Pyridopyrimidine compound containing morpholine structure as well as preparation method and application of pyridopyrimidine compound

Abstract

The invention discloses a pyridopyrimidine compound containing a morpholine structure as well as a preparation method and application of the pyridopyrimidine compound, and belongs to the technical field of medicines. The pyridopyrimidine compound containing the morpholine structure, provided by the invention, has relatively strong capability of inhibiting PI3K alpha kinase, so that the pyridopyrimidine compound can be used as an active component for preparing a medicine for treating diseases caused by abnormal activation of the PI3K alpha kinase; in addition, potential application value is shown in preparation of drugs for treating and / or preventing proliferative diseases and cancers. The invention provides important technical support for developing high-efficiency anti-cancer drugs with better selectivity.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a pyridopyrimidine compound containing a morpholine structure, a preparation method and an application thereof. Background Art

[0002] In terms of cancer types, lung cancer, female breast cancer, and colorectal cancer are the top three cancers with the highest number of newly diagnosed cases worldwide. Data reveals the serious threat cancer poses to human health, particularly 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 worldwide, accounting for a quarter of newly diagnosed cancer cases and one-sixth of cancer deaths in 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. The development of new drugs for breast cancer using molecularly targeted therapy strategies has become a current priority. Currently, personalized targeted therapies in clinical use primarily target aberrant activation of the PI3K signaling pathway for the treatment of breast cancer. Through the identification of clear molecular targets, target detection technologies, and marketed targeted drugs, clinical efficacy has been significantly improved. Phosphatidylinositol 3-kinases (Phosphatidylinositol 3-kinases) are a family of enzymes with serine / threonine (Ser / Thr) kinase activity that are widely involved in regulating key biological processes such as cell growth, differentiation, proliferation, and endocytosis. Based on their sequence homology, structural features, and substrate specificity, PI3Ks are classified into three major categories: type I, type II, and type III. Type I PI3Ks are the most intensively studied. Type I PI3Ks can be further divided into class IA and class IB: class IA includes PI3Kα, PI3Kβ, and PI3Kδ, while class IB contains only PI3Kγ. Class IA PI3Ks are heterodimers composed of the catalytic subunit p110α, p110β, or p110δ and the regulatory subunit p85, while class IB PI3Ks consist of the catalytic subunit p110γ and the regulatory subunits 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, they trigger autophosphorylation of tyrosine residues in the receptor's intracellular domain, thereby activating PI3K. In contrast, activation of PI3Kγ primarily relies on the interaction between GN-β (guanine nucleotide-binding protein-β) and GN-γ (guanine nucleotide-binding protein-γ) within G-protein-coupled receptors (GPCRs). PI3K activation can also be achieved through Ras proteins, which possess GTPase activity. Insulin can also indirectly activate PI3K through insulin receptor substrate 1 (IRS1). Furthermore, cytokines can mediate PI3K activation through Janus kinase 1 (Janus kinase 1). These diverse activation mechanisms make PI3K a crucial role in cellular signaling networks.

[0004] Research has shown that the PI3K-AKT-mTOR signaling pathway is a critical intracellular signaling network that plays a central role in multiple aspects of human life. Aberrant activation of this pathway is closely associated with the development, progression, 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 progression. Tumors may exploit the PI3K-AKT-mTOR pathway to suppress immune system attacks and achieve immune evasion. Sustained activation of the PI3K-AKT-mTOR pathway may contribute to tumor resistance to chemotherapy and targeted therapies. Furthermore, it interacts with other signaling pathways, such as MAPK and JAK, which can further promote tumor progression. Aberrant activation of the PI3K-AKT-mTOR pathway has been linked to multiple tumor types, including breast, colorectal, lung, prostate, and ovarian cancers. Due to its critical role in tumor progression, the PI3K-AKT-mTOR pathway has become an important target for cancer treatment. Currently available drugs have various limitations, and further improvements in efficacy are needed. Summary of the Invention

[0005] To solve the above problems, the present invention provides a pyridopyrimidine compound containing a morpholine structure, a preparation method and application thereof.

[0006] To achieve the above object, the present invention provides the following technical solutions:

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

[0008]

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

[0010] When R1 is When R2 is selected from

[0011] The names of the pyridopyrimidine compounds containing a morpholine structure in formula I of the present 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)hexanamide;

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

[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)heptylamide;

[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)cyclopropanecarboxamide;

[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-phenylpropanamide;

[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)propanamide;

[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)hexanamide;

[0040]

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

[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-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)cyclopropanecarboxamide;

[0045]

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

[0046]

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

[0047]

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

[0048]

[37] N-(2-cyano-4-(4-morpholino-7-(pyrazin-2-yl)-5,6,7,8-tetrahydropyrido[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-tetrahydropyrido[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-phenylpropanamide;

[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-tetrahydropyrido[3,4-d]pyrimidin-2-yl)phenyl)-3-methoxybenzamide.

[0055] The second technical solution of the present invention is to provide a method for preparing the above-mentioned pyridopyrimidine compound containing a morpholine structure, when R1 is When, the preparation route of the compound shown in Formula I is as follows:

[0056]

[0057] In the above route, compound 2 is prepared by Miyaura borylation reaction; compound 4 is prepared by nucleophilic substitution reaction; compound 5 is prepared by de-Boc reaction; compound 6-a is prepared by aromatic nucleophilic substitution reaction; compound 6-a and compound 2 are reacted by Suzuki coupling reaction to prepare compound 7-a; compound 8-a is prepared by amidation reaction;

[0058] When R1 is When, the preparation route of the compound shown in Formula I is as follows:

[0059]

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

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

[0062] The fourth technical solution of the present invention: a pharmaceutical preparation, the active ingredient of which is the above-mentioned pyridopyrimidine compound containing a morpholine structure or a pharmaceutically acceptable salt of the above-mentioned pyridopyrimidine compound containing a morpholine structure.

[0063] Optionally, the dosage form of the pharmaceutical preparation includes but is not limited to injection, tablet, capsule, aerosol, suppository, film, pellet, external application agent, ointment or enteric-coated tablet.

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

[0065] Technical solution six of the present invention: Use of the above-mentioned pyridopyrimidine compound containing a morpholine structure in the preparation of a drug for treating and / or preventing proliferative diseases.

[0066] Technical solution seven of the present invention: Use of a pharmaceutically acceptable salt of the above-mentioned pyridopyrimidine compound containing a morpholine structure in the preparation of a drug for treating and / or preventing cancer.

[0067] Technical solution eight of the present invention: Use of a pharmaceutically acceptable salt of the above-mentioned pyridopyrimidine compound containing a morpholine structure in the preparation of a drug for treating and / or preventing proliferative diseases.

[0068] Technical solution No. 9 of the present invention: Use of the above-mentioned pharmaceutical preparation in the preparation of drugs for treating and / or preventing cancer.

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

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

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

[0072] The morpholine-containing pyridopyrimidine compounds provided by this invention have a strong ability to inhibit PI3Kα kinase. Therefore, they can be used as active ingredients in the preparation of therapeutic drugs for diseases caused by abnormal activation of PI3Kα kinase. They also 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 selective anticancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 This is a diagram showing the molecular docking results of the pyridopyrimidine compound containing a morpholine structure prepared in Example 26 and PI3Kα.

[0074] Figure 2 The figure shows the effects of different concentrations of the morpholine-containing pyridopyrimidine compounds prepared in Example 26 on the hemolysis rate of red blood cells.

[0075] Figure 3 The analysis results 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 administration.

[0076] Figure 4 Figure 2 shows the body weight changes of KM mice during drug administration (a) and the organ indexes of KM mice after drug administration (b).

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

[0078] Figure 6 The time dependence of the action of the morpholine-containing pyridopyrimidine compounds prepared in Example 26 on MCF-7 at different concentrations.

[0079] Figure 7 This is a diagram showing the effect of the morpholine-containing pyrrolopyrimidine compounds prepared in Experimental Example 26 on the morphology of cancer cells.

[0080] Figure 8 This is a diagram showing the effect of the morpholine-containing pyrrolopyrimidine compounds prepared in Experimental Example 26 on the apoptosis of cancer cells detected by flow cytometry.

[0081] Figure 9 This is a diagram showing the effect of the morpholine-containing pyrrolopyrimidine compounds prepared in Experimental Example 26 on the cell cycle of cancer cells as detected by flow cytometry.

[0082] Figure 10 This is a graph showing the effect of the morpholine-containing pyrrolopyrimidine compounds prepared in Experimental Example 26 on cancer cell proliferation.

[0083] Figure 11 This figure shows the effect of the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 on the migration ability of MCF-7 cells detected by cell scratch test.

[0084] Figure 12 This is a diagram showing the effect of the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 on the mitochondrial membrane potential of cancer cells.

[0085] Figure 13This is a graph showing the effect of the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 on the reactive oxygen species content in MCF-7 cells as detected by the DCFH-DA probe. DETAILED DESCRIPTION

[0086] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention.

[0087] It should be pointed out that the matters not described in detail in the present invention are conventional operating means in this field and are not the focus of the present invention.

[0088] In addition, for numerical ranges in the present invention, it is understood that each intervening value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any stated value or stated range, and any other stated value or intervening value in the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

[0089] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

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

[0091] The hydrogen and carbon nuclear magnetic resonance spectra of the compounds in the examples of the present invention were measured using a Bruker ARX-400, and the mass spectra were measured using an Agilent 1100 LC / MSD; all reagents used were of analytical or chemical purity.

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

[0093] Route 1:

[0094]

[0095] The synthetic routes of the pyridopyrimidine compounds containing morpholine structures in Examples 26 to 43 of the present 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)hexanamide:

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

[0101]

[0102] Compound 1 (3.0 g, 15.23 mmol), bis(pinacolato)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 mixed solution was stirred at 80 ° C under nitrogen protection for 16 hours. After the reaction was completed, the solvent was removed by concentration and purified by silica gel column chromatography using an elution solvent of petroleum ether to ethyl acetate in a 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-morpholinyl-5,8-dihydropyrido[3,4-d]pyrimidine-7(6H)-carboxylate (Compound 4):

[0104]

[0105] Dissolve compound 3, morpholine, and N,N-diisopropylethylamine in N,N-dimethylformamide at a molar ratio of 1:2:2. Allow the solution to react at 60°C for 4 hours. Once the reaction is complete, add ice water and shake. Once a large amount of yellow precipitate appears, filter it. Place the filter cake in an oven at 50°C overnight. Wash it with dichloromethane. Dry the washings at 40°C and spin-dry it. Finally, recrystallize it using a solvent with a volume ratio of petroleum ether to ethyl acetate of 13:1. A light yellow precipitate forms. Filter it and dry it to obtain compound 4.

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

[0107]

[0108] Under ice bath conditions, compound 4 (8 g, equivalent to 22.6 mmol) was added to a pre-cooled dichloromethane (30 mL) solution containing HCl / 1,4-dioxane (60 mL). This pre-cooling step is intended to control the reaction rate and prevent excessive exotherm. Subsequently, the reaction mixture was stirred continuously overnight at room temperature to ensure sufficient reaction. After the reaction was completed, thin layer chromatography (TLC) analysis confirmed that the starting material had been completely consumed. Next, the reaction mixture was concentrated to remove the solvent, and the resulting solid was dissolved in a mixed solvent of methanol and dichloromethane (ratio of 10:1, total volume 200 mL). Then, an appropriate amount of sodium bicarbonate (NaHCO3) was added to the solution, and stirring was continued for 3 hours at constant temperature to promote the completion of the reaction and the removal of by-products. After the reaction was completed, the insoluble matter was removed by filtration, and the filtrate was concentrated to obtain 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. Under nitrogen protection, the reaction mixture was ventilated for 1 minute to remove the air in the container, ensure an oxygen-free environment, and avoid possible side reactions. Subsequently, this replacement step was repeated in another 120 mL of 1,4-dioxane to further ensure that the reaction environment was oxygen-free. Next, the reaction mixture was heated at 100 degrees Celsius for 18 hours to promote the reaction. After the reaction was completed, the mixture was concentrated to remove the solvent to obtain a solid residue. Subsequently, silica gel column chromatography was used for purification to separate the target product (compound 6) and by-products. The eluent used a mixed solvent system of dichloromethane / methanol with a volume ratio of 60:1 to optimize the elution efficiency of the target product.

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

[0113]

[0114] Weigh 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). These compounds were added to 15 ml of acetonitrile / water mixed solvent. Then, the reaction mixture was replaced for 1 minute under a nitrogen environment to exclude the air in the container and ensure that the reaction was carried out under anaerobic conditions. This step is crucial for preventing oxidative side reactions and improving reaction selectivity. Then, the reaction mixture was heated at 80 degrees Celsius for 16 hours to promote the complete reaction. After the reaction was completed, the mixture was concentrated by vacuum distillation or rotary evaporator to remove the solvent to obtain a residual solid product. Subsequently, silica gel column chromatography was used for purification, using an eluent system of n-hexane to dichloromethane / methanol with a volume ratio of 60:1 to optimize the elution efficiency and purity of the target product. Ultimately, compound 7 was successfully isolated.

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

[0116]

[0117] Take compound 7 (70 mg, 0.17 mmol) and dissolve it in 1 mL of pyridine solvent to form a uniform solution. The solution was placed in an ice bath and cooled for 2 minutes to reduce 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 requires careful operation to ensure that the acyl chloride can be evenly dispersed and fully react with compound 7. After the reaction is completed, the mixture is concentrated to remove excess solvent. Next, a mixed solvent system of dichloromethane / methanol is used for pre-chromatographic elution purification with a volume ratio set to 20:1. This step is intended to utilize the difference in solubility of the compound in different solvents to separate the target product and other by-products by a chromatographic column.

[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)propanamide:

[0121] According to the method of Example 1, in step 6, hexanoyl chloride is replaced by 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] Follow the method of Example 1, except that in step 6, 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)heptylamide:

[0129] Follow the method of Example 1, except that in step 6, hexanoyl chloride is replaced with an equimolar amount of heptanoyl 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] Follow the method of Example 1, except that in step 6, 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] Follow the method of Example 1, except that in step 6, hexanoyl chloride is replaced with an equimolar amount of furan-2-carbonyl 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] Follow the method of Example 1, except that in step 6, hexanoyl chloride is replaced with an equimolar amount of thiophene-2-carbonyl 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] Follow the method of Example 1, except that in step 6, hexanoyl chloride is replaced with an equimolar amount of tetrahydro-2H-pyran-4-carbonyl 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)cyclopropanecarboxamide:

[0149] Follow the method of Example 1, except that in step 6, hexanoyl chloride is replaced by an equimolar amount of cyclopropanecarbonyl 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)cyclobutanecarboxamide:

[0153] Follow the method of Example 1, except that in step 6, hexanoyl chloride is replaced by an equimolar amount of cyclobutanecarbonyl 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)cyclopentanecarboxamide:

[0157] According to the method of Example 1, in step 6, hexanoyl chloride is replaced by an equimolar amount of cyclopentanecarbonyl 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)cyclohexanecarboxamide:

[0161] Follow the method of Example 1, except that in step 6, hexanoyl chloride is replaced by an equal molar amount of cyclohexanecarbonyl 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] Follow the method of Example 1, except that in step 6, 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] Follow the method of Example 1, except that in step 6, hexanoyl chloride is replaced with an equal molar 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] Follow the method of Example 1, except that in step 6, 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] According to the method of Example 1, in step 6, the hexanoyl chloride is replaced by 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] According to the method of Example 1, in step 6, the hexanoyl chloride is replaced by 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-phenylpropanamide:

[0185] According to the method of Example 1, in step 6, hexanoyl chloride is replaced by 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] Follow the method of Example 1, except that in step 6, 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] Follow the method of Example 1, except that in step 6, hexanoyl chloride is replaced by 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] Follow the method of Example 1, except that in step 6, 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] Follow the method of Example 1, except that in step 6, 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] Follow the method of Example 1, except that in step 6, 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] Follow the method of Example 1, except that in step 6, 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] Follow the method of Example 1, except that in step 6, 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)propanamide:

[0217] According to the method of Example 1, the 3-iodopyridine in step 4 is replaced by an equal molar amount of 2-bromopyrazine, and the hexanoyl chloride in step 6 is replaced by an equal molar 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] Follow the method of Example 26, except that the isopropionyl chloride in step 6 is replaced by 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)hexanamide:

[0225] Follow the method of Example 26, except that the isopropionyl chloride in step 6 is replaced by 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)heptylamide:

[0229] Follow the method of Example 26, except that the isopropionyl chloride in step 6 is replaced by an equimolar amount of heptanoyl 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] Follow the method of Example 26, except that the isopropionyl chloride in step 6 is replaced by an equal molar 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] Follow the method of Example 26, except that the isopropionyl chloride in step 6 is replaced by an equimolar amount of furan-2-carbonyl 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] Follow the method of Example 26, except that the isopropionyl chloride in step 6 is replaced by an equimolar amount of thiophene-2-carbonyl 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)cyclopropanecarboxamide:

[0245] Follow the method of Example 26, except that the isopropanoyl chloride in step 6 is replaced by an equal molar amount of cyclopropanecarbonyl 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)cyclobutanecarboxamide:

[0249] Follow the method of Example 26, except that the isopropionyl chloride in step 6 is replaced by an equal molar amount of cyclobutanecarbonyl 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)cyclopentanecarboxamide:

[0253] Follow the method of Example 26, except that the isopropionyl chloride in step 6 is replaced by an equal molar amount of cyclopentanecarbonyl 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)cyclohexanecarboxamide:

[0257] Follow the method of Example 26, except that the isopropionyl chloride in step 6 is replaced by an equal molar amount of cyclohexanecarbonyl 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] Follow the method of Example 26, except that the isopropionyl chloride in step 6 is replaced by 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] Follow the method of Example 26, except that the isopropionyl chloride in step 6 is replaced by 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] According to the method of Example 26, the isopropionyl chloride in step 6 is replaced by 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-phenylpropanamide:

[0273] Follow the method of Example 26, except that the isopropionyl chloride in step 6 is replaced by 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] Follow the method of Example 26, except that the isopropionyl chloride in step 6 is replaced by 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] Follow the method of Example 26, except that the isopropionyl chloride in step 6 is replaced by 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] According to the method of Example 26, the isopropionyl chloride in step 6 is replaced by 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 morpholine-containing pyridopyrimidine compounds prepared in Examples 1 to 43 are shown in Table 1.

[0288] Table 1 Structural formula of pyridopyrimidine compounds containing morpholine structure prepared in Examples 1 to 43

[0289]

[0290]

[0291]

[0292]

[0293] The in vitro anti-tumor cell activity of the pyridopyrimidine compounds containing morpholine structure prepared in Examples 1 to 43 was determined.

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

[0295] (1) After the cells have recovered and been passaged 2-3 times to stabilize, use trypsin solution (0.25%) to digest them from the bottom of the culture flask. After the cell digestion solution is aspirated into a centrifuge tube, the culture medium is added to terminate the digestion. The centrifuge tube is centrifuged at 1000 r / min for 3 minutes, the supernatant is discarded, and 3 mL of culture medium is added. The cells are pipetted to mix, and 10 μL of the cell suspension is aspirated and added to a cell counting plate for counting. The cell concentration is adjusted to 10 4 180 μL of cell suspension was added to all wells of the 96-well plate, except for the top, bottom, and leftmost wells, which were blank wells. The 96-well plate was placed in an incubator and incubated for 24 h.

[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. Then dilute the sample in an EP tube to 1, 0.333, 0.111, 0.037, and 0.012 μg / mL.

[0297] Each concentration was added to 3 wells, 20 μL per well. The cells in the two rows and two columns surrounding the plate were significantly affected by the environment and served as blank wells. The 96-well plate was placed in an incubator and incubated for 72 h.

[0298] (3) Discard the drug-containing culture medium in the 96-well plate, rinse the cells twice with phosphate buffered saline (PBS), add 100 μL of MTT (tetrazolium) (0.5 mg / mL) to each well and place in an incubator for 4 hours. Discard the MTT solution and add 100 μL of dimethyl sulfoxide. Oscillate on a magnetic oscillator to fully dissolve the surviving cells and the MTT reaction product, formazan, and place in a microplate reader to measure the results at a wavelength of 492 nM. The drug IC can be calculated using the Bliss method. 50 value.

[0299] The anti-proliferative activities of the morpholine-containing pyridopyrimidine compounds prepared in Examples 1 to 43, namely the reference substance GDC-0941, against breast cancer cells MCF-7 are shown in Table 2, where ND represents compound IC 50 >50μM.

[0300] Table 2 Anti-MCF-7 cell proliferation activity of pyridopyrimidine compounds containing morpholine structure

[0301]

[0302]

[0303] The inhibitory activity of the pyridopyrimidine compounds containing morpholine structures prepared in Examples 1 to 43 on 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 containing test compounds, blank controls, and positive drug controls. 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 drug control wells. Add 2.5 μL of various compound concentrations to the test compound wells. Add 10 μL of PI3Kα kinase antibody and EDTA reagent to each well, mix thoroughly by centrifugation, and equilibrate at room temperature for 60 minutes. The final antibody concentration is 2 nM, and the final EDTA concentration is 8 mM. Fluorescence values ​​are read using Envision. Calculate the percent inhibition of the compound.

[0306] Percent 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 effect of the compound on PI3Kα kinase activity; Min is the average fluorescence signal value of the blank control wells, that is, the background signal in the absence of kinase (or substrate), representing 0% enzyme activity; Max is the average fluorescence signal value of the positive control wells, that is, the maximum signal generated under the conditions of kinase and no inhibitor, representing 100% enzyme activity.

[0308] The inhibitory activity of each compound on 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 morpholine-containing pyridopyrimidine compounds against PI3Kα kinase

[0310]

[0311]

[0312] From the test results in Table 2 and Table 3, it can be seen that the pyridopyrimidine compounds containing morpholine structures to be protected by the present invention have good in vitro anti-cell proliferation activity and PI3Kα kinase inhibition rate. Among them, the pyridopyrimidine compounds containing morpholine structures prepared in Examples 26 and 27 showed excellent inhibition rates on PI3Kα kinase. By further performing kinase IC on the compounds with higher inhibition rates, 50 The results of the assay showed that the morpholine-containing pyridopyrimidine compound prepared in Example 26 exhibited excellent cytotoxic activity against all selected cell lines, comparable to that of the positive drug. The morpholine-containing pyridopyrimidine compound prepared in the present invention is expected to become a promising inhibitor of PI3Kα kinase.

[0313] Structure-activity relationship analysis of the pyridopyrimidine compounds containing morpholine structure prepared in Example 26

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

[0315] Figure 1It is shown that the parent nucleus tetrahydropyrido [3,4-d] pyrimidine of the pyridopyrimidine compound containing a morpholine structure prepared in Example 26 forms important hydrogen bonds with amino acids ASP-933 and SER-774. These hydrogen bonds are crucial for stabilizing the binding between the compound and the protein, and help to enhance the binding affinity and selectivity of the compound. At the same time, the figure also shows that the cyano group of compound W-1 forms an important hydrogen bond with amino acid TYR-836. The formation of this hydrogen bond also enhances the binding ability of the compound to the protein, which may have a positive effect on improving the biological activity of the compound. And the distances marked in the figure (such as and ) shows that there is close contact between the pyrido-pyrimidine compound containing a morpholine structure prepared in Example 26 and the protein, and this close contact further enhances the binding affinity between the pyrido-pyrimidine compound containing a morpholine structure prepared in Example 26 and the protein.

[0316] Hemolytic toxicity evaluation

[0317] Hemolysis refers to the rupture of the cell membrane when red blood cells are stimulated by an inappropriate environment, releasing hemoglobin and other cellular components inside the cell. The hemolytic toxicity of the pyridopyrimidine compound containing a morpholine structure prepared in Example 26 was evaluated for its biosafety, which is an important part of preclinical research. The hemolytic toxicity experiment is a key indicator for evaluating the compound's effect on red blood cell membrane stability and blood compatibility. The present invention systematically evaluated the hemolytic toxicity of the pyridopyrimidine compound containing a morpholine structure prepared in Example 26 at different concentrations through an in vitro hemolysis test, in order to provide a safety basis for subsequent in vivo experiments and clinical applications. Generally, when the hemolysis rate of a compound is less than 5%, it can be considered that the compound has good hemolytic safety.

[0318] Figure 2 The figure shows the effects of different concentrations of the morpholine-containing pyridopyrimidine compounds prepared in Example 26 on the hemolysis rate of red blood cells.

[0319] Figure 2 It was shown that the hemolysis rate of the pyridopyrimidine compound containing a morpholine structure prepared in Example 26 was 4.9% at a concentration of 256 μg / mL. Therefore, it was considered that the hemolysis rate of the pyridopyrimidine compound containing a morpholine structure prepared in Example 26 was within a safe range.

[0320] KM mouse toxicity test

[0321] The KM mouse toxicity experiment is an important part of drug safety evaluation. It is mainly used to preliminarily evaluate the biosafety of candidate compounds and provide key data for subsequent preclinical studies. KM mice were randomly divided into 3 groups per group, which were set as a blank group (given 0.9% normal saline), a positive dosing group (GDC-0941, 75 mg / kg), and an Example 26 dosing group (75 mg / kg). The mice were administered the drug by gavage for 8 consecutive days (dosing every other day, a total of 4 times). After the administration, blood samples were collected by removing the mouse eyeballs and centrifuged to obtain serum. Subsequently, the liver and kidney functions of the mice were evaluated using a preoperative ten-item quantitative detection reagent disk and a biochemical analyzer.

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

[0323] Figure 3 The analysis results 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 administration.

[0324] Figure 3 The results show that compared with the blank group and the GDC-0941 positive group, the total protein (TP) level of the Example 26 (75 mg / kg) administration group was similar to that of the control group, indicating that the two compounds had no significant effect on the total protein level. The urea (UREA) and glucose (GLU) levels of the W-1 and W-2 treatment groups were slightly decreased compared with the control group, but the changes were not significant, indicating that the two compounds had little effect on the urea and glucose levels. The creatinine (CREA), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels of the Example 26 administration group were decreased compared with the control group, but were all 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) of the Example 26 administration group were significantly increased compared with the control group. In general, the pyridopyrimidine compounds containing morpholine structure prepared in Example 26 had little effect on most biochemical indicators and showed lower toxicity.

[0325] Figure 4 Figure 2 shows the body weight changes of KM mice during drug administration (a) and the organ indexes of KM mice after drug administration (b).

[0326] Figure 4 As shown in (a), the weight of the control group gradually increased, indicating 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 administered with Example 26 was also similar to that of the control group, indicating that at a dose of 75 mg / kg, the pyridopyrimidine compound containing a morpholine structure prepared in Example 26 had no significant effect on the weight of mice, which is an important indicator for evaluating drug safety. Figure 4 (b) shows that in all treatment groups, the indexes of organs such as the heart, spleen, and lungs were not significantly different from those in the control group, indicating that the pyridopyrimidine compounds containing morpholine structures 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, which may be due to the important role of the liver in metabolism and detoxification. The liver index of 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 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 kidney at the test dose. In short, the pyridopyrimidine compounds containing morpholine structures prepared in Example 26 did not cause significant adverse effects on the body weight and major organ indexes of mice at a dose of 75 mg / kg. This shows that the pyridopyrimidine compounds containing morpholine structures prepared in Example 26 have good safety at the test dose.

[0327] Figure 5 These are H&E staining images 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 compound prepared in Example 26 had relatively mild effects on mouse organs. At high doses, the liver and kidneys showed minimal cell degeneration and edema, but neither resulted in severe damage. The heart and lungs showed no significant damage or pathology, and overall organ tissue structure remained intact. These results demonstrate that the morpholine-containing pyridopyrimidine compound prepared in Example 26 did not cause significant organ damage at high doses, demonstrating a good safety profile and providing strong support for further preclinical studies.

[0329] Dose- and time-dependent testing

[0330] In experimental design, both the timing and dosage of drug treatment require careful optimization. Specifically, this optimization process is based on two aspects: time-dependence and dose-dependence. On the one hand, by treating cells with different drug concentrations over the same period of time, the effect of drug concentration on cellular responses can be evaluated; on the other hand, by treating cells with the same drug concentration for different periods of time, the differences in drug action duration on cellular effects can be explored. This systematic experimental design enables a more comprehensive understanding of the interaction between drugs and cells, providing a reliable experimental basis for subsequent research.

[0331] To evaluate the time-dependent inhibition of MCF-7 cell proliferation by the morpholine-containing pyridopyrimidine compounds prepared in Example 26, the MTT assay was used to assess cell viability at different exposure times (24, 48, and 72 hours). Following drug treatment, each group of cells was incubated with MTT for 4 hours, and the OD values ​​were measured after dissolution of the formazan crystals. The cell proliferation inhibition rate was calculated from three independent experiments.

[0332] Figure 6 The time dependence of the action of the morpholine-containing pyridopyrimidine compounds prepared in Example 26 on MCF-7 at different concentrations.

[0333] Figure 6 The results show that the antiproliferative effect of the pyridopyrimidine compounds containing morpholine structure prepared by Example 26 on MCF-7 cells is significantly enhanced with the increase of action concentration and the extension of action time. Specifically, for the pyridopyrimidine compounds containing morpholine structure prepared by Example 26, when the concentration is 11.1 μg / mL and the action is 48 hours, the inhibition rate has reached 56.63%. When the action time is extended to 72 hours, the inhibition rate is further increased to 61.5%. It is worth noting that when the concentration is increased to 100 μg / mL and the action is 72 hours, the inhibition rate of W-1 is significantly improved, reaching a maximum inhibition rate of 89.87%. The results show that the pyridopyrimidine compounds containing morpholine structure prepared by Example 26 have concentration- and time-dependent antiproliferative activity, and at higher concentrations and longer action times, their inhibitory effect is particularly significant.

[0334] AO staining experiment

[0335] Cell suspension was obtained from cells in logarithmic growth phase and cultured at 2.0 × 10 4 to 4.0×10 4The cells were seeded into 24-well plates at a density of 10 cells / mL and then placed in an incubator for 24 hours. Compound treatment: After washing the cells with PBS buffer, different concentrations of compound solution were added to each well, 1 mL was added to each well. For the blank control group, 1 mL of fresh culture medium was added, and then the 24-well plate was returned to the incubator for continued culture. After the drug acted for the specified time, the old culture medium was removed and the cells were washed twice with PBS buffer. Under light-proof conditions, AO staining solution diluted with PBS to a final concentration of micrograms per milliliter (μg / mL) was added, and then incubated in the dark in an incubator for 15 minutes. Finally, under blue light excitation, the cell morphology was observed and recorded by fluorescence inverted microscopy, and the images were photographed and saved to evaluate the effect of the compound on cell proliferation.

[0336] Figure 7 This is a diagram showing the effect of the morpholine-containing pyrrolopyrimidine compounds prepared in Experimental Example 26 on the morphology of cancer cells.

[0337] Figure 7 The results show that the MCF-7 cells of the control group are complete in morphology, and the nucleus and the top are uniformly green fluorescent, and the intercellular distribution is tight, and there is no obvious apoptosis feature. After the pyrrolopyrimidine compounds (4 μmol / L) containing morpholine structure prepared by experimental example 26 are processed for 24h, some cells show morphological changes, and the nucleus and the top are slightly reduced, and a small amount of cells present yellow fluorescence, prompting the occurrence of early apoptosis. As the pyrrolopyrimidine compounds concentration containing morpholine structure prepared by experimental example 26 is increased to 8 μmol / L, it can be observed that more cell volume is reduced, the edge is blurred, and the cells with orange-red fluorescence appear, indicating the increase of late apoptosis. In addition, the space between cells becomes larger, indicating that some cells are dead. The change of experimental example 26 (4 μmol / L) group is similar to GDC-0941 (4 μmol / L) group, showing a certain degree of apoptosis feature. Overall, the pyrrolopyrimidine compounds containing morpholine structure prepared by experimental example 26 can induce MCF-7 cell apoptosis in a dose-dependent manner, and the effect under high concentration is more obvious.

[0338] Annexin V-FITC / PI double staining to detect apoptotic cells

[0339] Prepare cell suspension from cells in logarithmic growth phase at 2.0 × 10 5The cells were seeded into 6-well plates at a density of 10 cells / 3 ml and then placed in an incubator for 24 hours. Culture medium containing a specific concentration of compound was added to each well and incubated in an incubator for a predetermined time. The cells were digested with EDTA-free trypsin, and after centrifugation to remove the supernatant, the cells were washed with PBS buffer. The centrifuge tube was wrapped with tin foil to ensure that it was protected 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 the two blank control tubes respectively, and a mixture of the two was added to the remaining tubes. After mixing, the mixture was incubated in the dark for 30 minutes. The cell suspension was filtered through a 200-mesh sieve, and the cells were counted by flow cytometry, recording 1.0×10 4 Data related to individual cells.

[0340] Figure 8 This is a diagram showing the effect of the morpholine-containing pyrrolopyrimidine compounds prepared in Experimental Example 26 on the apoptosis of cancer cells detected by flow cytometry.

[0341] Figure 8 The results showed that the total apoptosis rate of the blank control group was only 3.1%. However, when the 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 show 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, showing good anti-tumor activity.

[0342] Flow cytometry was used to determine the effect of the morpholine-containing pyrrolopyrimidine compounds 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: cells in the logarithmic growth phase were seeded in a 6-well plate and cultured for 24 hours. Then, solutions of the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 were added at concentrations of 4 μM and 8 μM, respectively, and incubated for a further 48 hours. The cells were then harvested and analyzed.

[0344] Figure 9 This is a diagram showing the effect of the morpholine-containing pyrrolopyrimidine compound prepared in Experimental Example 42 on the cell cycle of cancer cells as detected by flow cytometry.

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

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

[0347] The effect of Experimental Example 26 on the clonal proliferation ability of MCF-7 cells was evaluated using a cloning experiment. A cell suspension was prepared from cells in the logarithmic growth phase and seeded into a 6-well plate at a density of 5,000 cells per well, followed by culture in an incubator for 24 hours until the cells were completely attached. A culture medium containing a compound at a specified concentration was added to each well and cultured for 48 hours. The culture medium was replaced 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 aspirated, the cells were washed twice again with PBS buffer, and then 1 mL of crystal violet staining solution was added to each well and allowed to stand for 15 minutes. The crystal violet staining solution was aspirated, each well was washed with distilled water, and after air drying, photos were taken and recorded using a camera.

[0348] Figure 10 This is a graph showing the effect of the morpholine-containing pyrrolopyrimidine compounds prepared in Experimental Example 26 on cancer cell proliferation.

[0349] Figure 10 The results show that under low concentration (4 μM) treatment, the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 was able to significantly inhibit the colony formation of MCF-7 cells compared to the blank control group, indicating that even at relatively low concentrations, the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 also has certain anti-tumor activity. When the concentration is 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 is further enhanced. As the concentration is further increased, the inhibitory effect of the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 is more significant. When the concentration reaches 8 μM, the colony formation of MCF-7 cells is inhibited by nearly 95%, almost completely blocking the proliferation ability of the cells. However, compared with the positive control drug GDC-0941, the inhibitory effect of the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 at high concentrations is slightly inferior.

[0350] Cell scratch assay to measure cell migration and repair ability

[0351] The scratch test was used to evaluate the inhibitory effect of Experimental Example 26 on the migration ability of MCF-7 cells. 5The cells are seeded into a 6-well plate at a density of 10 cells / well. Before inoculation, we will draw parallel and equidistant lines in the center of the back of the 6-well plate for subsequent operations. When the cells grow to completely cover the well plate, use a vertical gun tip to scratch along the pre-drawn lines, scratching the entire well at one time. Subsequently, the well plate is washed twice with PBS buffer to remove cell debris that falls off during the scratching process, and replaced with new culture medium. After observing and recording the initial growth status of the cells under a microscope, the well plate is returned to the incubator for continued culture. After 24 hours of culture, the cell plate is removed, the morphological changes of the cells are observed under a microscope, and photographed and recorded.

[0352] Figure 11 This figure shows the effect of the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 on the migration ability of MCF-7 cells detected by cell scratch test.

[0353] Figure 11 Show, in blank control group, after cultivating 24 hours, MCF-7 cells show obvious trend to center migration.After using the pyrrolopyrimidine compounds containing morpholine structure prepared by experimental example 26 of low concentration (4 μ M) to process MCF-7 cells, the migration behavior of cells has been significantly suppressed. Compared with blank control group, the degree of cell migration to center in low concentration experimental example 26 treatment group significantly reduces, shows that the pyrrolopyrimidine compounds containing morpholine structure prepared by experimental example 26 can effectively suppress the migration ability of MCF-7 cells at low concentration. Generally speaking, the pyrrolopyrimidine compounds containing morpholine structure prepared by experimental example 26 show the ability of the inhibition MCF-7 cell migration of concentration-dependent. Along with the increase of concentration, the inhibitory effect of the pyrrolopyrimidine compounds containing morpholine structure prepared by experimental example 26 on cell migration and repair ability gradually strengthens.

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

[0355] The stable state 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 the cell is 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 mix. 5Cells were seeded into 6-well plates at a density of 10 cells / 3 ml and cultured in an incubator for 24 hours. After the culture was completed, the old culture medium in the wells was removed, the cells were washed twice with 1 ml of JC-1 staining buffer, and then 1 ml of complete culture medium was added. 1 ml of JC-1 staining working solution was added to the wells, gently shaken to mix evenly, and then incubated in the dark in an incubator for 15 minutes. After the incubation was complete, 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 the wells, and the cell morphology was observed under a fluorescence microscope and photographed.

[0357] Figure 12 This is a diagram showing the effect of the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 on the mitochondrial membrane potential of cancer cells.

[0358] Figure 12 The results show that the control group cells maintain a healthy state, and the mitochondrial membrane potential is stable, so the fluorescence signal is mainly red. As the drug concentration increases, the intracellular red fluorescence signal gradually weakens, and the green fluorescence intensity strengthens synchronously, prompting MMP to decline in a concentration-dependent manner. This phenomenon shows that the two compounds can activate the apoptosis pathway by inducing the destruction of mitochondrial membrane integrity. This transition from red fluorescence to green fluorescence is the direct evidence of the decline in cell membrane potential, which shows that these compounds can induce apoptosis in MCF-7 cells. When the pyrrolopyrimidine compounds containing morpholine structure prepared by embodiment 26 increase in concentration, they can cause the JC-1 fluorescence in MCF-7 cells to transition from red to green in a concentration-dependent manner. This transition not only shows the reduction of mitochondrial membrane potential, but also further confirms the ability of inducing cell apoptosis.

[0359] Detection of reactive oxygen species produced by MCF-7 cells using DCFH-DA probe

[0360] Reactive oxygen species (ROS) play a key role in the anti-tumor mechanisms of chemotherapeutic drugs. Elevated levels of ROS can trigger apoptosis signaling pathways and inhibit tumor proliferation by arresting the cell cycle. The present invention uses the DCFH-DA fluorescent probe to detect changes in ROS production in MCF-7 cells treated with the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 to elucidate its anti-tumor mechanism.

[0361] Prepare cell suspension from cells in logarithmic growth phase at 2.0 × 10 cells per well. 5Cells were seeded in a 6-well plate at a density of 10 cells / 3 ml and then cultured in an incubator for 24 hours. After the cells adhered, a culture medium containing a specific concentration of the compound was added to each well and cultured in the incubator for a certain period of time. The old culture medium was removed, the cells were washed twice with PBS buffer, and then Hoechst 33342 staining solution was added for 10 minutes of staining. After washing the cells twice with PBS buffer, 1 ml of culture medium containing DCFH-DA was added to each well. After absorbing the paraformaldehyde solution, the cells were washed twice with PBS buffer, and then 1 ml of crystal violet staining solution was added to each well and allowed to stand for 15 minutes. Finally, the cells were washed twice with PBS buffer, and the cell morphology was observed under a fluorescence microscope and photographed.

[0362] Figure 13 This is a graph showing the effect of the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 on the reactive oxygen species content in MCF-7 cells as detected by the DCFH-DA probe.

[0363] Figure 13 It was shown that almost no green fluorescence signal was detected in the MCF-7 cells in the blank control group. When the 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 shows that the low concentration of the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 can induce cells to produce a small amount of reactive oxygen species. Although the fluorescence signal is weak, this may be an early sign that the drug begins to work. As the concentration of the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 increases, the green fluorescence signal in the cells is significantly enhanced. In cells treated with high concentrations, the intensity and distribution range of the fluorescence signal are significantly expanded, indicating that the level of ROS in the cells is significantly increased. This concentration-dependent fluorescence enhancement phenomenon shows that the morpholine-containing pyrrolopyrimidine compound prepared in Example 26 can induce MCF-7 cells to produce more reactive oxygen species in a dose-dependent manner. It is worth noting that the fluorescence intensity in the high-concentration Example 26 treatment group is comparable to that of the positive control drug GDC-0941 treatment group.

[0364] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection 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 formula I, when R1 is When R2 is selected from When R1 is When R2 is selected from 2. A method for preparing the pyridopyrimidine compound containing a morpholine structure according to claim 1, characterized in that: When R1 is When, the preparation route of the compound shown in Formula I is as follows: In the above route, compound 2 is prepared by Miyaura borylation reaction; compound 4 is prepared by nucleophilic substitution reaction; compound 5 is prepared by de-Boc reaction; compound 6-a is prepared by aromatic nucleophilic substitution reaction; compound 6-a and compound 2 are reacted by Suzuki coupling reaction to prepare compound 7-a; compound 8-a is prepared by amidation reaction; When R1 is When, the preparation route of the compound shown in Formula I is as follows: In the above route, compound 2 is prepared by Miyaura borylation reaction; compound 4 is prepared by nucleophilic substitution reaction; compound 5 is prepared by de-Boc reaction; compound 6-b is prepared by aromatic nucleophilic substitution reaction; compound 6-b and compound 2 are reacted by Suzuki coupling reaction to prepare compound 7-b; and compound 8-b is prepared by amidation reaction.

3. A pharmaceutically acceptable salt of the pyridopyrimidine compound containing a morpholine structure according to claim 1.

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

5. Use of the pyridopyrimidine compound containing a morpholine structure according to claim 1 in the preparation of a drug for treating and / or preventing cancer.

6. Use of the pyridopyrimidine compound containing a morpholine structure according to claim 1 in the preparation of a medicament for treating and / or preventing proliferative diseases.

7. Use of a pharmaceutically acceptable salt of the morpholine-containing pyridopyrimidine compound according to claim 3 in the preparation of a drug for treating and / or preventing cancer.

8. Use of a pharmaceutically acceptable salt of the morpholine-containing pyridopyrimidine compound according to claim 3 in the preparation of a medicament for treating and / or preventing proliferative diseases.

9. Use of the pharmaceutical preparation according to claim 4 in the preparation of drugs for treating and / or preventing cancer.

10. Use of the pharmaceutical preparation according to claim 4 in the preparation of a medicament for treating and / or preventing a proliferative disease.

Images