Beta-carboline 3,6,9-position modified derivatives, processes for their preparation and uses thereof

Abstract

The present application relates to a kind of β-caproline 3,6,9-position modification derivatives and its preparation method and use, belong to natural medicine and pharmaceutical chemistry technical field.It is specifically related to a kind of β-caproline 3,6,9-position modification derivatives or its pharmaceutically acceptable salt, and further provides a kind of pharmaceutical composition, provides the preparation method of the β-caproline 3,6,9-position modification derivatives, simultaneously points out the application of β-caproline 3,6,9-position modification derivatives in preparation SIRT6 inhibitor or preparation treatment tumour drug.The β-caproline 3,6,9-position modification derivatives or its pharmaceutically acceptable salt shown in the present application is as shown in general formula I.Wherein, X, n, R 1 , R 2 And R 3 As described in claim and specification.

Description

Technical Field

[0001] This invention relates to a β-carboline 3,6,9-position modified derivative, its preparation method and uses, belonging to the field of natural medicine and medicinal chemistry technology. Background Technology

[0002] SIRT6 belongs to the family of class III histone deacetylases, mediating both histone and non-histone deacetylation. Furthermore, SIRT6 promotes the deacylation of long-chain fatty acid groups and possesses ADP-ribotransferase activity. Abnormally high expression of SIRT6 is associated with poor prognosis in various tumors. SIRT6 promotes breast cancer progression by enhancing oxidative phosphorylation and induces tumor drug resistance by enhancing DNA damage repair mechanisms. Although some compounds have shown inhibitory potential against SIRT6, the exploration of SIRT6 inhibitors is still in its early stages. Pharmacological inhibition of SIRT6 has shown advantages in the treatment of tumors and type 2 diabetes. While some compounds have shown some SIRT6 inhibitory potential, problems such as lack of subtype selectivity and poor drug-likeness remain, necessitating the search for new and promising SIRT6 inhibitors. Summary of the Invention

[0003] This invention provides a β-carboline 3,6,9-position modified derivative, its preparation method, and its use. The purpose is to provide a β-carboline 3,6,9-position modified derivative or a pharmaceutically acceptable salt thereof, and further to provide a pharmaceutical composition, a method for preparing the β-carboline 3,6,9-position modified derivative, and to indicate the application of the β-carboline 3,6,9-position modified derivative in the preparation of SIRT6 inhibitors or in the preparation of drugs for treating tumors.

[0004] Specifically, the present invention provides the following technical solution:

[0005] The first object of the present invention is to provide a β-carboline 3,6,9-position modified derivative or a pharmaceutically acceptable salt thereof, the general structural formula of which is shown below:

[0006]

[0007] in,

[0008] X is O or NH, and n is 1 or 2;

[0009] R 1 It is a 5- or 6-membered heteroaryl or substituted heteroaryl, wherein the heteroaryl contains 1-4 heteroatoms of N, O or S atoms in the aromatic heterocycle, and the substituted heteroaryl contains a halogen, or a hydroxyl, or a methoxy, or an alkyl containing 1-7 carbon atoms;

[0010] R 2 It is an aryl or substituted aryl containing 4-13 carbon atoms, or a hydrocarbon containing 1-10 carbon atoms, or a cycloalkyl containing 3-5 carbon atoms, or an alkoxy containing 1-4 carbon atoms, or a heteroaryl or substituted heteroaryl containing 2-10 carbon atoms, wherein the heteroaryl contains 1-4 N, O or S atoms in its aromatic heterocycle, and the substituent in the substituted aryl or substituted heteroaryl is a halogen, or a hydroxyl, or a cyano, or a trifluoromethyl, or a methoxy, or a trifluoromethoxy, or an alkyl containing 1-8 carbon atoms;

[0011] R 3 It is hydrogen, or halogen, or methoxy, or an alkyl group containing 1-9 carbon atoms.

[0012] The heteroaryl group described above can be derived from a heterocycle containing one, two, three or four identical or different heteroatoms, especially from a heterocycle containing one, two or three, especially one or two identical or different heteroatoms. The heterocycle can be a mono- or polycyclic ring, such as a mono-, bi- or tri-ring, preferably a mono- or bi-ring, especially a mono-ring.

[0013] The aryl group mentioned above is derived from an aromatic ring and can be a mono- or polycyclic ring, such as a mono-, bi-, or tri-ring, preferably a mono- or bi-ring.

[0014] The substituted heteroaryl or substituted aryl groups mentioned above can be monosubstituted or polysubstituted. In monosubstituted cases, the substituents can be in any position, while in polysubstituted cases, the substituents can be the same or different and can be located in any position.

[0015] The halogens mentioned above refer to fluorine, chlorine, bromine, or iodine atoms.

[0016] Preferably, X is NH and n is 1 or 2.

[0017] More preferably, X is NH and n is 1.

[0018] Preferably, the R 1 It is a 5- or 6-membered heteroaryl or substituted heteroaryl, wherein the heteroaryl contains 1, 2 or 3 heteroatoms of N, O or S atoms, and the substituted heteroaryl contains a halogen, a hydroxyl group, or an alkyl group containing 1-4 carbon atoms.

[0019] More preferably, the one is R 1 The substituents are pyridyl, furanyl, thiophene, triazolyl, pyrazinyl, pyrimidinyl, or thiazolyl, wherein the substituent is a halogen, a hydroxyl group, or a methyl group.

[0020] Most preferably, R 1 It is pyridyl.

[0021] Preferably, the R 2 It is a phenyl or substituted phenyl, or a naphthyl, or an alkenyl containing 2-4 carbon atoms, or a cycloalkyl containing 3 or 4 carbon atoms, or an alkoxy containing 2 or 3 carbon atoms, wherein the substituent in the substituted phenyl is a halogen, or a cyano, or a trifluoromethyl, or a methoxy, or a trifluoromethoxy, or an alkyl containing 1-4 carbon atoms.

[0022] More preferably, the R 2 It is phenyl or substituted phenyl, or naphthyl, or isobutylene, or cyclopropane, or ethoxy, wherein the substituent in the substituted aryl group is a fluorine atom, or a chlorine atom, or a cyano, or a trifluoromethyl, or a methoxy, or a trifluoromethoxy, or a tert-butyl.

[0023] Most preferably, the R 2 The substituted phenyl group is a cyano group.

[0024] Preferably, the R 3 It can be a hydrogen atom, or a chlorine atom, or a bromine atom.

[0025] Most preferably, the R 3 It is a chlorine atom.

[0026] Particularly preferably, X is NH, n is 1, and R 1 It is pyridinyl, R 2 For substituted phenyl groups, wherein the substituent is a cyano group, and R 3 It is a chlorine atom.

[0027] Furthermore, the structural formulas of the β-carboline 3,6,9-position modified derivatives and their pharmaceutically acceptable salts are shown below:

[0028]

[0029] A second object of the present invention is to provide a method for preparing the above-mentioned β-carboline 3,6,9-position modified derivatives or pharmaceutically acceptable salts thereof.

[0030] The β-carboline 3,6,9-position modified derivative of this invention can be prepared by the following method, including the following steps:

[0031] (1) Compound 1L-tryptophan was dissolved in sodium hydroxide solution, and 37% formaldehyde solution was added. The mixture was reacted at 37°C for 2 days to obtain compound 2. Compound 2 was dissolved in anhydrous methanol, and thionyl chloride was added dropwise under ice bath conditions. The mixture was refluxed at 70°C for 6 hours to obtain compound 3. Compound 3 was dissolved in N,N-dimethylformamide solution, and potassium permanganate was added under ice bath conditions. The mixture was reacted for 1 hour and then transferred to room temperature for 14 hours to obtain compound 4.

[0032] (2) Compound 4 was dissolved in methanol, and 1N NaOH solution was added. The mixture was heated at 78°C for 2 hours to obtain compound 5. Compound 5 was dissolved in N,N-dimethylformamide solution, and carbodiimide hydrochloride, 1-hydroxybenzotriazole and the corresponding ammonia were added. The compound was obtained by reacting at room temperature for 12 hours. Alternatively, carbodiimide hydrochloride, 4-dimethylaminopyridine, and the corresponding alcohol may be added. Compounds The obtained compound was dissolved in a tetrahydrofuran solution containing sodium hydride, and after stirring for 1 hour, the corresponding bromoalkane or benzyl bromo was added. The reaction was carried out at room temperature for 12 hours to obtain the compound. or

[0033] Alternatively, the β-carboline 3,6,9-position modified derivative of this invention can be prepared by the following method, including the following steps:

[0034] (1) Compound 1L-tryptophan was dissolved in sodium hydroxide solution, and 37% formaldehyde solution was added. The mixture was reacted at 37°C for 2 days to obtain compound 2. Compound 2 was dissolved in anhydrous methanol, and thionyl chloride was added dropwise under ice bath conditions. The mixture was refluxed at 70°C for 6 hours to obtain compound 3. Compound 3 was dissolved in N,N-dimethylformamide solution, and potassium permanganate was added under ice bath conditions. The mixture was reacted for 1 hour and then transferred to room temperature for 14 hours to obtain compound 4.

[0035] (2) Compound 4 was dissolved in dichloromethane, a halogenating agent was added, and the mixture was refluxed at 78°C for 12 h to obtain compound 4. The obtained compound was dissolved in methanol, and 1N NaOH solution was added. The mixture was heated at 78°C for 2 hours to obtain the compound. The above compound was dissolved in N,N-dimethylformamide solution, and carbodiimide hydrochloride, 1-hydroxybenzotriazole, and the corresponding ammonia were added. The compound was obtained by reacting at room temperature for 12 hours. Alternatively, carbodiimide hydrochloride, 4-dimethylaminopyridine, and the corresponding alcohol may be added. Compounds The obtained compound was dissolved in a tetrahydrofuran solution containing sodium hydride, and after stirring for 1 hour, the corresponding bromoalkane or benzyl bromo was added. The reaction was carried out at room temperature for 12 hours to obtain the compound.

[0036] In the above technical solution, compound 1, compound 2, compound 3, compound 4, and compound 5 each have the following structures:

[0037]

[0038] In the above technical solution, the reaction order in step (2) is not important; any order can achieve the purpose of the present invention.

[0039] Preferably, the corresponding ammonia is 2-methylaminopyridine, 2-methylaminothiophene, 2-methylaminofuran, 3-methylaminopyridine, 2-(2-aminoethyl)pyridine, 2-aminomethyl-5-chloropyridine, 2-aminomethyl-5-fluoropyridine, 6-methyl-2-pyridinemethylamine, 2-aminomethylpyrazine, or 4-aminomethylpyrimidine.

[0040] Preferably, the corresponding alcohols are 2-chloro-5-hydroxymethylpyridine, 2-hydroxymethylpyrazine, 5-hydroxymethylthiazole, 3-hydroxymethylpyridine, and 1-hydroxymethyl-1,2,4-triazole.

[0041] Preferably, the corresponding benzyl bromide or bromoalkyl group is 4-tert-butylbenzyl bromide, 1-bromo-trifluoro-p-xylene, 4-chlorobenzyl bromide, 3,4,5-trifluorobenzyl bromide, 4-chloro-3-fluorobenzyl bromide, p-cyanobenzyl bromide, 2-(bromomethyl)naphthalene, bromomethylcyclopropane, 1-bromo-3-methyl-2-butene, 2-bromoethylmethyl ether, 2,3,6-trifluorobenzyl bromide, 4-fluoro-3-trifluoromethylbenzyl bromide, 4-trifluoromethoxybenzyl bromide, and 3,5-dimethoxybenzyl bromide.

[0042] Preferably, the halogenating agents are N-chlorosuccinimide or N-bromosuccinimide.

[0043] Particularly preferably, the corresponding ammonia is dimethylaminopyridine, the corresponding benzyl bromo is 4-cyanobenzyl bromide, and the halogenating agent is N-chlorosuccinimide.

[0044] A third object of the present invention is to provide a pharmaceutical composition comprising a therapeutically effective amount of the β-carboline 3,6,9-position modified derivative of the general formula, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0045] A fourth object of the present invention is to provide the use of the β-carboline 3,6,9-position modified derivatives thereof and pharmaceutically acceptable salts thereof or the pharmaceutical compositions thereof in the preparation of SIRT6 inhibitors.

[0046] A fifth object of the present invention is to provide the use of the β-carboline 3,6,9-position modified derivatives thereof and pharmaceutically acceptable salts thereof or the pharmaceutical compositions thereof in the preparation of medicaments for treating tumor diseases.

[0047] Furthermore, the tumors are breast cancer, pancreatic cancer, leukemia, neuroblastoma, liver cancer, and lung cancer.

[0048] Furthermore, the breast cancer includes breast cancer cells MCF-7, MDA-MB-231, and MDA-MB-453; the pancreatic cancer includes pancreatic cancer cells BxPC-3, Panc-1, MIA-PaCa-2, and Capan-1; the leukemia includes leukemia cells HL-60, K562, U937, and THP-1; the neuroblastoma includes neuroblastoma cells SH-SY5Y; the liver cancer includes liver cancer cells HepG2, Huh7, and Hep3B; and the lung cancer includes lung cancer cells A549, BE1, and LH7.

[0049] The beneficial effects of the present invention: The β-carboline 3,6,9-position modified derivative provided by the present invention has a good inhibitory effect on the deacetylation of lysine in SIRT6 protein, and shows good anti-tumor activity against tumor cells such as MCF-7, HL-60, SH-SY5Y, and MIA-PaCa-2, and has the potential to be used as an anti-cancer drug. Detailed Implementation

[0050] The following non-limiting embodiments are intended to enable those skilled in the art to more fully understand the invention, but do not limit the invention in any way.

[0051] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0052] The β-carboline 3,6,9-position modified derivatives described in the following examples were prepared according to the following synthetic route:

[0053]

[0054]

[0055] Example 1

[0056] The structure of compound 10d is shown below:

[0057]

[0058] (1) 10 g of compound 1L-tryptophan (49.02 mmol) was dissolved in 10 mL of 0.5 N NaOH solution, and then 6 mL of 37% formaldehyde solution (73.94 mmol) was added. The reaction was carried out at 37 °C for two days. Thin-layer chromatography (TLC) was used to monitor the reaction. The reaction was basically complete. After cooling, glacial acetic acid was added to adjust the pH until a precipitate was formed. The precipitate was filtered, dried, and 29.28 g of compound was obtained.

[0059] (2) 9.28 g of compound 2 (42.96 mmol) was dissolved in 120 mL of anhydrous methanol, and 7.36 mL of SOCl2 (101.34 mmol) was added dropwise under ice bath conditions. The mixture was then refluxed at 70 °C for 6 h. The reaction was monitored by TLC until it was complete. After cooling, the reaction solution was concentrated, and then 100 mL of saturated sodium bicarbonate solution was added. The mixture was extracted three times with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 36 g of compound.

[0060] (3) 6 g of compound 3 (30.44 mmol) was dissolved in 100 mL of DMF. 14 g of potassium permanganate (88.58 mmol) was added under ice bath conditions. After reacting for 1 h, the mixture was transferred to room temperature and reacted for 14 h. The reaction was monitored by TLC and found to be complete. The mixture was filtered, and the filtrate was extracted three times with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (DCM:MeOH = 30:1) to obtain 44.0 g of compound.

[0061] (4) Dissolve 4 g of compound 4 (17.69 mmol) in dichloromethane, add 2.36 g (17.69 mmol) of N-chlorosuccinimide, reflux at 78 °C overnight, monitor by TLC, the reaction is complete, filter, evaporate the filtrate to dryness, extract with ethyl acetate 3 times, wash once with saturated brine, dry with anhydrous sodium sulfate, filter, concentrate, and separate by silica gel column chromatography (DCM:MeOH=40:1) to obtain 3.5 g of compound 4a.

[0062] (5) Dissolve 1 g of compound 4a (3.84 mmol) in dichloromethane, add 10 mL of 1N NaOH solution, and heat the reaction for 2 h. The reaction was monitored by TLC and found to be complete. After cooling, 2N hydrochloric acid solution was added to adjust the pH to 4-5, and a precipitate was formed. The precipitate was filtered, dried, and compound 8a 890 mg was obtained.

[0063] (6) 320 mg of compound 8a (1.3 mmol) was dissolved in DMF, and 320 mg (1.65 mmol) of carbodiimide hydrochloride (EDCI) and 225 mg (1.65 mmol) of 1-hydroxybenzotriazole (HOBT) were added. After stirring at room temperature for 30 min, 140.4 mg (1.3 mmol) of 2-aminomethylpyridine was added and the reaction was carried out for 12 h. The reaction was monitored by TLC and found to be complete. The reaction solution was extracted three times with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (DCM:MeOH = 100:1) to obtain 319 mg of compound 9a.

[0064] (7) 100 mg of compound 9a (0.29 mmol) was dissolved in anhydrous tetrahydrofuran, 90 mg of sodium hydride (3.75 mmol) was added, and the mixture was stirred at room temperature for 40 min. Then, 56.87 mg (0.29 mmol) of 4-cyanobenzyl bromide was added, and the mixture was reacted at room temperature for 2 h. The reaction was monitored by TLC until it was complete. The mixture was filtered, and the filtrate was evaporated to dryness. The filtrate was extracted three times with ethyl acetate, washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (DCM:MeOH = 100:1) to give 2.91 g of compound 10d as a white solid, with a yield of 59%. 1 H NMR (CDCl3, 600MHz) δ8.97 (1H, s), 8.88 (1H, d, J = 1.0Hz), 8.66 (1H, d, J = 1.0Hz), 8.5 9(1H,ddd,J=4.9,1.8,0.9Hz),8.15(1H,d,J=2.0Hz),7.67(1H,td,J=7.7,1.8Hz),7. 59–7.55(2H,m),7.54(1H,dd,J=8.7,2.0Hz),7.37(1H,d,J=7.8Hz),7.31(1H,d,J=8 .7Hz),7.20–7.18(2H,m),7.18(1H,d,J=1.8Hz),5.64(2H,s),4.85(2H,d,J=5.6Hz). 13 CNMR(CDCl3,151MHz)δ165.07,157.21,149.31,141.20,140.95,139.75,137.91,136.79,132.94,130.41,129.49,128 .53,127.00,126.92,122.89,122.32,122.11,121.99,118.17,114.61,112.27,110.75,46.85,44.83.HRMS(ESI)calcd for C 26 H 19 ClN5O,[M+H] + :m / z452.1273, found 452.1274.

[0065] Example 2

[0066] The structure of compound 7a is shown below:

[0067]

[0068] (1) 10 g of compound 1L-tryptophan (49.02 mmol) was dissolved in 10 mL of 0.5 N NaOH solution, and then 6 mL of 37% formaldehyde solution (73.94 mmol) was added. The reaction was carried out at 37 °C for two days. Thin-layer chromatography (TLC) was used to monitor the reaction. The reaction was basically complete. After cooling, glacial acetic acid was added to adjust the pH until a precipitate was formed. The precipitate was filtered, dried, and 29.28 g of compound was obtained.

[0069] (2) 9.28 g of compound 2 (42.96 mmol) was dissolved in 120 mL of anhydrous methanol, and 7.36 mL of SOCl2 (101.34 mmol) was added dropwise under ice bath conditions. The mixture was then refluxed at 70 °C for 6 h. The reaction was monitored by TLC until it was complete. After cooling, the reaction solution was concentrated, and then 100 mL of saturated sodium bicarbonate solution was added. The mixture was extracted three times with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 36 g of compound.

[0070] (3) 6 g of compound 3 (30.44 mmol) was dissolved in 100 mL of DMF. 14 g of potassium permanganate (88.58 mmol) was added under ice bath conditions. After reacting for 1 h, the mixture was transferred to room temperature and reacted for 14 h. The reaction was monitored by TLC and found to be complete. The mixture was filtered, and the filtrate was extracted three times with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (DCM:MeOH = 30:1) to obtain 44.0 g of compound.

[0071] (4) 1 g of compound 4 (4.42 mmol) was dissolved in dichloromethane, and 10 mL of 1 N NaOH solution was added. The mixture was heated for 2 h. The reaction was monitored by TLC and found to be complete. The mixture was cooled, and then 2 N hydrochloric acid solution was added to adjust the pH to 4-5. A precipitate was formed. The precipitate was filtered, dried, and 5920 mg of compound was obtained.

[0072] (5) 500 mg of compound 5 (2.36 mmol) was dissolved in DMF, and 543 mg (2.83 mmol) of carbodiimide hydrochloride (EDCI) and 600 mg (2.83 mmol) of 1-hydroxybenzotriazole (HOBT) were added. After stirring at room temperature for 30 min, 255 mg (2.36 mmol) of 2-aminomethylpyridine was added and the reaction was carried out for 12 h. The reaction was monitored by TLC and found to be complete. The reaction solution was extracted three times with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (DCM:MeOH = 100:1) to obtain 292 mg of compound 6a.

[0073] (6) 100 mg of compound 6a (0.33 mmol) was dissolved in anhydrous tetrahydrofuran, 150 mg of sodium hydride (6 mmol) was added, and the mixture was stirred at room temperature for 40 min. Then, 64.7 mg (0.33 mmol) of 4-tert-butylbenzyl bromide was added, and the mixture was reacted at room temperature for 2 h. The reaction was monitored by TLC until it was complete. The mixture was filtered, and the filtrate was evaporated to dryness. The filtrate was extracted three times with ethyl acetate, washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (DCM:MeOH = 100:1) to give 68 mg of compound 7a as a white solid, with a yield of 49%. 1 H NMR (CDCl3, 600MHz) δ9.01–8.95 (2H, m), 8.75 (1H, d, J = 1.0 Hz), 8.60 (1H, ddd, J = 4.9, 1. 8,0.9Hz),8.25(1H,dt,J=8.0,1.0Hz),7.66(1H,td,J=7.7,1.8Hz),7.61(1H,ddd,J=8. 3,7.1,1.2Hz),7.53–7.47(1H,m),7.41–7.32(2H,m),7.32–7.27(2H,m),7.19(1H,ddd, J=7.5,4.8,1.1Hz),7.11–7.06(2H,m),5.58(2H,s),4.87(2H,d,J=5.7Hz),1.26(9H,s). 13 CNMR(CDCl3,151MHz)δ165.67,157.58,151.08,149.28,141.92,140.33,137.92,136.80,133.02,130.58,129.27,128.93,126.25,125.9 5,122.36,122.24,122.00,121.75,120.72,114.54,109.98,77.26,77.04,76.83,46.88,44.89,34.53,31.26.HRMS(ESI)calculatedfor C 29 H 29 N4O, [M+H] + :m / z 449.2336, found 449.2335.

[0074] Example 3

[0075] The structure of compound 7f is shown below:

[0076]

[0077] The 4-tert-butylbenzyl bromide used in step (6) of Example 2 was replaced with 4-trifluoromethylbenzyl bromide, and the experimental operation of Example 1 was repeated under the same conditions to obtain compound 7f, a white solid with a yield of 61.3%. 1 H NMR (CDCl3, 600MHz) δ9.01–8.95 (2H, m), 8.75 (1H, d, J = 1.0 Hz), 8.60 (1H, ddd, J = 4.9, 1. 8,0.9Hz),8.25(1H,dt,J=8.0,1.0Hz),7.66(1H,td,J=7.7,1.8Hz),7.61(1H,ddd,J=8. 3,7.1,1.2Hz),7.53–7.47(1H,m),7.41–7.32(2H,m),7.32–7.27(2H,m),7.19(1H,ddd, J=7.5,4.8,1.1Hz),7.11–7.06(2H,m),5.58(2H,s),4.87(2H,d,J=5.7Hz),1.26(9H,s). 13 C NMR (CDCl3, 151MHz) δ165.67,157.58,151.08,149.28,141.92,140.33,137.92,136.80,133.02,130.58,129.27,128.93,126.25,125. 95,122.36,122.24,122.00,121.75,120.72,114.54,109.98,77.26,77.04,76.83,46.88,44.89,34.53,31.26.HRMS(ESI)calculated forC 29 H 29 N4O, [M+H] + :m / z 449.2336, found 449.2335.

[0078] Example 4

[0079] The structure of compound 7k is shown below:

[0080]

[0081] The 4-tert-butylbenzyl bromide used in step (6) of Example 2 was replaced with 4-chlorobenzyl bromide, and the experimental operation of Example 1 was repeated under the same conditions to obtain compound 7k, a white solid with a yield of 45.2%. 1H NMR(CDCl3,600MHz)δ9.01–8.96(2H,m),8.70(1H,d,J=1.0Hz),8.60(1H,dt,J=5.0,1.4Hz),8.24(1H,d,J=7.8Hz),7.69–7.63(1H,m),7.63–7.5 7(1H,m),7.43(1H,d,J=8.3Hz),7.41–7.34(2H,m),7.28–7.22(2H,m),7 .22–7.16(1H,m),7.08–7.04(2H,m),5.56(2H,s),4.87(2H,d,J=5.7Hz). 13 C NMR(CDCl3,151MHz)δ165.54,157.47,149.26,141.68,140.60,137.74,136.83,134.52,133.92,130.29,129.41, 129.25,129.10,127.83,122.46,122.28,122.00,121.80,120.99,114.57,109.80,46.56,44.86.HRMS(ESI)calcd for C 25 H 19 ClN4O,[M+H] + :m / z427.132,found427.1326.

[0082] Example 5

[0083] The structure of compound 7p is shown below:

[0084]

[0085] The 4-tert-butylbenzyl bromide used in step (6) of Example 2 was replaced with 3,4,5-trifluorobenzyl bromide, and the experimental operation of Example 1 was repeated under the same conditions to obtain compound 7p, a white solid with a yield of 66.2%. 1H NMR(CDCl3,600MHz)δ8.99(1H,t,J=5.7Hz),8.97(1H,d,J=0.9Hz),8.67(1H,d,J =1.0Hz),8.62–8.58(1H,m),8.26–8.21(1H,m),7.66(1H,td,J=7.6,1.8Hz),7.62 (1H,td,J=7.6,7.1,1.2Hz),7.39(3H,ddd,J=7.4,4.5,2.0Hz),7.20(1H,ddd,J= 7.6,4.9,1.1Hz),6.73(2H,dd,J=7.7,6.2Hz),5.53(2H,s),4.87(2H,d,J=5.6Hz) 13 C NMR (CDCl3, 151MHz) δ165.40,157.39,149.27,141.42,140.99,137.54,136.84,129.97,129.95,129.62,129.34,122. 60,122.31,122.01,121.87,121.33,114.63,110.64,110.61,110.53,110.50,109.51,46.02,44.86.HRMS(ESI)calcd for C 25 H 18 F3N4O,[M+H] + :m / z 447.1427,found447.1428.

[0086] Example 6

[0087] The structure of compound 7A is shown below:

[0088]

[0089] The 4-tert-butylbenzyl bromide used in step (6) of Example 2 was replaced with 4-cyano-benzyl bromide, and the experimental operation of Example 1 was repeated under the same conditions to obtain compound 7A, a white solid with a yield of 71.2%. 1H NMR (CDCl3, 600MHz) δ8.98 (2H, d, J = 9.1Hz), 8.67 (1H, s), 8.59 (1H, dd, J = 5.0, 1.7Hz), 8.24 (1H, d, J = 7.8Hz), 7.66 (1H, td, J = 7.7, 1.8Hz), 7.61(1H,td,J=7.7,7.0,1.2Hz),7.58–7.54(2H,m),7.42–7.36(3H,m),7.19(3H,dd,J=10.8,6.6Hz),5.65(2H,s),4.86(2H,d,J=5.6Hz). 13 C NMR (CDCl3, 151MHz) δ165.41,157.37,149.31,141.53,141.41,140.93,137.63,136.81,132.89,130.06,129.56,129. 29,127.08,122.57,122.31,122.00,121.86,121.27,118.28,114.61,112.11,109.60,46.71,44.86.HRMS(ESI)calcd for C 26 H 20 N5O, [M+H] + :m / z418.1663 found418.1658.

[0090] Example 7

[0091] The structure of compound 7B is shown below:

[0092]

[0093] By replacing 4-tert-butylbenzyl bromide in step (6) of Example 2 with 2-(bromomethyl)naphthalene, and keeping other conditions unchanged, the experimental operation of Example 1 was repeated to obtain compound 7B, a white solid with a yield of 49.2%. 1H NMR(CDCl3,600MHz)δ9.00(1H,s),8.97(1H,t,J=5.8Hz),8.77(1H,s),8.58(1H,dd,J=5.1,1.7Hz ),8.26(1H,d,J=7.8Hz),7.80–7.73(2H,m),7.70–7.66(1H,m),7.64(1H,td,J=7.7,1.8Hz),7.60 (1H,d,J=8.2Hz),7.57(1H,dd,J=6.7,1.4Hz),7.51(1H,d,J=8.2Hz),7.46–7.42(2H,m),7.37(2H ,t,J=7.9Hz),7.28–7.23(1H,m),7.17(1H,dd,J=7.4,4.9Hz),5.75(2H,s),4.86(2H,d,J=5.7Hz). 13 C NMR(CDCl3,151MHz)δ165.63,157.51,149.22,141.92,140.43,137.99,136.80,133.43,133.29,132.95,130.49,129.37,129.07,129.02, 127.78,127.73,126.58,126.27,125.33,124.23,122.40,122.23,121.96,121.81,120.85,114.56,110.00,47.46,44.85.HRMS(ESI)calcd for C 29 H 22 N4ONa,[M+Na] + :m / z465.1686,found465.1692.

[0094] Example 8

[0095] The structure of compound 7C is shown below:

[0096]

[0097] By replacing 4-tert-butylbenzyl bromide in step (6) of Example 2 with bromomethylcyclopropane, and keeping other conditions unchanged, the experimental operation of Example 1 was repeated to obtain compound 7C, a white solid with a yield of 32.6%. 1H NMR(CDCl3,600MHz)δ9.02(1H,t,J=5.8Hz),8.94(1H,s),8.79(1H,s),8.61(1H,dd,J=5.1,1.7 Hz),8.20(1H,d,J=7.8Hz),7.65(1H,td,J=7.7,1.8Hz),7.61(1H,ddd,J=8.2,6.9,1.2Hz),7.5 0(1H,d,J=8.3Hz),7.40(1H,d,J=7.8Hz),7.33(1H,t,J=7.5Hz),7.21–7.16(1H,m),4.88(2H,d ,J=5.6Hz),4.29(2H,d,J=6.6Hz),1.39–1.28(1H,m),0.61–0.55(2H,m),0.42(2H,q,J=5.3Hz). 13 C NMR(CDCl3,151MHz)δ165.76,157.63,149.30,141.73,139.92,137.86,136.77,130.36,128.96,12 8.72,122.24,121.97,121.59,120.42,114.47,109.89,47.70,44.92,10.84,4.18.HRMS(ESI)calcd for C 22 H 21 N4O, [M+H] + :m / z357.171,found 357.1712.

[0098] Example 9

[0099] The structure of compound 7D is shown below:

[0100]

[0101] By replacing 4-tert-butylbenzyl bromide in step (6) of Example 2 with 1-bromo-3-methyl-2-butene, and keeping other conditions unchanged, the experimental operation of Example 1 was repeated to obtain compound 7D, a white solid with a yield of 38.2%. 1H NMR(CDCl3,600MHz)δ9.00(1H,t,J=5.8Hz),8.94(1H,s),8.74(1H,s),8.63–8.59(1H,m),8.2 0(1H,d,J=7.8Hz),7.65(1H,td,J=7.7,1.7Hz),7.63–7.57(1H,m),7.46(1H,d,J=8.2Hz),7.3 9(1H,d,J=7.8Hz),7.32(1H,t,J=7.5Hz),7.18(1H,dd,J=7.5,4.9Hz),5.28(1H,ddd,J=8.2,5 .9, 1.5Hz), 4.95 (2H, d, J = 6.7Hz), 4.88 (2H, d, J = 5.7Hz), 1.93 (3H, s), 1.72 (3H, d, J = 1.6Hz). 13 C NMR(CDCl3,151MHz)δ165.78,157.64,149.29,141.48,139.90,137.55,136.76,136.63,130.38,129.05,128.67 ,122.25,122.23,121.95,121.66,120.40,118.86,114.47,109.84,44.92,41.67,25.62,18.25.HRMS(ESI)calcd for C 23 H 22 N4ONa,[M+Na] + :m / z 393.1686, found 393.1675.

[0102] Example 10

[0103] The structure of compound 7F is shown below:

[0104]

[0105] By replacing 4-tert-butylbenzyl bromide in step (6) of Example 2 with 2,3,6-trifluorobenzyl bromide, and keeping other conditions unchanged, the experimental operation of Example 1 was repeated to obtain compound 7F, a white solid with a yield of 67.9%. 1H NMR (CDCl3, 600MHz) δ9.00 (1H, t, J = 5.8Hz), 8.94 (2H, d, J = 12.1Hz), 8.65–8.6 0(1H,m),8.21(1H,d,J=7.7Hz),7.71–7.61(3H,m),7.41(1H,d,J=7.8Hz),7.3 7(1H,ddd,J=8.0,6.2,1.8Hz),7.26(1H,s),7.21(1H,dd,J=7.2,4.6Hz),7.12 (1H,dd,J=9.1,5.1Hz),6.90–6.84(1H,m),5.65(2H,s),4.88(2H,d,J=5.7Hz). 13 C NMR(CDCl3,151MHz)δ165.59,157.50,149.20,141.38,140.58,137.63,136.92, 130.53,130.51,130.48,129.51,129.04,122.30,122.23,122.07,121.84,121. 01,117.66,117.59,117.53,117.46,114.38,113.93,113.82,113.69,111.49,1 11.45,111.42,111.32,111.29,111.25,109.90,44.86,35.76.HRMS(ESI)calcd forC 25 H 18 F3N4O,[M+H] + :m / z 447.1427,found447.1430.

[0106] Example 11

[0107] The structure of compound 7G is shown below:

[0108]

[0109] The 4-tert-butylbenzyl bromide used in step (6) of Example 2 was replaced with 4-fluoro-3-trifluoromethylbenzyl bromide, and the experimental operation of Example 1 was repeated under the same conditions to obtain compound 7G, a white solid with a yield of 69.2%. 1H NMR(CDCl3,600MHz)δ9.00(1H,s),8.95(1H,d,J=0.9Hz),8.67(1H,d,J=0.9H z),8.58(1H,dt,J=4.9,1.3Hz),8.22(1H,d,J=7.9Hz),7.68–7.58(2H,m),7.5 0(1H,dd,J=6.5,2.3Hz),7.41(1H,d,J=8.3Hz),7.37(2H,dt,J=7.1,3.4Hz), 7.21–7.14(2H,m),7.07(1H,t,J=9.3Hz),5.59(2H,s),4.86(2H,d,J=5.7Hz). 13 C NMR(CDCl3,151MHz)δ165.43,157.37,149.29,141.49,140.85,137.58,136.79,132.46,132.44,131.79,131.74,130.02,129.54,129.29, 125.31,125.27,123.12,122.53,122.29,121.97,121.83,121.32,121.21,117.86,117.72,114.57,109.58,46.10,44.85.HRMS(ESI)calcd for C 26 H 19 F4N4O,[M+H] + :m / z 479.149,found479.1490.

[0110] Example 12

[0111] The structure of compound 7H is shown below:

[0112]

[0113] The 4-tert-butylbenzyl bromide used in step (6) of Example 2 was replaced with 4-trifluoromethoxybenzyl bromide, and the experimental operation of Example 1 was repeated under the same conditions to obtain compound 7H, a white solid with a yield of 45.4%. 1H NMR (CDCl3, 600MHz) δ9.00 (1H, t, J = 5.7Hz), 8.96 (1H, d, J = 0.9Hz), 8.68 (1H, d, J = 0.9Hz), 8.58 (1H, dt, J = 4.8, 1.4Hz), 8.22 (1H, d, J = 7.8Hz), 7.64 (1H,td,J=7.7,1.8Hz),7.59(1H,ddd,J=8.3,7.1,1.2Hz),7.44(1H,s),7 .40–7.33(2H,m),7.20–7.09(5H,m),5.57(2H,s),4.86(2H,d,J=5.7Hz). 13 C NMR(CDCl3,151MHz)δ165.53,157.45,149.27,148.85,141.63,140.61,137.68,136.79,134.74,130.25,129.39,129.14, 127.86,122.43,122.27,121.97,121.76,121.54,121.23,121.01,119.52,114.54,109.75,46.37,44.86.HRMS(ESI)calcd forC 26 H 19 F3N4O2Na,[M+Na] + :m / z 499.1352, found 499.1370.

[0114] Example 13

[0115] The structure of compound 7I is shown below:

[0116]

[0117] The 4-tert-butylbenzyl bromide used in step (6) of Example 2 was replaced with 3,5-dimethoxybenzyl bromide, and the experimental operation of Example 1 was repeated under the same conditions to obtain compound 7I, a white solid with a yield of 63.8%. 1H NMR(CDCl3,600MHz)δ8.98(2H,d,J=4.9Hz),8.74(1H,s),8.63–8.59(1H,m),8.25(1H ,d,J=7.8Hz),7.70–7.65(1H,m),7.63–7.57(1H,m),7.48(1H,d,J=8.3Hz),7.41(1H,d ,J=7.8Hz),7.36(1H,t,J=7.5Hz),7.26(1H,s),7.21(1H,dd,J=7.2,5.1Hz),6.34(1H ,t,J=2.3Hz), 6.28(2H,d,J=2.2Hz), 5.54(2H,s), 4.88(2H,d,J=5.7Hz), 3.68(6H,s). 13 C NMR(CDCl3,151MHz)δ165.68,161.39,157.52,149.14,141.95,140.40,138.48,138.00,136.95,130.50,129.34,129 .01,122.38,122.30,122.09,121.76,120.83,114.55,109.94,104.67,99.25,55.31,47.29,44.82.HRMS(ESI)calcd for C 27 H 25 N4O3,[M+H] + :m / z453.1921, found 453.1924.

[0118] Example 14

[0119] The structure of compound 10e is shown below:

[0120]

[0121] By replacing 4-cyanobenzyl bromide in step (7) of Example 1 with bromomethylcyclopropane, and keeping other conditions unchanged, the experimental operation of Example 1 was repeated to obtain compound 10e, a white solid with a yield of 41.8%. 1H NMR(CDCl3,600MHz)δ9.00(1H,t,J=5.6Hz),8.90(1H,dd,J=4.1,1.0Hz),8.82(1H,dd,J=16.9,1.0 Hz),8.63(1H,ddd,J=5.0,1.8,0.9Hz),8.17(1H,dd,J=3.6,2.0Hz),7.69(1H,td,J=7.6,1.8Hz),7 .59(1H,ddd,J=10.7,8.7,2.0Hz),7.41(1H,d,J=9.3Hz),7.26(1H,s),7.22(1H,ddd,J=6.3,5.1,1 .1Hz), 4.88 (2H, d, J = 5.7Hz), 4.32 (1H, d, J = 6.6Hz), 3.98 (1H, s), 1.25 (1H, s), 0.68–0.40 (3H, m). 13 C NMR(CDCl3,151MHz)δ165.49,165.46,157.47,149.23,140.46,140.43,14 0.30,140.05,138.53,138.23,136.91,130.75,130.38,129.06,128.99,12 8.09,127.99,126.09,122.72,122.59,122.31,122.10,121.89,121.86,11 4.60,114.55,111.01,110.63,47.98,44.86,10.82,4.24.HRMS(ESI)calcd for C 22 H 19 ClN4ONa,[M+Na] + :m / z 413.1139, found 413.1147.

[0122] Example 15

[0123] The structure of compound 10f is shown below:

[0124]

[0125] The 4-cyanobenzyl bromide used in step (7) of Example 1 was replaced with 2,3,6-trifluorobenzyl bromide, and the experimental operation of Example 1 was repeated under the same conditions to obtain compound 10f, a white solid with a yield of 68.9%. 1H NMR (CDCl3, 600MHz) δ8.99 (1H, t, J = 5.7Hz), 8.95 (1H, s), 8.87 (1H, d, J = 0.9H z), 8.63 (1H, dt, J = 4.9, 1.3Hz), 8.14 (1H, t, J = 1.3Hz), 7.68 (1H, td, J = 7.6, 1. 8Hz),7.58(2H,d,J=1.2Hz),7.41(1H,s),7.24–7.19(1H,m),7.13(1H,dd,J= 9.2, 5.1Hz), 6.88 (1H, dd, J = 3.6, 2.2Hz), 5.63 (2H, s), 4.88 (2H, d, J = 5.6Hz). 13 C NMR(CDCl3,151MHz)δ165.27,157.34,149.17,140.92,139.65,137.91,136.95,1 30.89,130.86,129.26,128.55,126.73,122.94,122.36,122.33,122.09,121.80, 117.87,117.80,117.74,117.68,114.41,113.59,113.49,113.45,111.58,111.55 ,111.51,111.42,111.39,111.09,111.07,111.04,44.81,35.89.HRMS(ESI)calcd forC 25 H 17 ClF3N4O,[M+H] + :m / z481.1038, found 481.1040.

[0126] Example 16

[0127] The structure of 10g of compound is shown below:

[0128]

[0129] The 4-cyanobenzyl bromide used in step (7) of Example 1 was replaced with 3,5-dimethoxybenzyl bromide, and the experimental operation of Example 1 was repeated under the same conditions to obtain 10 g of compound, a white solid, with a yield of 46.8%. 1H NMR (CDCl3, 600MHz) δ8.97 (1H, s), 8.88 (1H, d, J = 0.9Hz), 8.72 (1H, d, J = 1.0Hz), 8.59 ( 1H,ddd,J=4.9,1.8,0.9Hz),8.14(1H,d,J=2.0Hz),7.65(1H,td,J=7.6,1.8Hz),7.51(1 H,dd,J=8.7,2.1Hz),7.37(2H,d,J=8.9Hz),7.18(1H,ddd,J=7.6,4.9,1.1Hz),6.34(1 H,t,J=2.3Hz), 6.22(2H,d,J=2.2Hz), 5.48(2H,s), 4.86(2H,d,J=5.7Hz), 3.66(6H,s). 13 CNMR(CDCl3,151MHz)δ165.31,161.42,157.43,149.32,140.72,140.13,138.23,138.04,136.75,130.83,129.20,12 8.26,126.42,122.75,122.25,121.93,121.88,114.51,111.11,104.60,99.28,55.30,47.37,44.88.HRMS(ESI)calcd for C 27 H 24 ClN4O3,[M+H] + :m / z487.1532,found487.1530.

[0130] Example 17

[0131] The structure of compound 10h is shown below:

[0132]

[0133] The N-chlorosuccinimide used in step (4) of Example 1 was replaced with N-bromosuccinimide, and the 4-cyanobenzylbromobenzyl used in step (7) of Example 1 was replaced with 4-tert-butylbromobenzyl. The experimental operation of Example 1 was repeated under the same conditions to obtain compound 10h, a white solid with a yield of 76.9%. 1H NMR (Chloroform-d, 600MHz) δ9.19(1H,d,J=1.0Hz),8.99(1H,d,J=1.0Hz),8.77(1H,d,J=1.9Hz),7.82(1H,d,J=8.8Hz),7.80– 7.74(2H,m),7.37(1H,d,J=7.8Hz),7.33–7.28(3H,m),7.15(2H,d,J=8.4Hz),5.83(2H,s),4.69(2H,d,J=5.9Hz),1.20(10H,s). 13 C NMR (151MHz, DMSO-d6) δ165.16,158.70,150.58,149.29,140.88,140.59,138.15,137.23,134.37,132.42,131.88,130.12,127. 73,127.11,125.99,125.71,123.31,122.58,121.54,115.11,113.46,113.15,46.42,44.71,34.66,31.48.HRMS(ESI)calculated for C 27 H 23 BrN4O3,[M+H] + :m / z 531.1027,found531.1014.

[0134] Example 18

[0135] The structure of compound 10k is shown below:

[0136]

[0137] The N-chlorosuccinimide used in step (4) of Example 1 was replaced with N-bromosuccinimide, and the experimental operation of Example 1 was repeated under the same conditions to obtain compound 10k, a white solid with a yield of 43.8%. 1 HNMR(CDCl3,600MHz)δ8.97(1H,t,J=5.6Hz),8.87(1H,d,J=0.9Hz),8.66(1H,d,J=0.9Hz),8.59(1H,dt,J=4.7,1.3Hz),8.30(1H,d,J=1.8Hz) ,7.67(2H,td,J=8.8,8.2,1.8Hz),7.59–7.55(2H,m),7.38(1H,s),7.2 9–7.25(1H,m),7.22–7.15(3H,m),5.64(2H,s),4.85(2H,d,J=5.6Hz).13 C NMR (CDCl3, 151MHz) δ165.06,157.20,149.28,141.26,140.93,140.09,137.73,136.84,132.95,132.09,130.41,128. 39,127.02,125.17,123.45,122.35,122.01,118.18,114.59,114.12,112.27,111.17,46.83,44.81.HRMS(ESI)calcd for C 26 H 19 BrN5O,[M+H] + :m / z 496.0768, found 496.0776.

[0138] Example 19

[0139] The structure of compound 10l is shown below:

[0140]

[0141] The N-chlorosuccinimide used in step (4) of Example 1 was replaced with N-bromosuccinimide, and the 4-cyanobenzyl bromide used in step (7) of Example 1 was replaced with bromomethylcyclopropane. The experimental operation of Example 1 was repeated under the same conditions to obtain compound 10l, a white solid with a yield of 31.8%. 1 H NMR(CDCl3,600MHz)δ8.99(1H,s),8.86(1H,s),8.79(1H,s),8.62(1H,dd,J= 4.8,1.6Hz),8.28(1H,d,J=1.9Hz),7.67(2H,ddt,J=7.9,3.6,1.9Hz),7.39( 2H,d,J=17.8Hz),7.20(1H,dd,J=7.5,5.0Hz),4.87(2H,d,J=5.7Hz),4.28(2 H,d,J=6.6Hz),1.33–1.28(1H,m),0.63–0.58(2H,m),0.42(2H,d,J=5.1Hz). 13 C NMR(CDCl3,151MHz)δ165.42,157.48,149.29,140.30,137.97,136.81,131.53,130.70,127.87,12 4.85,123.23,122.28,122.01,114.48,113.23,111.41,47.91,44.89,10.79,4.23.HRMS(ESI)calcd for C22 H 20 BrN4O,[M+H] + :m / z435.0815,found435.0820.

[0142] Example 20

[0143] The structure of compound 7e is shown below:

[0144]

[0145] The 1-hydroxybenzotriazole used in step (5) of Example 2 was replaced with 4-dimethylaminopyridine, and the 2-aminomethylpyridine used in step (5) of Example 2 was replaced with 2-chloro-5-hydroxymethylpyridine. The experimental operation of Example 1 was repeated under the same conditions to obtain compound 7e 62 mg, white solid, yield 46%. 1 H NMR(CDCl3,600MHz)δ8.91(2H,d,J=11.8Hz),8.57(1H,d,J=2.4Hz),8.23(1H,d,J=7.9Hz),7.87(1H,dd,J=8.2,2.5Hz),7.5 4(1H,d,J=8.3Hz),7.40–7.33(2H,m),7.29(2H,d,J=8.3Hz),7.09(2H,d,J=8.2Hz),5.60(2H,s),5.49(2H,s),1.25(9H,s). 13 C NMR(CDCl3,151MHz)δ165.79,151.52,151.27,150.07,141.92,139.42,138.11,136.96,132.65,132.29,130.82,129.26 ,128.74,126.37,126.00,124.30,122.19,121.45,121.12,118.16,110.33,63.71,47.03,34.54,31.24.HRMS(ESI)calcd for C 29 H 27 ClN3O2,[M+H] + :m / z 484.1787,found 484.1786.

[0146] The following are the pharmacological activity test results of the compounds prepared in this invention:

[0147] Experimental instruments: Clean bench (Shanghai Lichen Instrument Technology Co., Ltd.), constant temperature incubator (Thermoelectron Corporation), microplate reader (Thermo Fisher Scientific), inverted biological microscope (OLYMPUS).

[0148] Experimental reagents: Cell culture media RPMI-1640, DMEM (GIBCO), fetal bovine serum (Hangzhou Sijiqing Co., Ltd.), CCK-8 (Biosharp), DMSO (Sigma), RHKK-Ac-AMC (Shanghai Jier Biochemical Co., Ltd.), NAD+ + (Sigma), trypsin (Shanghai Aladdin Biochemical Technology Co., Ltd.), nicotinamide (Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0149] Cell lines: human breast cancer cells MCF-7, human pancreatic cancer cells BxPC-3, Panc-1, MIA-PaCa-2, human leukemia cells HL-60, and human neuroblastoma cells SH-SY5Y.

[0150] SIRT6 deacetylation inhibition activity assay method:

[0151] SIRT6 deacetylation activity was analyzed using the Fluor-De-Lys (FDL) assay. 5 μM recombinant SIRT6 protein and 2.5 mM NAD+ were added to black 96-well plates. + Add 75 μM RHKK-Ac-AMC, DMSO, or the compound to SIRT6 detection buffer to bring the total volume to 100 μL. Incubate the reaction mixture at 37°C for 2 hours, then add 100 μL of stop solution (6 μg / μL trypsin and 40 mM nicotinamide) and incubate further at 37°C for 2 hours. Measure fluorescence intensity using a microplate reader at 360 nm excitation and 460 nm emission. Calculate the IC50 of the derivative using GraphPad Prism 8.0. 50 value.

[0152] Cell inhibitory activity assay method:

[0153] Cells were cultured routinely in an incubator at 37°C and 5% CO2 saturated humidity. Cells in the logarithmic growth phase were digested with trypsin cell digestion solution (0.25% trypsin + 0.01% EDTA), centrifuged, resuspended, and counted to prepare a concentration of 8 × 10⁻⁶ cells / mL. 5Cell suspensions of 100 μL / mL were seeded into 96-well plates and incubated overnight in a CO2 incubator. After cell adhesion, the drug compound was diluted to the required concentration using culture medium, and 100 μL of the corresponding drug-containing culture medium was added to each well. Three auxiliary wells were set up for each drug group, and blank control (containing only cell culture medium), negative control (DMSO group), and positive control (paclitaxel group) were also set up. The 96-well plates with the added drugs were incubated for 24 h. 10 μL of CCK-8 was added to each well of the 96-well plate and the plates were incubated for another 1 h. The OD value of each well was then measured at 450 nm using a microplate reader, and the inhibition rate was calculated.

[0154] Inhibition rate calculation formula:

[0155]

[0156] Relative OD value of experimental group = Absolute OD value of experimental group - Absolute OD value of blank control well

[0157] Experimental results:

[0158] Some of the compounds prepared in this invention can effectively inhibit the deacetylation activity of recombinant SIRT6 (Tables 1 and 2), among which compound 10d showed the strongest SIRT6 deacetylation inhibition (IC50). 50 =5.81μM).

[0159] Table 1. Inhibitory activity of the β-carboline disubstituted compounds prepared in this invention against the deacetylation activity of SIRT6.

[0160]

[0161]

[0162]

[0163] Table 2. Inhibitory activity of the β-carboline trisubstituted compounds prepared in this invention against the deacetylation activity of SIRT6.

[0164]

[0165]

[0166] The compound 10d prepared in this invention exhibits certain anticancer activity against human breast cancer cells MCF-7, human pancreatic cancer cells MIA-PaCa-2, human leukemia cells HL-60, and human neuroblastoma SH-SY5Y (Table 3).

[0167] Table 3. IC50 of the compound prepared in this invention against cancer cells at 10 days. 50 Value (μM)

[0168]

[0169] The above pharmacological tests show that the target derivative of the present invention has good SIRT6 inhibitory activity and certain anti-tumor cell proliferation activity, and has the potential to be used as a tool compound for SIRT6 function research and as an anticancer drug.

Claims

1. A kind β -Carboline 3,6,9-position modified derivatives or pharmaceutically acceptable salts thereof, characterized in that: The β -Carpolin 3, 6, 9-position modified derivatives are one of the following compounds: Compound 7a: X is NH, n is 1, R 1 for R 2 for R 3 For H; Compound 7f: X is NH, n is 1, R 1 for R 2 for R 3 For H; Compound 7k: X is NH, n is 1, R 1 for R 2 for R 3 For H; Compound 7x: X is 0, n is 1, R 1 for R 2 for R 3 For H; Compound 7y: X is 0, n is 1, R 1 for R 2 for R 3 For H; Compound 7z: X is NH, n is 1, R 1 for R 2 for R 3 For H; Compound 7p: X is NH, n is 1, R 1 for R 2 for R 3 For H; Compound 7q: X is NH, n is 1, R 1 for R 2 for R 3 For H; Compound 7A: X is NH, n is 1, R 1 for R 2 for R 3 For H; Compound 7B: X is NH, n is 1, R 1 for R 2 for R 3 For H; Compound 7C: X is NH, n is 1, R 1 for R 2 for R 3 For H; Compound 7D: X is NH, n is 1, R 1 for R 2 for R 3 For H; Compound 7E: X is NH, n is 1, R 1 for R 2 for R 3 For H; Compound 7F: X is NH, n is 1, R 1 for R 2 for R 3 For H; Compound 7G: X is NH, n is 1, R 1 for R 2 for R 3 For H; Compound 7H: X is NH, n is 1, R 1 for R 2 for R 3 For H; Compound 7I: X is NH, n is 1, R 1 for R 2 for R 3 For H; Compound 10a: X is NH, n is 1, R 1 for R 2 for R 3 -Cl; Compound 10b: X is NH, n is 1, R 1 for R 2 for R 3 -Cl; Compound 10c: X is NH, n is 1, R 1 for R 2 for R 3 -Cl; Compound 10d: X is NH, n is 1, R 1 for R 2 for R 3 -Cl; Compound 10e: X is NH, n is 1, R 1 for R 2 for R 3 -Cl; Compound 10f: X is NH, n is 1, R 1 for R 2 for R 3 -Cl; Compound 10g: X is NH, n is 1, R 1 for R 2 for R 3 -Cl; Compound 10h: X is NH, n is 1, R 1 for R 2 for R 3 -Br; Compound 10j: X is NH, n is 1, R 1 for R 2 for R 3 -Br; Compound 10k: X is NH, n is 1, R 1 for R 2 for R 3 -Br; Compound 10l: X is NH, n is 1, R 1 for R 2 for R 3 -Br; Compound 10m: X is NH, n is 1, R 1 for R 2 for R 3 It is -Br.

2. The claim 1 β A method for preparing a carpolin 3,6,9-position modified derivative, characterized in that: Includes the following steps: (1) Compound 1 L Tryptophan was dissolved in sodium hydroxide solution, and after adding 37% formaldehyde solution, the mixture was reacted at 37°C for 2 days to give compound 2. Compound 2 was dissolved in anhydrous methanol, and thionyl chloride was added dropwise under ice bath conditions. The mixture was then refluxed at 70°C for 6 hours to give compound 3. Compound 3 was dissolved in... N , N In a solution of dimethylformamide, potassium permanganate was added under ice bath conditions, and after reacting for 1 h, the mixture was transferred to room temperature and reacted for 14 h to obtain compound 4. Compound 1, compound 2, compound 3, and compound 4 each have the following structures: (2) Compound 4 was dissolved in methanol, and 1 N NaOH solution was added. The mixture was heated at 78 °C for 2 h to obtain compound 5. Compound 5 was dissolved in... N , N Carbodiimide hydrochloride, 1-hydroxybenzotriazole and the corresponding ammonia were added to a dimethylformamide solution. The compound was obtained by reacting at room temperature for 12 h. Alternatively, carbodiimide hydrochloride, 4-dimethylaminopyridine, and the corresponding alcohol may be added. Compounds The obtained compound was dissolved in a tetrahydrofuran solution containing sodium hydride, stirred for 1 h, and then the corresponding bromoalkane or benzyl bromo was added. The reaction was carried out at room temperature for 12 h to obtain the compound. or ; Compound 5 has the following structure: The n and R 1 R 2 n, R as described in claim 1 1 R 2 .

3. The claim 1 β A method for preparing a carpolin 3,6,9-position modified derivative, characterized in that: Includes the following steps: (1) Compound 1 L Tryptophan was dissolved in sodium hydroxide solution, and after adding 37% formaldehyde solution, the mixture was reacted at 37°C for 2 days to give compound 2. Compound 2 was dissolved in anhydrous methanol, and thionyl chloride was added dropwise under ice bath conditions. The mixture was then refluxed at 70°C for 6 hours to give compound 3. Compound 3 was dissolved in... N, N In a solution of dimethylformamide, potassium permanganate was added under ice bath conditions, and after reacting for 1 h, the mixture was transferred to room temperature and reacted for 14 h to obtain compound 4. Compound 1, compound 2, compound 3, and compound 4 each have the following structures: (2) Compound 4 was dissolved in dichloromethane, a halogenating agent was added, and the mixture was refluxed at 78°C for 12 h to obtain compound 4. The obtained compound was dissolved in methanol, and 1 N NaOH solution was added. The mixture was heated at 78°C for 2 h to obtain the compound. Dissolve the above compound in N, N- Carbodiimide hydrochloride, 1-hydroxybenzotriazole and the corresponding ammonia were added to a dimethylformamide solution. The compound was obtained by reacting at room temperature for 12 h. Alternatively, carbodiimide hydrochloride, 4-dimethylaminopyridine, and the corresponding alcohol may be added. Compounds The obtained compound was dissolved in a tetrahydrofuran solution containing sodium hydride, stirred for 1 h, and then the corresponding bromoalkane or benzyl bromo was added. The reaction was carried out at room temperature for 12 h to obtain the compound. or ; The n, R 1 R 2 R 3 n, R as described in claim 1 1 R 2 R 3 .

4. A pharmaceutical composition, characterized in that: The pharmaceutical composition contains a therapeutically effective amount as claimed in claim 1. β -Carboline 3, 6, 9-position modified derivatives thereof or pharmaceutically acceptable salts and pharmaceutically acceptable carriers.

5. The claim 1 β The use of a carboline 3, 6, 9-position modified derivative thereof or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 4 in the preparation of a SIRT6 inhibitor.

6. The claim 1 β The use of a carboline 3,6,9-position modified derivative or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 4 in the preparation of a medicament for treating tumor diseases, characterized in that: The tumors mentioned are breast cancer, pancreatic cancer, leukemia, and neuroblastoma.

Images