An oxazinane compound, preparation method, pharmaceutical composition and application

Abstract

The application provides an oxazinane compound, a preparation method, a pharmaceutical composition and application, and a general formula of the oxazinane compound or a pharmaceutically acceptable salt thereof is shown as formula (I). The oxazinane compound has obvious proliferation inhibition effect on cancer cells, especially human head and neck squamous cell carcinoma cell strains HSC4, HSC3, HSC2, CAL33, SCC4 and human glioma cell strain U251, can block the cell cycle, effectively inhibit the phosphorylation level of key proteins in the PI3K / AKT / mTOR pathway at the molecular level, has the potential to be prepared into a new antitumor drug, and has a good market prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to an oxazine alkyl compound, its preparation method, pharmaceutical composition, and its application. Background Technology

[0002] In recent years, the rapid development of modern industrialization has led to increasingly serious environmental pollution, increased work pressure, and unhealthy lifestyles, making cancer a leading factor threatening human health.

[0003] Traditional anti-tumor drugs are mainly cytotoxic, lacking specificity. While killing tumor cells, they also damage normal cells, causing serious toxic side effects such as bone marrow suppression, vomiting, and hair loss. Targeted anti-tumor drugs, on the other hand, can identify specific targets on the surface of tumor cells, blocking cell signaling pathways and inducing cell cycle arrest and apoptosis. Furthermore, appropriate therapeutic targets can be selected based on the patient's specific condition, enabling personalized treatment. Currently, the research and clinical application of targeted anti-tumor drugs has become one of the main development directions in the pharmaceutical industry.

[0004] Studies have found that the PI3K / AKT / mTOR signaling pathway is one of the important intracellular signaling pathways, closely related to the occurrence and development of various human tumors (such as head and neck cancer, non-small cell lung cancer, liver cancer, breast cancer, and colorectal cancer). The PI3K / AKT / mTOR pathway participates in the regulation of many important biological processes, affecting the function of multiple target molecules, and is closely related to tumor cell growth and proliferation, cell cycle regulation, invasion and metastasis, and tumor angiogenesis. The safety and efficacy of some small molecule inhibitors of the PI3K / AKT / mTOR pathway have been extensively studied in clinical trials, and their importance in inhibiting tumor progression has been confirmed. The development of anti-tumor drugs targeting this pathway has become a research hotspot in anti-tumor drugs and has received widespread attention internationally. Summary of the Invention

[0005] This invention addresses the technical problems of antitumor drugs lacking specificity, suffering from severe toxic side effects, and exhibiting low inhibitory activity. It provides an oxazine alkyl compound, its preparation method, pharmaceutical composition, and applications. The oxazine alkyl compound of this invention exhibits good proliferative inhibitory effects on cancer cells, especially squamous cell carcinomas such as CAL33 and gliomas such as U251, blocking the cell cycle and effectively inhibiting the phosphorylation level of key proteins in the PI3K / AKT / mTOR pathway at the molecular level. It has the potential to be developed into a novel antitumor drug and possesses good market prospects.

[0006] This invention provides an oxazine alkyl compound as shown in Formula I, or a pharmaceutically acceptable salt thereof.

[0007]

[0008] R1 and R2 are independently selected from hydrogen, halogen, nitro, amino, and C. 1-6 Alkyl, -OR 1a -OC(O)R 1a -OC(O)OR 1a -OC(O)NR 1b R 1c -NR 1b R 1c -NR 1a C(O)R 1d -NR 1a C(O)OR 1d -NR 1a C(O)NR 1b R 1c -NR 1a S(O)R 1d -NR 1a S(O)2R 1d -NR 1a S(O)NR 1b R 1c or -NR 1a S(O)2NR 1b R 1c ;

[0009] Among them, each R 1a R 1b R 1c and R 1d Independently selected from hydrogen and C 1-6 alkyl, C 3-7 cycloalkyl, C 6-14 Aryl, C 7-15 Aryl alkyl, heteroaryl or heterocyclic; or R 1b and R 1c Together with the nitrogen atoms attached to them, they form heterocyclic groups;

[0010] R3 is hydrogen, C 1-6 Alkyl, C 1-6 Alkyl or CH n X 3-n , where X is a halogen and n is 0, 1 or 2;

[0011] R4 can be halogen, homomorpholino, morpholino, oxazinyl, piperazinyl, oxazolyl, piperidinyl, pyrrolidinyl, or C. 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-7 cycloalkyl, C 3-7 cycloalkyl-C 1-6Alkyl, C 6-14 Aryl, C 7-15 Aryl groups, heteroaryl groups, heteroaryl-C 1-6 Alkyl, heterocyclic, heterocyclic-C 1-6 Alkyl or -NR 1e R 1f or -NR 1g - Heterocyclic group;

[0012] Among them, R 1e R 1f and R 1g Independently selected from hydrogen and C 1-6 Alkyl, C2-C6 alkenyl, C 2-6 alkynyl group, C 3-7 cycloalkyl, C 6-14 Aryl, C 7-15 Aryl alkyl, heteroaryl, C 1-6 alkyl-aryl or C 1-6 Alkyl-heterocyclic group.

[0013] Furthermore, R1 and R2 are independently selected from hydrogen atoms and amino groups;

[0014] Furthermore, in the halogen group, R1 and R2 are preferably bromine atoms;

[0015] Furthermore, R1 and R2 are in the -OR group 1a -OC(O)R 1a -OC(O)OR 1a -OC(O)NR 1b R 1c In this context, -OR is preferred independently. 1a ;where R 1a R 1b and R 1c Selected independently from C 1-6 Alkyl groups, preferably methyl groups;

[0016] Furthermore, R1 and R2 in the group -NR 1b R 1c -NR 1a C(O)R 1d -NR 1a C(O)OR 1d -NR 1a C(O)NR 1b R 1c -NR 1a S(O)R 1d -NR 1a S(O)2R 1d -NR 1a S(O)NR 1b R1c or -NR 1a S(O)2NR 1b R 1c In this context, -NR is preferred independently. 1b R 1c or -NR 1a C(O)OR 1d ;where R 1a R 1b R 1c For hydrogen, R 1d C 1-6 Alkyl group, preferably R 1d For tert-butyl;

[0017] Furthermore, R1 and R2 are independently selected from hydrogen atoms, bromine atoms, amino groups, or -OCH3.

[0018] Furthermore, R3 is C 1-6 Alkyl or CH n X 3-n , where X is preferably F.

[0019] Furthermore, R3 is difluoromethyl.

[0020] Furthermore, R4 can be halogenated, homomorpholino, or morpholino.

[0021] Furthermore, R1 and R2 are hydrogen atoms, and R3 is a difluoromethyl group;

[0022] Alternatively, R1 and R2 can be hydrogen atoms, bromine atoms, amino groups, or -OCH3, and R3 can be difluoromethyl.

[0023] Furthermore, R1 and R2 are one or two of hydrogen atoms, bromine atoms, amino groups or -OCH3, R3 is difluoromethyl, and R4 is morpholino or homomorpholino.

[0024] Furthermore, any one of the following compounds:

[0025] Compound 1: 3-(4-(2-(difluoromethyl)-1H-benzo[d]imidazol-1-yl)-6-morpholino-1,3,5-triazin-2-yl)-1,3-oxazine;

[0026] Compound 2: 3-(4-(6-bromo-2-(difluoromethyl)-4-methoxy-1H-benzo[d]imidazol-1-yl)-6-morpholino-1,3,5-triazin-2-yl)-1,3-oxazine;

[0027] Compound 3: 2-(difluoromethyl)-4-methoxy-1-(4-morpholino-6-(1,3-oxazinyl-3-yl)-1,3,5-triazinyl-2-yl)-1H-benzo[d]imidazol-6-amine;

[0028] Compound 4: 4-(4-(2-(difluoromethyl)-1H-benzo[d]imidazol-1-yl)-6-(1,3-oxazinyl-3-yl)-1,3,5-triazinyl-2-yl)-1,4-homomorpholine;

[0029] When the compounds of the present invention contain asymmetric carbon atoms, the isomers and mixtures thereof (racemates) caused by the asymmetric carbon atoms are also included within the scope of the compounds of the present invention.

[0030] The compounds of the present invention may be in the form of acid addition salts that are pharmaceutically acceptable salts.

[0031] In another aspect, the present invention provides a method for preparing oxazine alkyl compounds as shown in general formula (I), wherein the preparation method is based on the following synthetic route:

[0032] The synthetic route includes the following steps: in an organic solvent, compound II is used as the starting material to obtain compound III; compound III reacts with compound IV to obtain compound V; compound V reacts with compound VI to obtain compound VII; compound VII reacts with compound VIII to obtain oxazine alkyl compound I.

[0033]

[0034] The definitions of R1, R2, R3, and R4 in the synthetic route are as described in the oxazine alkyl compounds represented by the general formula I.

[0035] Furthermore, in the synthetic route:

[0036] Preferably, the molar ratio of compound II to ammonium chloride is 1:(20-22); the molar ratio of compound III to compound V is 1:15; the molar ratio of compound V to compound VI is 1:1; and the molar ratio of compound VII to compound VIII is 1:(20-22).

[0037] Preferably, the solvent used for the reaction of compound II with ammonium chloride is ethanol; the solvent used for the reaction of compound III with compound IV is 6 mol / L hydrochloric acid solution; the solvent used for the reaction of compound V with compound VI is DMF; and the solvent used for the reaction of compound VII with compound VIII is THF or oxazine.

[0038] The reaction temperature of compound II with ammonium chloride is 70℃~90℃; the reaction temperature of compound III with compound IV is 95℃~115℃; the reaction temperature of compound V with compound VI is 20℃~30℃; and the reaction temperature of compound VII with compound VIII is 60℃~85℃.

[0039] The synthetic route includes the following post-processing steps: such as neutralization, solvent removal, or filtration; the neutralization, solvent removal, or filtration can be performed using conventional methods of this type of operation in the art.

[0040] The synthetic route includes the following separation and purification steps: such as column chromatography, thin-layer chromatography, extraction, and vacuum distillation; the separation and purification methods can adopt conventional methods of this type of operation in the art.

[0041] In another aspect, the present invention provides a pharmaceutical composition comprising an oxazine alkyl compound as shown in general formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0042] The carrier includes pharmaceutical excipients, the selection of which varies depending on the route of administration and characteristics of action, and is usually a filler, diluent, binder, wetting agent, disintegrant, lubricant, emulsifier or suspending agent.

[0043] The pharmaceutical composition may be prepared using any method known to those skilled in the art, as disclosed, such as conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, or lyophilization processes.

[0044] Furthermore, the mass percentage of the oxazine alkyl compound or its pharmaceutically acceptable salt as represented by formula (I) in the pharmaceutical composition is 0.1% to 99.9%, whereby the mass percentage refers to the percentage of the oxazine alkyl compound or its pharmaceutically acceptable salt as represented by formula (I) to the total mass of the pharmaceutical composition. The sum of the mass fractions of the oxazine alkyl compound or its pharmaceutically acceptable salt as represented by formula (I) and the pharmaceutical excipients is 100%.

[0045] The pharmaceutical compositions of the present invention can be administered orally, by injection (intravenous, intramuscular, subcutaneous, and intracoronary), sublingually, buccally, rectally, urethra, vaginally, nasally, by inhalation, or topically.

[0046] The present invention provides the use of oxazine alkyl compounds as shown in general formula (I) or pharmaceutically acceptable salts thereof or the pharmaceutical compositions thereof as active ingredients in the preparation of antitumor drugs.

[0047] Furthermore, its application in the preparation of antitumor drugs with inhibitory activity against human head and neck squamous cell carcinoma HSC2, HSC3, HSC4, Cal33, SCC4 and human glioma cells U251.

[0048] The antitumor drugs described above that exhibit inhibitory activity against human head and neck squamous cell carcinoma HSC2, HSC3, HSC4, Cal33, and SCC4 are all drugs for treating human head and neck squamous cell carcinoma. The antitumor drug described above that exhibits inhibitory activity against human glioma cells U251 is a drug for treating human glioma.

[0049] Compared with existing technologies, the oxazine alkyl compounds, preparation methods, pharmaceutical compositions, and applications described in this invention have the following advantages:

[0050] The compounds provided by this invention have an oxazine alkyl skeleton, and the preparation method and potential applications of this type of compound have not been previously reported. Our experimental results show that the compounds of this invention have significant inhibitory effects on the proliferation of human cancer cells, especially human head and neck squamous cell carcinomas such as HSC2, HSC3, HSC4, Cal33, and SCC4, and human gliomas such as U251. Furthermore, the compounds of this invention have no significant effect on other tumor cells or normal cells, and exhibit stronger specificity, showing better therapeutic effects on human head and neck squamous cell carcinomas such as HSC2, HSC3, HSC4, Cal33, and SCC4, and human gliomas such as U251. These compounds have the potential to be developed into novel anti-tumor drugs and have good market prospects. Attached Figure Description

[0051] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0052] Figure 1 A schematic diagram illustrating the effect of compound 1 prepared in Example 1 on the CAL33 cell cycle;

[0053] Figure 2 A schematic diagram illustrating the effect of compound 1 prepared in Example 1 on the SCC4 cell cycle;

[0054] Figure 3 A schematic diagram illustrating the effect of compound 3 prepared in Example 3 on the cell cycle of CAL33 cells;

[0055] Figure 4 A schematic diagram illustrating the effect of compound 3 prepared in Example 3 on the HSC4 cell cycle;

[0056] Figure 5 This is a schematic diagram illustrating the effect of compound 1 prepared in Example 1 on the phosphorylation level of proteins in the PI3K / Akt / mTOR pathway.

[0057] Figure 6The diagram shows the results of the in vivo antitumor test of compound 1 prepared in Example 1 in the CAL33 nude mouse xenograft model (A is the curve of tumor volume change in each group of mice, B is the comparison of tumor weight in each group of mice, and C is the curve of body weight change in each group of mice). Detailed Implementation

[0058] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0059] Unless otherwise specified, all descriptions in the following examples are in units of mass (grams), and room temperature refers to 20°C to 30°C.

[0060] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0061] Synthesis route:

[0062]

[0063] Example 1

[0064] Preparation of Compound 1: 3-(4-(2-(difluoromethyl)-1H-benzo[d]imidazol-1-yl)-6-morpholino-1,3,5-triazin-2-yl)-1,3-oxazine:

[0065] 2-Difluoromethyl-1H-benzimidazole (0.38 g, 2.28 mmol), 4-(4,6-dichloro-1,3,5-triazin-2-yl)morpholine (0.54 g, 2.28 mmol), and potassium carbonate (1.26 g, 9.12 mmol) were dissolved in DMF (9 mL). After addition, the mixture was stirred at room temperature. TLC was performed, and 5 mL of water was added to the reaction solution. The precipitate was filtered, washed with DMF, and dried to give 0.26 g (yield 33.28%) of 4-(4-chloro-6-(2-difluoromethyl-1H-benzimidazole-1-yl)-1,3,5-triazin-2-yl)morpholine.

[0066] 0.037 g (0.1 mmol) of 4-(4-chloro-6-(2-difluoromethyl-1H-benzo[d]imidazol-1-yl)-1,3,5-triazin-2-yl)morpholine was added to 1,3-oxazolidine (0.087 g, 1 mmol). After the addition was complete, the mixture was heated to reflux, cooled, diluted with water, and subjected to TLC. The mixture was then filtered to give 0.024 g (76.8% yield) of 3-(4-(2-(difluoromethyl)-1H-benzo[d]imidazol-1-yl)-6-morpholino-1,3,5-triazin-2-yl)-1,3-oxazinidine.

[0067] The obtained target product was tested, and the results are as follows:

[0068] 1 H NMR (400MHz, CDCl3): δ8.34 (s, 1H), 7.90 (d, J = 7.5Hz, 1H), 7.76–7.36 (m, 3H), 5.35 (s, 2H), 3.93 (dd, J = 68.7, 31.8Hz, 12H), 1.83 (s, 2H).

[0069] MS m / z: 418.18 (M+1).

[0070] Example 2

[0071] Preparation of compound 2: 3-(4-(6-bromo-2-(difluoromethyl)-4-methoxy-1H-benzo[d]imidazol-1-yl)-6-morpholino-1,3,5-triazin-2-yl)-1,3-oxazine;

[0072] Replacing 2-difluoromethyl-1H-benzimidazole with 6-bromo-2-(difluoromethyl)-4-methoxy-1H-benzi[d]imidazole, and using the same raw materials, reagents, and preparation methods as in Example 1, yielded 3-(4-(6-bromo-2-(difluoromethyl)-4-methoxy-1H-benzi[d]imidazole-1-yl)-6-morpholino-1,3,5-triazin-2-yl)-1,3-oxazineane in a yield of 75.8%.

[0073] The obtained target product was tested, and the test results are as follows:

[0074] 1 H NMR (400MHz, CDCl3): δ8.07 (s, 1H), 7.46 (d, J = 56.5Hz, 1H), 6.87 (s, 1H), 5.27 (s, 2H), 4.13–3.56 (m, 15H), 1.77 (s, 2H). MS m / z:526.1(M+1).

[0075] Example 3

[0076] Compound 3: 2-(difluoromethyl)-4-methoxy-1-(4-morpholino-6-(1,3-oxazin-3-yl)-1,3,5-triazin-2-yl)-1H-benzo[d]imidazol-6-amine

[0077] 0.50 g of 4-bromo-2-methoxy-6-nitroaniline was dissolved in 10 mL of ethanol, and 2 g of zinc powder and 2.3 g of ammonium chloride were added. The mixture was refluxed for 2 h, cooled to room temperature, and the zinc powder was removed by filtration. 10 mL of 4 M hydrochloric acid / methanol solution was added to the ethanol solution, and the mixture was stirred for 30 min and then evaporated to dryness. 20 mL of 6 M hydrochloric acid and 2 g of difluoroacetic acid were added to the evaporated solid, and the mixture was heated and stirred at 105 °C for 16 h. The mixture was subjected to TLC, evaporated to dryness, and the pH was adjusted to 9-10 with ammonia solution. The mixture was extracted with ethyl acetate and evaporated to dryness to give 0.41 g (73.21%) of 5-bromo-3-methoxyphenyl-1,2-diamine.

[0078] 6-Bromo-2-(difluoromethyl)-4-methoxy-1H-benzo[d]imidazolium (0.10 g, 0.36 mmol), 4-(4,6-dichloro-1,3,5-triazin-2-yl)morpholine (0.085 g, 0.36 mmol), and potassium carbonate (0.20 g, 1.44 mmol) were dissolved in DMF (1.4 mL). After addition, the mixture was stirred at room temperature. TLC was performed, and 2 mL of water was added to the reaction solution. The mixture was filtered to obtain a precipitate, which was washed with DMF and dried to give 0.13 g (76% yield) of 4-(4-(6-bromo-2-(difluoromethyl)-4-methoxy-1H-benzo[d]imidazol-1-yl)-6-chloro-1,3,5-triazin-2-yl)morpholine.

[0079] 2-Methoxy-4-nitroaniline (20 g, 120 mmol) and glacial acetic acid (60 mL) were stirred in an ice bath, and concentrated sulfuric acid (100 mL) was slowly added dropwise. The temperature was controlled at 0-10 °C in an ice bath. A mixed acid (70% nitric acid (7.8 mL, 120 mmol) and concentrated sulfuric acid (4.6 mL, 80 mmol)) was added dropwise to the system. After the addition was complete, the reaction was stirred at 0-10 °C for 15 min. The temperature was then raised to room temperature and the reaction was monitored by TLC. After the reaction was complete, the system was poured into ice water and stirred for 20 min. The mixture was filtered, washed with water, and dried. The dried product was then slurried with ethyl acetate, dried under vacuum, and dried to obtain 8.1 g (yield 31.6%) of 2-methoxy-4,6-dinitroaniline.

[0080] 2-Methoxy-4,6-dinitroaniline (8 g, 37.5 mmol) was dissolved in triethylamine (24 mL) and acetonitrile (20 mL), and then 10% Pd / C (0.37 g) was added. The mixture was cooled to 15 °C, and the prepared formic acid-acetonitrile solution (96% formic acid (8 mL) and acetonitrile (20 mL)) was added dropwise to the system. The temperature was controlled at 20–30 °C. After the addition was complete, the mixture was refluxed. The reaction was performed by TLC, filtered, the catalyst was removed, the solvent was removed under reduced pressure, and column chromatography was used to obtain 2.90 g (yield 42.2%) of 3-methoxy-5-nitrobenzene-1,2-diamine.

[0081] 3-Methoxy-5-nitrobenzene-1,2-diamine (2.90 g, 15.80 mmol) and difluoroacetic acid (1.67 g, 17.40 mmol) were added to HCl (4 mol / L, 30 mL), and the mixture was heated to reflux and reacted by TLC. After the reaction was completed, the mixture was cooled to room temperature, and the hydrochloric acid in the system was neutralized with saturated sodium carbonate solution. The mixture was filtered, the solid was washed with an appropriate amount of water, and dried at 50 °C to give 2.30 g (yield 59.9%) of 2-(difluoromethyl)-4-methoxy-6-nitro-1H-benzo[d]imidazole.

[0082] 2-(difluoromethyl)-4-methoxy-6-nitro-1H-benzo[d]imidazole (2.30 g, 9.50 mmol) was added to methanol (100 mL) and 10% Pd / C (100 mg). The mixture was hydrogenated and subjected to TLC. After the reaction was complete, the mixture was filtered to remove the catalyst and the solvent was removed under reduced pressure. Boc anhydride (di-tert-butyl dicarbonate) (6.40 g) was added to the system, followed by dioxane (40 mL). The mixture was refluxed and the solvent was removed under reduced pressure. Methanol (60 mL) was added, and sodium hydroxide solution (2 mol / L, 25 mL) was added under ice bath conditions. The mixture was reacted at 20 °C for 1 h. The pH was adjusted to neutral with acetic acid, and the mixture was concentrated under reduced pressure. The mixture was extracted with ethyl acetate, and the organic phases were combined, washed with water, dried, concentrated, and column chromatography was performed to obtain 1.36 g (yield 45.6%) of tert-butyl (2-(difluoromethyl)-4-methoxy-1H-benzo[d]imidazole-6-yl)carbamate.

[0083] 1.36 g (4.30 mmol) of tert-butyl (2-(difluoromethyl)-4-methoxy-1H-benzo[d]imidazol-6-yl)carbamate, 1.02 g (4.30 mmol) of 4-(4,6-dichloro-1,3,5-triazin-2-yl)morpholine, and 2.37 g (17.20 mmol) of potassium carbonate were added to DMF (25 mL). The mixture was stirred at room temperature and the reaction was carried out by TLC. After the reaction was completed, the mixture was poured into ice water (100 mL), stirred, filtered, washed with water and methanol, and dried to obtain 1.10 g (yield 50.0%) of tert-butyl (1-(4-chloro-6-morpholino-1,3,5-triazin-2-yl)-2-(difluoromethyl)-4-methoxy-1H-benzo[d]imidazol-6-yl)carbamate.

[0084] THF (20 mL) was added to (1-(4-chloro-6-morpholino-1,3,5-triazin-2-yl)-2-(difluoromethyl)-4-methoxy-1H-benzo[d]imidazol-6-yl)carbamate (0.21 g, 0.40 mmol) and 1,3-oxazolidine (0.35 g, 4 mmol). The reaction was carried out at room temperature and measured by TLC. After the reaction was completed, the reaction solution was poured into water (50 mL), stirred, and a solid precipitated. The solid was filtered, washed with an appropriate amount of methanol, and dried to give 0.18 g (79% yield) of (2-(difluoromethyl)-4-methoxy-1-(4-morpholino-6-(1,3-oxazolidine-3-yl)-1,3,5-triazin-2-yl)-1H-benzo[d]imidazol-6-yl)carbamate.

[0085] Dichloromethane (2 mL) and trifluoroacetic acid (2 mL) were added to 0.174 g (0.31 mmol) of tert-butyl carbamate. After the reaction was complete, the solvent was removed under reduced pressure, and column chromatography was performed to obtain 0.06 g (53.55%) of 2-(difluoromethyl)-4-methoxy-1-(4-morpholino-6-(1,3-oxazolidine-3-yl)-1,3,5-triazin-2-yl)-1H-benzo[d]imidazol-6-amine.

[0086] The obtained target product was tested, and the test results are as follows:

[0087] 1 H NMR (400MHz, CDCl3) δ7.61–7.33(m,2H),7.11(ddd,J=48.5,29.4, 13.6Hz,1H),6.27(s,1H),5.35(d,J=16.3Hz,3H),4.04–3.71(m,17H). MS m / z:463.2(M+1).

[0088] Example 4

[0089] Compound 4: 4-(4-(2-(difluoromethyl)-1H-benzo[d]imidazol-1-yl)-6-(1,3-oxazinyl-3-yl)-1,3,5-triazinyl-2-yl)-1,4-homomorpholine

[0090] Replacing 4-(4,6-dichloro-1,3,5-triazin-2-yl)morpholine with 4-(4,6-dichloro-1,3,5-triazin-2-yl)-1,4-homomorpholine, and using the same raw materials, reagents, and preparation methods as in Example 1, yielded 4-(4-(2-(difluoromethyl)-1H-benzo[d]imidazol-1-yl)-6-(1,3-oxazinan-3-yl)-1,3,5-triazin-2-yl)-1,4-homomorpholine in 73.5% yield.

[0091] The obtained target product was tested, and the test results are as follows: 1 H NMR (400MHz, CDCl3) δ8.48–8.24(m,1H),8.04–7.33(m,4H),5.36(s, 2H),4.51–3.42(m,12H),2.19–1.95(m,2H),1.83(s,2H). MS m / z:432.19(M+1).

[0092] Performance testing

[0093] In the following text, the target products obtained in Examples 1 to 4 will be referred to as compounds 1 to 4.

[0094] 1. Tests on the inhibitory effect on cancer cell proliferation

[0095] The effects of oxazine derivatives on the viability of HSC2, HSC3, HSC4, CAL33, SCC4, U251, and HUVEC cells were investigated using the MTT assay. Seven cell lines in logarithmic growth phase were trypsinized to obtain cell suspensions, which were then counted and adjusted to suitable cell densities. 200 μL of cells were seeded into each well of a 96-well plate and incubated at 37°C and 5% CO2 for 24 h. Different concentrations of the compound were then added, with three replicates for each concentration. The control group was incubated with the corresponding volume of solvent (DMSO) and continued incubation at 37°C and 5% CO2 for another 48 h. Finally, 20 μL of 3-(4,5-dimethylthiazol-2)-2 (MTT) at a final concentration of 5 mg / mL was added, and the cells were incubated for 4 h. The absorbance of each well was measured at 490 nm using a Bio-Rad microplate reader. Cell count (% of blank group) = 100 × (absorbance value of compound treatment group - absorbance value of blank group) / (absorbance value of control group - absorbance value of blank group). Each experiment was repeated 3 times. GraphPad Prism software was used to plot the data and calculate the IC50 value.

[0096] The inhibitory effects of the compound on the proliferation of HSC4, HSC3, HSC2, CAL33, SCC4, U251 and HUVEC cells are shown in Table 1.

[0097] Table 1

[0098]

[0099] The inhibitory effects of compounds 1 and 3 on the proliferation of human prostate cancer PC3, human breast cancer BT549 and MDA-MB-231, human non-small cell lung cancer A549 and human liver cancer HEPG2 were further investigated. The specific results are shown in Table 2.

[0100] Table 2

[0101]

[0102] The inhibitory effects of compounds 1 and 3 on the proliferation of normal human umbilical vein endothelial cells were further investigated, and the specific results are shown in Table 3.

[0103] Table 3

[0104] Test compound <![CDATA[Cell IC 50 (μM)]]> 1 >10 3 >10

[0105] Table 1 shows that the compounds of this invention have significant inhibitory effects on the proliferation of human head and neck squamous cell carcinoma and human glioma cells. Table 2 shows that compound 1 of this invention has a weak inhibitory effect on the proliferation of human prostate cancer PC3, human breast cancer BT549 and MDA-MB-231, human non-small cell lung cancer A549, and human liver cancer HEPG2 cells. Compound 3 has a weak inhibitory effect on the proliferation of human prostate cancer PC3, human breast cancer BT549 and MDA-MB-231, and human non-small cell lung cancer A549 cells, but a significant inhibitory effect on the proliferation of human liver cancer HEPG2 cells. Table 3 shows that compounds 1 and 3 of this invention have a weak inhibitory effect on the proliferation of normal human umbilical vein endothelial cells, further confirming that compounds 1 and 3 may cause less damage to normal human cells while exerting anti-tumor effects.

[0106] Flow cytometry to detect the effects of compounds on cell cycle arrest

[0107] Effects of Compound 1 on Cell Cycle Arrest in CAL33 and HSC4

[0108] (1) CAL33 and SCC4 were seeded in 6-well plates at a suitable cell density and incubated at 37°C for 24 hours. After changing the culture medium, the control group was given DMSO and different concentrations of compound 1 and incubated for 24 hours.

[0109] (2) Collect cells in EP tubes and wash twice with ice-cold PBS;

[0110] (3) Add 250 μL of ice-cold PBS to the lower layer of cells, mix well by pipetting, and slowly fix the cells in 750 μL of ice-cold ethanol. Place the cells in a 4°C refrigerator overnight.

[0111] (4) After centrifugation to remove ethanol, wash twice with ice-cold PBS. Add 100 μL of PBS to the lower cell pellet to resuspend the cells. Add 200 μL of pre-prepared PI staining solution to each tube, wrap with aluminum foil, and stain at 4°C for 1 hour. Filter through a nylon mesh and analyze using a flow cytometer.

[0112] (5) Analyze the flow cytometry results using FlowJo 7.6.1 software.

[0113] Effect of compound 1 on cell cycle arrest in CAL33 cells

[0114] After 24 hours of treatment with compound 1 (concentrations: 2.5, 5, 10 μM) on CAL33 cells, the G0 / G1 phase percentages in the 5 μM and 10 μM groups were significantly increased compared to the blank control group (47.2%), reaching 57.7% and 63.0%, respectively. This indicates that compound 1 can arrest the CAL33 cell cycle at the G0 / G1 phase. Figure 1 .

[0115] Effect of compound 1 on SCC4 cell cycle arrest

[0116] After SCC4 cells were treated with compound 1 (concentrations: 3, 6, 12 μM) for 24 h, the G2 / M phase percentages in the groups with compound 2 concentrations of 6 μM and 12 μM were significantly increased compared to the blank control group (30.9%), reaching 41.2% and 59.2%, respectively. This indicates that compound 1 can arrest the SCC4 cell cycle at the G2 / M phase. Figure 2 .

[0117] Effects of compound 3 on cell cycle arrest in CAL33 and HSC4 cells

[0118] (1) CAL33 and HSC4 were seeded in 6-well plates at a suitable cell density and incubated at 37°C for 24 hours. After changing the culture medium, the control group was given DMSO and different concentrations of compound 3 and incubated for 24 hours.

[0119] (2) Collect cells in EP tubes and wash twice with ice-cold PBS;

[0120] (3) Add 250 μL of ice-cold PBS to the lower layer of cells, mix well by pipetting, and slowly fix the cells in 750 μL of ice-cold ethanol. Place the cells in a 4°C refrigerator overnight.

[0121] (4) After centrifugation to remove ethanol, wash twice with ice-cold PBS. Add 100 μL of PBS to the lower cell pellet to resuspend the cells. Add 200 μL of pre-prepared PI staining solution to each tube, wrap with aluminum foil, and stain at 4°C for 1 hour. Filter through a nylon mesh and analyze using a flow cytometer.

[0122] (5) Analyze the flow cytometry results using FlowJo 7.6.1 software.

[0123] Effect of compound 3 on cell cycle arrest in CAL33 cells

[0124] After 24 hours of treatment with compound 3 (concentrations: 1, 2, 4 μM) on CAL33 cells, the G0 / G1 65.5% phase was significantly increased in the 2 μM and 4 μM groups compared to the blank control group, reaching 86.1% and 84.9%, respectively. This indicates that compound 3 can arrest the CAL33 cell cycle at the G0 / G1 phase. Figure 3 .

[0125] Effect of compound 3 on HSC4 cell cycle arrest

[0126] After 24 hours of treatment of HSC4 cells with compound 3 (concentrations: 0.5, 1, and 2 μM), the G0 / G1 phase percentages in the 1 μM and 2 μM groups were significantly increased compared to the blank control group (70.9%), reaching 76.8% and 82.1%, respectively. This indicates that compound 3 can arrest the HSC4 cell cycle at the G0 / G1 phase. Figure 4 .

[0127] 3. Kinase experiment

[0128] The total volume of the kinase reaction system was 10 μL, containing reaction buffer with different concentrations of compound 1, PI3K kinase, 2 mM DTT, 0.1 mM substrate, and 0.02 mM ATP. The reaction was carried out at room temperature for 1 hour, followed by the addition of 5 μL of buffer containing 30 mM EDTA, 6 nM antibody, and 12 nM tracer, and incubation at room temperature for 0.5 hours. The absorbance of the reaction system was analyzed, and the concentration at which compound 1 achieved 50% inhibitory activity against PI3K kinase was calculated based on the absorbance, as shown in Table 4. The concentrations of compound 1 at which 50% inhibitory activity against PI3Kα / β / δ / γ kinases was 675.1, 78.48, 5.54, and 161.3 nM, respectively. Compound 1 is a selective PI3Kδ inhibitor with an IC50 of 5.54 nM; its selectivity for PI3Kδ is 14.2 to 121.9 times that for PI3Kα / β / γ.

[0129] Table 4

[0130]

[0131] 4. Western blot analysis to detect the effect of compound 1 on the expression of PI3K / Akt / mTOR pathway proteins.

[0132] (1) Cell treatment: CAL33 and SCC4 cells in the logarithmic growth phase were selected and prepared into cell suspensions. The cell suspensions were evenly seeded into 6-well plates at 2 mL per well. After incubation for 24 h, the culture medium was changed, and DMSO and different concentrations of compound 1 were added respectively. The plates were then incubated at 37 °C and 5% CO2 for 48 h.

[0133] (2) Protein extraction: Remove the 6cm culture dish from the incubator and place it on ice. Use a scraper to scrape the bottom of the culture dish to remove all adherent cells. Transfer the cells to multiple 1.5mL EP tubes using a pipette. Pre-cool the centrifuge and centrifuge at 3000rpm for 5min. Discard the old culture medium, wash and mix the cells with 1mL of ice-cold PBS, and centrifuge again at 3000rpm for 5min. After removing the supernatant, repeat the same steps. Remove the supernatant and prepare the lysis buffer. Mix RIPA and a broad-spectrum phosphatase inhibitor at a ratio of 100:1, vortex to mix, and add 50μL to each EP tube. Freeze at -80℃. After 2h, remove and place on ice. After the protein thaws, vortex every 5min for at least 10s, for a total of 6 vortexes. Pre-cool the centrifuge, centrifuge at 12000 rcf for 15 min, and transfer the supernatant to a new 1.5 mL EP tube, which is the total protein.

[0134] (3) Protein quantification: Different concentrations of BSA standard samples A, B, C, D, E, F, G, H, and I were prepared using BSA and ultrapure water. The protein samples were appropriately diluted 10-fold (1 μL protein sample + 9 μL ultrapure water) according to the cell concentration. In a 96-well plate, 10 μL of each of the prepared BSA standard samples A, B, C, D, E, F, G, H, and I were added sequentially from top to bottom. The diluted protein sample was added to the blank wells. The required amount of working solution was calculated and prepared at a ratio of 50:1 (A solution to B solution). After vortexing, the solution was poured into the loading tank. 200 μL of working solution was added to each well using a pipette, thoroughly mixed, shaken for 30 seconds, and incubated in an incubator for 30 minutes at 37°C. The samples were then placed in a microplate reader and detected at a wavelength of 570 nm. Data were recorded, a standard curve was plotted, and the required protein concentration was calculated.

[0135] (4) Gel preparation: Clean the glass plate thoroughly to prevent adhesive from adhering to it. Then, align the bottom and sides and place it in the clamp, securing both sides simultaneously. Place it vertically on the rack and pour ultrapure water until it overflows from the upper edge of the small glass plate to check for leaks. At the same time, prepare to prepare a 10% separating gel. After adding 30% acrylamide, 1.5 M Tris (pH 8.8), 10% SDS, and ultrapure water, if the water level in the glass plate does not drop, pour out the water and blot dry with paper. Continue to prepare the 10% separating gel. After adding TEMED and APS, mix immediately. Use a pipette to evenly add the gel up to the center line height of the glass plate. If uneven edges or air bubbles appear, evenly add a layer of isopropanol on the gel. Let it stand for 40 minutes until the separating gel solidifies. Rinse off the upper layer of isopropanol with ultrapure water and blot dry with paper. Prepare a 1.5mm 10-well comb in advance. Accurately prepare a 4% concentrated resin solution. Add TEMED and APS, mix well immediately, and pour into a glass plate up to the top edge. After removing air bubbles, insert the comb vertically. Let it stand for about 20 minutes until the concentrated resin solidifies.

[0136] (5) Sample processing: According to the calculated data, first add ultrapure water, vortex centrifuge the protein sample and then add it, vortex centrifuge again, add a certain amount of 5×loading loading buffer to make its final concentration 1×loading, vortex centrifuge, place it in a 95℃ water bath and heat for 5 minutes to denature the protein, take it out and let it cool before centrifuging again.

[0137] (6) Sample loading: Rinse the gel plates with water to remove any adhesive residue from the outside. Clamp the plates together and place them in the electrophoresis tank. Fill the space between the two gel plates with electrophoresis buffer. Hold the comb by both sides and gently pull it out vertically. Remove any air bubbles in the lanes. After loading the samples, slowly add an appropriate amount of electrophoresis buffer.

[0138] (7) Electrophoresis;

[0139] (8) Transfer: 1. Preparations for transfer during electrophoresis: Preheat the transfer buffer to ice. Cut a PVDF membrane to 8cm × 5.5cm and activate it in methanol before transferring it to the transfer buffer. Soak appropriately sized filter paper and sponges in the transfer buffer. 2. Place a layer of sponge in the transfer apparatus, then lay four layers of filter paper on top of the sponge. Pry open the glass plate, gently scrape off the stacking gel, peel off the separating gel, and place it in the transfer buffer. Lay the separating gel on the filter paper, then lay the PVDF membrane on top of the separating gel, removing any air bubbles. Avoid letting the separating gel and PVDF membrane dry out during the process. Place four layers of filter paper and one layer of sponge on top of the PVDF membrane, and secure the clamps. During the transfer process, the following order should be placed in the transfer tank: positive electrode (white) — sponge — four layers of filter paper — PVDF membrane — separating gel — four layers of filter paper — sponge — negative electrode (black). After installing the transfer apparatus and connecting the positive and negative electrodes, place it in an ice basin and transfer the membrane for about 2.5 hours at a current of 220mA.

[0140] (9) Blocking: Prepare a 5% skim milk powder (skim milk powder: TTBS = 1:20) blocking solution. After the transfer is completed, take out the PVDF membrane with tweezers, and put the two membranes in contact with the blocking solution. Shake slowly on a shaker at room temperature for 1 hour.

[0141] (10) Immunological reaction: Incubation with primary antibody: Prepare primary antibody according to the ratio (antibody: blocking buffer = 1:1000). After blocking, according to the molecular weight shown by the marker, cut out the region containing the target protein and place it in a hybridization bag. Add an appropriate amount of the prepared primary antibody according to the width of the band, remove air bubbles, seal the hybridization bag, and place it on a shaker. Incubate overnight at 4°C with gentle shaking. The next day, remove the band and place it in 1×TTBS. Wash it 4 times on a shaker, changing the TTBS every 10 minutes. Incubation with secondary antibody: Prepare secondary antibody according to the ratio (antibody: TTBS = 1:2000). Place the band in a hybridization bag, add the prepared secondary antibody, seal the hybridization bag, and incubate at room temperature on a shaker with gentle shaking for 1 hour. Remove the band and place it in 1×TTBS. Wash it 4 times on a shaker, changing the TTBS every 10 minutes.

[0142] (11) ECL development: Prepare the developing solution (solution A:solution B = 1:1) and use the Bio-Rad chemiluminescence system for imaging. Place the strips face up in the imaging area, add the developing solution dropwise onto the PVDF membrane, about 200 μL of developing solution per strip, and select appropriate parameters for development.

[0143] Effect of compound 1 on the expression of proteins in the PI3K / Akt / mTOR pathway

[0144] In CAL33 and SCC4 cells, compound 1 dose-dependently induced a decrease in the expression levels of p-PDK1, p-Akt, and p-mTOR, see [see details]. Figure 5 .

[0145] 5. In vivo antitumor assay of compound 1 in a CAL33 nude mouse xenograft model

[0146] Tumor-bearing mice were induced by subcutaneous injection of CAL33 cells into male BALB / c nude mice. Once the tumor volume reached 5mm × 5mm × 5mm, the mice were randomly divided into the following groups: control (model) group, low-dose compound 1 group (100mg / kg), and high-dose compound 1 group (200mg / kg). The mice were then administered the compound 1 at the above doses via gavage once daily for 16 days. After 16 days, the animals were photographed, the nude mice were dissected, the tumors were removed, photographed, and their volume and weight were measured.

[0147] In vivo antitumor assay of compound 1 in a CAL33 nude mouse xenograft model.

[0148] Sixteen days after oral administration of the compound, tumor volume and weight in the treated group were significantly smaller compared to the control group. There was no significant difference in body weight among the treatment groups, demonstrating that no significant toxic side effects were observed in any treatment group. Overall, compound 1 shows a significant antitumor effect. Figure 6 .

Claims

1. An oxazine alkyl compound as shown in general formula (I) or a pharmaceutically acceptable salt thereof: in, R1 and R2 are independently selected from hydrogen, halogen, and -OR 1a amino; Among them, R 1a Selected from C 1-6 Alkyl groups; R3 is hydrogen, C 1-6 Alkyl or CH n X 3-n , where X is a halogen and n is 0, 1 or 2; R4 can be homomorpholino, morpholino, oxazinyl, piperazinyl, oxazolyl, piperidinyl, or pyrrolidinyl.

2. The oxazine alkyl compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: in, R1 and R2 are hydrogen atoms, and R3 is a difluoromethyl group; Alternatively, R1 and R2 may be either bromine atoms or -OCH3, and R3 may be a difluoromethyl group; R1 and R2 may be either amino or -OCH3, and R3 may be difluoromethyl.

3. The oxazine alkyl compound or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: in, R1 and R2 are hydrogen atoms, R3 is difluoromethyl, and R4 is homomorpholino or morpholino. Alternatively, R1 and R2 may be either bromine atoms or -OCH3, R3 may be difluoromethyl, and R4 may be morpholino; R1 and R2 may be either amino or -OCH3, R3 may be difluoromethyl, and R4 may be morpholino.

4. The oxazine alkyl compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: This compound is 3-(4-(2-(difluoromethyl)-1H-benzo[d]imidazol-1-yl)-6-morpholino-1,3,5-triazin-2-yl)-1,3-oxazine; Or 3-(4-(6-bromo-2-(difluoromethyl)-4-methoxy-1H-benzo[d]imidazol-1-yl)-6-morpholino-1,3,5-triazin-2-yl)-1,3-oxazine; Or 2-(difluoromethyl)-4-methoxy-1-(4-morpholino-6-(1,3-oxazinyl-3-yl)-1,3,5-triazinyl-2-yl)-1H-benzo[d]imidazol-6-amine; Or 4-(4-(2-(difluoromethyl)-1H-benzo[d]imidazol-1-yl)-6-(1,3-oxazinyl-3-yl)-1,3,5-triazinyl-2-yl)-1,4-homomorpholine.

5. The method for preparing an oxazine alkyl compound according to claim 4, characterized in that: The following synthetic routes are included: 。 6. The method for preparing an oxazine alkyl compound according to claim 5, characterized in that: The molar ratio of compound II to ammonium chloride is 1:(15-25); The molar ratio of compound III to compound IV is 1:(15-25); The molar ratio of compound V to compound VI is 1:1; the molar ratio of compound VII to compound VIII is 1:(1-10). The reaction temperature of compound II with ammonium chloride is 70℃-90℃; the reaction temperature of compound III with compound IV is 95℃-115℃. The reaction temperature of compound V with compound VI is 20℃-30℃; the reaction temperature of compound VII with compound VIII is 60℃-85℃.

7. The method for preparing an oxazine alkyl compound according to claim 6, characterized in that: Compound II reacts with ammonium chloride using ethanol as the solvent; Compound III reacts with Compound IV using 6 mol / L hydrochloric acid solution as the solvent.

8. The method for preparing an oxazine alkyl compound according to claim 6, characterized in that: Compound V reacts with compound VI using DMF as the solvent; compound VII reacts with compound VIII using THF or oxazine as the solvent.

9. A pharmaceutical composition comprising a pharmaceutically acceptable carrier or an oxazine compound as described in any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof.

10. The use of the oxazine alkyl compound of any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 7 in the preparation of an antitumor drug.

11. The application according to claim 10, characterized in that, The tumor is one of human head and neck squamous cell carcinoma HSC2, HSC3, HSC4, Cal33, SCC4, and human glioma cell U251.

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