CN113200969B

CN113200969B - PI3K alpha selective inhibitor and preparation method and application thereof

Info

Publication number: CN113200969B
Application number: CN202110544311.4A
Authority: CN
Inventors: 杨鹏; 张芳; 后毅; 闵文剑; 袁凯; 谢胜男
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2021-05-19
Filing date: 2021-05-19
Publication date: 2022-11-25
Anticipated expiration: 2041-05-19
Also published as: CN113200969A; WO2022242321A1

Abstract

The invention discloses a PI3K alpha selective inhibitor, a preparation method and application thereof, and a triazine benzo oxazole compound, a preparation method and application thereof, wherein the structural formula of the compound is shown as a formula I, or pharmaceutically acceptable salts thereof. According to the application, the compounds have good inhibitory activity on PI3K alpha through biochemical activity tests, so that the compounds can provide effective and selective inhibitors for disease treatment regulated and controlled by PI3K, and further, targeted drugs for treating diseases related to PI3K signal pathways, such as gastric cancer, breast cancer, ovarian cancer, leukemia and the like, are expected to be developed.

Description

PI3K alpha selective inhibitor and preparation method and application thereof

Technical Field

The invention belongs to the field of medicinal chemistry, and particularly relates to a PI3K alpha selective inhibitor, and a preparation method and application thereof.

Background

Cancer has been recognized as a leading cause of death in every national population of the world and is a serious threat to human life and health. According to the latest research report of the international cancer research institute, 1930 ten thousand new cancer cases and nearly 1000 ten thousand cancer deaths occur in 2020 worldwide. This disease is a significant cause of morbidity and mortality worldwide, regardless of the human development level. Currently, clinical therapeutic drugs for cancer are mainly classified into traditional cytotoxic drugs and novel molecular targeted drugs. In recent years, targeting molecule drugs have begun to enter the central stage of the global anticancer market due to the fact that targeting molecule drugs have a relatively definite action target, thereby improving selectivity to cancer cells, reducing toxicity to normal tissues and the like. The molecular targeted therapy of cancer uses the marker molecule of cancer cell as target spot, and intervenes the process of canceration of cell, such as inhibiting cancer cell proliferation, interfering cell cycle, inducing cancer cell differentiation, inhibiting cancer cell metastasis, inducing cancer cell apoptosis and inhibiting tumor angiogenesis, etc. to achieve the purpose of treating cancer. Growth factor receptors of tumors, signal transduction molecules, cyclin, apoptosis regulators, proteolytic enzymes, vascular endothelial growth factors, and the like can be used as molecular targets for cancer treatment.

Phosphatidylinositol-3-kinase (PI 3K) -protein kinase B (Akt/PKB) -mammalian target of rapamycin (mTOR) pathway is an important signaling pathway in mammalian tumor immunity, and is closely related to cell proliferation, cell cycle progression, cell survival, cell growth, angiogenesis and the like. The activation of the pathway is closely related to the apoptosis and migration of tumor cells, the occurrence and development of tumors and drug resistance. The upstream member of this pathway, PI3K, is a highly conserved family of lipid kinases that are capable of integrating growth factors, cytokines and other environmental signals, converting them into intracellular signals, regulating multiple signaling pathways and thereby modulating human cellular and biological functions. In vivo, PI3K can phosphorylate phosphatidylinositol-4, 5-diphosphate (PIP 2) to generate phosphatidylinositol-3, 4, 5-triphosphate (PIP 3), and PIP3 is used as a second messenger to activate downstream AKT, mTOR and the like, thereby playing a role in regulating and controlling cellular processes and physiological functions. Activating mutations, amplification, etc. of PI3K are often found in various cancers, making it a major drug target for anticancer therapy.

PI3K can be classified into type I, type II and type III according to structural characteristics and substrate molecules. Class I PI3 ks are further classified into class IA and class IB according to their regulation. Among them, the IA type PI3K has the closest relationship with tumors, and is a very active field in drug development. It can be divided into 3 subtypes such as PI3K alpha, PI3K beta and PI3K delta according to the difference of catalytic subunits. Wherein PI3K α consists of a catalytic subunit p110 α and a regulatory subunit p 85. Cancer genome map (TCGA) the mutation profile of over 3000 cancers was studied, with the gene PIK3CA encoding PI3K α being listed as the second most common mutant oncogene. Its hot spot mutations are mainly concentrated in the helical region and the kinase domain, including E542K, E545K and H1047R. After mutation, the mutant can inhibit the expression of cancer suppressor PTEN while abnormally activating PI3K alpha, so that PI3K alpha is an important target in the research and development of various anti-cancer drugs.

The PI3K inhibitors studied at present mainly comprise pan-PI 3K inhibitors and specific selective PI3K inhibitors. pan-PI 3K inhibitors can act on all subtype proteins and they lack targeting to a single subtype and therefore have varying degrees of potential toxicity, most of which have stopped development. In contrast, inhibitors which tend to be specific for the subtype, which bind to one or both subtypes, have less off-target toxicity. Up to now, PI3K has 4 classes of inhibitors on the market, namely the PI3K δ inhibitor Idelalisib approved in 2014, the PI3K α/δ inhibitor copalisib marketed in 2017, the PI3K δ/γ inhibitor Duvelisib approved in 2018, and the PI3K α inhibitor Alpelisib marketed in 2019. In addition, there are several dozen inhibitors that are in clinical research, but most of them are specific inhibitors that are on the market and in clinical research. With the difficulty of adverse reactions, research is now more biased towards subtype-specific PI3K inhibitors. Currently, only one selective inhibitor of PI3K α is on the market, and Alpelisib, developed by nova, was approved in 2019 for use in combination with fulvestrant in the treatment of patients with HR carrying mutations in the PIK3CA gene ⁺ /HER ^2- Postmenopausal female and male patients with advanced or metastatic breast cancer who undergo disease progression during or after endocrine treatment regimens. In addition, 6 PI3K α inhibitors are in clinical study, including CH-5132799, CYH-33, inavolisib, serabelisib, ASN-003, and LX-086. However, in addition to the significant effect on breast cancer, clinical feedback regarding solid tumors from PI3K α inhibitors currently under investigation is not ideal for clinical outcome of other solid tumors, and therefore, researchers need to explore new selective inhibitors of PI3K α for the treatment of more cancers.

Disclosure of Invention

The invention aims to: aiming at the prior art, the invention provides a brand-new parent nucleus triazine benzo oxazole compound which has obvious inhibition effect on PI3K alpha, so that the compound can be used for preparing an inhibitor with high selectivity on the PI3K alpha, and further is expected to develop a targeted medicament for treating diseases related to a PI3K signal pathway, such as gastric cancer, breast cancer, ovarian cancer, leukemia and the like. The invention also provides a specific preparation method and medical application of the compound and an intermediate thereof.

The technical scheme is as follows: the invention provides a triazine benzo oxazole compound shown as the following formula (I) or pharmaceutically acceptable salt thereof:

wherein X is selected from N and S; y is selected from O and N;

R ₁ independently selected from one of the following:

R ₂ independently selected from one of the following:

R ₃ independently selected from hydrogen, C ₁ -C ₈ Alkyl radical, C ₃ -C ₈ Cycloalkyl, -C (O) R ₄ 、-S(O) ₂ R ₅ 、

R ₄ Independently selected from C ₁ -C ₈ Alkyl radical, C ₃ -C ₈ Cycloalkyl, unsubstituted or para-R ₇ Substituted phenyl;

R ₅ independently selected from C ₁ -C ₈ An alkyl group;

R ₆ independently selected from hydrogen, hydroxy, mercapto, cyano, nitro, trifluoromethyl, C ₁ -C ₈ Alkyl radical, C ₁ -C ₈ Alkoxy, -C (O) OR ₈ 、-C(O)NHR ₉ ；

R ₇ 、R ₈ 、R ₉ Each independently selected from hydrogen, C ₁ -C ₈ An alkyl group.

Preferably, X is selected from N, S; y is selected from O and N;

preferably, R ₁ Independently selected from one of the following:

preferably, R ₂ Independently selected from one of the following:

preferably, R ₃ Independently selected from hydrogen, isopropyl, cyclopentane, -C (O) R ₄ 、-S(O) ₂ R ₅ 、

R ₄ Selected from methyl, isopropyl, cyclohexane, unsubstituted benzene rings; r ₅ Is selected from methyl; r ₆ Selected from hydrogen, cyano, nitro, trifluoromethyl, isopropyl, methoxy, -C (O) OR ₈ 、-C(O)NHR ₉ ，R ₈ Selected from hydrogen, methyl, R ₉ Selected from hydrogen.

The present invention provides the compound or a pharmaceutically acceptable salt thereof: particularly preferably from I-1 to I-46:

the chemical reaction route of the preparation method of the compound or the pharmaceutically acceptable salt thereof is as follows:

the compound I is prepared by the coupling reaction of a compound A and a compound B under the action of a palladium catalyst.

The compound described herein, or a pharmaceutically acceptable salt thereof, wherein the acid used to form the salt comprises inorganic acids including hydrochloric acid, sulfuric acid, phosphoric acid, and methanesulfonic acid, and organic acids including acetic acid, trichloroacetic acid, propionic acid, butyric acid, maleic acid, p-toluenesulfonic acid, malic acid, malonic acid, cinnamic acid, citric acid, fumaric acid, camphoric acid, digluconic acid, aspartic acid, and tartaric acid. Preferably, the pharmaceutically acceptable salts of the present invention are predominantly hydrochloride salts.

The application also provides a pharmaceutical composition, which comprises the compound shown in the general formula (I) or pharmaceutically acceptable salt, tautomer and stereoisomer, metabolite, metabolic precursor, solvate or prodrug thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition can be in the form of conventional preparations in pharmaceutics such as tablets, capsules, granules, powder, syrup, oral liquid or injection.

The application also provides application of the compound shown in the general formula (I) or pharmaceutically acceptable salt thereof in preparing a PI3K alpha target inhibitor. Further, the compound or the pharmaceutically acceptable salt thereof can be used for preparing medicines for treating diseases regulated by PI 3K. Preferably, the treatment of diseases associated with the PI3K signaling pathway is cancer, immune disorders or inflammatory disorders, and the cancer mainly includes gastric cancer, breast cancer, prostate cancer, lung cancer, liver cancer, bone cancer, brain cancer, head and neck cancer, intestinal cancer, pancreatic cancer, bladder cancer, testicular cancer, ovarian cancer, endometrial cancer, multiple myeloma, and the like.

The compound or the pharmaceutically acceptable salt thereof can also be combined with other medicines with specific targets, such as the compound and the paclitaxel which are combined and applied to the gastric cancer, so that the compound and the paclitaxel have a synergistic treatment effect.

Has the advantages that: compared with the prior art, the application provides a brand-new parent nucleus triazine ring benzo oxazole compound, and the compound has good inhibitory activity on PI3K alpha through biochemical activity test, so that the compound can provide an effective inhibitor with better selectivity for disease treatment regulated by PI3K, and further is expected to develop a targeted drug for treating diseases related to PI3K signal pathways, such as gastric cancer, breast cancer, ovarian cancer, leukemia and the like.

Drawings

FIG. 1 is a graph of the growth of human gastric carcinoma cells MGC-803 xenograft tumors in mice inhibited by Compound I-7;

FIG. 2 is the body weight change of human gastric carcinoma cells MGC-803 xenograft tumor mice during administration of Compound I-7.

Detailed Description

The present application will be described in detail with reference to specific examples.

The raw materials used for synthesis in the invention are all directly purchased or prepared by conventional operation, and the commercially available raw materials are directly used for chemical reaction without being treated unless specified.

Synthesis of intermediate reactants

1) Synthesis of 4- (4, 6-dichloro-1, 3, 5-triazin-2-yl) morpholine (M1)

Cyanuric chloride (10.0 g,1.0 eq.) as a raw material was dissolved in acetone, triethylamine (10.9 g,2.0 eq.) was added, a morpholine (4.72g, 1.0 eq.) solution was diluted with acetone and placed in a dropping funnel, and the solution was slowly dropped into the reaction flask at-10 ℃. After the morpholine solution is dripped, the reaction is monitored by a TLC plate immediately, and the reaction is stopped when the raw materials are basically reacted. The solution is poured into crushed ice, filtered and washed by water to obtain 11.67g of white solid which is directly fed to the next step. The equivalent of morpholine in the step is controlled to be 1.0 time, otherwise trisubstituted byproducts are generated, and the post-treatment step is increased. Second, the morpholine solution needs to be diluted with acetone and added slowly at low temperature, otherwise trisubstituted by-products also appear. White solid, yield 92%, MS (ESI) m/z 236.1, [ M ] +H] ⁺ .

2) Synthesis of 4- (4-chloro-6- (4- (methylsulfonyl) piperazin-1-yl) -1,3, 5-triazin-2-yl) morpholine (M2)

Intermediate M1 (235.07mg, 1.0 eq.) was dissolved in tetrahydrofuran, DIPEA (193.88mg, 1.5 eq.) was added as a base, the starting material N-methylsulfonylpiperazine (164.23mg, 1.0 eq.) was diluted with tetrahydrofuran and added dropwise to the reaction flask under ice-bath conditions, the reaction was monitored by TLC, water was added after the reaction was completed, extraction was performed 3 times with DCM, the organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and further purified by flash preparative liquid chromatography. Obtain white solid 185mg, yield 51%, MS (ESI) m/z 363.8M + H] ⁺ .

3) Synthesis of 4- (4-chloro-6- (4-ethylpiperazin-1-yl) -1,3, 5-triazin-2-yl) morpholine (M3)

Reference is made to the synthesis of intermediate compound M2, a white solid, in 82% yield, MS (ESI) M/z 313.8, M + H] ⁺ .

4) Synthesis of 4- (4-chloro-6- (4-isopropylpiperazin-1-yl) -1,3, 5-triazin-2-yl) morpholine (M4)

Referring to the synthesis of intermediate compound M2, white solid, yield 85%, MS (ESI) M/z 327.8[ 2 ], [ M + H ]] ⁺ .

5) Synthesis of 1- (4- (4-chloro-6-morpholino-1, 3, 5-triazin-2-yl) piperazin-1-yl) ethan-1-one (M5)

Referring to the synthesis of intermediate compound M2, a white solid, yield 67%, MS (ESI) M/z 327.8, [ M ] +H ] +.

6) Synthesis of tert-butyl 4- (4-chloro-6-morpholino-1, 3, 5-triazin-2-yl) piperazine-1-carboxylate (M6)

Referring to the synthesis of intermediate compound M2, a white solid, yield 80%, MS (ESI) M/z 385.9[ 2 ], [ M + H ] +.

7) Synthesis of 4- (4-chloro-6- (4- (trifluoromethyl) piperidin-1-yl) -1,3, 5-triazin-2-yl) morpholine (M7)

Referring to the synthesis method of intermediate compound M2, white solid, yield 68%, MS (ESI) M/z 352.8, [ M + H ] +.

8) Synthesis of 4,4' - (6-chloro-1, 3, 5-triazine-2, 4-diyl) dimorpholine (M8)

With reference to the synthesis of intermediate compound M1, a white solid, yield 95%, MS (ESI) M/z 286.7, [ M + H ] +.

9) Synthesis of 4-chloro-6-morpholino-N- (tetrahydro-2H-pyran-4-yl) -1,3, 5-triazin-2-amine (M9)

With reference to the synthesis of intermediate compound M2, a white solid, yield 55%, MS (ESI) M/z 300.8, [ M ] +H ] +.

10 Synthesis of 4-chloro-6-morpholino-N- (3- (trifluoromethyl) phenyl) -1,3, 5-triazin-2-amine (M10)

With reference to the synthesis of intermediate compound M2, a white solid, yield 78%, MS (ESI) M/z 360.7, [ M + H ] +.

11 Synthesis of N- (benzo [ d ] [1,3] dioxa-5-yl) -4-chloro-6-morpholino-1, 3, 5-triazin-2-amine (M11)

Referring to the synthesis of intermediate compound M2, a white solid, yield 58%, MS (ESI) M/z 336.7, [ M ] +H ] +.

12 Synthesis of 5- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzo [ d ] oxazol-2-amine (M12)

Raw materials of 2-amino-5-bromobenzoxazole (100mg, 1.0eq.) and pinacol diboron (131mg, 1.1eq.), potassium acetate (138.4mg, 3.0eq.) as a base, 1' -bis (diphenylphosphine ferrocene) palladium dichloride (34.4mg, 0.1eq.) as a catalyst, 1, 4-dioxane as a solvent are added into a three-port reaction bottle, the three-port reaction bottle is protected by argon gas, the reaction is carried out for 12 hours at 105 ℃, a reaction solution is taken by a long needle head, the reaction is monitored by TLC, and the reaction of the raw materials is stopped after the reaction is finished. Filtering while hot, filtering to remove salt and metal catalyst, concentrating the residual reaction solution under reduced pressure, and purifying the crude product by rapid preparative liquid chromatography. The reaction time is important in this step, because potassium acetate is used as a base, and if the reaction time exceeds 12h, the amino terminal of the product is easily provided with an acetyl group, and a byproduct is generated. Light brown solid 67mg, yield 55%, MS (ESI) m/z 261.1M + H ] +.

13 Synthesis of 6- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzo [ d ] thiazol-2-amine (M13)

Reference is made to the synthesis of intermediate compound M12. However, the benzothiazole ring has reduced reactivity, most of the raw material remains by using the original 1,1' -bis diphenylphosphino ferrocene palladium dichloride as a catalyst, and the product has similar polarity with the raw material and is difficult to separate by aftertreatment. So in the case of trying 1,1 '-bis diphenylphosphino ferrocene palladium dichloride as catalyst and potassium acetate or cesium carbonate as base, we exchanged for [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride dichloromethane complex as catalyst and potassium acetate or cesium carbonate as base, which is better than potassium acetate. Reacting at 105 ℃ for 12 hours by using 1, 4-dioxane as a solvent under the protection of argon, taking reaction liquid by using a long needle, monitoring the reaction by using TLC, and stopping the reaction after the raw materials are reacted. Filtering while hot, filtering to remove salt and metal catalyst, concentrating the residual reaction solution under reduced pressure, and purifying the crude product by rapid preparative liquid chromatography. Light brown solid, yield 50%, MS (ESI) m/z 277.2[ 2 ], [ M + H ] +.

14 Synthesis of 2, 5-dibromobenzo [ d ] oxazole (M14)

Dissolving raw materials of 2-amino-5-bromobenzoxazole (229mg, 1.0eq.) and copper bromide (335mg, 1.5eq.) in acetonitrile, dropwise adding an acetonitrile solution of isoamyl nitrite (140mg, 1.2eq.) at 0 ℃, keeping the reaction at 0 ℃ for 1h, monitoring the reaction by TLC, adding water after the reaction is finished, precipitating a solid, filtering and drying. White solid, yield 58%, MS (ESI) m/z 277.9, [ M + H ] +.

15 Synthesis of 5-bromo-N-cyclopentylbenzo [ d ] oxazol-2-amine (M15)

Intermediate M14 (90mg, 1.0 eq.) and cyclopentylamine (77mg, 3.0 eq.) were dissolved in 1,4 dioxane, reacted overnight at 80 ℃, monitored by TLC, and after completion of the reaction, the reaction solution was concentrated under reduced pressure, and the crude product was further purified by flash preparative liquid chromatography. White solid, yield 40%, MS (ESI) m/z 282.2, [ M ] +H ] +.

16 Synthesis of 5-bromo-N-isopropylbenzo [ d ] oxazol-2-amine (M16)

Reference is made to the synthesis of intermediate compound M15. White solid, yield 52%, MS (ESI) m/z 256.1[ m + H ] +.

17 Synthesis of N-cyclopentyl-5- (4, 5-tetramethyl-1, 3-dioxolan-2-yl) benzo [ d ] oxazol-2-amine (M17)

Reference is made to the synthesis of intermediate compound M12. White solid, yield 65%, MS (ESI) m/z 331.4, (+) M + H ] +.

18 Synthesis of N-isopropyl-5- (4, 5-tetramethyl-1, 3-dioxolan-2-yl) benzo [ d ] oxazol-2-amine (M18)

Reference is made to the synthesis of intermediate compound M12. White solid, yield 65%, MS (ESI) m/z 305.4[ 2 ], [ M + H ] +.

19 Synthesis of 6-bromo-N, N-dimethylbenzo [ d ] thiazol-2-amine (M19)

Raw materials of 4-bromo-2-iodobenzylamine (19.4g, 1.0eq.) and thiram (21g, 2.0eq.) were subjected to a direct one-pot method in the presence of ketoacetate (12g, 1.0eq.) and potassium carbonate (18.4g, 2.0eq.) by using N, N-dimethylformamide as a solvent, subjected to a reaction at 120 ℃ for 6 hours, subjected to tlc monitoring, added with water after the reaction is completed, extracted with DCM 3 times, and an organic layer was dried over sodium sulfate anhydrous, concentrated under reduced pressure, and further purified by flash preparative liquid chromatography. White solid, yield 49%, MS (ESI) m/z 257.0[ 2 ], [ M + H ] +.

20 Synthesis of N, N-dimethyl-6- (4, 5-tetramethyl-1, 3-dioxolan-2-yl) benzo [ d ] thiazol-2-amine (M20)

Reference is made to the synthesis of intermediate compound M12. White solid, yield 52%, MS (ESI) m/z 307.4, [ M + H ] +.

21 Synthesis of 2-chloro-4, 6-bis (4- (methylsulfonyl) piperazin-1-yl) -1,3, 5-triazine (M21)

With reference to the synthesis of M2, white solid, yield 76%, MS (ESI) M/z 441.0[ M + H ] +, this step is controlled to control the equivalent of the corresponding amine to 2.0eq. And solid may be directly separated out in the reaction process, and the solid can be directly filtered out by suction and then further purified by fast preparative liquid chromatography.

22 Synthesis of N2, N4-bis (benzo [ d ] [1,3] dioxa-5-yl) -6-chloro-1, 3, 5-triazine-2, 4-diamine (M22)

Reference to the synthesis of M2, a white solid, yield 71%, MS (ESI) M/z 386.8[ 2 ], [ M + H ] +.

23 Synthesis of 1,1' - (((6-chloro-1, 3, 5-triazine-2, 4-diyl) bis (piperazin-4, 1-diyl)) bis (eth-1-one) (M23)

Reference is made to the synthesis of intermediate compound M2 as a white solid, in 82% yield, MS (ESI) M/z 368.8[ 2 ], [ M + H ] +.

Synthesis of Compounds I-1 to I-46

Example 1

1) Synthesis of 5- (4- (4- (methylsulfonyl) piperazin-1-yl) -6-morpholino-1, 3, 5-triazin-2-yl) benzo [ d ] oxazol-2-amine (I-1):

the intermediates M2 (100mg, 1.0eq.) and M12 (91mg, 1.0eq.) were dissolved in ethylene glycol dimethyl ether, sodium carbonate (74.2mg, 2.0eq.) was dissolved in a small amount of water as a base, and then added to a reaction flask, tetrakis (triphenylphosphine) palladium (40.5mg, 0.1eq.) was added as a catalyst, and the mixture was reacted at 92 ℃ for 24 hours under argon protection. TLC monitoring reaction, after the reaction was completed, filtering while hot, filtering to remove salts and metal catalyst, extracting the remaining reaction solution with DCM for 3 times, drying the organic layer with anhydrous sodium sulfate, concentrating under reduced pressure, further purifying by fast preparative liquid chromatography to obtain a white solid of 109mg, yield of 68%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.16(d,J＝1.7Hz,1H),8.08(dd,J＝8.3,1.7Hz,1H),7.53(s,2H),7.39(d,J＝8.3Hz,1H),4.02–3.66(m,12H),3.20(d,J＝6.0Hz,4H),2.89(s,3H)ppm； ¹³ C NMR(126MHz,DMSO-d ₆ )δ170.19,165.06,164.94,163.79,150.96,144.19,132.87,121.48,115.36,108.39,66.50,45.76,43.75,42.75,34.27ppm；HRMS(ESI)m/z calcd.for C ₁₉ H ₂₄ N ₈ O ₄ S(M+H)+461.1714,found:461.1712.

example 2

2) Synthesis of 5- (4- (4-ethylpiperazin-1-yl) -6-morpholino-1, 3, 5-triazin-2-yl) benzo [ d ] oxazol-2-amine (I-2):

the synthesis according to example 1, white solid, yield 82%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.13(d,J＝1.6Hz,1H),8.06(dd,J＝8.4,1.7Hz,1H),7.52(s,2H),7.37(d,J＝8.4Hz,1H),3.79–3.64(m,12H),2.41–2.35(m,6H),1.03(t,J＝7.1Hz,3H)ppm；HRMS(ESI)m/z calcd.for C ₂₀ H ₂₆ N ₈ O ₂ (M+H)+411.2251,found:411.2247.

example 3

3) Synthesis of 5- (4- (4-isopropylpiperazin-1-yl) -6-morpholino-1, 3, 5-triazin-2-yl) benzo [ d ] oxazol-2-amine (I-3):

the synthesis of reference example 1, white solid, yield 67%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.14(d,J＝1.6Hz,1H),8.06(dd,J＝8.4,1.7Hz,1H),7.52(s,2H),7.38(d,J＝8.4Hz,1H),3.86–3.65(m,12H),2.73-2.66(m,1H),2.51-2.47(m,4H),0.99(d,J＝6.5Hz,6H)ppm；HRMS(ESI)m/z calcd.for C ₂₁ H ₂₈ N ₈ O ₂ (M+H) ⁺ 425.2408,found:425.2406.

example 4

4) Synthesis of 1- (4- (4- (2-aminobenzo [ d ] oxazol-5-yl) -6-morpholino-1, 3, 5-triazin-2-yl) piperazin-1-yl) ethyl-1-one (I-4):

the synthesis of reference example 1, white solid, yield 81%, ¹ H NMR(300MHz,DMSO-d ₆ )δ8.15(d,J＝1.6Hz,1H),8.08(dd,J＝8.7,1.4Hz,1H),7.55(s,2H),7.39(d,J＝8.4Hz,1H),3.83–3.53(m,12H),3.53(s,4H),2.06(s,3H)ppm； ¹³ C NMR(126MHz,DMSO)δ170.07,168.90,165.05,165.01,163.79,150.93,144.20,132.95,121.45,115.35,108.37,66.50,45.89,43.75,43.36,21.79ppm；HRMS(ESI)m/z calcd.for C ₂₀ H ₂₄ N ₈ O ₃ (M+H) ⁺ 425.2044,found:425.2044.

example 5

5) Synthesis of 4- (4- (2-aminobenzo [ d ] oxazol-5-yl) -6-morpholino-1, 3, 5-triazin-2-yl) piperazine-1-carboxylic acid tert-butyl ester (I-5):

the synthesis according to example 1, white solid, yield 53%, ¹ H NMR(300MHz,CDCl ₃ )δ8.41(d,J＝1.6Hz,1H),8.22(dd,J＝8.5,1.7Hz,1H),7.31(d,J＝8.4Hz,1H),5.41(s,2H),3.92-3.49(m,16H),1.50(s,9H)ppm；HRMS(ESI)m/z calcd.for C ₂₃ H ₃₀ N ₈ O ₄ (M+H) ⁺ 483.2463,found:483.2453.

example 6

6) Synthesis of 5- (4-morpholino-6- (4- (trifluoromethyl) piperidin-1-yl) -1,3, 5-triazin-2-yl) benzo [ d ] oxazol-2-amine (I-6):

synthesis of white solid according to example 1, yieldThe rate of the reaction is 62.5 percent, ¹ H NMR(300MHz,DMSO-d ₆ )δ8.14(d,J＝1.6Hz,1H),8.07(dd,J＝8.4,1.7Hz,1H),7.52(s,2H),7.38(d,J＝8.4Hz,1H),4.92(d,J＝46.4Hz,2H),3.82–3.65(m,8H),2.94(t,J＝12.9Hz,2H),2.74–2.65(m,1H),1.92(d,J＝12.6Hz,2H),1.45–1.33(m,2H)ppm；HRMS(ESI)m/z calcd.for C ₂₀ H ₂₂ F ₃ N ₇ O ₂ (M+H) ⁺ 450.1860,found:450.1841.

example 7

7) Synthesis of 6- (4, 6-dimorpholino-1, 3, 5-triazin-2-yl) benzo [ d ] oxazol-2-amine (I-7):

the synthesis according to example 1, white solid, yield 68%, ¹ H NMR(300MHz,DMSO-d ₆ )δ8.15(d,J＝1.6Hz,1H),8.08(dd,J＝8.4,1.7Hz,1H),7.55(s,2H),7.38(d,J＝8.4Hz,1H),3.84-3.65(m,16H). ¹³ C NMR(126MHz,DMSO)δ169.67,164.68,163.43,150.56,143.85,132.60,121.08,114.99,107.97,66.13,43.37ppm；HRMS(ESI)m/z calcd.for C ₁₈ H ₂₁ N ₇ O ₃ (M+H) ⁺ 384.1779,found:384.1767.

example 8

8) Synthesis of 5- (4-morpholino-6- (((tetrahydro-2H-pyran-4-yl) amino) -1,3, 5-triazin-2-yl) benzo [ d ] oxazol-2-amine (I-8):

the synthesis of reference example 1, white solid, yield 85%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.13(dd,J＝5.1,1.6Hz,1H),8.07-8.01(m,1H),7.51(s,2H),7.46(d,J＝7.7Hz,1H),7.41–7.27(m,1H),4.16(dt,J＝7.7,3.9Hz,1H),3.88–3.64(m,10H),3.47–3.38(m,2H),1.84(dd,J＝12.6,5.7Hz,2H),1.58–1.48(m,2H)ppm；HRMS(ESI)m/z calcd.for C ₁₉ H ₂₃ N ₇ O ₃ (M+H)+398.1935,found:398.1922.

example 9

9) Synthesis of 5- (4-morpholino-6- ((3- (trifluoromethyl) phenyl) amino) -1,3, 5-triazin-2-yl) benzo [ d ] oxazol-2-amine (I-9):

the synthesis of reference example 1, white solid, yield 51%, ¹ H NMR(400MHz,DMSO-d ₆ )δ10.03(s,1H),8.49(s,1H),8.19(d,J＝1.6Hz,1H),8.11(dd,J＝8.3,1.7Hz,1H),7.89(d,J＝8.3Hz,1H),7.57(d,J＝6.3Hz,3H),7.44(d,J＝8.4Hz,1H),7.35(d,J＝7.8Hz,1H),3.96–3.70(m,8H)ppm；HRMS(ESI)m/z calcd.for C ₂₁ H ₁₈ F ₃ N ₇ O ₂ (M+H) ⁺ 458.1547,found:458.1540.

example 10:

10 Synthesis of 5- (4- (benzo [ d ] [1,3] dioxazol-5-ylamino) -6-morpholino-1, 3, 5-triazin-2-yl) benzo [ d ] oxazol-2-amine (I-10):

the synthesis of reference example 1, white solid, yield 49%, ¹ H NMR(400MHz,DMSO-d ₆ )δ9.57(s,1H),8.16(s,1H),8.07(dd,J＝8.4,1.7Hz,1H),7.55(s,2H),7.42(d,J＝8.4Hz,1H),7.14(dd,J＝8.5,2.1Hz,1H),6.88(d,J＝8.4Hz,1H),6.00(s,2H),3.91–3.69(m,8H)ppm； ¹³ C NMR(126MHz,DMSO-d ₆ )δ170.04,164.70,164.11,163.49,150.64,147.07,143.92,142.39,134.24,132.49,120.98,114.94,112.83,108.13,108.05,102.32,100.96,66.08,43.50ppm；HRMS(ESI)m/z calcd.for C ₂₁ H ₁₉ N ₇ O ₄ (M+H) ⁺ 434.1571,found:434.1564.

example 11

11 Synthesis of 5- (4-morpholino-6- (piperazin-1-yl) -1,3, 5-triazin-2-yl) benzo [ d ] oxazol-2-amine hydrochloride (I-11):

putting the compound of example 5 into a reaction bottle, introducing hydrochloric acid gas by taking dichloromethane as a solvent, reacting for 2h, decompressing and evaporating the redundant solvent to obtain a white solid with the yield of 100 percent, ¹ H NMR(400MHz,D ₂ O)δ8.08–8.04(m,1H),7.99–7.90(m,1H),7.49–7.42(m,1H),4.13(s,4H),3.85–3.77(m,8H),3.35–3.32(m,4H)ppm；HRMS(ESI)m/z calcd.for C ₁₈ H ₂₂ N ₈ O ₂ (M+H) ⁺ 383.1938,found:383.1935.

example 12

12 Synthesis of 4- (4- (2- (benzylamino) benzo [ d ] oxazol-5-yl) -6-morpholino-1, 3, 5-triazin-2-yl) piperazine-1-carboxylic acid tert-butyl ester (I-12):

the compound of example 5 (300mg, 1.0 eq.) was dissolved in N, N-dimethylformamide, naH (28mg, 1.1 eq.) was added to the solution for 2h while cooling, bromobenzyl (117mg, 1.1 eq.) was diluted with N, N-dimethylformamide and slowly dropped into the flask, reacted at room temperature for 3h, followed by tlc monitoring, water was added to quench the reaction after completion of the reaction, DMF was washed off with a large amount of water, extracted 3 times with DCM, the organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and further purified by flash preparative liquid chromatography. 141mg of white solid, 40% yield, ¹ H NMR(300MHz,DMSO-d ₆ )δ8.01(dd,J＝8.3,1.7Hz,1H),7.73(d,J＝1.7Hz,1H),7.43–7.40(m,2H),7.38–7.29(m,3H),7.22(d,J＝8.4Hz,1H),6.94(s,1H),5.07(s,2H),3.78–3.64(m,12H),3.42–3.38(m,4H),1.43(s,9H)ppm；HRMS(ESI)m/z calcd.for C ₃₀ H ₃₆ N ₈ O ₄ (M+H) ⁺ 573.2932,found:573.2916.

example 13

13 Synthesis of N-cyclopentyl-5- (4, 6-dimorpholino-1, 3, 5-triazin-2-yl) benzo [ d ] oxazol-2-amine (I-13):

the synthesis according to example 1, white solid, yield 59%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.22(d,J＝1.6Hz,1H),8.08–8.06(m,2H),7.39(d,J＝8.4Hz,1H),4.05(d,J＝6.2Hz,1H),3.88–3.65(m,16H),1.95(q,J＝5.1Hz,2H),1.70(dq,J＝9.0,3.2,2.8Hz,2H),1.59(dq,J＝13.2,5.1Hz,4H)ppm；HRMS(ESI)m/z calcd.for C ₂₃ H ₂₉ N ₇ O ₃ (M+H) ⁺ 452.2405,found:452.2405.

example 14

14 Synthesis of 5- (4, 6-dimorpholino-1, 3, 5-triazin-2-yl) -N-isopropylbenzo [ d ] oxazol-2-amine (I-14):

the synthesis according to example 1, white solid, yield 75%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.21(d,J＝1.6Hz,1H),8.06(dd,J＝8.4,1.7Hz,1H),7.98(d,J＝7.5Hz,1H),7.38(d,J＝8.3Hz,1H),3.90–3.65(m,16H),1.23(d,J＝6.5Hz,6H)ppm；HRMS(ESI)m/z calcd.for C ₂₁ H ₂₇ N ₇ O ₃ (M+H) ⁺ 426.2248,found:426.2240.

example 15

15 Synthesis of N-benzyl-5- (4, 6-dimorpholino-1, 3, 5-triazin-2-yl) benzo [ d ] oxazol-2-amine (I-15):

the synthesis of reference example 12, white solid, yield 55%, ¹ H NMR(300MHz,DMSO-d ₆ )δ8.01(dd,J＝8.3,1.7Hz,1H),7.72(d,J＝1.7Hz,1H),7.43–7.27(m,5H),7.22(d,J＝8.3Hz,1H),6.90(s,1H),5.05(s,2H),3.78–3.64(m,16H)ppm； ¹³ C NMR(126MHz,DMSO)δ169.18,164.91,146.85,136.54,133.27,132.78,129.17,128.22,122.18,108.46,107.75,66.45,60.23,45.87,43.71ppm；HRMS(ESI)m/z calcd.for C ₂₅ H ₂₇ N ₇ O ₃ (M+H) ⁺ 474.2248,found:474.224.

example 16

16 Synthesis of 6- (4, 6-dimorpholino-1, 3, 5-triazin-2-yl) -N- (4-methoxybenzyl) benzo [ d ] oxazol-2-amine (I-16):

the synthesis of reference example 12, a white solid, yield 45%, ¹ H NMR(300MHz,DMSO-d ₆ )δ7.99(dd,J＝8.3,1.7Hz,1H),7.74(d,J＝1.6Hz,1H),7.36(d,J＝8.6Hz,2H),7.20(d,J＝8.3Hz,1H),6.95(s,1H),6.91(d,J＝8.6Hz,2H),4.99(s,2H),3.72(s,3H),3.79–3.66(m,16H)ppm； ¹³ C NMR(126MHz,DMSO)δ168.84,164.54,158.87,146.44,132.87,132.29,129.31,128.08,121.62,114.11,107.89,107.29,66.06,55.14,44.90,43.33ppm；HRMS(ESI)m/z calcd.for C ₂₆ H ₂₉ N ₇ O ₄ (M+H) ⁺ 504.2354,found:504.2347.

example 17

17 Synthesis of 6- (4, 6-dimorpholino-1, 3, 5-triazin-2-yl) -N- (4-isopropylbenzyl) benzo [ d ] oxazol-2-amine (I-17):

the synthesis of reference example 12, white solid, yield 50%, ¹ H NMR(300MHz,DMSO-d ₆ )δ8.01(dd,J＝8.3,1.6Hz,1H),7.79(d,J＝1.6Hz,1H),7.32(d,J＝7.8Hz,2H),7.21(d,J＝8.2Hz,3H),6.89(s,1H),5.02(s,2H),3.84-3.64(m,16H),2.89-2.80(m,1H),1.16(d,J＝6.9Hz,6H)ppm；HRMS(ESI)m/z calcd.for C ₂₈ H ₃₃ N ₇ O ₃ (M+H) ⁺ 516.2718,found:516.2713.

example 18

18 Synthesis of 5- (4, 6-dimorpholino-1, 3, 5-triazin-2-yl) -N- (4-nitrobenzyl) benzo [ d ] oxazol-2-amine (I-18):

the synthesis of reference example 12, a white solid, yield 48%, ¹ H NMR(300MHz,DMSO-d ₆ )δ8.25(d,J＝2.0Hz,1H),8.23(d,J＝1.9Hz,1H),8.04(dd,J＝8.4,1.6Hz,1H),7.76(s,1H),7.66(d,J＝8.4Hz,2H),7.26(d,J＝8.3Hz,1H),7.00(s,1H),5.24(s,2H),3.78–3.63(m,16H)ppm； ¹³ C NMR(126MHz,DMSO-d ₆ )δ169.19,164.91,147.43,146.93,144.68,133.20,132.87,129.17,124.30,122.37,108.54,107.35,66.45,45.20,43.71ppm；HRMS(ESI)m/z calcd.for C ₂₅ H ₂₆ N ₈ O ₅ (M+H) ⁺ 519.2099,found:519.2094.

example 19

19 Synthesis of 4- (((5- (4, 6-dimorpholino-1, 3, 5-triazin-2-yl) benzo [ d ] oxazol-2-yl) amine) methyl) benzonitrile (I-19):

the synthesis of reference example 12, a white solid, yield 51%, ¹ H NMR(300MHz,DMSO-d ₆ )δ8.04(dd,J＝8.4,1.6Hz,1H),7.86(d,J＝8.0Hz,2H),7.72(d,J＝1.6Hz,1H),7.58(d,J＝8.0Hz,2H),7.25(d,J＝8.3Hz,1H),7.01(s,1H),5.19(s,2H),3.81–3.63(m,16H)ppm； ¹³ C NMR(126MHz,DMSO-d ₆ )δ169.20,164.92,146.91,142.55,133.20,133.11,132.86,128.93,122.35,119.10,110.89,108.54,107.41,66.46,49.06,45.42,43.72ppm；HRMS(ESI)m/z calcd.for C ₂₆ H ₂₆ N ₈ O ₃ (M+H) ⁺ 499.2201,found:499.2186.

example 20

20 Synthesis of 5- (4, 6-dimorpholino-1, 3, 5-triazin-2-yl) -N- (4- (trifluoromethyl) benzyl) benzo [ d ] oxazol-2-amine (I-20):

the synthesis of reference example 12, a white solid, yield 52%, ¹ H NMR(300MHz,DMSO-d ₆ )δ8.03(dd,J＝8.3,1.7Hz,1H),7.74(d,J＝7.6Hz,3H),7.61(d,J＝8.1Hz,2H),7.24(d,J＝8.4Hz,1H),6.96(s,1H),5.17(s,2H),3.77–3.63(m,16H)ppm；HRMS(ESI)m/z calcd.for C ₂₆ H ₂₆ F ₃ N ₇ O ₃ (M+H) ⁺ 542.2122,found:542.2106.

example 21

21 Synthesis of 4- (((5- (4, 6-dimorpholino-1, 3, 5-triazin-2-yl) benzo [ d ] oxazol-2-yl) amino) methyl) benzamide (I-21):

the synthesis of reference example 12, a white solid, yield 35%, ¹ H NMR(300MHz,DMSO-d ₆ )δ8.02–7.97(m,2H),7.86(d,J＝7.9Hz,2H),7.68(s,1H),7.47(d,J＝7.9Hz,2H),7.38(s,1H),7.22(d,J＝8.3Hz,1H),6.93(s,1H),5.10(s,2H),3.76–3.63(m,16H)ppm； ¹³ C NMR(126MHz,DMSO)δ168.81,167.50,164.53,146.51,139.47,133.72,132.89,132.36,127.97,127.60,121.78,108.04,107.24,66.08,45.22,43.34ppm；HRMS(ESI)m/z calcd.for C ₂₆ H ₂₈ N ₈ O ₄ (M+H) ⁺ 517.2306,found:517.2301.

example 22

22 Synthesis of methyl 4- (((5- (4, 6-dimorpholino-1, 3, 5-triazin-2-yl) benzo [ d ] oxazol-2-yl) amine) methyl) benzoate (I-22):

the synthesis of reference example 12, a white solid, 62% yield, ¹ H NMR(300MHz,DMSO-d ₆ )δ8.01(dd,J＝8.3,1.7Hz,1H),7.95(d,J＝8.2Hz,2H),7.69(s,1H),7.54(d,J＝8.0Hz,2H),7.23(d,J＝8.3Hz,1H),6.95(s,1H),5.14(s,2H),3.84(s,3H),3.79–3.62(m,16H)ppm； ¹³ C NMR(126MHz,DMSO)δ169.18,166.38,164.90,146.89,142.23,133.26,132.77,130.05,129.39,128.39,122.22,108.46,107.50,66.44,52.62,45.57,43.70ppm；HRMS(ESI)m/z calcd.for C ₂₇ H ₂₉ N ₇ O ₅ (M+H) ⁺ 532.2303,found:532.2299.

example 23

23 Synthesis of 4- (((5- (4, 6-dimorpholino-1, 3, 5-triazin-2-yl) benzo [ d ] oxazol-2-yl) amino) methyl) benzoic acid (I-23):

dissolving the compound of example 22 in anhydrous tetrahydrofuran, adding lithium aluminum hydride and water, stirring overnight at normal temperature, adjusting pH to weak acidity with dilute hydrochloric acid, precipitating white solid with yield of 90%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.01(dd,J＝8.3,1.7Hz,1H),7.93(d,J＝8.2Hz,2H),7.66(d,J＝1.7Hz,1H),7.50(d,J＝8.0Hz,2H),7.23(d,J＝8.3Hz,1H),5.15(s,2H),3.76–3.63(m,16H)ppm； ¹³ C NMR(126MHz,DMSO-d ₆ )δ168.01,166.97,164.44,159.00,146.49,138.43,134.47,130.87,130.69,129.84,128.51,124.55,110.84,110.67,66.04,46.93,43.33ppm；HRMS(ESI)m/z calcd.for C ₂₆ H ₂₇ N ₇ O ₅ (M+H) ⁺ 518.2146,found:518.2146.

example 24

24 Synthesis of 5- (4, 6-dimorpholino-1, 3, 5-triazin-2-yl) -N, N-dimethylbenzo [ d ] oxazol-2-amine (I-24):

the compound (600mg, 1.0 eq.) of example 7 was dissolved in N, N-dimethylformamide, sodium hydride (69mg, 1.1 eq.) was slowly added to the solution under ice-cooling to react for 2 hours, and methyl iodide was added to N, N-dimethylformamideDiluting formamide, slowly dropping into the reaction bottle in a dropping funnel, monitoring the reaction process by a TLC plate at any time, adding water to quench the reaction after the reaction is finished, extracting by dichloromethane for 3 times, concentrating the reaction solution under reduced pressure, further purifying the crude product by fast preparative liquid chromatography to obtain 138mg of white solid with the yield of 21 percent, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.27(dd,J＝8.7,2.1Hz,1H),8.21(d,J＝2.1Hz,1H),7.22(d,J＝8.7Hz,1H),3.94(s,3H),3.86–3.65(m,16H),3.29(s,3H)ppm；HRMS(ESI)m/z calcd.for C ₂₀ H ₂₅ N ₇ O ₃ (M+H) ⁺ 412.2092,found:412.2071.

example 25

25 Synthesis of N- (5- (4, 6-dimorpholino-1, 3, 5-triazin-2-yl) benzo [ d ] oxazol-2-yl) acetamide (I-25):

the compound of example 7 (100mg, 1.0eq.) was dissolved in anhydrous pyridine, and acetyl chloride (22.7mg, 1.1eq.) was slowly added dropwise under ice bath to react, the progress of the reaction was monitored by a TLC plate as needed, after completion of the reaction, the reaction was quenched by addition of 1N hydrochloric acid, extracted 3 times with dichloromethane, the reaction solution was concentrated under reduced pressure, and the crude product was further purified by flash preparative liquid chromatography. 62mg of white solid is obtained, the yield is 56 percent, ¹ H NMR(400MHz,DMSO-d ₆ )δ11.75(s,1H),8.51(d,J＝1.6Hz,1H),8.33(dd,J＝8.5,1.7Hz,1H),7.68(d,J＝8.5Hz,1H),3.90–3.65(m,16H),2.24(s,3H)ppm； ¹³ C NMR(126MHz,DMSO-d ₆ )δ169.56,165.05,156.45,150.38,134.01,124.40,118.28,110.03,66.51,43.80,24.40ppm；HRMS(ESI)m/z calcd.for C ₂₀ H ₂₃ N ₇ O ₄ (M+H) ⁺ 426.1884,found:426.1875.

example 26

26 Synthesis of N- (5- (4, 6-dimorpholino-1, 3, 5-triazin-2-yl) benzo [ d ] oxazol-2-yl) isobutyramide (I-26):

the synthesis of reference example 25, a white solid, yield 55%, ¹ H NMR(400MHz,DMSO-d ₆ )δ11.70(s,1H),8.50(d,J＝1.6Hz,1H),8.33(dd,J＝8.6,1.7Hz,1H),7.68(d,J＝8.5Hz,1H),3.90–3.66(m,16H),2.84–2.79(m,1H),1.14(d,J＝6.8Hz,6H)ppm；HRMS(ESI)m/z calcd.for C ₂₂ H ₂₇ N ₇ O ₄ (M+H) ⁺ 454.2197,found:454.2184.

example 27

27 Synthesis of N- (5- (4, 6-dimorpholino-1, 3, 5-triazin-2-yl) benzo [ d ] oxazol-2-yl) cyclohexanecarboxamide (I-27):

referring to the synthesis of example 25, white solid, yield 53%, ¹ H NMR(400MHz,DMSO-d ₆ )δ11.66(s,1H),8.50(d,J＝1.7Hz,1H),8.33(dd,J＝8.6,1.7Hz,1H),7.67(d,J＝8.6Hz,1H),3.90–3.66(m,16H),1.86(d,J＝12.5Hz,2H),1.78–1.75(m,2H),1.65(d,J＝10.7Hz,1H),1.45-1.34(m,2H),1.30–1.21(m,4H)ppm；HRMS(ESI)m/z calcd.for C ₂₅ H ₃₁ N ₇ O ₄ (M+H) ⁺ 494.251,found:494.2505.

example 28

28 Synthesis of N- (5- (4, 6-dimorpholino-1, 3, 5-triazin-2-yl) benzo [ d ] oxazol-2-yl) benzamide (I-28):

the synthesis of reference example 25, a white solid, yield 58%, ¹ H NMR(400MHz,DMSO-d ₆ )δ12.18(s,1H),8.57(s,1H),8.38(d,J＝8.7Hz,1H),8.03(d,J＝7.6Hz,2H),7.75–7.65(m,2H),7.57(t,J＝7.6Hz,2H),3.91–3.67(m,16H)ppm；HRMS(ESI)m/z calcd.for C ₂₅ H ₂₅ N ₇ O ₄ (M+H) ⁺ 488.2041,found:488.2041.

example 29

29 Synthesis of N- (5- (4, 6-dimorpholino-1, 3, 5-triazin-2-yl) benzo [ d ] oxazol-2-yl) ethanesulfonamide (I-29):

the compound (100mg, 1.0 eq.) of example 7 was dissolved in dichloromethane, triethylamine (52.6 mg,2.0 eq.) was added, and ethylsulfonyl chloride (40.2 mg,1.2 eq.) was added dropwise under ice bath and reacted at room temperature overnight. TLC plate to monitor reaction progress, after reaction, quenching reaction with water, extracting with dichloromethane for 3 times, concentrating reaction solution under reduced pressure, further purifying crude product by fast preparative liquid chromatography to obtain white solid 41mg, yield 33%, ¹ H NMR(300MHz,DMSO-d ₆ )δ12.52(s,1H),8.28–8.25(m,2H),7.59(d,J＝8.7Hz,1H),3.87–3.65(m,16H),3.17(q,J＝7.3Hz,2H),1.27(t,J＝7.3Hz,3H)ppm；HRMS(ESI)m/z calcd.for C ₂₀ H ₂₅ N ₇ O ₅ S(M+H) ⁺ 476.1711,found:476.1711.

example 30

30 Synthesis of 6- (4, 6-dimorpholino-1, 3, 5-triazin-2-yl) benzo [ d ] thiazol-2-amine (I-30):

the synthesis of reference example 7, a white solid, yield 35%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.67(d,J＝1.7Hz,1H),8.24(dd,J＝8.4,1.8Hz,1H),7.76(s,2H),7.36(d,J＝8.5Hz,1H),3.86–3.65(m,16H)ppm；HRMS(ESI)m/z calcd.for C ₁₈ H ₂₁ N ₇ O ₂ S(M+H) ⁺ 400.155,found:400.1538.

example 31

31 Synthesis of N- (6- (4, 6-dimorpholino-1, 3, 5-triazin-2-yl) benzo [ d ] thiazol-2-yl) acetamide (I-31):

the synthesis of reference example 25, a white solid, yield 54%, ¹ H NMR(300MHz,DMSO-d ₆ )δ12.50(s,1H),8.99(d,J＝1.6Hz,1H),8.42(dd,J＝8.6,1.7Hz,1H),7.77(d,J＝8.5Hz,1H),3.91–3.66(m,16H),2.23(s,3H)ppm；HRMS(ESI)m/z calcd.for C ₂₀ H ₂₃ N ₇ O ₃ S(M+H) ⁺ 442.1656,found:442.1655.

example 32

32 Synthesis of 6- (4, 6-dimorpholino-1, 3, 5-triazin-2-yl) -N, N-dimethylbenzo [ d ] thiazol-2-amine (I-32):

the synthesis of reference example 1, white solid, yield 39%, ¹ H NMR(400MHz,CDCl ₃ )δ8.66(d,J＝1.7Hz,1H),8.38(dd,J＝8.5,1.7Hz,1H),7.57(d,J＝8.5Hz,1H),3.92–3.76(m,16H),3.24(s,6H)ppm；HRMS(ESI)m/z calcd.for C ₂₀ H ₂₅ N ₇ O ₂ S(M+H) ⁺ 428.1863,found:428.1858.

example 33

33 Synthesis of tert-butyl 4- (4- (2-aminobenzo [ d ] thiazol-6-yl) -6-morpholino-1, 3, 5-triazin-2-yl) piperazine-1-carboxylate (I-33):

the synthesis according to example 1, white solid, yield 38%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.67(d,J＝1.7Hz,1H),8.24(dd,J＝8.5,1.8Hz,1H),7.76(s,2H),7.36(d,J＝8.5Hz,1H),3.94–3.65(m,12H),3.41(t,J＝5.3Hz,4H),1.43(s,9H)ppm；HRMS(ESI)m/z calcd.for C ₂₃ H ₃₀ N ₈ O ₃ S(M+H) ⁺ 499.2234,found:499.2236.

example 34

34 Synthesis of 6- (4-morpholino-6- (4- (trifluoromethyl) piperidin-1-yl) -1,3, 5-triazin-2-yl) benzo [ d ] thiazol-2-amine (I-34):

the synthesis of reference example 1, white solid, yield 35%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.66(d,J＝1.6Hz,1H),8.24(dd,J＝8.4,1.8Hz,1H),7.76(s,2H),7.37(d,J＝8.5Hz,1H),4.93(d,J＝87.8Hz,2H),3.84–3.65(m,8H),2.92(t,J＝12.8Hz,2H),2.69–2.62(m,1H),1.92(d,J＝12.6Hz,2H),1.44-1.33(m,2H)ppm；HRMS(ESI)m/z calcd.for C ₂₀ H ₂₂ F ₃ N ₇ OS(M+H) ⁺ 466.1631,found:466.1621.

example 35

35 Synthesis of 1- (4- (4- (2-aminobenzo [ d ] thiazol-6-yl) -6-morpholino-1, 3, 5-triazin-2-yl) piperazin-1-yl) ethan-1-one (I-35):

the synthesis according to example 1, white solid, yield 45%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.67(d,J＝1.8Hz,1H),8.25(dd,J＝8.5,1.8Hz,1H),7.77(s,2H),7.37(d,J＝8.5Hz,1H),3.88–3.65(m,12H),3.55–3.51(m,4H),2.06(s,3H)ppm；HRMS(ESI)m/z calcd.for C ₂₀ H ₂₄ N ₈ O ₂ S(M+H) ⁺ 441.1816,found:441.1811.

example 36

36 Synthesis of 6- (4- (4- (methylsulfonyl) piperazin-1-yl) -6-morpholino-1, 3, 5-triazin-2-yl) benzo [ d ] thiazol-2-amine (I-36):

the synthesis according to example 1, white solid, yield 32%, ¹ H NMR(300MHz,DMSO-d ₆ )δ8.68(d,J＝1.7Hz,1H),8.25(dd,J＝8.5,1.8Hz,1H),7.77(s,2H),7.37(d,J＝8.5Hz,1H),4.02–3.65(m,12H),3.18(t,J＝5.1Hz,4H),2.90(s,3H)ppm； ¹³ C NMR(126MHz,DMSO-d ₆ )δ169.42,168.76,164.66,164.55,155.98,131.08,129.27,126.09,121.19,117.02,66.13,45.38,43.37,42.35,33.91ppm；HRMS(ESI)m/z calcd.for C ₁₉ H ₂₄ N ₈ O ₃ S ₂ (M+H) ⁺ 477.1486,found:477.1479.

example 37

37 Synthesis of 4- (4- (2- (dimethylamino) benzo [ d ] thiazol-6-yl) -6-morpholino-1, 3, 5-triazin-2-yl) piperazine-1-carboxylic acid tert-butyl ester (I-37):

the synthesis of reference example 1, white solid, yield 51%, ¹ H NMR(400MHz,CDCl ₃ )δ8.66(d,J＝1.7Hz,1H),8.38(dd,J＝8.5,1.7Hz,1H),7.57(d,J＝8.5Hz,1H),3.92-3.52(m,12H),3.51(t,J＝4.9Hz,4H),3.24(s,6H),1.50(s,9H)ppm；HRMS(ESI)m/z calcd.for C ₂₅ H ₃₄ N ₈ O ₃ S(M+H) ⁺ 527.2547,found:527.2541.

example 38

38 Synthesis of N, N-dimethyl-6- (4-morpholino-6- (piperazin-1-yl) -1,3, 5-triazin-2-yl) benzo [ d ] thiazol-2-amine hydrochloride (I-38):

the synthesis of reference example 11, a white solid, with a yield of 100%, ¹ H NMR(400MHz,D ₂ O)δ8.52–8.48(m,1H),8.30–8.26(m,1H),7.46(t,J＝8.6Hz,1H),4.17(s,4H),3.89(s,4H),3.79(t,J＝4.7Hz,4H),3.36–3.32(m,10H)ppm；HRMS(ESI)m/z calcd.for C ₂₀ H ₂₆ N ₈ OS(M+H) ⁺ 427.2023,found:427.2023.

example 39

39 Synthesis of N, N-dimethyl-6- (4- (4- (methylsulfonyl) piperazin-1-yl) -6-morpholino-1, 3, 5-triazin-2-yl) benzo [ d ] thiazol-2-amine (I-39):

the synthesis of reference example 1, white solid, yield 37%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.75(s,1H),8.30(d,J＝8.5Hz,1H),7.48(d,J＝8.5Hz,1H),4.03–3.65(m,12H),3.19(s,10H),2.89(s,3H)ppm；HRMS(ESI)m/z calcd.for C ₂₁ H ₂₈ N ₈ O ₃ S ₂ (M+H) ⁺ 505.1799,found:505.1798.

example 40

40 Synthesis of N, N-dimethyl-6- (4-morpholino-6- ((3- (trifluoromethyl) phenyl) amino) -1,3, 5-triazin-2-yl) benzo [ d ] thiazol-2-amine (I-40):

the synthesis of reference example 1, white solid, yield 47%, ¹ H NMR(400MHz,DMSO-d ₆ )δ10.00(s,1H),8.74(d,J＝1.8Hz,1H),8.48(s,1H),8.32(dd,J＝8.5,1.8Hz,1H),7.91(d,J＝9.2Hz,1H),7.57(t,J＝8.0Hz,1H),7.52(d,J＝8.5Hz,1H),7.35(d,J＝7.7Hz,1H),4.04–3.70(m,8H),3.20(s,6H)ppm；HRMS(ESI)m/z calcd.for C ₂₃ H ₂₂ F ₃ N ₇ OS(M+H) ⁺ 502.1631,found:502.1626.

EXAMPLE 41

41 Synthesis of N, N-dimethyl-6- (4-morpholino-6- (4- (trifluoromethyl) piperidin-1-yl) -1,3, 5-triazin-2-yl) benzo [ d ] thiazol-2-amine (I-41):

the synthesis of reference example 1, white solid, yield 51%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.74(d,J＝1.7Hz,1H),8.29(dd,J＝8.5,1.8Hz,1H),7.48(d,J＝8.5Hz,1H),5.05(s,1H),4.82(s,1H),3.86–3.65(m,8H),3.18(s,6H),2.92(t,J＝13.0Hz,2H),2.68–2.65(m,1H),1.94–1.90(m,2H),1.44-1.33(m,2H)ppm；HRMS(ESI)m/z calcd.for C ₂₂ H ₂₆ F ₃ N ₇ OS(M+H) ⁺ 494.1944,found:494.1933.

example 42

42 Synthesis of 6- (4, 6-bis (4- (methylsulfonyl) piperazin-1-yl) -1,3, 5-triazin-2-yl) benzo [ d ] oxazol-2-amine (I-42):

the synthesis according to example 1, white solid, yield 56%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.17(d,J＝1.7Hz,1H),8.09(dd,J＝8.4,1.7Hz,1H),7.55(s,2H),7.41(d,J＝8.4Hz,1H),3.98(d,J＝42.8Hz,8H),3.19(d,J＝5.3Hz,8H),2.90(s,6H)ppm；HRMS(ESI)m/z calcd.for C ₂₀ H ₂₇ N ₉ O ₅ S ₂ (M+H) ⁺ 538.1649,found:538.1645.

example 43

43 Synthesis of 6- (2-aminobenzo [ d ] oxazol-6-yl) -N2, N4-bis (benzo [ d ] [1,3] dioxa-5-yl) -1,3, 5-triazine-2, 4-diamine (I-43):

the synthesis of reference example 1, white solid, yield 53%, ¹ H NMR(400MHz,DMSO-d ₆ )δ9.66(s,2H),8.17(s,1H),8.07(d,J＝8.4Hz,1H),7.57(s,3H),7.47(d,J＝8.4Hz,2H),7.16(s,2H),6.88(s,2H),6.01(s,4H)ppm；HRMS(ESI)m/z calcd.for C ₂₄ H ₁₇ N ₇ O ₅ (M+H) ⁺ 484.1364,found:484.1363.

example 44

44 Synthesis of 1,1' - (((6- (2-aminobenzo [ d ] oxazol-6-yl) -1,3, 5-triazine-2, 4-diyl) bis (piperazine-4, 1-diyl)) bis (eth-1-one) (I-44):

the synthesis of reference example 1, white solid, yield 60%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.16(d,J＝1.7Hz,1H),8.08(dd,J＝8.4,1.7Hz,1H),7.55(s,2H),7.40(d,J＝8.4Hz,1H),4.02-3.52(m,16H),2.06(s,6H)ppm；HRMS(ESI)m/z calcd.for C ₂₂ H ₂₇ N ₉ O ₃ (M+H) ⁺ 466.2310,found:466.2304.

example 45

45 Synthesis of methyl 4- ((((6- (4, 6-bis (4-acetylpiperazin-1-yl) -1,3, 5-triazin-2-yl) benzo [ d ] oxazol-2-yl) amino) methyl) benzoate (I-45):

the synthesis of reference example 12, a white solid, yield 45%, ¹ H NMR(300MHz,DMSO-d ₆ )δ8.04–7.96(m,3H),7.69(s,1H),7.56(d,J＝7.9Hz,2H),7.25(d,J＝8.3Hz,1H),6.96(s,1H),5.15(s,2H),3.87-3.74(m,11H),3.52–3.50(m,8H),2.07(s,6H)ppm；HRMS(ESI)m/z calcd.for C ₃₁ H ₃₅ N ₉ O ₅ (M+H) ⁺ 614.2834,found:614.2829.

example 46

46 Synthesis of 4- ((((6- (4, 6-bis (4-acetylpiperazin-1-yl) -1,3, 5-triazin-2-yl) benzo [ d ] oxazol-2-yl) amino) methyl) benzoic acid (I-46):

the synthesis of reference example 11, a white solid, yield 85%, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.75(s,1H),8.30(d,J＝8.7Hz,1H),7.99(d,J＝7.9Hz,3H),7.70(d,J＝8.0Hz,3H),5.64(s,2H),3.81-3.73(m,8H),3.53–3.50(m,8H),2.08(s,6H)ppm；HRMS(ESI)m/z calcd.for C ₃₀ H ₃₃ N ₉ O ₅ (M+H) ⁺ 600.2677,found:600.2677.

application example biological evaluation experiment

PI3K alpha kinase activity detection method

1. Principle of testing

The PI3K alpha kinase Activity test method used in this experiment employed the ADP-Glo method, which performed lipid kinase reaction by incubating lipid substrate with recombinase and ATP, and used ADP-Glo ^TM Kinase assays measure kinase activity. ADP-Glo ^TM The kinase assay was performed in two steps: first, after the kinase reaction, an ATP consuming reagent is added to terminate the lipid kinase reaction and consume any remaining ATP, leaving only ADP. Next, a detection reagent is added to simultaneously convert ADP to ATP and the newly synthesized ATP to a fluorescent signal using a coupled luciferase/luciferin reaction.

2. Test procedure

(1) Preparation of the Compounds

The compounds were dissolved in DMSO at 100% to 10mM stock solution and stored under nitrogen in dark. PI103 was used as a positive control compound. Compounds were screened on PI3K α kinase at an initial concentration of 30 μ M, 3-fold dilutions, 10 concentrations, and duplicate wells.

(2) Reaction process

1) A1 XKinase buffer was prepared.

2) Preparation of compound concentration gradient: test compound concentrations were 30000nM starting, 3-fold dilutions, 10 concentrations, and duplicate wells. The 10 different concentration solutions were diluted in a 384 well plate in steps to 100 fold final concentration. Transfer 50nl to compound wells of 384-well plates with Echo; 50nl of DMSO was added to each of the negative control well and the positive control well.

3) A2-fold final concentration of Kinase solution was prepared using a 1 XKinase buffer.

4) Add 2.5. Mu.L of 2-fold final concentration kinase solution to the compound well and the positive control well, respectively; to the negative control well was added 2.5. Mu.L of 1 XKinase buffer.

5) Centrifuged at 1000rpm for 30 seconds, shaken and mixed and incubated at room temperature for 10 minutes.

6) A mixed solution of ATP and substrate P1P2 was prepared at 2-fold final concentration using 1 XKinase buffer.

7) The reaction was initiated by adding 2.5. Mu.L of a 2-fold final ATP and substrate mixture.

8) The 384 well plate was centrifuged at 1000rpm for 30 seconds, and after shaking and mixing, the reaction was carried out at room temperature for 60 minutes.

9) 5 μ of LADP-Glo Reagent was added, centrifuged at 1000rpm for 30 seconds, shaken well and incubated at room temperature for 180 minutes.

10 10 μ LKING ase Detection Reagent was added, centrifuged at 1000rpm for 30 seconds, shaken well mixed and incubated at room temperature for 30 minutes.

11 Luminescence RLU was read with an Envision plate reader.

(3) Data analysis

1) Formula for calculation

Inhibition％＝100-((RLU-Mean(NC))/(Mean(PC)-Mean(NC)))×100

Wherein: RLU: the chemiluminescence value of the sample; mean (NC): negative control well mean; mean (PC): positive control well ratio means.

2) Fitted dose-effect curve

The log value of the concentration is taken as an X axis, the percent inhibition rate is taken as a Y axis, and the log (inhibitor) v.response-Variable slope of GraphPad Prism 5 of analysis software is adopted to fit a dose-effect curve, so that the IC of each compound to the enzyme activity is obtained ₅₀ The value is obtained.

The formula is as follows: y = Bottom + (Top-Bottom)/(1 +10^ ((LogicC 50-X) > HillSlope))

Measured IC ₅₀ The values are shown in table 1 below, and it can be seen from the experimental results that the compounds of the examples of the present invention have strong inhibitory activity against PI3K α kinase activity.

Table 1 IC of compounds of the present application for PI3K alpha kinase activity ₅₀ Measured value

(II) selectivity test on four I-type PI3K subtypes

IC of PI3K (p 110. Delta./p 85. Alpha.), PI3K (p 110. Beta./p 85. Alpha.) and PI3K (p 120. Gamma.) kinases were performed by selecting the compounds of the examples having the best PI 3K. Alpha. Kinase activity ₅₀ Value testing, testing whether the compound in the invention is a selective inhibitor of PI3K alpha. PI103 was tested as a positive control compound. The test method is the same as in (1), except that the compound screening was performed on PI3K (p 110. Delta./p 85. Alpha.), PI3K (p 110. Beta./p 85. Alpha.) and PI3K (p 120. Gamma.) kinases, starting at 10. Mu.M, at 3-fold dilution, at 10 concentrations, and in single well assay.

Measured IC ₅₀ The values are shown in table 2 below, and it can be seen from the experimental results that the compounds of the examples of the present invention have better selectivity for PI3K α kinase activity.

TABLE 2 IC of the Compounds of the present application on the kinase Activity of four subtypes of PI3K ₅₀ Measured value

(III) measurement of inhibition of proliferation of various cancer cells

The inhibitory effects of the compounds on the proliferation of 10 cancer cells such as a human gastric cancer (MGC-803) cell line, a prostate cancer (PC 3) cell line, a breast cancer (MCF-7) cell line, a triple negative breast cancer (MDA-MB-231) cell line, a liver cancer (HepG 2) cell line, an ovarian cancer (SKOV-3) cell line, a multiple myeloma (RPMI 8226) cell line, a human chronic myelogenous leukemia (K562) cell line, a human glioma (U251) cell line and a human acute lymphoblastic leukemia (MOLT-4) cell line were examined by the following methods.

1. Principle of testing

This experiment was conducted using the Cell Counting Kit-8 (CCK 8) method to test the inhibitory effect of compounds on the proliferation of various cancer cells. Rapid assay for cell proliferation/toxicity using WST-8. WST is a tetrazolium salt, which is reduced by dehydrogenase in living cells in the presence of an electron carrier to form water-soluble orange Formazan. And detecting the generation amount of Formazan by a colorimetric method to reflect the number of living cells in the experiment.

2. Testing procedure

(1) Plate preparation: cells in logarithmic growth phase were grown at 1X 10 ⁵ cells/well seeded in 96-well plates, placed at 37 ℃ C. 5% CO ₂ Culturing under the condition until the cells are 90% fused, and then culturing and incubating for 2h by using a serum-free DMEM medium, an RPMI-1640 medium, an F12K medium or other corresponding cell culture media to synchronize the cells.

(2) Adding medicine: adding 100 μ L of test compound solution of different concentrations diluted in a gradient to the plate, incubating the plate at 37 deg.C, 5% CO ₂ Incubating for 72 hours under incubator conditions;

(3) And (3) CCK8 detection: after the incubation is finished, 10 mu L of CCK8 solution is added into each hole, the culture box is placed for 4 hours at 37 ℃, the OD value of the hole under OD450 is detected by a microplate reader, and the cell proliferation inhibition rate is calculated. Inhibition rate = (control OD value-experimental OD value)/control OD value × 100%;

(4) After data were obtained, graphPad Prism 6 was fitted to obtain IC ₅₀ 。

The compound of example 7 of the present invention was tested for the proliferation activity of various cancer cells and the IC was determined ₅₀ The values are shown in Table 3. The results show that the compound can effectively inhibit the growth of various cancer cells, IC ₅₀ The values are all between 0.43 and 1.33. Mu.M. The inhibitor has better inhibition effect on blood tumor, the inhibition effect on a human chronic myelogenous leukemia cell line K562 can reach 0.429 mu M, the inhibition effect on a multiple myeloma cell line RPMI8226 can reach 0.537 mu M, the inhibition effect on human acute lymphoblastic leukemia MOLT-4 can reach 0.606 mu M, the inhibitor also has better inhibition effect on solid tumor, and the inhibition effect on a gastric cancer cell line MGC-803 can reach 1.089 mu M.

TABLE 3 inhibition of proliferative Activity of example 7 Compounds on various cancer cells IC ₅₀

(IV) acute toxicity assay of Compounds

Acute toxicity testing of example compound 7 was conducted to establish a safe dosing window. The preliminary experiment shows that the tested drug has certain toxicity, the drug gavage of 800mg/kg in the preliminary experiment can cause the death of 4/4 mice, and the drug gavage of 350mg/kg in the preliminary experiment causes the death of 0/4 mice. Therefore, on the basis of preliminary experiments, the test uses ICR female mice, seven dose concentrations of 720, 648, 583.2, 524.9, 472.4, 425.2 and 382.6 and a saline control group, 10 mice in each dose group are administrated in a gavage way for 1 time, toxic symptoms and death conditions of each group of mice are recorded, and dead animals are necropsied for 14 days. The details are shown in Table 4.

The results show that compound 7 is toxic to mice and causes death after administration at higher doses. Intragastric administered LD ₅₀ The value was 496.9116 (474.5824-520.2914) mg/kg. Dead mice are subjected to autopsy, mice in the groups with the lowest dose of 382.6mg/kg and the highest dose of 720mg/kg are selected to perform HE staining on heart, liver, spleen, lung and kidney organs, and the result shows that the drug has no obvious toxicity on the organs.

Table 4 acute toxicity test data for the compound of example 7

The regression equation is: y = -15.7287+7.6879X; (r = 0.9977)

When a =0.01, r is found to be: 0.8740

(wherein Y is the value of the probability unit plus 5 and X is the logarithm of the dose logx)

So LD ₅₀ ＝496.9116mg/kg

The confidence interval when the confidence coefficient a =0.05 is: 474.5824 and 520.2914, LD50 is more than or equal to or less than 0.0200

(V) Compound pharmacokinetic assay

The pharmacokinetic profile tests of example 7 were performed using a total of 6 SD rats divided into two groups, and the groups were administered to example 7 by tail vein injection (1 mg/kg) and intragastric administration (10 mg/kg), and at 11 time points before and after administration, the groups were administered intravenously at 2min, 5min, 15min, 30min, 1h, 2h, 4h, 6h, 8h, 12h, and 24h after administration; the gavage group collected about 0.25mL of blood sample from the retroorbital venous plexus at 5min, 15min, 30min, 1h, 2h, 4h, 6h, 8h, 12h and 24h after administration. The concentration of the compound in the plasma samples of SD rats was determined by LC-MS/MS method and pharmacokinetic parameters were calculated using WinNolin software. The results are shown in Table 5.

The results show that the main pharmacokinetic parameters for intravenous administration after a single intravenous injection of 1mg/kg example 7 and gastric gavage of 10mg/kg in SD rats are: half life (t) _1/2 ) 6.29h, time to peak (T) _max ) 0.033h, maximum blood concentration (C) _max ) 454.7ng/mL, area under the curve (AUC) _0-∞ ) 2922.4hr ng/mL, clearance (Cl) 342.9mL/hr/kg, mean Residence Time (MRT) _0-∞ ) The time is 8.53h. The major pharmacokinetic parameters of intragastric administration are as follows: half life (t) _1/2 ) 3.89h, time to peak (T) _max ) At 9.33h, maximum blood concentration (C) _max ) 2256.7ng/mL, area under the curve (AUC) _0-∞ ) 38114.3hr ng/mL, mean Residence Time (MRT) _0-∞ ) The time is 9.50h. Most importantly, the compound of example 7 has an oral bioavailability F of 130.42%. Wherein the oral bioavailability F exceeds 100% because the oral administration dosage is 10 times of the intravenous administration dosage, thereby causing the orally administered blood drug concentration to be higher than that of the intravenous administration, and when the blood drug concentration is higher, the body can reach the saturation of the clearance of the drug, namely nonlinear PK appears, in this case, the F can be caused>100 percent. The pharmacokinetic data show that the compound in example 7 has good pharmacokinetic characteristics, better metabolism, better absorption and exposure and higher bioavailability in SD rats.

Table 5 pharmacokinetic parameters of the compound of example 7

(VI) determination of anti-gastric cancer Activity of Compound in vivo

1. Test consumable

(1) Laboratory animal

Source, strain: SPF-grade BALB/c nude mice, supplied by Changzhou Kavens laboratory animals Co. Production license of experimental animals: SCXK (threo) 2016-0010; license for use of experimental animal: SYXK (threo) 2017-0040. The age in days: 4-6 weeks at procurement and 6-8 weeks at the start of dosing. Weight: the weight is 16-18g at the time of purchase and 18-20g at the time of administration. Sex: and (4) male. Number of animals per group: 5, the number of the main body is only 5.

(2) Cell line

Human gastric carcinoma cells MGC-803, supplied by Shanghai cells of Chinese academy of sciences, were cultured in DF12 medium containing 10% fetal bovine serum.

(3) Test drug

Compound I7 and positive medicine taxol.

2. Test method

(1) Drug configuration

Positive drug paclitaxel (10 mg/kg): weighing about 2mg of positive control compound powder, dissolving in 2mL of normal saline to prepare a drug with the concentration of 1mg/mL, and carrying out intraperitoneal injection administration with the administration volume of 0.2mL/20g of body weight once every three days. Compound I7 (30 mg/kg): about 6mg of the powder of the compound to be tested was weighed and dissolved in 2mL of physiological saline to prepare a drug with a concentration of 3mg/mL, and the drug was orally administered by gavage in a volume of 0.2mL/20g of body weight once a day. I7 (30 mg/kg) + paclitaxel (10 mg/kg) as a positive drug in combination: i7 (3 mg/mL drug) administered orally by gavage in a volume of 0.2mL/20g body weight once a day. Paclitaxel (1 mg/mL drug) as a positive drug was administered by intraperitoneal injection at a dose volume of 0.2mL/20g body weight once every three days.

(2) Test method

The human gastric cancer cell MGC-803 xenotransplantation tumor model is established by inoculating the human gastric cancer cell MGC-803 subcutaneously under the armpit of a nude mouse. Taking MGC-803 cells in logarithmic growth phase, inoculating the cells under the right axillary skin of 28 BALB/c nude mice under the aseptic condition, wherein the inoculation amount of the cells is 5×10 ⁶ One/only. Measuring the diameter of the transplanted tumor by using a vernier caliper until the tumor grows to 85mm ³ On the left and right, 20 tumor-bearing nude mice with good growth state and good tumor size uniformity are selected, randomly divided into 4 groups, and each group comprises 5 mice, namely a negative blank group, a positive drug paclitaxel control group, a compound I7 administration group and a compound I7+ positive drug paclitaxel combination group. Positive drug paclitaxel (10 mg/kg), intraperitoneal injection, once every three days, 7 times in total; compound I7 (30 mg/kg) was administered orally once daily by gavage, i.e. once every 1 day for 22 days; the compound I7 (30 mg/kg) and the positive drug paclitaxel (10 mg/kg) are taken together, wherein the compound I7 is taken orally and is administrated by gastric lavage once, namely once every 1 day and once every 21 days, and the positive drug paclitaxel (10 mg/kg) (intraperitoneal injection and once every three days) is administrated for 7 times; meanwhile, the tumor volume and the animal weight of the nude mice are measured and recorded every other day, and the death rate of the mice is recorded at any time. On day 22, the mice were sacrificed, tumor mass was surgically removed and weighed, and tumor tissues were preserved in a liquid nitrogen tank to calculate the tumor inhibition rate (%).

(3) Results of the experiment

The experimental results are shown in fig. 1, when the drug is administered on day 9, the tumors of the mice in the blank group grow well, and the tumors of the mice in the drug administration group, the positive drug group and the combination drug group are obviously reduced compared with the tumors of the mice in the drug administration group, the positive drug group and the combination drug group. When the drug is administered for 22 days, compared with the blank group, the tumor growth inhibition rates of the MGC-803 nude mouse transplanted tumor of the human gastric cancer cell in the I7 drug administration group, the positive drug group and the combined drug group are respectively 41.5%, 60.3% and 63.9%, and the relative tumor proliferation rates T/C (%) are respectively 53.2%, 39.6% and 30.2%. During the drug administration period, no mice died, and the mice of each administration group grew normally, as shown in fig. 2, and the body weight of the mice did not differ significantly from that of the mice of the control group.

In general, the compound I7 can effectively inhibit the growth of gastric cancer tumors at the administration concentration of 30mg/kg, and the combined administration of the compound I7 and the paclitaxel which is a common medicament for traditional gastric cancer patients can play a synergistic role in vivo, and the tumor inhibition rate reaches 63.9%. At the same time, the mice did not die during the dosing period, grew normally, and the body weight did not drop significantly, indicating that the toxicity of the drug at therapeutic doses was acceptable.

Claims

1. A triazinocyclobenzoxazole compound represented by general formula I, or a pharmaceutically acceptable salt thereof:

wherein X is selected from N; y is selected from O;

R ₁ independently selected from:

R ₂ independently selected from:

R ₃ independently selected from hydrogen, -C (O) R ₄ 、

R ₄ Independently selected from C ₁ -C ₈ An alkyl group;

R ₆ independently selected from hydrogen, cyano, nitro, C ₁ -C ₈ Alkoxy, -C (O) OR ₈ 、-C(O)NHR ₉ ；

R ₈ 、R ₉ Each independently selected from hydrogen and C ₁ -C ₈ An alkyl group.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein: r is ₃ Independently selected from hydrogen, -C (O) R ₄ 、

R ₄ Is methyl; r ₆ Is hydrogen, cyano, nitro, methoxy, -C (O) OR ₈ 、-C(O)NHR ₉ ；R ₈ Hydrogen and methyl; r ₉ Is hydrogen.

3. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein: the pharmaceutically acceptable salt is an acid addition salt of the compound of the general formula I, wherein the acid for salt formation comprises inorganic acids and organic acids, the inorganic acids comprise hydrochloric acid, sulfuric acid, phosphoric acid and methanesulfonic acid, and the organic acids comprise acetic acid, trichloroacetic acid, propionic acid, butyric acid, maleic acid, p-toluenesulfonic acid, malic acid, malonic acid, cinnamic acid, citric acid, fumaric acid, camphoric acid, digluconic acid, aspartic acid and tartaric acid.

4. A triazine-benzo-oxazole compound is characterized in that: a compound selected from any one of:

5. a pharmaceutical composition characterized by: comprising a compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

6. Use of a compound according to any one of claims 1 to 4 or a pharmaceutical composition according to claim 5 for the preparation of a PI3K alpha target inhibitor and a medicament for diseases modulated by PI 3K.

7. Use of a compound according to any one of claims 1 to 4 or a pharmaceutical composition according to claim 5 for the preparation of a medicament for a PI 3K-regulated tumor.

8. Use of a compound according to any one of claims 1 to 4 in combination with paclitaxel in the manufacture of a medicament for the treatment of gastric cancer.