1Hz ,1H),7.65(dd,J=5.0,1.1Hz,1H),7.19(dd,J=5.0,3.8Hz,1H),4.74(s,2H),3.9 4(t,J=5.4Hz,2H),3.91-3.84(m,4H),3.63-3.53(m,4H),2.84(t,J=5.4Hz,2H). 13 C NMR (150MHz, CDCl3-d) δ165.12,160.06,159.01,154.24,142.26,141.93,138.58,134.38,134.21,133.62,132.77,132.14,130.9 4,129.55,128.59,120.61,116.83,114.73,101.93,67.10(2C),50.15,48.65(2C),41.21,26.62.HRMS(ESI)(m / z):[M+H]+:calcd for C 27 H 24 N8O2S:525.1821,found:525.1801.

[0243] Example 33

[0244] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)cyclopropaneformamide:

[0245] Following the method of Example 26, simply replace the isopropionyl chloride in step six with an equimolar amount of cyclopropaneformyl chloride.

[0246] 1 H NMR (400MHz, CDCl3) δ8.26(s,1H),8.14(s,1H),7.93(s,1H),7.47(d,J=8.3Hz,1H),3.95(d,J=5.1Hz,2H) ,3.87(t,J=4.4Hz,4H),3.58(t,J=4.5Hz,4H),2.85(s,2H),1.25(t,J=5.4Hz,6H),0.96(d,J=7.8Hz,4H). 13 C NMR (150MHz, CDCl3-d) δ176.47,164.92,162.01,158.44,153.99,143.22,142.05,139.26,133.49,133.37,133.23,130.72,13 0.67,115.92,115.06,113.90,66.85(2C),49.91(2C),48.42,40.99,26.48,16.53,11.59(2C).HRMS(ESI)(m / z):[M+H]+:calcd for C 26 H 26 N8O2:483.2257, found:483.2248.

[0247] Example 34

[0248] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)cyclobutane formamide:

[0249] Following the method of Example 26, simply replace the isopropionyl chloride in step six with an equimolar amount of cyclobutaneformyl chloride.

[0250] 1H NMR (400MHz, DMSO) δ10.10(s,1H),8.63-8.54(m,2H),8.47-8.42(m,1H),8.14(t,J=2.1Hz,1H),7.89(d,J=2.6Hz,1H),7.74(d,J=8.6Hz,1H), 4.73(s,2H),3.87(t,J=5.3Hz,2H),3.75(t,J=4.5Hz,4H),3.54(t,J=4.6Hz,4H),2.82(t,J=5.4Hz,2H),2.34-2.06(m,5H),2.06-1.74(m,2H). 13 C NMR (150MHz, CDCl3-d) δ165.10,162.00,159.07,154.26,142.30,142.14,134.07(d,J=4.6Hz),133.48,132.16,130.85,120.67,116.82 ,114.65,101.65,67.09(2C),50.13,48.63(2C),41.35,41.21,37.95,26.60(2C),25.67,25.57,18.37.HRMS(ESI)(m / z):[M+H]+:calcd for C 27 H 28 N8O2:497.2413, found:497.2396.

[0251] Example 35

[0252] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)cyclopentaneformamide:

[0253] Following the method of Example 26, simply replace the isopropionyl chloride in step six with an equimolar amount of cyclopentaneformyl chloride.

[0254] 1H NMR (400MHz, CDCl3) δ8.67-8.60 (m, 2H), 8.57 (d, J = 8.7Hz, 1H), 8.24 (s, 1H ),8.15-8.10(m,1H),7.91(d,J=2.6Hz,1H),7.77(s,1H),4.74(s,2H),3.93 (t,J=5.3Hz,2H),3.86(t,J=4.5Hz,4H),3.56(t,J=4.6Hz,4H),2.89-2.76 (m,3H),2.10-1.88(m,3H),1.87-1.71(m,4H),1.66(dt,J=7.1,2.9Hz,1H). 13 C NMR (150MHz, CDCl3-d) δ175.25,165.10,162.00,159.08,154.24,142.28(d,J=10.4Hz),134.04(d,J=8.2Hz),133.60,132.14,130.94,120 .71,116.90,114.65,101.65,67.09(2C),50.14,48.64(2C),47.49,41.20,30.81(2C),26.60,26.32(2C).HRMS(ESI)(m / z):[M+H]+:calcd for C 28 H 30 N8O2:511.2570, found:511.2573.

[0255] Example 36

[0256] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)cyclohexaneformamide:

[0257] Following the method of Example 26, simply replace the isopropionyl chloride in step six with an equimolar amount of cyclohexaneformyl chloride.

[0258] 1H NMR (400MHz, CDCl3) δ8.64(d,J=9.1Hz,2H),8.58(d,J=8.7Hz,1H),8.25(s,1H ),8.13(s,1H),7.92(d,J=2.6Hz,1H),7.79(s,1H),4.76(s,2H),3.93(s,2H), 3.87(t,J=4.6Hz,4H),3.57(t,J=4.6Hz,4H),2.42-2.30(m,1H),2.04(d,J=12 .9Hz,2H),1.74(d,J=12.5Hz,6H),1.57(q,J=11.9Hz,2H),1.40-1.26(m,2H). 13 C NMR (150MHz, CDCl3-d) δ174.93,165.10,162.00,159.07,154.23,142.24(d,J=1.9Hz),134.07,133.62,132.12,130.95,120.78,116.91, 114.66,101.71,67.09(2C),50.14,48.63(2C),46.95,41.20,29.90(2C),26.60,25.90(2C)(d,J=4.4Hz).HRMS(ESI)(m / z):[M+H]+:calcd forC 29 H 32 N8O2: 525.2726, found: 525.2720.

[0259] Example 37

[0260] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-4-methylbenzamide:

[0261] Following the method of Example 26, simply replace the isopropionyl chloride in step six with an equimolar amount of 4-methylbenzoyl chloride.

[0262] 1H NMR (400MHz, CDCl3) δ8.81-8.71(m,3H),8.54(s,1H),8.27(d,J=1.5Hz,1H),8.16(dd,J=2.7,1.4Hz,1H),7.93(d,J=2.6Hz,1H),7.90-7.83(m ,2H),7.35(d,J=7.9Hz,2H),4.84(s,2H),3.95(t,J=5.3Hz,2H),3.92-3.85(m,4H),3.63(t,J=4.6Hz,4H),2.85(t,J=5.4Hz,2H),2.46(s,3H). 13 C NMR (150MHz, CDCl3-d) δ165.66,165.11,162.03,159.08,154.25,143.93,142.37,142.26,134.24,134.18,133.59,132.17,131.12,130.93,1 30.17(2C),127.62(2C),120.68,116.98,114.69,102.00,67.10(2C),50.15,48.65(2C),41.21,26.62,21.97.HRMS(ESI)(m / z):[M+H]+:calcd for C 30 H 28 N8O2:533.2413, found:533.2415.

[0263] Example 38

[0264] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-4-ethylbenzamide:

[0265] Following the method of Example 26, simply replace the isopropionyl chloride in step six with an equimolar amount of 4-ethylbenzoyl chloride.

[0266] 1H NMR (400MHz, CDCl3) δ8.75 (d, J = 9.5Hz, 1H), 8.72-8.67 (m, 2H), 8.53 (s, 1H ),8.25(s,1H),8.16-8.10(m,1H),7.95-7.86(m,3H),7.40-7.33(m,2H),4 .74(s,2H),3.94(t,J=5.3Hz,2H),3.88(t,J=4.6Hz,4H),3.57(t,J=4.6Hz ,4H),2.84(t,J=5.4Hz,2H),2.75(q,J=7.6Hz,2H),1.28(q,J=7.2Hz,3H). 13 C NMR (150MHz, CDCl3-d) δ165.70,165.11,162.03,159.08,154.24,150.10, 142.38,142.25,134.21(d,J=8.3Hz),133.61,132.17,131.35,130.94,12 9.00(2C),127.73(2C),120.67,116.98,114.69,101.99,67.10(2C),50.1 5,48.6(2C)5,41.21,29.25,26.62,15.60.HRMS(ESI)(m / z):[M+H]+:calcd forC 31 H 30 N8O2: 547.2570, found: 547.2585.

[0267] Example 39

[0268] Preparation method of 4-(tert-butyl)-N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)benzamide:

[0269] Following the method of Example 26, the isopropionyl chloride in step six can be replaced with an equimolar amount of 4-(tert-butyl)-benzoyl chloride.

[0270] 1H NMR (400MHz, CDCl3) δ8.76(d,J=9.0Hz,1H),8.71(s,2H),8.53(s,1H),8.26(s,1H),8.16-8.11(m,1H),7.91(dd,J=9.3,2.7Hz, 3H),7.60-7.52(m,2H),4.76(s,2H),3.95(s,2H),3.88(s,4H),3.58(s,4H),2.84(s,2H),1.37(s,8H),1.27(d,J=12.0Hz,1H). 13 CNMR(150MHz, CDCl3-d)δ165.69,165.12,162.03,159.09,156.92,154.25, 142.39,142.26,134.21(d,J=7.6Hz),133.60,132.16,131.11,130.94,127. 48(2C),126.49(2C),120.64,116.96,114.69,101.99,67.10(2C),50.15,4 8.65(2C),41.21,35.49,31.47(2C),26.62.HRMS(ESI)(m / z):[M+H]+:calcd for C 33 H 34 N8O2: 575.2883, found: 575.2898.

[0271] Example 40

[0272] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-3-phenylpropionamide:

[0273] Following the method of Example 26, simply replace the isopropionyl chloride in step six with an equimolar amount of 3-phenylpropionyl chloride.

[0274] 1H NMR (400MHz, CDCl3) δ8.63 (dq, J=4.4, 2.1Hz, 2H), 8.54 (d, J=9.4Hz, 1H), 8.25 (s,1H),8.13(s,1H),7.92(s,1H),7.64(s,1H),7.32(dd,J=8.0,6.7Hz,2H),7 .24-7.18(m,1H),4.74(s,2H),3.93(t,J=5.4Hz,2H),3.90-3.83(m,4H),3.56 (t,J=4.6Hz,4H),3.10(t,J=7.6Hz,2H),2.86-2.77(m,4H),1.32-1.23(m,2H). 13 C NMR (150MHz, CDCl3-d) δ170.97,165.10,162.02,159.02,154.23,142.25,1 41.85,140.33,134.29,134.04,133.61,132.21,130.94,129.10,128.70(2 C),126.92(2C),120.88,116.72,114.69,101.82,67.08(2C),50.13,48.63 (2C),41.20,39.88,31.51,26.60,0.35.HRMS(ESI)(m / z):[M+H]+:calcdfor C 31 H 30 N8O2: 547.2570, found: 547.2595.

[0275] Example 41

[0276] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-3-fluorobenzamide:

[0277] Following the method of Example 26, simply replace the isopropionyl chloride in step six with an equimolar amount of 3-fluorobenzoyl chloride.

[0278] 1H NMR (400MHz, CDCl3) δ8.72(s,3H),8.49(s,1H),8.26(s,1H),8.14(s,1H),7.93(s,1H),7.71(t,J=6.9Hz,2H),7.59-7.51(m ,1H),4.77(s,2H),3.95(t,J=5.4Hz,2H),3.88(t,J=4.6Hz,4H),3.59(t,J=4.7Hz,4H),3.49(s,1H),2.84(d,J=6.6Hz,2H). 13 C NMR(150MHz, CDCl3-d)δ165.12,164.43(d,J=2.7Hz),164.19,162.54,162.07,158.95,1 54.24,142.26,141.80,136.24(d,J=7.0Hz),134.21,133.63,132.21,131.25(d,J=8.1H z),130.94,122.73(d,J=3.2Hz),120.92,120.34,120.20,116.83,115.43,115.28,114. 80,102.38,67.09(2c),50.15,48.65(2c),41.22,26.63.HRMS(ESI)(m / z):[M+H]+:calcd for C 29 H 25 FN8O2:537.2163,found:537.2160.

[0279] Example 42

[0280] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-2-methoxybenzamide:

[0281] Following the method of Example 26, simply replace the isopropionyl chloride in step six with an equimolar amount of 2-methoxybenzoyl chloride.

[0282] 1H NMR (400MHz, CDCl3) δ9.08-8.54(m,3H),8.41-8.10(m,4H),8.03-7.76(m,2H),5.89(d,J=2.1Hz,2H),4.75(s,2H),4. 00(d,J=2.1Hz,3H),3.90(d,J=5.1Hz,4H),3.84(s,2H),3.72(s,3H),3.11-2.77(m,2H),1.18(dd,J=7.9,6.0Hz,2H). 13 C NMR (150MHz, CDCl3-d) δ165.11,164.07,161.97,159.28,158.00,154.25,143.41,142.25,134.57,134.10,133.67,133.57,133.08,132.49,130 .94,121.92,120.91(d,J=7.1Hz),114.55,111.92,101.86,67.11(2C),56.45,50.16,48.66(2C),41.20,26.60.HRMS(ESI)(m / z):[M+H]+:calcd for C 30 H 28 N8O3:549.2362, found:549.2388.

[0283] Example 43

[0284] Preparation of N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-3-methoxybenzamide:

[0285] Following the method of Example 26, simply replace the isopropionyl chloride in step six with an equimolar amount of iso-3-methoxybenzoyl chloride.

[0286] 1 H NMR (400MHz, CDCl3) δ8.83(s,1H),8.64(s,1H),8.25(s,1H),8.17(s,1H),7.92(s,1H),7.40(d,J=7.5Hz,2H),7.3 4(s,2H),7.27(q,J=9.8Hz,2H),7.00(dd,J=7.8,2.6Hz,2H),3.95(s,2H),3.87(s,4H),3.79(s,5H),3.58(s,4H). 13C NMR (151MHz, CDCl3-d) δ172.98,165.09,162.15,160.01,158.56,154.19 ,144.12,142.25,138.68,135.86,133.76,133.69,133.47,130.92,130. 11,129.93,121.85,119.74,116.83,115.20,114.19,112.60,67.05(2C) ,55.78,50.09,48.61(2C),41.17,26.68.HRMS(ESI)(m / z):[M+H]+:calcd forC 30 H 28 N8O3:549.2363, found:549.2361.

[0287] The structural formulas of the pyridine-pyrimidine compounds containing morpholine structures prepared in Examples 1-43 are shown in Table 1.

[0288] Table 1. Structural formulas of pyridine-pyrimidine compounds containing morpholine structures prepared in Examples 1-43

[0289]

[0290]

[0291]

[0292]

[0293] The in vitro antitumor cell activity of the pyridine-pyrimidine compounds containing morpholine structures prepared in Examples 1-43 was determined.

[0294] The selected cancer cells were MCF-7 breast cancer cells, and the control was GDC-0941. The method is as follows:

[0295] (1) After cell resuscitation and stabilization through 2-3 passages, digest the cells from the bottom of the culture flask using trypsin solution (0.25%). Transfer the digestion solution to a centrifuge tube, then add culture medium to stop the digestion. Centrifuge the tube at 1000 rpm for 3 minutes, discard the supernatant, add 3 mL of culture medium, mix the cells by pipetting, and add 10 μL of the cell suspension to a cell counting chamber for counting. Adjust the cell concentration to 102. 4 Cells / well. In a 96-well plate, except for the top, bottom, and leftmost wells which are blank (no cells added), 180 μL of cell suspension was added to all other wells. The 96-well plate was then incubated for 24 hours.

[0296] (2) Dissolve the test sample in 20 μL of dimethyl sulfoxide, then add 980 μL of culture medium to dissolve the sample into a 1 mg / mL solution, and then dilute the sample in an EP tube to 1, 0.333, 0.111, 0.037, 0.012 μg / mL.

[0297] Add 20 μL of each concentration to 3 wells. The two outer rows and two columns of cells are more susceptible to environmental influences and are used as blank cells. Incubate the 96-well plate in an incubator for 72 hours.

[0298] (3) Discard the drug-containing culture medium in the 96-well plate, wash the cells twice with phosphate-buffered saline (PBS), add 100 μL of MTT (tetrazazole) (0.5 mg / mL) to each well, incubate for 4 h, discard the MTT solution, and add 100 μL of dimethyl sulfoxide. Shake on a magnetic oscillator to fully dissolve the surviving cells and the MTT reaction product formazan, then measure the results at 492 nM using a microplate reader. The IC50 of the drug can be determined using the Bliss method. 50 value.

[0299] The antiproliferative activities of the pyridopyrimidine compounds containing morpholine structures prepared in Examples 1-43, i.e., control GDC-0941, against breast cancer cells MCF-7 are shown in Table 2, where ND indicates the IC50 of the compound. 50 >50μM.

[0300] Table 2. Anti-MCF-7 cell proliferation activity of pyridine-pyrimidine compounds containing morpholine structures.

[0301]

[0302]

[0303] The inhibitory activity of the pyridine-pyrimidine compounds containing morpholine structures prepared in Examples 1-43 against PI3Kα kinase was determined.

[0304] The positive control is GDC-0941, and the method is as follows:

[0305] Prepare a 384-well plate with wells for the test compound, blank control, and positive control. Add 2.5 μL of PI3Kα kinase solution to the test compound wells, 2.5 μL of PI3Kα kinase buffer to the blank control wells, and 2.5 μL of PI3Kα kinase solution to the positive control wells. Add 2.5 μL of different concentrations of the compound to each test compound well. Add 10 μL of PI3Kα kinase antibody and EDTA reagent to each well. Centrifuge to mix and incubate at room temperature for 60 min. The final antibody concentration is 2 nM, and the final EDTA concentration is 8 mM. Read the fluorescence values ​​using Envision. Calculate the percentage inhibition rate of the compound.

[0306] Percentage inhibition rate = (Lance signal value - Min) / (Max - Min) × 100.

[0307] In the above formula, the Lance signal value refers to the fluorescence signal intensity measured in each well of the plate at a specific compound concentration, reflecting the degree of influence of the compound on the PI3Kα kinase activity; Min is the average fluorescence signal value of the blank control well, that is, the background signal when there is no kinase (or no substrate), representing 0% enzyme activity; Max is the average fluorescence signal value of the positive control well, that is, the maximum signal produced under the condition of kinase presence and no inhibitor, representing 100% enzyme activity.

[0308] The inhibitory activity of each compound against PI3Kα kinase was tested using the Kinase-Glo enzyme activity evaluation method. The results are shown in Table 3, where ND indicates an inhibition rate of less than 1%.

[0309] Table 3. Inhibitory activity of PI3Kα kinase pairs of pyridine compounds containing morpholine structures.

[0310]

[0311]

[0312] As can be seen from the experimental results in Tables 2 and 3, the morpholine-containing pyridopyrimidine compounds to be protected in this invention exhibit good in vitro anti-cell proliferation activity and PI3Kα kinase inhibition rate. Among them, the morpholine-containing pyridopyrimidine compounds prepared in Examples 26 and 27 showed excellent inhibition rates against PI3Kα kinase. Further kinase IC50 assays were conducted on the compounds with higher inhibition rates. 50 The assays revealed that the morpholine-containing pyridopyrimidine compounds prepared in Example 26 exhibited excellent toxic activity against all selected cell lines, comparable to positive control drugs. The morpholine-containing pyridopyrimidine compounds prepared in this invention show promise as potential inhibitors of PI3Kα kinase.

[0313] Structure-activity relationship analysis was performed on the pyridopyrimidine compounds containing morpholine structures prepared in Example 26.

[0314] The morpholine-containing pyridopyrimidine compounds prepared in Example 26 were subjected to molecular docking with PI3Kα (PDB code: 4L23) to study their binding mode with PI3Kα. The molecular docking results are shown in [Figure number missing]. Figure 1 .

[0315] Figure 1The figure shows that the tetrahydropyrido[3,4-d]pyrimidine core of the morpholine-containing pyridopyrimidine compound prepared in Example 26 forms important hydrogen bonds with the amino acids ASP-933 and SER-774. These hydrogen bonds are crucial for stabilizing the binding between the compound and the protein, contributing to enhanced binding affinity and selectivity. The figure also shows that the cyano group of compound W-1 forms an important hydrogen bond with the amino acid TYR-836. This hydrogen bond formation also enhances the binding ability of the compound to the protein, potentially having a positive impact on improving the compound's biological activity. Furthermore, the distances marked in the figure (e.g.) and This indicates that the morpholine-containing pyridopyrimidine compounds prepared in Example 26 have a close contact with proteins, which further enhances the binding affinity of the morpholine-containing pyridopyrimidine compounds prepared in Example 26 to proteins.

[0316] hemolytic toxicity evaluation

[0317] Hemolysis refers to the rupture of red blood cells due to unsuitable environmental stimuli, resulting in the release of intracellular hemoglobin and other cellular components. The hemolytic toxicity evaluation of the morpholine-containing pyridopyrimidine compounds prepared in Example 26 aims to assess biosafety, a crucial step in preclinical research. Hemolytic toxicity testing is a key indicator for evaluating the compound's effect on red blood cell membrane stability and blood compatibility. This invention systematically evaluated the hemolytic toxicity of the morpholine-containing pyridopyrimidine compounds prepared in Example 26 at different concentrations using an in vitro hemolysis assay system, aiming to provide a safety basis for subsequent in vivo experiments and clinical applications. Generally, a hemolysis rate below 5% indicates good hemolytic safety.

[0318] Figure 2 The effect of pyridine-pyrimidine compounds containing morpholine structures prepared in Example 26 at different concentrations on the hemolysis rate of erythrocytes.

[0319] Figure 2 The results showed that the hemolysis rate of the morpholine-containing pyridopyrimidine compound prepared in Example 26 was 4.9% at a concentration of 256 μg / mL. Therefore, the hemolysis rate of the morpholine-containing pyridopyrimidine compound prepared in Example 26 was considered to be within a safe range.

[0320] KM mouse toxicity test

[0321] The KM mouse toxicity assay is a crucial step in drug safety evaluation, primarily used to preliminarily assess the biosafety of candidate compounds and provide key data for subsequent preclinical studies. KM mice were randomly divided into three groups of three: a control group (administered 0.9% saline), a positive control group (GDC-0941, 75 mg / kg), and the group receiving the drug as described in Example 26 (75 mg / kg). Mice were administered the drug via gavage for eight consecutive days (administered every other day, for a total of four times). After administration, blood samples were collected by enucleation and centrifugation to obtain serum. Subsequently, liver and kidney function were assessed using a preoperative ten-item quantitative assay and a biochemical analyzer.

[0322] In addition, mice were dissected, and histological analysis of their major organs (including heart, liver, spleen, lungs, and kidneys) was performed using hematoxylin and eosin (H&E staining). Serum samples from Kunming mice after drug administration were collected for biochemical analysis to further evaluate the in vivo biosafety of Example 26. Liver function was evaluated using total protein (TP), alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and glucose (GLU) levels; liver and myocardial function were evaluated using lactate dehydrogenase (LDH) and creatine kinase (CK) levels; and kidney function was evaluated using creatinine (CREA) and urea (UREA) levels.

[0323] Figure 3 The results show the analysis of total protein, urea, glucose, creatinine, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, and creatine kinase in the blood of KM mice in each group after drug administration.

[0324] Figure 3 The results showed that, compared with the blank group and the GDC-0941 positive group, the total protein (TP) level in the Example 26 (75 mg / kg) treatment group was similar to that in the control group, indicating that the two compounds had no significant effect on the total protein level. The urea (UREA) and glucose (GLU) levels in the W-1 and W-2 treatment groups were slightly lower than those in the control group, but the changes were not significant, indicating that the two compounds had little effect on urea and glucose levels. The creatinine (CREA), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels in the Example 26 treatment group were lower than those in the control group, but all remained within the normal range. This may indicate that the two compounds have a certain protective effect on liver function. The alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and creatine kinase (CK) levels in the Example 26 treatment group were significantly higher than those in the control group. Overall, the morpholine-containing pyridopyrimidine compounds prepared in Example 26 had little effect on most biochemical indicators, showing low toxicity.

[0325] Figure 4 The changes in body weight of KM mice during drug administration (a) and the organ index of KM mice after drug administration (b).

[0326] Figure 4 As shown in Figure (a), the weight of the control group gradually increased, showing a normal growth trend. The weight growth trend of the positive GDC-0941 group was similar to that of the control group. The weight growth trend of the group treated in Example 26 was also similar to that of the control group. This indicates that at a dose of 75 mg / kg, the pyridopyrimidine compound containing the morpholine structure prepared in Example 26 did not have a significant effect on the weight of mice. This is an important indicator for assessing drug safety. Figure 4 As shown in Figure (b), the indices of the heart, spleen, and lungs were not significantly different from those of the control group in all treatment groups, indicating that the morpholine-containing pyridopyrimidine compounds prepared in Example 26 did not cause significant hypertrophy or atrophy of these organs at the test dose. The liver index was higher than that of the heart, spleen, and lungs in all treatment groups, likely due to the important role of the liver in metabolism and detoxification. The liver index in the Example 26 administration group was not significantly different from that of the control group, indicating that these compounds did not cause significant pathological changes in the liver at the test dose. The kidney index was relatively low in all treatment groups, and there was no significant difference between the Example 26 administration group and the control group, indicating that these compounds did not cause significant pathological changes in the kidneys at the test dose. In summary, the morpholine-containing pyridopyrimidine compounds prepared in Example 26 did not cause significant adverse effects on the body weight and major organ indices of mice at a dose of 75 mg / kg. This indicates that the morpholine-containing pyridopyrimidine compounds prepared in Example 26 have good safety at the test dose.

[0327] Figure 5 The images show the H&E staining of the five internal organs of KM mice in each group after drug administration.

[0328] Figure 5 The results showed that the morpholine-containing pyridopyrimidine compounds prepared in Example 26 had minimal effects on mouse organs. At high doses, the liver and kidneys exhibited very mild cellular degeneration and edema, but none reached the level of severe damage; the heart and lungs showed no obvious damage or lesions, and the overall organ tissue structure remained intact. These results indicate that the morpholine-containing pyridopyrimidine compounds prepared in Example 26 did not cause serious organ damage at high doses, demonstrating good safety and providing strong support for further preclinical studies.

[0329] Dose-time dependence test

[0330] In experimental design, both the duration and dosage of drug treatment require meticulous optimization. Specifically, this optimization process is primarily based on two aspects: time dependence and dose dependence. On the one hand, by treating cells with different concentrations of drug within the same time period, the effect of drug concentration on cellular response can be assessed. On the other hand, treating cells with the same concentration of drug for different durations can explore the differences in cellular effects caused by drug action time. Through this systematic experimental design, a more comprehensive understanding of the interaction between drugs and cells can be achieved, providing reliable experimental evidence for subsequent research.

[0331] To evaluate the time-dependent inhibition of MCF-7 cell proliferation by the morpholine-containing pyridopyrimidine compound prepared in Example 26, cell viability at different treatment times (24h, 48h, and 72h) was detected using the MTT assay. After drug treatment, cells in each group were incubated with MTT for 4h, and the OD value was measured after dissolving formazan crystals. The cell proliferation inhibition rate was calculated through three independent experiments.

[0332] Figure 6 Time-dependent effects of morpholine-containing pyridopyrimidine compounds prepared in Example 26 at different concentrations on MCF-7.

[0333] Figure 6 The results show that the antiproliferative effect of the morpholine-containing pyridopyrimidine compounds prepared in Example 26 against MCF-7 cells significantly increased with increasing concentration and treatment time. Specifically, for the morpholine-containing pyridopyrimidine compounds prepared in Example 26, the inhibition rate reached 56.63% at a concentration of 11.1 μg / mL and an action time of 48 hours. When the action time was extended to 72 hours, the inhibition rate further increased to 61.5%. Notably, when the concentration was increased to 100 μg / mL and the action time was 72 hours, the inhibition rate of W-1 cells significantly increased, reaching a maximum inhibition rate of 89.87%. The results indicate that the morpholine-containing pyridopyrimidine compounds prepared in Example 26 exhibit concentration- and time-dependent antiproliferative activity, and their inhibitory effect is particularly significant at higher concentrations and longer action times.

[0334] AO staining experiment

[0335] Cell suspensions were obtained from cells in the logarithmic growth phase and cultured at 2.0 × 10⁻⁶ cells / mL. 4 Up to 4.0×10 4Cells were seeded at a density of 100 cells / mL into 24-well plates and then incubated for 24 hours. Compound treatment: After washing cells with PBS buffer, 1 mL of compound solution of different concentrations was added to each well. For the blank control group, 1 mL of fresh culture medium was added. The 24-well plates were then returned to the incubator for further incubation. After the designated treatment time, the old culture medium was removed, and the cells were washed twice with PBS buffer. Under light-protected conditions, AO staining solution diluted to a final concentration of μg / mL with PBS was added, followed by incubation in the dark for 15 minutes. Finally, under blue light excitation, cell morphology was observed and recorded using a fluorescence inverted microscope, and images were photographed and saved to assess the effect of the compound on cell proliferation.

[0336] Figure 7 The figure shows the effect of pyrrolopyrimidine compounds containing morpholine structures prepared in Experiment Example 26 on the morphology of cancer cells.

[0337] Figure 7 The results showed that the control group's MCF-7 cells were morphologically intact, with uniform green fluorescence in the nucleus and cytoplasm, and were tightly distributed among cells, showing no obvious apoptotic characteristics. After treatment with the morpholine-containing pyrrolopyrimidine compound (4 μmol / L) prepared in Experiment 26 for 24 h, some cells showed morphological changes, with slight shrinkage of the nucleus and cytoplasm, and a small number of cells exhibiting yellow fluorescence, indicating early apoptosis. As the concentration of the morpholine-containing pyrrolopyrimidine compound prepared in Experiment 26 increased to 8 μmol / L, more cells were observed to shrink in size, have blurred edges, and exhibit orange-red fluorescence, indicating an increase in late apoptosis. In addition, the gaps between cells increased, indicating that some cells had died. The changes in the Experiment 26 (4 μmol / L) group were similar to those in the GDC-0941 (4 μmol / L) group, showing a certain degree of apoptotic characteristics. Overall, the morpholine-containing pyrrolopyrimidine compound prepared in Experiment 26 was able to induce apoptosis in MCF-7 cells in a dose-dependent manner, and the effect was more pronounced at higher concentrations.

[0338] Annexin V-FITC / PI double staining method for detecting apoptotic cells

[0339] Cell suspensions were prepared from cells in the logarithmic growth phase at a concentration of 2.0 × 10⁻⁶. 5Cells were seeded at a density of 10 cells / 3 mL into 6-well plates and incubated for 24 hours. Culture medium containing a specific concentration of the compound was added to each well and incubated for the predetermined time. Cells were digested with EDTA-free trypsin, and after centrifugation to remove the supernatant, the cells were washed with PBS buffer. The centrifuge tubes were wrapped in aluminum foil to protect them from light. Binding buffer was added to the cell suspension and the cells were mixed by pipetting. 5.0 μL of Annexin V-FITC and 5.0 μL of PI were added to two blank control tubes, and a mixture of both was added to the remaining tubes. After mixing, the cells were incubated in the dark for 30 minutes. The cell suspension was filtered through a 200-mesh sieve, and cell counting was performed using flow cytometry, recording a result of 1.0 × 10⁶ cells / mL. 4 Data related to individual cells.

[0340] Figure 8 The effect of morpholine-containing pyrrolopyrimidine compounds prepared for flow cytometry analysis on cancer cell apoptosis is shown in Figure 26.

[0341] Figure 8 The results showed that the total apoptosis rate in the blank control group was only 3.1%. However, when cells were treated with the morpholine-containing pyrrolopyrimidine compound prepared in Example 26, the apoptosis rate increased significantly. At concentrations of 4 μM and 8 μM, the apoptosis rate induced by Example 26 increased from 8.12% to 12.98%, respectively. In particular, at the highest concentration, the late apoptosis rate reached 11.0%, indicating that the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 has a strong apoptosis-inducing ability at higher concentrations. These results demonstrate that the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 can significantly induce apoptosis in MCF-7 cells and mainly promotes the cells to enter the late apoptosis stage, exhibiting good antitumor activity.

[0342] Flow cytometry analysis was used to determine the effect of pyrrolopyrimidine compounds containing morpholine structures prepared in Example 26 on the cancer cell cycle.

[0343] The effect of the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 on the cell cycle of breast cancer MCF-7 cells was analyzed by flow cytometry. The specific procedure was as follows: Logarithmic growth phase cells were seeded into 6-well plates and cultured for 24 h as usual. Then, solutions of the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 at concentrations of 4 μM and 8 μM were added, and the cells were incubated for another 48 h. Cells were then collected and analyzed by flow cytometry.

[0344] Figure 9 The effect of morpholine-containing pyrrolopyrimidine compounds prepared for flow cytometry detection on the cell cycle of cancer cells is shown in Figure 42.

[0345] Figure 9The results showed that the proportion of S-phase cells in the blank control group was 20.96%, which increased to 35.94% in the 4μM treatment group and further increased to 40.92% at the 8μM concentration, exhibiting a significant dose-dependent arrest.

[0346] Cell cloning assay to detect anti-cell proliferation ability

[0347] The effect of Experiment 26 on the clonal proliferation capacity of MCF-7 cells was evaluated using a clonal assay. Cell suspensions were prepared from cells in the logarithmic growth phase and seeded into 6-well plates at a density of 5000 cells per well. The plates were then incubated for 24 hours until complete cell adhesion. Culture medium containing the specified concentration of the compound was added to each well, and the plates were incubated for another 48 hours. The culture medium was changed every other day for 7 days. After removing the culture medium, the cells were washed twice with PBS buffer, and then 2 mL of 4% paraformaldehyde solution was added to each well to fix the cells for 15 minutes. The paraformaldehyde solution was removed, and the cells were washed twice again with PBS buffer. Then, 1 mL of crystal violet staining solution was added to each well, and the plates were allowed to stand for 15 minutes. The crystal violet staining solution was removed, and each well was washed with distilled water. After air drying, photographs were taken for recording the data.

[0348] Figure 10 The figure shows the effect of pyrrolopyrimidine compounds containing morpholine structures prepared in Experiment Example 26 on cancer cell proliferation.

[0349] Figure 10 The results showed that, at a low concentration (4 μM), the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 significantly inhibited the colony formation of MCF-7 cells compared to the blank control group, indicating that even at low concentrations, the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 possessed certain antitumor activity. When the concentration was increased to 6 μM, the inhibitory effect of the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 on the colony formation of MCF-7 cells was further enhanced. With further increases in concentration, the inhibitory effect of the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 became more significant. When the concentration reached 8 μM, the colony formation of MCF-7 cells was inhibited by nearly 95%, almost completely blocking cell proliferation. However, compared to the positive control drug GDC-0941, the inhibitory effect of the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 at high concentrations was slightly inferior.

[0350] Cell scratch assay to determine cell migration and repair capabilities

[0351] The inhibitory effect of Experiment 26 on the migration ability of MCF-7 cells was evaluated using a scratch assay. First, approximately 5 × 10⁶ cells were scratched... 5Cells were seeded at a density of 100 cells / well into 6-well plates. Before seeding, parallel and equidistant lines were drawn on the center of the back of the 6-well plate for subsequent operations. Once the cells had grown to completely cover the wells, the wells were scored using a vertical pipette tip along the pre-drawn lines in one stroke. The plates were then washed twice with PBS buffer to remove cell debris detached during scoring, and the medium was replaced with fresh culture medium. After observing and recording the initial growth status of the cells under a microscope, the plates were returned to the incubator for further culture. After 24 hours of culture, the plates were removed, and the morphological changes of the cells were observed and photographed under a microscope.

[0352] Figure 11 The figure shows the effect of morpholine-containing pyrrolopyrimidine compounds prepared in Example 26 on the migration ability of MCF-7 cells, as detected by the cell scratch assay.

[0353] Figure 11 The results showed that, in the blank control group, MCF-7 cells exhibited a significant tendency to migrate towards the center after 24 hours of culture. Treatment of MCF-7 cells with a low concentration (4 μM) of the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 significantly inhibited cell migration. Compared to the blank control group, the degree of cell migration towards the center was significantly reduced in the low-concentration Example 26 treatment group, indicating that the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 could effectively inhibit the migration ability of MCF-7 cells even at low concentrations. In summary, the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 exhibited a concentration-dependent inhibitory effect on MCF-7 cell migration. With increasing concentration, the inhibitory effect of the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 on cell migration and repair gradually increased.

[0354] JC-1 method for detecting mitochondrial membrane potential in cells

[0355] The homeostasis of mitochondrial membrane potential (MMP) is fundamental to maintaining cellular energy metabolism and homeostasis. The JC-1 fluorescent probe can effectively reflect this change. When the mitochondrial membrane potential is normal, the cell exhibits red fluorescence, while when the potential decreases, the fluorescence turns green. Therefore, it can be used to visually determine whether cells are damaged or undergoing apoptosis.

[0356] Preparation of JC-1 staining working solution: Take 2 μL of JC-1 solution (500×), add 900 μL of JC-1 diluent, mix well, then add 900 μL of JC-1 staining buffer (10×), and finally vortex to mix. Pour the cell suspension into each well at a concentration of 2.0 × 10⁻⁶ cells / well. 5Cells were seeded at a density of 100 cells / 3 mL into 6-well plates and incubated for 24 hours. After incubation, the old culture medium was removed from the wells, and the cells were washed twice with 1 mL of JC-1 staining buffer, followed by the addition of 1 mL of complete culture medium. 1 mL of JC-1 staining working solution was added to each well, and the mixture was gently shaken to ensure homogeneity. The cells were then incubated in the dark for 15 minutes. After incubation, the supernatant was removed, and the cells were washed twice again with 1 mL of JC-1 staining buffer. Finally, 2 mL of JC-1 staining buffer was added to each well, and cell morphology was observed and photographed under a fluorescence microscope.

[0357] Figure 12 The graph shows the effect of pyrrolopyrimidine compounds containing morpholine structures prepared in Example 26 on the mitochondrial membrane potential of cancer cells.

[0358] Figure 12 The results showed that the control group cells remained healthy with stable mitochondrial membrane potential, thus exhibiting predominantly red fluorescence. As drug concentration increased, the intracellular red fluorescence gradually weakened, while the green fluorescence intensity increased synchronously, indicating a concentration-dependent decrease in MMP. This phenomenon suggests that both compounds can induce apoptosis pathway activation by disrupting mitochondrial membrane integrity. This transition from red to green fluorescence is direct evidence of a decrease in cell membrane potential, indicating that these compounds can induce apoptosis in MCF-7 cells. When the concentration of the morpholine-containing pyrrolopyrimidine compounds prepared in Example 26 increased, they were able to induce a concentration-dependent shift in JC-1 fluorescence from red to green in MCF-7 cells. This shift not only indicates a decrease in mitochondrial membrane potential but also further confirms their ability to induce apoptosis.

[0359] DCFH-DA probe detects reactive oxygen species produced by MCF-7 cells

[0360] Reactive oxygen species (ROS) play a crucial role in the antitumor mechanism of chemotherapeutic drugs. Elevated ROS levels can trigger apoptosis signaling pathways and inhibit tumor proliferation by arresting the cell cycle. This invention uses the DCFH-DA fluorescent probe to detect changes in ROS production in MCF-7 cells treated with pyrrolopyrimidine compounds containing morpholine structures prepared in Example 26, in order to elucidate their antitumor mechanism.

[0361] Cell suspensions were prepared from cells in the logarithmic growth phase at a density of 2.0 × 10⁶ cells per well. 5Cells were seeded at a density of 3 cells / mL in 6-well plates and incubated for 24 hours. After cell attachment, culture medium containing a specific concentration of the compound was added to each well, and the cells were incubated for a further period. The old culture medium was removed, and the cells were washed twice with PBS buffer, followed by staining with Hoechst 33342 staining solution for 10 minutes. After washing the cells twice with PBS buffer, 1 mL of culture medium containing DCFH-DA was added to each well. After removing the paraformaldehyde solution, the cells were washed twice with PBS buffer, followed by adding 1 mL of crystal violet staining solution to each well and incubating for 15 minutes. Finally, the cells were washed twice with PBS buffer, and cell morphology was observed and photographed under a fluorescence microscope.

[0362] Figure 13 The effect of morpholine-containing pyrrolopyrimidine compounds prepared in Example 26 on reactive oxygen species content in MCF-7 cells, as detected by the DCFH-DA probe.

[0363] Figure 13 The results showed that almost no green fluorescence signal was detected in MCF-7 cells in the blank control group. When MCF-7 cells were treated with a low concentration (4 μM) of the morpholine-containing pyrrolopyrimidine compound prepared in Example 26, a small amount of green fluorescence signal began to appear in the cells. This indicates that the low concentration of the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 can induce the cells to produce a small amount of reactive oxygen species (ROS). Although the fluorescence signal was weak, this may be an early sign that the drug is beginning to exert its effect. As the treatment concentration of the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 increased, the green fluorescence signal in the cells significantly increased. In the cells treated with high concentrations, the intensity and distribution range of the fluorescence signal were significantly expanded, indicating a significant increase in the level of intracellular ROS. This concentration-dependent fluorescence enhancement phenomenon indicates that the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 can induce MCF-7 cells to produce more ROS in a dose-dependent manner. It is noteworthy that the fluorescence intensity in the high-concentration Example 26 treatment group was comparable to that in the positive control drug GDC-0941 treatment group.

[0364] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A pyridopyrimidine compound containing a morpholine structure, characterized in that, The structural formula is shown in Formula I: ； In Equation I, when R1 is When R2 is selected , , , , , , , , , , , , , , , , , , , , or ; When R1 is When R2 is selected , , , , , , , , , , , , , , , , or .

2. A method for preparing a pyridopyrimidine compound containing a morpholine structure as described in claim 1, characterized in that, When R1 is The preparation route for the compound represented by Formula I is as follows: ； In the above routes, compound 2 was prepared by the Miyaura borylation reaction; compound 4 was prepared by the nucleophilic substitution reaction; compound 5 was prepared by the de-Boc reaction; compound 6-a was prepared by the aromatic nucleophilic substitution reaction; compound 6-a and compound 2 were combined by the Suzuki coupling reaction to prepare compound 7-a; and compound 8-a was prepared by the amidation reaction. When R1 is The preparation route for the compound represented by Formula I is as follows: ； In the above routes, compound 2 was prepared by the Miyaura borylation reaction; compound 4 was prepared by the nucleophilic substitution reaction; compound 5 was prepared by the de-Boc reaction; compound 6-b was prepared by the aromatic nucleophilic substitution reaction; compound 6-b and compound 2 were combined by the Suzuki coupling reaction to prepare compound 7-b; and compound 8-b was prepared by the amidation reaction.

3. A pharmaceutically acceptable salt of a pyridopyrimidine compound containing a morpholine structure as described in claim 1.

4. A pharmaceutical preparation, characterized in that, The active ingredient is a pharmaceutically acceptable salt of a pyridopyrimidine compound containing a morpholine structure as described in claim 1 or a pyridopyrimidine compound containing a morpholine structure as described in claim 3.

5. The use of the pyridopyrimidine compound containing a morpholine structure as described in claim 1 in the preparation of a medicament for treating and / or preventing cancer, characterized in that, The cancer in question is breast cancer.

6. The use of a pharmaceutically acceptable salt of a pyridopyrimidine compound containing a morpholine structure as described in claim 3 in the preparation of a medicament for treating and / or preventing cancer, characterized in that... The cancer in question is breast cancer.

7. The use of the pharmaceutical preparation of claim 4 in the preparation of a medicament for treating and / or preventing cancer, characterized in that, The cancer in question is breast cancer.

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