An imidazo[1,5-a]pyrazine antitumor compound and its preparation method and application

Abstract

The present invention belongs to the field of pharmaceutical preparation technology and relates to an imidazo[1,5-a]pyrazine anti-tumor compound, a preparation method and an application thereof. The imidazo[1,5-a]pyrazine anti-tumor compound shown in Formula I provided by the present invention can effectively inhibit the growth of lung cancer cells A549 based on the proliferation inhibition test of human small cell lung cancer cells A549. Among them, the imidazo[1,5-a]pyrazine anti-tumor compound NJ-12 provided by the present invention can significantly inhibit the growth of tumors in the A549 nude mouse transplant tumor model. At the same time, there was no death or weight loss in the mice after 3 weeks of administration, indicating that the imidazo[1,5-a]pyrazine anti-tumor compound NJ-12 provided by the present invention has low toxicity. The imidazo[1,5-a]pyrazine compound provided by the present invention is an mTOR inhibitor and can be used to prepare drugs for treating diseases related to the activation of this target. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparation, and relates to an imidazo[1,5-a]pyrazine anti-tumor compound, a preparation method and an application thereof. Background Art

[0002] Cancer is a large class of diseases characterized by abnormal cell proliferation and metastasis. The incidence and mortality of cancer have been on the rise. Traditional anti-cancer drugs are mainly cytotoxic drugs. Most of these drugs are non-selective. While killing cancer cells, they also kill normal cells of the body. They have the disadvantages of poor selectivity, strong toxic side effects, and easy to develop drug resistance.

[0003] With the rapid development of tumor biology and related disciplines, people have gradually realized that the essence of cell carcinogenesis is the dysregulation of cell signaling pathways, leading to unlimited cell proliferation. This has led to a major shift in the concept of anti-tumor drug research and development. The focus of research and development is shifting from traditional cytotoxic drugs to specific anti-tumor drugs that target abnormal signaling systems within tumor cells.

[0004] mTOR is a serine / threonine protein kinase that has been shown to be closely associated with the development and progression of numerous human diseases. Inhibiting the mTOR signaling pathway can effectively block the abnormal signal transduction of various growth factors, thereby preventing the onset and progression of diseases.

[0005] The development and progression of many diseases are linked to abnormal mTOR signaling, most of which manifest as overactivation or overexpression of mTOR. Sustained overactivation of mTOR signaling leads to elevated cellular metabolism, sustained growth and proliferation, prolonged cell lifespan, and even cellular immortalization, which can directly or indirectly induce cancer, metabolic diseases, and aging-related diseases. Inhibiting this state can effectively delay or treat diseases such as cancer and cardiovascular damage caused by mTOR overactivation. Inhibiting the mTOR signaling pathway can block the transmission of multiple abnormal signals, thereby preventing and controlling the occurrence of diseases. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to address the deficiencies of the existing technology and provide an imidazo[1,5-a]pyrazine anti-tumor compound and its preparation method and application. The anti-tumor compound is an mTOR inhibitor that can effectively inhibit the growth of tumors such as non-small cell lung cancer and can be used to prepare drugs for treating diseases related to the activation of this target.

[0007] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0008] The present invention discloses an imidazo[1,5-a]pyrazine anti-tumor compound represented by formula I or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, pharmaceutically acceptable salts or prodrugs;

[0009]

[0010] Wherein, R is selected from C2-C6 alkyl, halogenated C2-C6 alkyl, C3-C6 cycloalkyl, heterocycloalkyl or aromatic group.

[0011] In some embodiments, R is selected from C3-C5 alkyl, C3-C5 cycloalkyl, or five-membered heterocyclyl.

[0012] In some embodiments, preferably, R is selected from C3 alkyl, C4 alkyl, C5 alkyl, C3 cycloalkyl, C5 cycloalkyl or oxygen-containing five-membered heterocyclic group.

[0013] In some embodiments, further preferably, R is selected from isopropyl, tert-butyl, pentyl, cyclopropyl, cyclopentyl or tetrahydrofuranyl.

[0014] In some embodiments, more preferably, R is selected from isopropyl, tert-butyl, pent-3-yl, cyclopropyl, cyclopentyl or tetrahydrofuran-2-yl.

[0015] In some embodiments, the imidazo[1,5-a]pyrazine anti-tumor compound is selected from any one of the following compounds:

[0016]

[0017] Furthermore, the present invention discloses a method for preparing the imidazo[1,5-a]pyrazine anti-tumor compound represented by the above-mentioned formula I, comprising the following steps:

[0018] Compound 1 undergoes a first reaction with a formic acid compound under the action of a first coupling agent and a first base to obtain compound 2; compound 2 undergoes a second reaction under the action of phosphorus oxychloride to obtain compound 3; compound 3 undergoes a third reaction under the action of an iodination reagent to obtain compound 4; compound 4 undergoes a fourth reaction under the action of an amination reagent to obtain compound 5; compound 5 undergoes a fifth reaction with a boric acid compound under the action of a palladium catalyst and a second base to obtain an imidazo[1,5-a]pyrazine anti-tumor compound I;

[0019]

[0020] wherein R is selected from C2-C6 alkyl, halogenated C2-C6 alkyl, C3-C6 cycloalkyl, heterocycloalkyl or aromatic group; X is selected from Cl or Br;

[0021] Wherein, the boric acid compound is

[0022] Among them, preferably, R is selected from C3-C5 alkyl, C3-C5 cycloalkyl or five-membered heterocyclic group; further preferably, R is selected from C3 alkyl, C4 alkyl, C5 alkyl, C3 cycloalkyl, C5 cycloalkyl or oxygen-containing five-membered heterocyclic group; further preferably, R is selected from isopropyl, tert-butyl, pentyl, cyclopropyl, cyclopentyl or tetrahydrofuranyl; most preferably, R is selected from isopropyl, tert-butyl, pent-3-yl, cyclopropyl, cyclopentyl or tetrahydrofuran-2-yl.

[0023] In some embodiments, the first coupling agent is any one or a combination of HATU, HOBt, EDCI, BOP and DCC; the first base is any one or a combination of N-methylmorpholine, triethylamine, diethylamine and diisopropylethylamine; the molar ratio of the compound 1 to the formic acid compound, the first coupling agent and the first base is 1.0-1.1:1.0-1.5:2.0-5.0:2.0-5.0; the reaction temperature of the first reaction is 0°C to 60°C.

[0024] In some embodiments, preferably, the first coupling agent is a composition of HOBt and EDCI in any molar ratio, more preferably a composition with a molar ratio of 1:1; the first base is N-methylmorpholine; the molar ratio of the compound 1 to the formic acid compound, the first coupling agent, and the first base is 1:1.1:3.0:3.0; and the reaction temperature of the first reaction is room temperature.

[0025] The room temperature, unless otherwise specified, is approximately 25°C.

[0026] Wherein, the HATU is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; the HOBt is 1-hydroxybenzotriazole; the EDCI is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; the BOP is benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate; and the DCC is dicyclohexylcarbodiimide.

[0027] Wherein, the N-methylmorpholine is abbreviated as NMM; the diisopropylethylamine is abbreviated as DIPEA.

[0028] The solvent used in the first reaction includes but is not limited to dichloromethane, dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, n-butanol, dioxane, tetrahydrofuran, preferably dichloromethane; there is no special requirement for the amount of the solvent, as long as it can dissolve the raw materials and has a moderate viscosity.

[0029] In some embodiments, the molar ratio of compound 2 to phosphorus oxychloride is 1.0-1.2:3.0-6.0; and in the second reaction, the reaction temperature is 40°C-80°C.

[0030] In some embodiments, preferably, the molar ratio of the compound 2 to phosphorus oxychloride is 1.0:4.0; and the reaction temperature of the second reaction is 60°C.

[0031] Wherein, a small amount of N,N-dimethylformamide can be added into the second reaction to catalyze the reaction.

[0032] The solvent used in the second reaction includes but is not limited to acetonitrile, dioxane, and tetrahydrofuran, preferably acetonitrile; there is no special requirement for the amount of the solvent, as long as it can dissolve the raw materials and has a moderate viscosity.

[0033] In some embodiments, the iodine reagent is N-iodosuccinimide; the molar ratio of compound 3 to the iodine reagent is 1.0-1.1:1.1-2.0; the reaction temperature of the third reaction is 20° C. to 80° C.;

[0034] In some embodiments, preferably, the iodine reagent is N-iodosuccinimide; the molar ratio of compound 3 to the iodine reagent is 1.0-1.1:1.1-1.5, more preferably 1.0:1.2; the reaction temperature of the third reaction is 60-80°C, preferably 70°C.

[0035] The solvent used in the third reaction includes but is not limited to DMF and DMSO, preferably DMF; there is no special requirement for the amount of the solvent, as long as it can dissolve the raw materials and have a moderate viscosity.

[0036] In some embodiments, the aminating agent is aqueous ammonia; and the reaction temperature of the fourth reaction is 90° C. to 130° C.

[0037] In some embodiments, preferably, the fourth reaction has a reaction temperature of 110°C.

[0038] The concentration of the ammonia water is 28 wt %; the amount of the aminating agent is excessive.

[0039] Wherein, a solvent may or may not be added to the fourth reaction; when a solvent is added to the fourth reaction, the solvent used includes but is not limited to dioxane, isopropanol, and tetrahydrofuran, preferably dioxane; there is no special requirement for the amount of the solvent used, as long as the raw materials are dissolved and the viscosity is moderate.

[0040] In some embodiments, the palladium catalyst is any one or a combination of tetrakistriphenylphosphine palladium, tert-butylphosphine palladium, dichloro[1,1'-bis(diphenylphosphino)ferrocene]palladium, tris(dibenzylacetone)dipalladium and palladium acetate; the second base is any one or a combination of sodium carbonate, potassium carbonate and sodium bicarbonate; the molar ratio of the compound 5 to the boric acid compound, the palladium catalyst and the second base is 1.0-1.2:1.0-2.0:0.04-0.20:2.0-8.0.

[0041] In some embodiments, preferably, the palladium catalyst is dichloro[1,1'-bis(diphenylphosphino)ferrocene]palladium; the second base is sodium carbonate; the molar ratio of the compound 5 to the boric acid compound, the palladium catalyst, and the second base is 1.0-1.2:1.0-1.2:0.06-0.12:3.0-5.0, and more preferably 1.0:1.0:0.08:4.0.

[0042] Wherein, the dichloro[1,1'-bis(diphenylphosphino)ferrocene]palladium is abbreviated as PdCl2(dppf); the tris(dibenzylacetone)dipalladium is abbreviated as Pd2(dba)3.

[0043] Among them, the fifth reaction is a reflux reaction carried out under the protection of inert gas, and the reaction temperature is determined by the boiling point of the solvent used, and the reaction temperature allows the solvent to reflux; the solvent used includes but is not limited to mixed solvents such as dioxane-water, DMF-water, ethanol-water, ethylene glycol dimethyl ether-water and toluene-water, preferably a mixed solvent of dioxane and water in any volume ratio, and further preferably a mixed solvent of dioxane and water with a volume ratio of 28:7; there is no special requirement for the amount of the solvent, as long as the raw materials are dissolved and the viscosity is moderate.

[0044] Furthermore, the present invention discloses a pharmaceutical composition comprising the above-mentioned imidazo[1,5-a]pyrazine anti-tumor compound represented by Formula I or any one of its stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, pharmaceutically acceptable salts or prodrugs, and one or more pharmaceutically acceptable carriers.

[0045] Specifically, the above-mentioned pharmaceutical composition is in the form of one or more of subcutaneous injection, intradermal injection, spray, powder aerosol, external solution, lotion, liniment, ointment, plaster, paste, patch, granule, tablet, capsule, and liquid preparation.

[0046] The use of the imidazo[1,5-a]pyrazine anti-tumor compound represented by the above-mentioned formula I or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, pharmaceutically acceptable salts or prodrugs, or the above-mentioned pharmaceutical composition in the preparation of mTOR kinase inhibitors is also within the scope of protection of the present invention.

[0047] The use of the imidazo[1,5-a]pyrazine anti-tumor compound represented by the above-mentioned formula I or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, pharmaceutically acceptable salts or prodrugs, or the above-mentioned pharmaceutical compositions in the preparation of drugs for treating diseases related to mTOR target activation is also within the scope of protection of the present invention.

[0048] Specifically, the related disease is non-small cell lung cancer.

[0049] Beneficial effects:

[0050] (1) The imidazo[1,5-a]pyrazine anti-tumor compounds provided by the present invention have not been reported in the literature and are new compounds. The parent ring of this type of compound is imidazo[1,5-a]pyrazine. From the perspective of the proliferation inhibition test on human small cell lung cancer cells A549, the compounds are significantly stronger than the reference substance PI-103. This type of compound can effectively inhibit the growth of lung cancer cells A549. Among them, the present invention conducted an in vivo pharmacodynamic evaluation of the NJ-12 compound (1-(2-aminobenzo[d]oxazol-5-yl)-3-isopropylimidazo[1,5-a]pyrazin-8-amine) in an A549 nude mouse transplant tumor model and found that the NJ-12 compound had a significant inhibitory effect on the tumor at a low dose.

[0051] (2) When the imidazo[1,5-a]pyrazine antitumor compound (NJ-12) provided by the present invention was administered to an A549 nude mouse transplanted tumor model, the mice did not die or lose weight 3 weeks after administration, indicating that the imidazo[1,5-a]pyrazine antitumor compound (NJ-12) provided by the present invention has low toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0053] Figure 1 This is a graph showing the inhibition of mTOR kinase by compound NJ-12.

[0054] Figure 2 This is the curve of the mTOR kinase inhibition by the control substance PI-103.

[0055] Figure 3This is a graph showing the in vivo efficacy (time / tumor volume) of the compound NJ-12 administration group and the solvent control group in the A549 nude mouse transplanted tumor model.

[0056] Figure 4 This is a curve diagram of the compound NJ-12 administration group and the solvent control group after administration to A549 nude mice (time / nude mouse weight). DETAILED DESCRIPTION

[0057] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0058] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0059] Example 1: Synthesis of Compound NJ-12

[0060] The synthetic route of compound NJ-12 is as follows:

[0061]

[0062] (1) Synthesis of N-((3-chloropyrazin-2-yl)methyl)isobutyramide (Compound 2a)

[0063] (3-Chloropyrazin-2-yl)methylamine hydrochloride (5.4 g, 30 mmol) was suspended in dichloromethane (80 mL), and isobutyric acid (2.91 g, 33 mmol), HOBt (6.08 g, 45 mmol), EDCI (8.62 g, 45 mmol) and N-methylmorpholine (9.10 g, 90 mmol) were added and stirred at room temperature overnight. After the reaction, the reaction solution was concentrated to dryness under reduced pressure, and water (50 mL) and dichloromethane (200 mL) were added for extraction. The mixture was washed with water (2 x 100 mL), saturated sodium carbonate solution (100 mL), and saturated brine (100 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a light yellow solid N-((3-chloropyrazin-2-yl)methyl)isobutyramide, designated as compound 2a, 4.6 g, 21.5 mmol, with a yield of 71.7%.

[0064] Compound 2a was detected by mass spectrometry, EI-MS MS (m / z): 214.3 [M+H] + ; The NMR data of compound 2a are as follows: 1H-NMR (CDCl3, 400MHz): δ8.44(d,J=4.0Hz,1H), 8.30(d,J=4.0Hz,1H), 6.87(br,s,1H), 4.67(d,J=4.0Hz,2H),2.55~2.48(m,1H),1.21(d,J=8.0Hz,3H),1.16(d,J=8.0Hz,3H).

[0065] (2) Synthesis of 8-chloro-3-isopropylimidazo[1,5-a]pyrazine (Compound 3a)

[0066] N-((3-chloropyrazin-2-yl)methyl)isobutyramide (Compound 2a) (1.5 g, 7.0 mmol) was dissolved in acetonitrile (22.5 mL), and phosphorus oxychloride (4.3 g, 28.0 mmol) and DMF (3 drops) were added. The mixture was heated to 60°C in an oil bath and stirred for 1 hour. After the reaction, the reaction solution was concentrated to dryness under reduced pressure, ice water was added, shaken, extracted with dichloromethane (200 mL), washed three times with ice water (80 mL), washed with saturated brine (80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a low-melting-point product, 8-chloro-3-isopropylimidazo[1,5-a]pyrazine, designated as Compound 3a, 840 mg, 4.3 mmol, with a yield of 61.4%.

[0067] Compound 3a was detected by mass spectrometry, EI-MS MS (m / z): 196.3 [M+H] + ; The NMR data of compound 3a are as follows: 1 H-NMR (CDCl3, 400MHz): δ7.80 (s, 1H), 7.62 (d, J = 8.0Hz, 1H), 7.34 (d, J = 8.0Hz, 1H), 3.35 ~ 3.29 (m, 1H), 1.52 (d, J = 8.0Hz, 3H), 1.47 (d, J = 8.0Hz, 3H).

[0068] (3) Synthesis of 8-chloro-1-iodo-3-isopropylimidazo[1,5-a]pyrazine (Compound 4a)

[0069] 8-Chloro-3-isopropylimidazo[1,5-a]pyrazine (Compound 3a) (822 mg, 4.2 mmol) was dissolved in DMF (10 mL), and NIS (1.13 g, 5.04 mmol) was added. The mixture was heated to 70°C in an oil bath and stirred for 3 hours. After completion of the reaction, the reaction solution was concentrated to dryness under reduced pressure, extracted with dichloromethane (200 mL), washed with saturated Na2SO3 solution (50 mL), and washed three times with saturated brine (80 mL). The product was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a solid. The solid was purified by silica gel flash column chromatography (dichloromethane → dichloromethane:methanol = 80:1) to give 8-chloro-1-iodo-3-isopropylimidazo[1,5-a]pyrazine as a pale yellow solid, designated as Compound 4a, 1.0 g, 3.11 mmol, in a yield of 74.0%.

[0070] Compound 4a was detected by mass spectrometry: EI-MS MS (m / z): 322.0 [M+H] + ; The NMR data of compound 4a are as follows: 1 H-NMR (CDCl3, 400MHz): δ7.64 (d, J = 4.0Hz, 1H), 7.30 (d, J = 4.0Hz, 1H), 3.27~3.22 (m, 1H), 1.44 (d, J = 8.0Hz, 6H).

[0071] (4) Synthesis of 1-iodo-3-isopropylimidazo[1,5-a]pyrazin-8-amine (Compound 5a)

[0072] 8-Chloro-1-iodo-3-isopropylimidazo[1,5-a]pyrazine (Compound 4a) (380 mg, 1.18 mmol) was placed in a thick-walled pressure-resistant glass bottle. Dioxane (5 mL) and concentrated aqueous ammonia (28 wt%, 5 mL) were added. The mixture was heated to 110°C in an oil bath and stirred for 7 hours. After the reaction, the reaction solution was cooled, decompressed to dryness, and water (50 mL) was added. The mixture was shaken, filtered, washed with water, and dried to obtain an off-white solid, 1-iodo-3-isopropylimidazo[1,5-a]pyrazin-8-amine, designated as Compound 5a, with a yield of 75.4% (270 mg, 0.89 mmol).

[0073] Compound 5a was detected by mass spectrometry: EI-MS MS (m / z): 303.1 [M+H] + ; The NMR data of compound 5a are as follows: 1 H-NMR (CDCl3, 400MHz): δ7.23 (d, J = 4.0Hz, 1H), 7.00 (d, J = 4.0Hz, 1H), 3.22 ~ 3.17 (m, 1H), 1.41 (d, J = 8.0Hz, 6H).

[0074] (5) Synthesis of 1-(2-aminobenzo[d]oxazol-5-yl)-3-isopropylimidazo[1,5-a]pyrazin-8-amine (Compound NJ-12)

[0075] 1-iodo-3-isopropylimidazo[1,5-a]pyrazin-8-amine (compound 5a) (580 mg, 1.92 mmol), 2-amino-benzoxazole-5-boronic acid hydrochloride (412 mg, 1.92 mmol), PdCl2(dppf) (110 mg, 0.15 mmol), sodium carbonate (814 mg, 7.68 mmol) and dioxane-water (28 mL-7 mL) were refluxed under nitrogen protection for 7 hours. After the reaction, the reaction solution was cooled and concentrated to dryness. Water (50 mL) was added, and the mixture was shaken and filtered to obtain a solid. The solid was purified by flash column chromatography on silica gel (eluted with dichloromethane: methanol = 10:1) and concentrated to obtain an off-white solid 1-(2-aminobenzo[d]oxazol-5-yl)-3-isopropylimidazo[1,5-a]pyrazin-8-amine, designated as compound NJ-12, 350 mg, 1.14 mmol, with a yield of 59.4%.

[0076] Compound NJ-12 was detected by mass spectrometry: EI-MS MS (m / z): 309.4 [M+H] + ; The NMR data of compound NJ-12 are as follows: 1 H-NMR (DMSO-d6, 400MHz): δ7.56(d,J=4.0Hz,1H),7.48(s,2H),7.43(d,J=8.0Hz,1H),7.34(s,1H),7. 17(d,J=8.0Hz,1H),7.01(d,J=4.0Hz,1H),5.98(br,s,2H),3.45-3.38(m,1H),1.34(d,J=8.0Hz,6H). 13 C-NMR(DMSO-d6,100MHz): δ163.83,152.14,148.13,145.34,144.42,134.8 3,130.99,127.96,121.95,116.56,113.55,108.75,106.66,25.58,21.07.

[0077] Example 2: Synthesis of 1-(2-aminobenzo[d]oxazol-5-yl)-3-cyclopentylimidazo[1,5-a]pyrazin-8-amine (Compound NJ-10)

[0078]

[0079] The preparation method was the same as that in Example 1, except that the starting material isobutyric acid in step (1) was replaced by cyclopentanecarboxylic acid, and 1-(2-aminobenzo[d]oxazol-5-yl)-3-cyclopentylimidazo[1,5-a]pyrazin-8-amine was finally synthesized, which was recorded as compound NJ-10.

[0080] Compound NJ-10 was detected by mass spectrometry: EI-MS MS (m / z): 335.1 [M+H] + ; The NMR data of compound NJ-10 are as follows: 1 H-NMR (DMSO-d6, 400MHz): δ7.59(d,J=4.0Hz,1H),7.52(s,2H),7.45(d,J=8.0Hz,1H),7.36(s,1H),7.19(d,J=8.0Hz,1H),7.03( d,J=4.0Hz,1H),6.04(br,s,2H),3.57-3.49(m,1H),2.14-2.10(m,2H),1.95-1.90(m,2H),1.82-1.79(m,2H),1.71-1.68(m,2H).

[0081] Example 3: Synthesis of 1-(2-aminobenzo[d]oxazol-5-yl)-3-cyclopropylimidazo[1,5-a]pyrazin-8-amine (Compound NJ-11)

[0082]

[0083] The preparation method was the same as that in Example 1, except that the starting material isobutyric acid in step (1) was replaced by cyclopropylcarboxylic acid, and 1-(2-aminobenzo[d]oxazol-5-yl)-3-cyclopropylimidazo[1,5-a]pyrazin-8-amine was finally synthesized, which was recorded as compound NJ-11.

[0084] Compound NJ-11 was detected by mass spectrometry: EI-MS MS (m / z): 307.3 [M+H] +; the synthesis data of compound NJ-11 are as follows: 1 H-NMR (DMSO-d6, 400MHz): δ7.81(d,J=8.0Hz,1H),7.54(s,2H),7.44(d,J=8.0Hz,1H),7.35(s,1H),7 .19(d,J=8.0Hz,1H),7.08(d,J=8.0Hz,1H),2.42-2.37(m,1H),1.12-1.08(m,2H),1.05-1.03(m,2H).

[0085] Example 4: Synthesis of 1-(2-aminobenzo[d]oxazol-5-yl)-3-(pentan-3-yl)imidazo[1,5-a]pyrazin-8-amine (Compound NJ-13)

[0086]

[0087] The preparation method was the same as that in Example 1, except that the starting material isobutyric acid in step (1) was replaced by 2-ethylbutyric acid, and 1-(2-aminobenzo[d]oxazol-5-yl)-3-(pentan-3-yl)imidazo[1,5-a]pyrazin-8-amine was finally synthesized, which was recorded as compound NJ-13.

[0088] Compound NJ-13 was detected by mass spectrometry: EI-MS MS (m / z): 337.2 [M+H] +; the NMR data of compound NJ-13 are as follows: 1 H-NMR (DMSO-d6, 400MHz): δ7.64(d,J=4.0Hz,1H),7.52(s,2H),7.44(d,J=8.0Hz,1H),7.35(d,J=4.0Hz,1H),7.16(d ,J=8.0Hz,1H),6.99(d,J=4.0Hz,1H),6.01(br,s,2H),3.10-3.08(m,1H),1.81-1.71(m,4H),0.77(t,J=8.0Hz,6H).

[0089] Example 5: Synthesis of 3-tert-butyl-1-(2-aminobenzo[d]oxazol-5-yl)imidazo[1,5-a]pyrazin-8-amine (Compound NJ-14)

[0090]

[0091] The preparation method was the same as that in Example 1, except that the starting material isobutyric acid in step (1) was replaced by 2,2-dimethylpropionic acid, and 3-tert-butyl-1-(2-aminobenzo[d]oxazol-5-yl)imidazo[1,5-a]pyrazin-8-amine was finally synthesized, which was recorded as compound NJ-14.

[0092] Compound NJ-14 was detected by mass spectrometry: EI-MS MS (m / z): 323.4 [M+H] + ; The NMR data of compound NJ-14 are as follows: 1H-NMR(DMSO-d6,400MHz): δ7.75(d,J=4.0Hz,1H),7.52(s,2H),7.46(d,J=8.0Hz,1H),7 .36(s,1H),7.19(d,J=8.0Hz,1H),7.00(d,J=4.0Hz,1H),5.99(br,s,2H),1.52(s,9H).

[0093] Example 6: Synthesis of 1-(2-aminobenzo[d]oxazol-5-yl)-3-(tetrahydrofuran-2-yl)imidazo[1,5-a]pyrazin-8-amine (Compound NJ-15)

[0094]

[0095] The preparation method was the same as that in Example 1, except that the starting material isobutyric acid in step (1) was replaced by tetrahydrofuran-2-carboxylic acid, and 1-(2-aminobenzo[d]oxazol-5-yl)-3-(tetrahydrofuran-2-yl)imidazo[1,5-a]pyrazin-8-amine was finally synthesized, which was recorded as compound NJ-15.

[0096] Compound NJ-15 was detected by mass spectrometry: EI-MS MS (m / z): 337.3 [M+H] + ; The NMR data of compound NJ-15 are as follows: 1 H-NMR (DMSO-d6, 400MHz): δ7.65(d,J=4.0Hz,1H),7.54(s,2H),7.45(d,J=8.0Hz,1H),7.38(d,J=4.0Hz,1H),7.20(d,J=8.0Hz,1H),7.0 9(d,J=8.0Hz,1H),6.09(br,s,2H),5.35(t,J=8.0Hz,1H),3.88-3.85(m,2H),2.66-2.61(m,1H),2.30-2.26(m,1H),2.12-2.00(m,2H).

[0097] Example 7: Detection of the inhibitory activity of compound NJ-12 on mTOR kinase

[0098] The compound NJ-12 prepared in Example 1 was used in this example.

[0099] Experimental methods:

[0100] 1. Reagents

[0101] Kinase buffer: 50 mM HEPES, pH 7.5, 1 mM EGTA, 0.01% Tween-20, 10 mM MgCl2, 3 mM MnCl2.

[0102] 2. Experimental steps:

[0103] The test compound NJ-12 and the control compound PI-103 were dissolved in DMSO and diluted fourfold to a predetermined starting concentration, for example, 10 μM. These solutions were then added to the enzyme reaction system. A DMSO control and a control without mTOR kinase were also established. The enzyme reaction system was prepared using kinase buffer to optimize the concentrations of mTOR enzyme, substrate (Ulight-4E-BP1), and ATP. The reaction system consisted of kinase buffer, ATP (8 μM), kinase substrates (50 nM, Ulight-4E-BP1), and mTOR kinase (4 nM). The drug solutions were added to the enzyme reaction system at varying concentrations (final drug concentrations are shown in Table 1). The reaction system was then incubated at room temperature for 30 minutes. Pre-treated detection buffer containing EDTA and an antibody (Eu-anti-phospho-4E-BP1) was added, the mixture was centrifuged, and the reaction was equilibrated at room temperature for 60 minutes. Signal data were collected using an Envision instrument, and the fluorescence readings were measured as the ratio (665 nm / 615 nm). The inhibition rate was calculated according to the following formula: % inhibition rate = (the numerical ratio of the fluorescence reading of the DMSO control - the numerical ratio of the fluorescence reading of the sample) / (the numerical ratio of the fluorescence reading of the DMSO control - the numerical ratio of the fluorescence reading of the control without kinase addition). The curve was fitted using the formula Y = Bottom + (Top - Bottom) / (1 + (IC50 / X)^HillSlope) to obtain the IC50. 50 value.

[0104] Table 1 Inhibition rate of compound NJ-12 on mTOR kinase at different concentrations

[0105]

[0106] Table 2 Inhibition rate of mTOR kinase by reference substance PI-103 at different concentrations

[0107]

[0108]

[0109] From Table 1, Table 2, Figure 1 、 Figure 2 The data show that compound NJ-12 inhibits mTOR kinase with IC 50 The IC value of the control substance PI-103 for inhibiting mTOR kinase is 3.61nM. 50The inhibitory activity of compound NJ-12 on mTOR kinase target was significantly stronger than that of the reference substance PI-103.

[0110] Example 8: Inhibitory effect of the test samples on the proliferation of human small cell lung cancer cells A549

[0111] Compounds NJ-10 to NJ-15 prepared in Examples 1 to 6 were used in this example.

[0112] The reference substance PI-103 was purchased commercially.

[0113] 1. Tumor cell culture

[0114] Tumor cell line: Human small cell lung cancer A549 cells were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences. They were cultured at 37°C in F-12K culture medium containing 10% fetal bovine serum and passaged every 2 to 3 days.

[0115] Preparation of drug solution: Compounds NJ-10 to NJ15 prepared in Examples 1 to 6 and the reference substance PI-103 were mixed with F-12K culture medium to prepare drug solutions with concentrations of 1000 nM, 333 nM, 111 nM, 37.0 nM, 12.4 nM, 4.12 nM, 1.37 nM and 0.457 nM, respectively.

[0116] 2. Alamar Blue assay for cell proliferation

[0117] 1) Collect A549 cells in the logarithmic phase, adjust the cell suspension concentration, and plate the cells in a 96-well plate. Add 100 μL of the suspension to each well to achieve a cell density of 3000 cells / well (fill the edge wells with 0.01 M PBS, pH 7.4; the cell density is adjusted according to the cell growth rate);

[0118] 2) Incubate at 5% CO2, 37°C until cells adhere. After 24 h, remove the original culture medium and add the drug solutions prepared above at different concentration gradients, with the concentrations of each drug being 1000 nM, 333 nM, 111 nM, 37.0 nM, 12.4 nM, 4.12 nM, 1.37 nM, and 0.457 nM, with two replicate wells for each concentration.

[0119] 3) Incubate the culture plate in an incubator at 37°C for 72 hours;

[0120] 4) Add 22 μL of Lamar Blue (Resazurin) solution to each well;

[0121] 5) Incubate the culture plate in an incubator at 37°C for 5 hours;

[0122] 6) Measure the absorbance (fluorescence intensity) at 530 / 590 nm using a microplate reader.

[0123] 3. Data Analysis

[0124] Graphpadprism software was used for graphic analysis and IC was calculated from the measured fluorescence intensity data. 50 ,The specific experimental results are shown in Table 3.

[0125] Table 3 Inhibitory effects of the tested samples on the proliferation of human small cell lung cancer cells A549 (IC 50 )

[0126]

[0127]

[0128] From the data in Table 3, it can be seen that the prepared NJ series compounds have a significantly stronger inhibitory effect on the proliferation of human small cell lung cancer cells A549 than the reference substance PI-103. Among them, the inhibitory activity of compound NJ-12 on A549 cells is about 30 times that of the reference substance PI-103.

[0129] Example 9: In vivo efficacy evaluation of compound NJ-12 in a nude mouse xenograft model of human small cell lung cancer cells A549

[0130] 1. Experimental Materials

[0131] 1.1 Reagents

[0132] F-12K culture medium; fetal bovine serum; penicillin-streptomycin double antibody (100U penicillin and 100ug / mL streptomycin), trypsin (containing 0.25% EDTA), PBS (pH=7.4, 0.01M), etc.

[0133] 1.2 Experimental Animals

[0134] Female BALB / c nude mice (18 mice, 6-8 weeks old, weighing 20-24 g) were purchased from Cavensburg (Suzhou) Model Animal Research Co., Ltd. and housed in the SPF animal room at Suzhou Shengsu New Drug Development Co., Ltd. at a temperature of 20-25°C, a relative humidity of 40%-70%, and a 12-hour light and dark cycle. The animals had free access to water and food. After approximately 5 days of normal feeding, mice that were in good physical condition after veterinary examination were enrolled in this experiment. Before grouping, the animals were identified at the base of their tails with a marker pen. After grouping, each animal was identified by ear notching.

[0135] 1.3 Transplantable Tumor Cells

[0136] Human small cell lung cancer A549 cells were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences (CAS, cryopreserved in liquid nitrogen in our laboratory).

[0137] 2. Experimental methods

[0138] 2.1 A549 cell culture

[0139] A549 cells were routinely cultured in F-12K complete medium (containing 10% fetal bovine serum and 1% cyan-chain double antibody) under 5% CO2, 37°C, and saturated humidity conditions. Depending on cell growth, cells were washed with PBS and then digested with trypsin (containing 0.25% EDTA) and passaged 1 to 2 times per week at a passage ratio of 1:3 to 1:6.

[0140] 2.2 Animal Model Preparation

[0141] A549 cells in the logarithmic growth phase were collected, counted, and resuspended in serum-free F-12K medium to adjust the cell concentration to 8×10 7 cells / mL; pipette the cells to disperse them evenly and then put them into a 50-mL centrifuge tube, which was placed in an ice box; use a 1-mL syringe to draw up the cell suspension and inject it into the subcutaneous part of the right axilla of the nude mice. Each animal was inoculated with 100 μL (8×10 6 A549 nude mouse xenograft tumor model was established. The animal status and tumor growth were regularly observed after inoculation. The tumor diameter was measured using an electronic vernier caliper, and the data was entered into an Excel spreadsheet to calculate the tumor volume. When the tumor volume reached 100-300 mm 3 Animals in good health and with similar tumor volumes were selected and randomly divided into two groups (n=6). The day of grouping was designated as the first day of the experiment (D1). After the start of the experiment, tumor diameters were measured twice a week, and tumor volumes were calculated. Animal body weights were also weighed and recorded.

[0142] 2.3 Preparation of drug delivery preparations

[0143] Before each administration, an appropriate amount of compound NJ-12 was weighed, and an appropriate volume of PEG400 was added thereto. The mixture was vortexed to mix the liquid evenly to obtain a final concentration of 0.5 mg mL -1 The dosing preparation was prepared once a week and stored in a 4°C refrigerator.

[0144] 2.4 Animal Grouping and Dosing

[0145] Drug administration began on the day of grouping, and was administered orally orally, once a day. The dosage was 5.0 mg / kg in the first week and 10.0 mg / kg in the next two weeks. The experiment was terminated after 3 weeks.

[0146] Among them, the solvent control group: A549 nude mouse transplanted tumor model mice were given an equal volume of PEG400.

[0147] from Figure 3 It can be seen that after administration, compound NJ-12 can significantly inhibit the growth of small cell lung cancer (A549) transplanted tumors in nude mice.

[0148] from Figure 4 It can be seen that after 3 weeks of administration, the weight of A549 nude mouse transplanted tumor model mice did not decrease after administration of compound NJ-12, and the nude mice did not die compared with the body weight of the solvent control group, indicating that compound NJ-12 has little toxic side effects.

[0149] The present invention provides an imidazo[1,5-a]pyrazine antitumor compound, its preparation method, and its application. Numerous methods and approaches exist for implementing this technical solution. The foregoing description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. An imidazo[1,5-a]pyrazine anti-tumor compound of formula I or a pharmaceutically acceptable salt or prodrug thereof; in, R is selected from C2-C6 alkyl, halogenated C2-C6 alkyl, C3-C6 cycloalkyl or heterocycloalkyl.

2. The compound according to claim 1, characterized in that R is selected from isopropyl, tert-butyl, pent-3-yl, cyclopropyl, cyclopentyl or tetrahydrofuran-2-yl.

3. The compound according to claim 1, characterized in that The imidazo[1,5-a]pyrazine anti-tumor compound is selected from any one of the following compounds:

4. The method for preparing the imidazo[1,5-a]pyrazine antitumor compound of formula I according to claim 1, characterized in that: The steps include: Compound 1 undergoes a first reaction with a formic acid compound under the action of a first coupling agent and a first base to obtain compound 2; compound 2 undergoes a second reaction under the action of phosphorus oxychloride to obtain compound 3; compound 3 undergoes a third reaction under the action of an iodination reagent to obtain compound 4; compound 4 undergoes a fourth reaction under the action of an amination reagent to obtain compound 5; compound 5 undergoes a fifth reaction with a boric acid compound under the action of a palladium catalyst and a second base to obtain an imidazo[1,5-a]pyrazine anti-tumor compound I; wherein R is selected from C2-C6 alkyl, halogenated C2-C6 alkyl, C3-C6 cycloalkyl or heterocycloalkyl; X is selected from Cl or Br; Wherein, the boric acid compound is 5. The preparation method according to claim 4, characterized in that The first coupling agent is any one or a combination of HATU, HOBt, EDCI, BOP and DCC; the first base is any one or a combination of N-methylmorpholine, triethylamine, diethylamine and diisopropylethylamine; the molar ratio of the compound 1 to the formic acid compound, the first coupling agent and the first base is 1.0-1.1:1.0-1.5:2.0-5.0:2.0-5.0; the reaction temperature of the first reaction is 0°C-60°C; the molar ratio of the compound 2 to phosphorus oxychloride is 1.0-1.2:3.0-6.0; the reaction temperature of the second reaction is 40°C-80°C; the iodine reagent is N-iodosuccinimide; the compound 3 The molar ratio of the compound 5 to the iodine reagent is 1.0-1.1:1.1-2.0; the reaction temperature of the third reaction is 20°C-80°C; the aminating reagent is aqueous ammonia; the reaction temperature of the fourth reaction is 90°C-130°C; the palladium catalyst is any one or a combination of tetrakistriphenylphosphine palladium, tert-butylphosphine palladium, dichloro[1,1'-bis(diphenylphosphino)ferrocene]palladium, tris(dibenzylacetone)dipalladium and palladium acetate; the second base is any one or a combination of sodium carbonate, potassium carbonate and sodium bicarbonate; the molar ratio of the compound 5 to the boric acid compound, the palladium catalyst and the second base is 1.0-1.2:1.0-2.0:0.04-0.20:2.0-8.

0.

6. A pharmaceutical composition, characterized in that The invention comprises an imidazo[1,5-a]pyrazine antitumor compound or a pharmaceutically acceptable salt thereof represented by formula I as claimed in claim 1, and one or more pharmaceutically acceptable carriers.

7. The pharmaceutical composition according to claim 6, characterized in that The dosage form of the pharmaceutical composition is one or more of subcutaneous injection, intradermal injection, spray, powder aerosol, external solution, lotion, liniment, ointment, plaster, paste, patch, granule, tablet, capsule, and liquid preparation.

8. Use of the imidazo[1,5-a]pyrazine antitumor compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, or the pharmaceutical composition according to claim 6 in the preparation of an mTOR kinase inhibitor.

9. Use of the imidazo[1,5-a]pyrazine anti-tumor compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, or the pharmaceutical composition according to claim 6, in the preparation of a drug for treating diseases associated with mTOR target activation.

10. The use according to claim 9, characterized in that The related disease is non-small cell lung cancer.

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