Novel substituted benzoyl pyridinone compounds and uses thereof

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to novel substituted benzoylpyridinone compounds and their applications. These novel substituted benzoylpyridinone compounds have the general structural formulas shown in (Ⅰ), (Ⅱ), (Ⅲ), (Ⅳ), (Ⅴ), (Ⅵ), (Ⅶ), (Ⅷ), or (Ⅸ), and as antitumor drugs, they exhibit significant EZH2 histone methyltransferase inhibitory activity. This invention provides methods for preparing these compounds, as well as pharmaceutical compositions containing them and their uses. The novel substituted benzoylpyridinone compounds obtained by this invention possess more significant antitumor activity and safety, and are of great value, particularly as antitumor agents, in EZH2, PRC2, and EZH2 / PRC2-mediated diseases.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a novel class of compounds having substituted benzoylpyridinone structures, their pharmaceutically acceptable salts, preparation methods, and pharmaceutical uses. The compounds can effectively inhibit EZH2, PRC2, or EZH2 / PRC2-mediated disease progression and exhibit significant antitumor activity in inhibiting tumor cell growth. Background Technology

[0002] Cancer development involves multi-layered regulation, including genetic variations, epigenetics, cell signaling, and complex interactions within the tumor microenvironment. Epigenetic regulation has been proven to be a key driver of malignant tumors acquiring malignant phenotypes such as proliferation, invasion, and metastasis. Epigenetic modifications, by regulating gene transcription and post-transcriptional processes, are widely involved in vital activities such as cell differentiation, metabolism, apoptosis, and immune responses. Abnormal regulation of epigenetics is closely related to the occurrence and development of various diseases, including tumors, autoimmune diseases, and infectious diseases. EZH2 is a histone methyltransferase involved in cell cycle, DNA damage repair, cell differentiation, autophagy, apoptosis, and immune regulation. Its main function is to catalyze the H3 histone methylation of H3K27Me3, thereby inhibiting the transcription of target genes. Studies have shown that EZH2 is overexpressed in various tumor tissues, and its expression level is significantly correlated with tumor progression and poor prognosis, thus making it an important molecule for epigenetic targeted therapy. Tazemetostat is the world's first FDA-approved oral EZH2 small molecule inhibitor. Tazemetostat is used to treat epithelioid sarcoma (ES) and relapsed or refractory follicular lymphoma (FL). However, it has unsatisfactory pharmacokinetic properties. Therefore, the development of EZH2 inhibitors with novel structures, good safety profiles, and excellent efficacy has become an important direction in current antitumor drug research. Summary of the Invention

[0003] The purpose of this invention is to provide a novel class of substituted benzoylpyridinone compounds and their use in EZH2, PRC2, and EZH2 / PRC2-mediated disease drugs. After in vitro activity screening, this compound exhibits significant antitumor activity.

[0004] The technical solution adopted in this invention is:

[0005] Novel substituted benzoylpyridinone compounds and their pharmaceutically acceptable salts, having the general structural formulas shown as (Ⅰ), (Ⅱ), (Ⅲ), (Ⅳ), (Ⅴ), (Ⅵ), (Ⅶ), (Ⅷ) or (Ⅸ):

[0006] ;

[0007] in,

[0008] A is selected from primary amine compounds;

[0009] R1 or R2 is selected from secondary amines or structures containing secondary amine substitutions.

[0010] Preferably, the above-mentioned novel substituted benzoylpyridinone compounds and their pharmaceutically acceptable salts,

[0011] A is selected from

[0012] ;

[0013] R1 is selected from

[0014] ;

[0015] R2 is selected from

[0016] .

[0017] More preferably, the above-described novel substituted benzoylpyridinone compounds and their pharmaceutically acceptable salts, wherein the novel substituted benzoylpyridinone compounds have the following structural formula:

[0018]

[0019]

[0020] .

[0021] A pharmaceutical composition comprising, as an active ingredient, any one of the novel substituted benzoylpyridinone compounds and their pharmaceutically acceptable salts, combined with a pharmaceutically acceptable carrier.

[0022] Furthermore, in the above-mentioned pharmaceutical composition, the pharmaceutically acceptable carrier is one or more of a solid dosage form carrier and a liquid dosage form carrier.

[0023] Furthermore, the above-mentioned pharmaceutical composition is formulated into dosage forms such as tablets, capsules, granules, sprays, or injections.

[0024] The use of any of the novel substituted benzoylpyridinone compounds and their pharmaceutically acceptable salts, or any of the pharmaceutical compositions described above, in the preparation of medicaments for treating EZH2, PRC2, or EZH2 / PRC2-mediated diseases.

[0025] The use of any of the novel substituted benzoylpyridinone compounds and their pharmaceutically acceptable salts, or any of the pharmaceutical compositions described above, in the preparation of drugs targeting EZH2 as a molecular therapeutic target.

[0026] The use of any of the novel substituted benzoylpyridinone compounds and their pharmaceutically acceptable salts, or any of the pharmaceutical compositions described above, in the preparation of antitumor drugs.

[0027] Furthermore, in the above applications, the tumors mentioned are: liver cancer, nasopharyngeal carcinoma, colorectal cancer, melanoma, bladder cancer, leukemia, esophageal cancer, breast cancer, gastric cancer, prostate cancer, pancreatic cancer, lung cancer, ovarian cancer, epithelioid sarcoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, and follicular lymphoma.

[0028] The beneficial effects of this invention are:

[0029] 1. Broad antitumor spectrum and good safety profile: The compounds have shown excellent antitumor activity and good safety profile in preclinical studies, and have potential therapeutic value for various solid tumors such as epithelioid sarcoma, lymphoma, prostate cancer, and breast cancer, as well as hematologic malignancies and some autoimmune diseases.

[0030] 2. Significant Targeted Inhibition of EZH2: These compounds are highly effective selective inhibitors of EZH2 kinase with a well-defined mechanism. Experiments have shown that they significantly inhibit EZH2 enzyme activity, making them particularly suitable for developing drugs to treat and prevent diseases driven by abnormally high expression of EZH2 kinase, especially showing promising applications in the field of anti-tumor drugs.

[0031] 3. Novel structure and outstanding in vitro activity: The compounds of this invention possess a completely new core skeleton. In in vitro biological evaluations, most compounds not only exhibited potent inhibitory activity against EZH2 kinase, but also significant anti-proliferative effects against various tested tumor cell lines. This lays a solid chemical and biological foundation for the development of novel cancer treatment and prevention drugs. Detailed Implementation

[0032] The examples and preparation methods provided below further illustrate and demonstrate the compounds of the present invention and their preparation methods. It should be understood that the scope of the following examples and preparation methods does not limit the scope of the present invention in any way.

[0033] The following synthetic routes describe the preparation methods of novel substituted benzoylpyridinone compounds of general formulas (Ⅰ), (Ⅱ), (Ⅲ), (Ⅳ), (Ⅴ), (Ⅵ), (Ⅶ), (Ⅷ) or (Ⅸ) of this invention.

[0034] All raw materials were prepared by methods well known to those skilled in the art of organic chemistry, as described in the synthetic routes below, or were commercially available. All final compounds of this invention were prepared by methods described in the synthetic routes below or by similar methods well known to those skilled in the art of organic chemistry. All variable factors used in the synthetic routes below are as defined below or as defined in the foregoing specification.

[0035] The synthetic routes of compounds 1 to 38 according to the general formulas (Ⅰ), (Ⅱ), (Ⅲ), (Ⅳ), (Ⅴ), (Ⅵ), (Ⅶ), (Ⅷ) or (Ⅸ) of the present invention are as follows, wherein the functional groups of R1 and R2 are as shown in the above description.

[0036] 1) As shown in the synthetic route 1 of the target compound, 2-methyl-3-nitrobenzoic acid was reacted with dibromohydantoin at 25 °C to obtain intermediate 2; intermediate 2 underwent esterification to obtain intermediate 3; intermediate 3 was reduced by iron powder to obtain intermediate 4; intermediate 4 was reacted with cyclopropionyl chloride in acetonitrile solution to obtain intermediate 5; intermediate 5 was reacted with iodoethane in DMF solution to obtain intermediate 6; intermediate 6 was coupled with boric acid to obtain intermediate 7; intermediate 7 and intermediate 9 were coupled to obtain intermediate 10; intermediate 10 was hydrolyzed in methanol aqueous solution to obtain intermediate 11; intermediate 11 was reacted with 3-(aminomethyl)-4,6 -Dimethylpyridin-2(1H)-one yielded the target compounds (compound 1, compound 2, compound 3, compound 4, compound 13, compound 14, compound 15, compound 17, compound 22, compound 23, compound 24, compound 28, compound 35, compound 36, compound 37, compound 38); intermediate 11 and 3-(aminomethyl)-4-methoxy-6-methylpyridin-2(1H)-one yielded the target compounds (compound 31, compound 32, compound 33).

[0037]

[0038] Synthetic route 1 of the target compound

[0039] Among them, (a) DBDMH, H2SO4, RT; (b) CH3OH, H2SO4, 60℃; (c) Fe, CH3COOH, EA,H2O, 80℃; (d) cyclopropanecarbonyl chloride, DMAP, DIPEA, THF, RT; (e) iodoethane, Cs2CO3, DMF, 90℃; (f) B2Pin2, CH3COOK, Pd(pph3)2Cl2, 1,4-Diox, H2O,100℃; (g)HATU, TEA, DMF, RT or POCl3, ACN, 80℃; (h) CH3COOK, Pd(pph3)2Cl2,1,4-Diox, H2O, 100℃; (i)NaOH, EtOH, H2O,60℃; (j) HATU, TEA, DMF,RT.

[0040] 2) As shown in the synthetic route 2 of the target compound, methyl 6-bromo-4-indolecarboxylate was used as a starting material and reacted with 2-iodopropane in acetonitrile solution to obtain intermediate 15; intermediate 15 was coupled with boric acid to obtain intermediate 16; intermediate 16 and intermediate 9 were coupled to obtain intermediate 17; intermediate 17 was reacted with 3-(aminomethyl)-4,6-dimethylpyridin-2(1H)-one to obtain the target compound (compound 5, compound 6, compound 7, compound 9, compound 16, compound 18, compound 25, compound 27, compound 34); intermediate 17 was reacted with 3-(aminomethyl)-4-methoxy-6-methylpyridin-2(1H)-one to obtain the target compound (compound 26).

[0041]

[0042] Synthetic route 2 of the target compound

[0043] Among them, (a) 2-iodopropane, Cs2CO3, DMF, 90℃; (b) CH3COOK, Pd(pph3)2Cl2, 1,4-Diox, H2O, 100℃; (c) HATU, TEA, DMF, RT or POCl3, ACN, 80℃; (d) CH3COOK, Pd(pph3)2Cl2, 1,4-Diox, H2O, 100℃; (e)NaOH, EtOH, H2O, 60℃; (f) HATU, TEA, DMF,RT.

[0044] 3) As shown in the synthetic route 3 of the target compound, methyl 6-bromo-4-indazolecarboxylate was used as a starting material and reacted with 2-iodopropane in acetonitrile solution to obtain intermediate 21; intermediate 21 was coupled with boric acid to obtain intermediate 22; intermediate 22 and intermediate 9 were coupled to obtain intermediate 23; intermediate 23 was hydrolyzed to obtain intermediate 24; intermediate 24 was reacted with 3-(aminomethyl)-4,6-dimethylpyridin-2(1H)-one to obtain the target compound (compound 8, compound 10, compound 11, compound 12, compound 19, compound 20, compound 21, compound 29); intermediate 24 was reacted with 3-(aminomethyl)-4-methoxy-6-methylpyridin-2(1H)-one to obtain the target compound (compound 30).

[0045]

[0046] Synthetic route 3 of the target compound

[0047] Among them, (a) 2-iodopropane, Cs2CO3, DMF, 90℃; (b) CH3COOK, Pd(pph3)2Cl2, 1,4-Diox, H2O, 100℃; (c) HATU, TEA, DMF, RT or POCl3, ACN, 80℃; (d) CH3COOK, Pd(pph3)2Cl2, 1,4-Diox, H2O, 100℃; (e) NaOH, EtOH, H2O, 60℃; (f) HATU, TEA, DMF, RT.

[0048] Example 1 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(N-ethylcyclopropanecarbamate)-2-methyl-5-(1-(2-morpholinoacetyl)indoline-5-yl)benzamide (Compound 1)

[0049]

[0050] Step 1: Synthesis of 5-bromo-2-methyl-3-nitrobenzoic acid

[0051]

[0052] 2-Methyl-3-nitrobenzoic acid (30 g, 0.17 mol) was dissolved in 50 mL of concentrated sulfuric acid in a 1000 mL round-bottom flask. Stirring was started, and dibromohydantoin (8.7 g, 8.3 mmol) was added. The reaction was allowed to proceed at room temperature for 5 h until complete. The reaction solution was then added to cold water, precipitating a large amount of white solid. After drying, 41.10 g of product was obtained, with a yield of 95.8%.

[0053]

[0054] Step 2: Synthesis of methyl 5-bromo-2-methyl-3-nitrobenzene

[0055] 5-Bromo-2-methyl-3-nitrobenzoic acid (30 g, 0.12 mol) was added to a 1000 mL pear-shaped flask and dissolved in 150 mL of methanol solution. Then, 100 mL of concentrated sulfuric acid was slowly added dropwise into a cold trap and the mixture was refluxed at 60 °C. After 6 h, the reaction was completed. The pH of the reaction solution was adjusted to about 9 with NaOH solution, and a large amount of pale yellow solid precipitated. After drying, 30.04 g of solid was obtained, with a yield of 94.7%.

[0056] Step 3: Synthesis of methyl 3-amino-5-bromo-2-methylbenzoate

[0057]

[0058] 1000 mL of flask was filled with methyl 5-bromo-2-methyl-3-nitrobenzene (30 g, 0.11 mol), dissolved in 280 mL of methanol-water solution (3:1), and CH3COOH (90 g, 2.81 mol) was added. Then, iron powder (61.31 g, 1.1 mol) was added to the reaction system. The reaction was carried out at 80 °C for 9 h until complete. The mixture was filtered using diatomaceous earth and the filtrate was collected. Extraction was performed with EA, and the organic phase was washed three times with 300 mL of water, followed by two washes with 100 mL of saturated Na2CO3 solution. The organic phase was then completely dried using anhydrous sodium sulfate. Sodium sulfate was removed by filtration. The filtrate was distilled under reduced pressure until completely dry. Column chromatography yielded 22.31 g of solid, with a yield of 83.6%.

[0059] Step 4: Synthesis of methyl 5-bromo-3-(cyclopropanecarbamate)-2-methylbenzoate

[0060]

[0061] Take a 500 mL pear-shaped flask, add methyl 3-amino-5-bromo-2-methylbenzoate (20 g, 0.08 mol) and DMAP (2 g, 0.016 mol), dissolve in 100 mL THF solution, and add cyclopropionyl chloride (11.98 g, 0.12 mol) dropwise while stirring. React at room temperature for 4 h until complete. Filter and collect the filtrate. Then, add 100 mL of dichloromethane and 50 mL of water for washing. Wash the mother liquor three times with water and twice with salt to further purify the organic phase. After washing, dry the organic phase with anhydrous sodium sulfate. Then, remove the sodium sulfate by filtration. Finally, evaporate the filtrate under reduced pressure until completely dry to obtain the desired crude product. Separate by column chromatography, then distill under reduced pressure and dry to obtain 21.03 g of solid, yield 82.1%.

[0062] Step 5: Synthesis of methyl 5-bromo-3-(N-ethylcyclopropanecarbamate)-2-methylbenzoate

[0063]

[0064] In a 250 mL three-necked flask, methyl 5-bromo-3-(cyclopropanecarbamate)-2-methylbenzoate (21 g, 0.067 mol) and Cs₂CO₃ (44.08 g, 0.135 mol) were added and dissolved in 80 mL of DMF. Iodoethane (12.65 g, 0.81 mol) was added dropwise while stirring. The reaction system was heated to 90 °C in an oil bath and refluxed for 4 hours until complete. The reaction solution was collected and filtered. The filtrate was then washed with 100 mL of dichloromethane and 50 mL of salt. The mother liquor was washed three times with water and twice with salt to further purify the organic phase. After washing, the organic phase was dried using anhydrous sodium sulfate. The sodium sulfate was then removed by filtration. Finally, the filtrate was evaporated under reduced pressure until completely dry to obtain the desired crude product. Crystallization upon cooling yielded 22.1 g of solid, with a yield of 96.5%.

[0065] Step 6: Synthesis of methyl 3-(N-ethylcyclopropanecarbamate)-2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxoborhexacyclopentan-2-yl)benzoate

[0066]

[0067] In a 250 mL three-necked flask, add methyl 5-bromo-3-(N-ethylcyclopropanecarbamate)-2-methylbenzoate (22 g, 0.065 mol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxane-pentaborane) (33 g, 0.13 mol), and potassium acetate (12.7 g, 0.13 mol). Dissolve in 80 mL of Diox, then purge with nitrogen for 30 min. Add Pd(PPh3)Cl2 (2.27 g, 0.003 mol), evacuate three times, and react in an oil bath at 100 °C under nitrogen protection for 6 h. The reaction is complete as monitored by TLC. Cool the reaction system to room temperature, then remove the solids from the reaction solution by filtration. Wash the filter cake with a small amount of DCM and collect the filtrate. Add 100 mL of [unspecified liquid] to the filtrate. Extracted with DCM and 50 mL of water, the organic phases were combined and washed three times with water, then twice with saturated NaCl solution, each wash for further purification. The organic phase was then completely dried using anhydrous sodium sulfate. Sodium sulfate was removed by vacuum filtration. The filtrate was distilled under reduced pressure until completely dry. Subsequent column chromatography purified the solution to 13.4 g of a pale yellow solid, yield 48.2%.

[0068] Step 7: Synthesis of 1-(5-bromoindololin-1-yl)-2-morpholin-1-one

[0069]

[0070] Take a 50 mL pear-shaped flask, add 5-bromo-indoline (1.8 g, 0.009 mol), morpholine acetic acid (1.3 g, 0.006 mol), and HATU (4.6 g, 0.012 mol), dissolve in 10 mL DMF, then add triethylamine (1.85 g, 0.018 mol) dropwise, and stir the mixture at 30 °C. After 4 h, the reaction is complete. Add dichloromethane and sodium carbonate solution to the reaction solution for extraction. After washing twice with water and Litsch salt in the aqueous phase, add anhydrous sodium sulfate, evaporate to dryness under reduced pressure, and purify by column chromatography to obtain 2.8 g, with a yield of 94.5%.

[0071] Step 8: Synthesis of methyl 3-(N-ethylcyclopropanecarbamate)-2-methyl-5-(1-(2-morpholinoacetyl)indoline-5-yl)benzoate

[0072]

[0073] Take a 50 mL three-necked flask, add potassium acetate (550 mg, 5.6 mmol), methyl 3-(N-ethylcyclopropanecarbamate)-2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)benzoate (1 g, 2.6 mmol), and 1-(5-bromoindololin-1-yl)-2-morpholin-1-one (600 mg, 1.85 mmol), and purge with N2 for 30 min; add Pd(PPh3)Cl2 (65 mg, 0.093 mmol), evacuate three times, place in an oil bath at 100 ℃ under nitrogen protection, connect to a reflux tube and react for 6 h. TLC monitoring showed that the reaction was complete. The reaction system was cooled to room temperature. Solids in the reaction solution were removed by vacuum filtration. The filter cake was washed with a small amount of DCM to obtain the filtrate. 100 mL of DCM was added three times to the filtrate for extraction, yielding the organic phase. The organic phase was washed three times with 300 mL of water, followed by two more washes with 30 mL of saturated NaCl solution to further purify the organic phase. The organic phase was then completely dried using anhydrous sodium sulfate. Sodium sulfate was removed by vacuum filtration. The filtrate was distilled under reduced pressure until completely dry. Column chromatography yielded 436 mg, with a yield of 52.4%.

[0074] Step 9: Synthesis of 3-(N-ethylcyclopropanecarbamate)-2-methyl-5-(1-(2-morpholinoacetyl)indoline-5-yl)benzoic acid

[0075]

[0076] The methyl 3-(N-ethylcyclopropanecarbamate)-2-methyl-5-(1-(2-morpholinoacetyl)indololin-5-yl)benzoate (400 mg, 0.89 mmol) obtained above was mixed with 5 V / mol EtOH and 5 V / mol water. After thorough mixing, NaOH (53 mg, 1.33 mmol) was added to the mixture. The reaction solution was placed in an oil bath and refluxed at 60 °C for 4 h. The reaction was monitored by TLC and found to be complete. A portion of the ethanol solution in the reaction solution was then rotary evaporated, 10 mL of H2O was added, and the mixture was extracted once with DCM. The aqueous phase was collected, and the pH was adjusted to acidic with 10-fold diluted hydrochloric acid. The precipitated solid was filtered to obtain 352 mg, with a yield of 80.9%.

[0077] Step 10: Synthesis of N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(N-ethylcyclopropanecarbamate)-2-methyl-5-(1-(2-morpholinoacetyl)indoline-5-yl)benzamide

[0078]

[0079] Using 3-(N-ethylcyclopropanecarbamate)-2-methyl-5-(1-(2-morpholinoacetyl)indololin-5-yl)benzoic acid (100 mg, 0.20 mmol) and 3-(aminomethyl)-4,6-dimethylpyridin-2(1H)-one (34 mg, 0.22 mmol) as starting materials, the mixture was dissolved in 5 mL of DMF. Then, HATU (155.3 mg, 41 mmol) and triethylamine (62.7 mg, 0.62 mmol) were added. The reaction was carried out at room temperature for 4 h, and TLC monitoring showed that the reaction was complete. The organic phases were extracted with 100 mL of DCM and sodium carbonate aqueous solution, and the combined organic phases were washed three times with 50 mL of water, followed by two more washes with 20 mL of saturated NaCl solution. The organic phase was then completely dried using anhydrous sodium sulfate. Sodium sulfate was removed by vacuum filtration. The filtrate was distilled under reduced pressure until completely dry. After thin-layer chromatography, 54 mg of the compound was obtained, which was compound 1, with a yield of 42.2%.

[0080] MS (ESI), m / z: 625.33 [M+H] + ; 1H NMR (500 MHz, CDCl3) δ 11.71 (s, 1H), 8.19 (d, J = 9.0 Hz, 1H), 7.54 (s, 1H), 7.38 – 7.32 (m, 4H), 5.94 (s, 1H), 4.56 – 4.50 (m, 2H), 4.18 (t, J = 8.5 Hz, 2H), 3.80 (s, 4H), 3.39 – 3.31 (m,3H), 3.24 – 3.20 m, 2H), 2.75 (s, 3H), 2.40 (s, 3H), 2.30 (s, 3H), 2.18(s,3H),1.33 – 1.25 (m, 2H), 1.20 – 1.16 (s, 1H), 1.13 – 1.10 (m, 3H), 0.98 – 0.96 (m, 2H), 0.60 – 0.53 (m, 2H).

[0081] By replacing appropriate raw materials and reagents, and following the preparation scheme of Example 1, Examples 1-4, 13-15, 17, 22-24, 28, 31-33, and 35-38 (compounds 1-4, 13-15, 17, 22-24, 28, 31-33, and 35-38) were finally obtained.

[0082] Example 2 N-(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(N-ethylcyclopropanecarbamate)-2-methyl-5-(1-(2-morpholinoacetyl)-1,2,3,4-tetrahydroquinoline-6-yl)benzamide (Compound 2)

[0083]

[0084] MS (ESI), m / z: 639.34 [M+H] + ; 1 H NMR (500 MHz, CDCl3) δ 11.16 (s, 1H), 7.55 (s, 2H), 7.40 – 7.32 (m, 3H), 5.94 (s, 1H), 4.56 (s, 2H), 3.82 – 3.76(m, 6H), 3.44 – 3.31 0.99- 0.97 (m, 2H), 0.61 – 0.64 m, 2H).

[0085] Example 3 N-(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(1-(dimethylglyceryl)-1,2,3,4-tetrahydroquinoline-6-yl)-3-(N-ethylcyclopropanecarbamate)-2-methylbenzamide (Compound 3)

[0086]

[0087] MS (ESI), m / z: 583.32 [M+H] + ; 1H NMR (500 MHz, CDCl3) δ 11.27 (s, 1H),7.55 (s, 1H), 7.43 – 7.29 (m, 4H), 5.95 (s, 1H), 4.55 (d, J = 5.6 Hz, 2H), 3.81 –3.79 (m, 2H), 3.58 (s, 2H), 2.80 – 2.87 (m, 2H), 2.52 (s, 6H), 2.40 (s,3H), 2.32 (s, 3H), 2.21 (s, 3H), 2.03 – 1.97 (m, 3H), 1.33 – 1.25 (m, 2H),1.20 – 1.16 (m, 1H),1.14 –1.11 (m, 3H), 0.99 – 0.94 (m, 2H), 0.62 – 0.54 (m, 2H).

[0088] Example 4 N-[(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl]-1-isopropyl-6-(4-(4-methylpiperidin-1-yl)quinolin-7-yl)-1H-indazole-4-carboxamide (Compound 4)

[0089]

[0090] MS (ESI), m / z: 583.32 [M+H] + ; 1 H NMR (500 MHz, CDCl3) δ 11.73 (s, 1H),8.15 (d, J = 9.5 Hz, 1H), 7.52 (s, 1H), 7.38 – 7.34 (m, 3H), 5.93 (s, 1H),4.55 – 4.54 (m, 2H),4.15 – 4.12 (m, 2H),3.68 (s, 2H), 3.20 – 3.16 (m, 2H),2.98 (s, 1H), 2.67 (s, 6H), 2.39 – 2.31 (m, 6H), 2.19 (s, 3H), 1.34 – 1.09(m, 5H), 1.00 – 0.96 (m, 2H), 0.60 – 0.54 (m, 2H).

[0091] Example 5 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-isopropyl-6-(1-(2-morpholinoacetyl)-1,2,3,4-tetrahydroquinoline-6-yl)-1H-indole-4-carboxamide (Compound 5)

[0092]

[0093] Step 1: Synthesis of methyl 6-bromo-1-isopropyl-1H-indole-4-carboxylate

[0094]

[0095] Take a 100 mL pear-shaped flask, add methyl 6-bromo-4-indolecarboxylate (10 g, 0.039 mol), dissolve in 40 mL acetonitrile, then add iodopropane (10 g, 0.59 mol) and Cs₂CO₃ (20.3 g, 0.063 mol), and mix thoroughly. Then, incubate at 90 °C in a reflux tube for 4 h, monitoring the reaction by TLC until complete. Remove the solid from the reaction solution by filtration, wash with a small amount of DCM, and obtain the filtrate; add 500 mL of DCM and 100 mL of water to the filtrate for extraction, wash the mother liquor three times with water, then wash twice with saturated NaCl solution, and then completely dry the organic phase with anhydrous sodium sulfate. Distill under reduced pressure to obtain an orange-yellow oil, then cool and crystallize to obtain 11.02 g of solid. Yield: 94.6%.

[0096] Step 2: Synthesis of methyl 6-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)-1H-indole-4-carboxylic acid

[0097]

[0098] Potassium acetate (6.6 g, 0.067 mol), intermediate 19 (10 g, 0.034 mol), and 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxane) (17.2 g, 0.68 mol) were added to a 100 mL three-necked flask and dissolved in 1,4-dioxane. The mixture was then purged with N2 for 15 min. Next, 0.05 eq (1.2 g, 0.002 g) of Pd(dppf)Cl2 was added, and the mixture was evacuated three times. The mixture was then placed in an oil bath at 80 °C under nitrogen protection and connected to a reflux duct for 6 h. The reaction was monitored by TLC until complete. The solid in the reaction solution was removed by filtration and washed with a small amount of DCM to obtain the filtrate. The filtrate was washed three times with 100 mL of DCM and 300 mL of water, followed by two more washes with 30 mL of saturated NaCl solution each time. The organic phase was then completely dried with anhydrous sodium sulfate, followed by vacuum distillation and purification by column chromatography to obtain 8.3 g of pale yellow solid, with a yield of 65.2%.

[0099] Step 3: Synthesis of 1-(6-bromo-3,4-dihydroquinolin-1(2H)-yl)-2-morpholino-1-one

[0100]

[0101] Take a 50 mL pear-shaped flask, add 6-bromo-1,2,3,4-tetrahydroquinoline (3 g, 0.014 mol), morpholine acetic acid, and HATU (10.75 g, 0.028 mol), dissolve in 20 mL DMF, then add triethylamine (4.29 g, 0.042 mol) dropwise, and stir the mixture at 30 °C. After 4 h, the reaction is complete. Add dichloromethane and sodium carbonate solution to the reaction solution for extraction. After washing twice with water and Litsch salt in the aqueous phase, add anhydrous sodium sulfate, evaporate to dryness, distill under reduced pressure, and purify by column chromatography to obtain 2.35 g, with a yield of 73.7%.

[0102] Step 4: Synthesis of methyl 1-isopropyl-6-(1-(2-morpholinoacetyl)-1,2,3,4-tetrahydroquinolino-6-yl)-1H-indole-4-carboxylic acid

[0103]

[0104] Take a 50 mL three-necked flask, add potassium acetate (464 mg, 4.73 mmol), methyl 6-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)-1H-indole-4-carboxylic acid (1.2 g, 3.50 mmol), and 1-(6-bromo-3,4-dihydroquinoline-1(2H)-yl)-2-morpholino-1-one (800 mg, 2.37 mmol), and purge with N2 for 30 min; add Pd(dppf)Cl2 (83 mg, 0.118 mmol), evacuate three times, place in an oil bath at 100 ℃ under nitrogen protection, connect to a reflux tube and react for 6 h. TLC monitoring showed that the reaction was complete. The reaction system was cooled to room temperature. Solids in the reaction solution were removed by vacuum filtration. The filter cake was washed with a small amount of DCM to obtain the filtrate. 100 mL of DCM was added three times to the filtrate for extraction, yielding the organic phase. The organic phase was washed three times with 300 mL of water, followed by two more washes with 30 mL of saturated NaCl solution to further purify the organic phase. The organic phase was then completely dried using anhydrous sodium sulfate. Sodium sulfate was removed by vacuum filtration. The filtrate was distilled under reduced pressure until completely dry. Column chromatography yielded 754 mg, with a yield of 38.3%.

[0105] Step 5: Synthesis of 1-isopropyl-6-(1-(2-morpholinoacetyl)-1,2,3,4-tetrahydroquinolino-6-yl)-1H-indole-4-carboxylic acid

[0106]

[0107] The obtained isopropyl-6-(1-(2-morpholinoacetyl)-1,2,3,4-tetrahydroquinolino-6-yl)-1H-indole-4-carboxylic acid methyl ester (300 mg, 0.63 mmol) was mixed with 5 V / mol EtOH and 5 V / mol water. After thorough mixing, NaOH (38 mg, 0.95 mmol) was added to the mixture. The reaction solution was placed in an oil bath and refluxed at 60 °C for 4 h. The reaction was monitored by TLC and found to be complete. A portion of the ethanol solution in the reaction solution was then rotary evaporated, 10 mL of H2O was added, and the mixture was extracted once with DCM. The aqueous phase was collected, and the pH was adjusted to acidic with 10-fold diluted hydrochloric acid. The precipitated solid was filtered to obtain 234 mg, with a yield of 80.4%.

[0108] Step 6: Synthesis of N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-isopropyl-6-(1-(2-morpholinoacetyl)-1,2,3,4-tetrahydroquinoline-6-yl)-1H-indole-4-carboxamide

[0109]

[0110] Using 1-isopropyl-6-(1-(2-morpholinoacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)-1H-indole-4-carboxylic acid (174 mg, 0.37 mmol) and 3-(aminomethyl)-4,6-dimethylpyridin-2(1H)-one (86 mg, 0.565 mmol) as starting materials, the mixture was dissolved in 5 mL of DMF. Then, HATU (286.7 mg, 0.75 mmol) and triethylamine (114.3 mg, 1.13 mmol) were added. The reaction was carried out at room temperature for 4 h, and TLC monitoring showed that the reaction was complete. The organic phases were extracted with 100 mL of DCM and sodium carbonate aqueous solution, and the combined organic phases were washed three times with 50 mL of water, followed by two more washes with 20 mL of saturated NaCl solution. The organic phase was then completely dried using anhydrous sodium sulfate. Sodium sulfate was removed by vacuum filtration. The filtrate was subjected to vacuum distillation until completely dry. Thin-layer chromatography then purified the solution to obtain 99 mg of compound 1, with a yield of 44.2%.

[0111] MS (ESI), m / z: 595.32 [M+H] + ; 1 H NMR (500 MHz, CDCl3) δ 12.17 (s, 1H), 7.82 (s, 2H), 7.62 (s, 1H), 7.44 (s, 2H), 7.29 (s, 1H), 6.92 (s, 1H), 5.92 (s, 1H), 4.65 (s, 2H), 3.76 (s, 6H), 3.41 (s, 2H), 2.80 – 2.68 (m, 6H), 2.43(s, 3H), 2.15 (s, 3H), 1.98 – 1.95 (m, 2H), 1.53 (d, J = 6.5 Hz, 6H), 1.25(s, 2H).

[0112] By replacing appropriate raw materials and reagents, and following the preparation scheme of Example 5, Examples 5-7, 9, 16, 18, 19, 25-27, and 34 (compounds 5-7, 9, 16, 18, 19, 25-27, and 34) were finally obtained.

[0113] Example 6 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(1-(dimethylglycine)-1,2,3,4-tetrahydroquinoline-6-yl)-1-isopropyl-1H-indole-4-carboxamide (Compound 6)

[0114]

[0115] MS (ESI), m / z: 553.31 [M+H] + ; 1 H NMR (500 MHz, CDCl3) δ 12.19 (s, 1H),7.90 (s, 1H), 7.78 (s, 1H), 7.62 (s, 1H), 7.42 (s, 2H), 7.30 –7.29 (m, 1H),6.94 (s, 1H), 5.92 (s, 1H), 4.78 – 4.73 (m, 1H), 4.65 (d, J = 6.5 Hz, 2H), 3.76 (s, 2H), 3.51 (s, 2H), 2.76 – 2.73 (m, 2H), 2.50 –2.43 (m, 9H), 2.15 (s,3H), 1.96 –1.92 (m, 2H), 1.54 – 1.53 (m, 6H), 1.32 –1.25 (m, 1H).

[0116] Example 7 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-isopropyl-6-(1-(2-morpholinoacetyl)indololin-5-yl)-1H-indol-4-carboxamide (Compound 7)

[0117]

[0118] MS (ESI), m / z: 581.30[M+H]+; 1 H NMR (500 MHz, CDCl3) δ 12.73 (s, 1H),8.01 – 7.58 (m, 4H), 7.36 – 7.29 (m, 3H), 6.90 (s, 1H), 5.95 (s, 1H), 4.74 –4.66 (m, 3H), 4.18 – 3.83 (m, 6H), 3.28 – 3.08 (m, 3H), 2.73 (s, 3H), 2.44 (s,3H), 2.18 (s, 3H), 1.53 (d, J = 7.0 Hz, 6H), 1.25 (s, 2H).

[0119] Example 8 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-isopropyl-6-(1-(2-morpholinoacetyl)-1,2,3,4-tetrahydroquinoline-6-yl)-1H-indazole-4-carboxamide (Compound 8)

[0120]

[0121] Step 1: Synthesis of methyl 6-bromo-1-isopropyl-1H-indazole-4-carboxylate

[0122]

[0123] Take a 100 mL pear-shaped flask, add methyl 6-bromo-4-indazolecarboxylate (20 g, 0.078 mol), dissolve in 40 mL acetonitrile, then add iodopropane (20 g, 0.12 mol) and Cs₂CO₃ (51 g, 0.156 mol), and mix thoroughly. Then, connect a reflux tube at 90 °C and react for 4 h. The reaction was monitored by TLC and found to be complete. The solid in the reaction solution was removed by filtration, washed with a small amount of DCM, and the filtrate was obtained. Add 50 mL of DCM to the filtrate and extract with 100 mL of water. Wash the mother liquor three times with water, then wash twice with saturated NaCl solution. The organic phase was then completely dried with anhydrous sodium sulfate, and distilled under reduced pressure to obtain an orange-yellow oil, which was then cooled and crystallized to give 22.4 g of solid. The yield was 96.6%.

[0124] Step 2: Synthesis of methyl 1-isopropyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)-1H-indazole-4-carboxylate

[0125]

[0126] Potassium acetate (15.2 g, 0.155 mol), methyl 6-bromo-1-isopropyl-1H-indazole-4-carboxylate (22 g, 0.074 mol), and 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxane) (39 g, 0.154 mol) were added to a 250 mL three-necked flask and dissolved in 1,4-dioxane. The mixture was then purged with N2 for 30 min. Pd(dppf)Cl2 (2.8 g, 0.004 mol) was added, and the mixture was evacuated three times. The mixture was then placed in an oil bath at 80 °C under nitrogen protection and connected to a reflux tube for 6 h. The reaction was monitored by TLC and found to be complete. The solid in the reaction solution was removed by filtration and washed with a small amount of DCM to obtain the filtrate. 100 mL of DCM and 300 mL of water were added to the filtrate, and the mixture was washed three times. Then, it was washed twice with saturated NaCl solution, 30 mL each time. The organic phase was then completely dried with anhydrous sodium sulfate, distilled under reduced pressure, and purified by column chromatography to obtain 22 g of a pale yellow solid, with a yield of 88%.

[0127] Step 3: Synthesis of 1-(6-bromo-3,4-dihydroquinolin-1(2H)-yl)-2-morpholino-1-one

[0128]

[0129] Take a 50 mL pear-shaped flask, add 6-bromo-1,2,3,4-tetrahydroquinoline (3 g, 0.014 mol), morpholine acetic acid, and HATU (10.75 g, 0.028 mol), dissolve in 20 mL DMF, then add triethylamine (4.29 g, 0.042 mol) dropwise, and stir the reaction at 30 °C. After 4 h, the reaction is complete. Add dichloromethane and sodium carbonate solution to the reaction solution for extraction. After washing with water and Litsch salt twice with the aqueous phase, add anhydrous sodium sulfate, evaporate to dryness, distill under reduced pressure, and purify by column chromatography to obtain 2.35 g, with a yield of 73.7%.

[0130] Step 4: Synthesis of methyl 1-isopropyl-6-(1-(2-morpholinoacetyl)-1,2,3,4-tetrahydroquinoline-6-yl)-1H-indazole-4-carboxylate

[0131]

[0132] Take a 50 mL three-necked flask, add potassium acetate (464 mg, 4.93 mmol), methyl 6-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)-1H-indazole-4-carboxylate (0.85 g, 2.46 mmol), and 1-(6-bromo-3,4-dihydroquinoline-1(2H)-yl)-2-morpholino-1-one (1 g, 2.58 mmol), and purge with N2 for 30 min; add Pd(dppf)Cl2 (90 mg, 0.123 mmol), evacuate three times, place in an oil bath at 100 ℃ under nitrogen protection, connect to a reflux tube and react for 6 h. The reaction is monitored by TLC and is complete. The reaction system was cooled to room temperature. Solids in the reaction solution were removed by vacuum filtration. The filter cake was washed with a small amount of DCM to obtain the filtrate. 100 mL of DCM was added three times to the filtrate for extraction, yielding the organic phase. The organic phase was washed three times with 300 mL of water, followed by two more washes with 30 mL of saturated NaCl solution to further purify the organic phase. The organic phase was then completely dried using anhydrous sodium sulfate. Sodium sulfate was removed by vacuum filtration. The filtrate was distilled under reduced pressure until completely dry. Column chromatography yielded 553 mg, with a yield of 39.5%.

[0133] Step 5: Synthesis of 1-isopropyl-6-(1-(2-morpholinoacetyl)-1,2,3,4-tetrahydroquinoline-6-yl)-1H-indazole-4-carboxylic acid

[0134]

[0135] The obtained isopropyl-6-(1-(2-morpholinoacetyl)-1,2,3,4-tetrahydroquinoline-6-yl)-1H-indazole-4-carboxylic acid methyl ester (460 mg, 0.96 mmol) was mixed with 5 V / mol EtOH and 5 V / mol water. After thorough mixing, NaOH (58 mg, 1.45 mmol) was added to the mixture. The reaction solution was placed in an oil bath and refluxed at 60 °C for 4 h. The reaction was monitored by TLC until it was complete. A portion of the ethanol solution in the reaction solution was then rotary evaporated, 10 mL of H2O was added, and the mixture was extracted once with DCM. The aqueous phase was collected, and the pH was adjusted to acidic with 10-fold diluted hydrochloric acid. The precipitated solid was filtered to obtain 382 mg, with a yield of 85.6%.

[0136] Step 6: Synthesis of N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-isopropyl-6-(1-(2-morpholinoacetyl)-1,2,3,4-tetrahydroquinoline-6-yl)-1H-indazole-4-carboxamide

[0137]

[0138] Using 1-isopropyl-6-(1-(2-morpholinoacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)-1H-indazole-4-carboxylic acid (300 mg, 0.65 mmol) and 3-(aminomethyl)-4,6-dimethylpyridin-2(1H)-one (148 mg, 0.972 mmol) as starting materials, the mixture was dissolved in 5 mL of DMF. Then, HATU (493 mg, 1.30 mmol) and triethylamine (196.7 mg, 1.95 mmol) were added. The reaction was carried out at room temperature for 4 h, and TLC monitoring showed that the reaction was complete. The organic phases were extracted with 100 mL of DCM and sodium carbonate aqueous solution, and the combined organic phases were washed three times with 50 mL of water, followed by two more washes with 20 mL of saturated NaCl solution. The organic phase was then completely dried using anhydrous sodium sulfate. Sodium sulfate was removed by vacuum filtration. The filtrate was distilled under reduced pressure until completely dry. After thin-layer chromatography purification, 261 mg of compound 1 was obtained, with a yield of 61.4%.

[0139] MS (ESI), m / z: 596.31[M+H]+; 1 H NMR (500 MHz, CDCl3) δ 12.34 (s, 1H), 8.41 (s, 1H), 7.95 (t, J = 5.5 Hz, 1H), 7.80 (s, 1H), 7.65 (s, 1H), 7.47–7.45 (m, 2H), 5.93 (s, 1H), 4.93– 4.88 (m, 1H), 4.66 (d, J = 5.5 Hz, 2H), 3.82 (t, J = 6.5 Hz, 2H), 3.76 (s, 4H), 3.45 (s, 2H), 2.81 – 2.66 (m, 6H), 2.42 (s, 3H), 2.17 (s, 3H), 2.02 – 1.96 m, 2H), 1.60 – 1.59 (d, J = 6.5 Hz, 7H).

[0140] By replacing appropriate raw materials and reagents, and following the preparation scheme of Example 1, Examples 8, 10-12, 19-21, 29, and 30 (compounds 8, 10-12, 19-21, 29, and 30) were finally obtained.

[0141] Example 9 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(1-(dimethylglycine)indololin-5-yl)-1-isopropyl-1H-indol-4-carboxamide (Compound 9)

[0142]

[0143] MS (ESI), m / z: 539.29[M+H]+; 1 H NMR (500 MHz, CDCl3) δ 12.79 (s, 1H), 8.02 (s, 2H), 7.65 (s, 1H), 7.56 (s, 1H), 7.34 – 7.30 (m, 3H), 6.92– 6.91 (m, 1H), 5.96 (s, 1H), 4.66 (s, 2H), 3.94 (s, 1H), 3.55 (s, 2H), 3.07 (t, J = 8.5Hz, 2H), 2.55 – 2.45 (m, 9H), 2.19 (s, 3H), 1.54 (d, J = 7.0 Hz, 6H), 1.25(s, 3H).

[0144] Example 10 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-isopropyl-6-(1-(2-morpholinoacetyl)indololin-5-yl)-1H-indazole-4-carboxamide (Compound 10)

[0145]

[0146] MS (ESI), m / z: 582.30[M+H]+; 1 H NMR (500 MHz, CDCl3) δ 12.33 (s, 1H), δ8.39 (s, 1H), 8.24 (s, 1H), 7.85 (s, 1H), 7.73 (s, 1H), 7.63 (s, 1H), 7.48 (s, 2H), 6.00 (s, 1H), 4.62 (s, 2H), 4.18 – 4.15 (m, 2H), 3.79 (s, 4H), 3.33 –3.20(m, 4H), 2.70 (s, 3H), 2.44 (s, 3H), 2.22 (s, 3H), 2.09 (s, 2H), 1.60 (d,J = 6.5 Hz, 6H).

[0147] Example 11 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(1-(dimethylglycine)indoline-5-yl)-1-isopropyl-1H-indazole-4-carboxamide (Compound 11)

[0148]

[0149] MS (ESI), m / z: 540.28[M+H]+; 1 H NMR (500 MHz, CDCl3) δ 12.52 (s, 1H), 8.41 (s, 1H), 8.12 – 8.05 (m, 2H), 7.63 (d, J = 26.5 Hz, 2H), 7.38 (d, J =11.0 Hz, 2H), 5.94 (s, 1H),4.91 – 4.87 (m, 1H), 4.64 (d, J = 5.5 Hz, 2H),4.00 (s, 2H), 3.44 (s, 2H), 3.08–3.05 (m, 2H), 2.60 (s, 6H), 2.41 (s, 3H),2.17 (s, 3H), 1.58 (d, J = 6.5 Hz, 6H).

[0150] Example 12 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(1-(dimethylglycine)-1,2,3,4-tetrahydroquinoline-6-yl)-1-isopropyl-1H-indazole-4-carboxamide (Compound 12)

[0151]

[0152] MS (ESI), m / z: 540.28[M+H]+; 1 H NMR (500 MHz, CDCl3) δ 11.66 (s, 1H),8.45 (s, 1H), 8.02 (s, 1H), 7.72 (s, 1H), 7.64 (s, 1H), 7.45 (s, 2H), 5.94(s, 1H), 4.94 – 4.88 (m, 1H), 4.65 (d, J = 5.5 Hz, 2H), 3.86 – 3.79 (m, 4H), 2.97 (s, 1H), 2.81 – 2.78(m, 2H), 2.72 (s, 6H), 2.41 (s, 3H), 2.22 (s, 3H),2.02 – 1.97 (m, 2H), 1.60 (d, J = 6.5 Hz, 6H).

[0153] Example 13 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(N-ethylcyclopropanecarbamate)-5-(1-(4-methoxypiperidin-1-yl)phenanthridine[3,2-but]isoxazol-6-yl)-2-methylbenzamide (Compound 13)

[0154]

[0155] MS (ESI), m / z: 622.33[M+H]+; 1 H NMR (500 MHz, CDCl3) δ 11.45 (s, 1H),9.18 (s, 1H), 8.08 – 8.01 (m, 3H), 7.79 (s, 1H), 7.65 (s, 1H), 7.54 (s, 1H),5.89 (s, 1H), 4.58 (d, J = 6.5 Hz, 2H), 3.82 – 3.80 (m, 2H), 3.43 – 3.38 (s,4H), 3.30 – 3.26 (m, 2H), 2.40 (d, J = 5.5 Hz, 6H), 2.18 – 2.12 m, 5H), 1.88– 1.80 (m, 2H), 1.32–1.23 (m, 2H), 1.20 – 1.12 (m, 4H), 1.03 – 0.98 (m, 2H), 0.64 – 0.53 (m, 2H).

[0156] Example 14 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(N-ethylcyclopropanecarbamate)-2-methyl-5-(1-(4-methylpiperazin-1-yl)phthalazin-6-yl)benzamide (Compound 14)

[0157]

[0158] MS (ESI), m / z: 607.33[M+H]+; 1H NMR (500 MHz, CDCl3) δ 11.54 (s, 1H), 9.11 (s, 1H), 8.07 –8.05 (m, 1H), 8.00 – 7.99 (m, 2H), 7.78 (s, 1H), 7.63 –7.61 m, 1H), 7.54 (s, 1H), 5.87 (s, 1H), 4.59 (d, J = 5.5 Hz, 2H), 4.10 –4.04 (m, 1H), 3.61 (s, 4H), 3.43 –3.36 (m, 1H), 2.76 (s, 4H), 2.45 (s, 3H),2.40 –2.39 (m, 6H), 2.16 (s, 3H), 1.21 – 1.13 (m, 4H), 1.02 – 0.97 (m, 2H), 0.61 – 0.54 (m, 2H).

[0159] Example 15 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(N-ethylcyclopropanecarbamate)-2-methyl-5-(1-(4-methylpiperidin-1-yl)phthalazin-6-yl)benzamide (Compound 15)

[0160]

[0161] MS (ESI), m / z: 606.33[M+H]+; 1 H NMR (500 MHz, CDCl3) δ 12.01 (s, 1H),9.21 (s, 1H), 8.10 – 8.01 (m, 3H), 7.75 (s, 1H), 7.56 (s, 1H), 6.05 (s, 1H),4.57 (s, 2H), 3.95 (d, J = 13.0 Hz, 2H), 3.12 (t, J = 12.0 Hz, 2H), 2.45 (s,3H), 2.37 (s, 3H), 2.27 (s, 3H), 2.08 (s, 3H), 1.86 (d, J = 12.0Hz, 2H), 1.57 – 1.51 (m, 2H), 1.26 – 1.21 (m, 1H), 1.18 – 1.15 (m, 3H), 1.07 – 1.06 (m, 5H), 0.66 – 0.58 (m, 2H).

[0162] Example 16 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-isopropyl-6-(1-(4-methylpiperidin-1-yl)phthalazin-6-yl)-1H-indole-4-carboxamide (Compound 16)

[0163]

[0164] MS (ESI), m / z: 562.72[M+H]+; 1 H NMR (400 MHz, CDCl3) δ 11.94 (s, 1H),9.15 (s, 1H), 8.14 – 7.99 (m, 4H), 7.75 (s, 1H), 7.36 (s, 1H), 7.00 (s, 1H),5.91 (s, 1H), 4.80 – 4.64(m, 3H), 3.95 – 3.92 (m, 2H), 3.11 – 3.05 (m, 2H), 2.43 (s, 3H), 2.17 – 2.10 (s, 3H), 1.85 – 1.82(m, 2H), 1.57 (d, J =6.4Hz,9H), 1.25 (s, 1H),1.05 (d, J = 6.4Hz, 3H).

[0165] Example 17 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(1-((2S,6R)-2,6-dimethylmorpholino)phthalazin-6-yl)-3-(N-ethylcyclopropanecarbamate)-2-methylbenzamide (Compound 17)

[0166]

[0167] MS (ESI), m / z: 622.33[M+H]+; 1H NMR (500 MHz, CDCl3) δ 11.87 (s, 1H),9.18 (s, 1H), 8.08 – 8.01 m, 3H), 7.78 (s, 1H), 7.62 (s, 1H), 7.54 (s, 1H),5.89 (s, 1H), 4.58 – 4.57 (m, 2H), 3.99 – 3.96 (m, 2H), 3.76 (d, J = 12.5 Hz,2H), 2.91 – 2.87 (m, 2H), 2.39 (s, 6H), 2.17 (s, 3H), 1.24 – 1.12 (m, 12H),1.00 – 0.98 (m, 2H), 0.61 – 0.54 (m, 2H).

[0168] Example 18 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(1-((2S,6R)-2,6-dimethylmorpholino)phthalazin-6-yl)-1-isopropyl-1H-indole-4-carboxamide (Compound 18)

[0169]

[0170] MS (ESI), m / z: 578.30[M+H]+; 1 H NMR (400 MHz, CDCl3) δ 12.09 (s, 1H), 9.24 (s, 1H), 8.19 – 8.06 (m, 3H), 7.96 (s, 1H), 7.78 (s, 1H), 7.42 (d, J =3.6Hz, 1H), 6.98 (d, J = 3.2Hz, 1H), 6.03 (s, 1H), 4.64 (d, J =5.6 Hz, 1H), 4.06 – 4.02 (m, 2H), 3.81 (d, J = 12.8Hz, 1H), 2.97 – 2.88 (m, 2H), 2.48 (s,2H), 2.24 (s, 2H), 2.09 (s, 3H), 1.59 (d, J = 5.6Hz, 6H), 1.28 – 1.25 (m,9H).

[0171] Example 19 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-isopropyl-6-(1-(4-methylpiperazin-1-yl)phthalazin-6-yl)-1H-indazole-4-carboxamide (Compound 19)

[0172]

[0173] MS (ESI), m / z: 564.30[M+H]+; 1 H NMR (400 MHz, CDCl3) δ 12.49 (s, 1H),9.09 (s, 1H), 8.52 (s, 1H), 8.31– 8.28(m, 1H), 8.14 – 8.05 (m, 3H), 8.00 (s,1H), 7.80 (s, 1H), 5.85 (s, 1H), 4.98 – 4.92 (m, 1H), 4.66 (d, J =5.6 Hz,2H), 3.63 – 3.59 (m, 4H), 2.74 (s, 4H), 2.43 – 2.38 (s, 8H), 2.12 (s, 3H),1.62 (d, J = 6.8 Hz, 6H).

[0174] Example 20 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-isopropyl-6-(1-(4-methylpiperidin-1-yl)phthalazin-6-yl)-1H-indazole-4-carboxamide (Compound 20)

[0175]

[0176] MS (ESI), m / z: 563.30[M+H]+; 1 H NMR (400 MHz, CDCl3) δ 12.34 (s, 1H),9.15 (s, 1H), 8.50 (s, 1H), 8.14 – 7.94 (m, 4H), 7.80 (s, 1H), 5.94 (s, 1H),4.95 (s, 1H), 4.64 (s, 2H), 3.94 (d, J = 13.2 Hz, 2H), 3.10 –3.04 (s, 2H),2.41 (s, 3H), 2.18– 2.09 (m, 4H), 1.85 –1.82 (m, 2H),1.64 – 1.50 (m, 8H), 1.25 (s, 2H), 1.06 (s, 2H).

[0177] Example 21 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-(1-((2S,6R)-2,6-dimethylmorpholino)phthalazin-6-yl)-1-isopropyl-1H-indazole-4-carboxamide (Compound 21)

[0178]

[0179] MS (ESI), m / z: 579.30[M+H]+; 1 H NMR (400 MHz, CDCl3) δ 12.21 (s, 1H),9.17 (s, 1H), 8.49 (s, 1H), 8.20 – 8.04 (m, 3H), 7.95 (s, 1H), 7.80 (s, 1H),5.95 (s, 1H), 4.64 (d, J = 6.0 Hz, 2H), 4.02 – 3.98 (m, 2H), 3.78 (d, J =12.8Hz, 2H), 2.94 – 2.88(m, 2H), 2.42 (s, 2H), 2.19 (s, 2H), 2.07 (s, 1H),1.63 (d, J = 6.4 Hz, 6H), 1.27 – 1.25 (m, 9H).

[0180] Example 22 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(N-ethylcyclopropanecarbamate)-2-methyl-5-(1-(2-(4-methylpiperazin-1-yl)acetyl)-1,2,3,4-tetrahydroquinoline-6-yl)benzamide (Compound 22)

[0181]

[0182] MS (ESI), m / z: 652.37[M+H]+; 1H NMR (400 MHz, CDCl3) δ 11.58 (s, 1H),7.63 – 7.54 (m, 2H), 7.40 (d, J = 2.0 Hz, 1H), 7.34 – 7.27 (m, 3H), 5.94 (s,1H), 4.56 – 4.55 m, 2H), 4.10 – 4.01 (m, 1H), 3.83 – 3.80 (m, 2H), 3.38 –3.31 (m, 3H), 2.80 – 2.76 (m, 2H), 2.62 – 2.52 (m, 8H), 2.40 (s, 3H), 2.31(d, J =6.8 Hz, 6H), 2.17 (s, 3H), 2.02 – 1.96 (m, 2H), 1.21 – 1.10 (m, 4H), 0.98 – 0.97 (m, 2H), 0.63 – 0.53 (m, 2H).

[0183] Example 23 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(N-ethylcyclopropanecarbamate)-2-methyl-5-(1-(2-(4-methylpiperazin-1-yl)acetyl)indoline-5-yl)benzamide (Compound 23)

[0184]

[0185] MS (ESI), m / z: 638.81[M+H]+; 1 H NMR (400 MHz, CDCl3) δ 11.70 (s, 1H),8.23 (d, J = 8.8 Hz, 1H), 7.54 (d, J = 2.0 Hz, 1H), 7.38 – 7.29 (m, 4H),5.94 (s, 1H), 4.55 (d, J = 6.0 Hz, 2H), 4.17 (t, J = 8.4Hz, 2H), 3.37 – 3.20(m, 5H), 2.71– 2.64 (m, 9H), 2.40 – 2.38 m, 6H), 2.30 (s, 3H), 2.17 (s, 3H),1.25–1.10 (m, 4H), 0.99 – 0.94 (m, 2H), 0.62–0.51(m, 2H).

[0186] Example 24 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(N-ethylcyclopropanecarbamate)-2-methyl-5-(1-(3-morpholinopropionyl)indoline-5-yl)benzamide (Compound 24)

[0187]

[0188] MS (ESI), m / z: 639.34[M+H]+; 1 H NMR (400 MHz, CDCl3) δ 11.92 (s, 1H), 8.17 (d, J =5.2 Hz, 1H), 7.54 (s, 1H), 7.38 – 7.34 (m,3H), 5.92 (s, 1H), 4.55(d, J = 6.0 Hz, 2H), 4.12 – 4.00 (m, 3H), 3.77 – 3.75(m, 4H), 3.23 – 3.19(m, 2H), 2.89 (t, J = 7.2 Hz, 2H), 2.72– 2.68 (m, 2H), 2.60 (s, 4H), 2.39 (s,3H), 2.30 (s, 3H), 2.16 (s, 3H), 1.28 – 1.10 (m, 6H), 0.97 (d, J =6.0 Hz, 2H), 0.59 – 0.52 (m, 2H).

[0189] Example 25 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-isopropyl-6-(1-(2-(4-methylpiperazin-1-yl)acetyl)-1,2,3,4-tetrahydroquinoline-6-yl)-1H-indole-4-carboxamide (Compound 25)

[0190]

[0191] MS (ESI), m / z: 608.35[M+H]+; 1H NMR (400 MHz, CDCl3) δ 12.16 (s, 1H), 7.84 (s, 1H), 7.75 – 7.72 (m, 1H), 7.63 (s, 1H), 7.48 – 7.46(m, 2H), 7.30 (d,J = 3.2Hz, 1H), 6.92 (d, J = 3.2 Hz, 1H), 5.95 (s, 1H), 4.82 – 4.72 (m, 1H), 4.65 (d, J = 5.6 Hz, 2H), 3.82 (t, J = 6.4Hz, 2H), 3.33 (s, 2H), 2.79–2.76(m,2H), 2.62 – 2.46 (m, 9H), 2.31 (s, 3H), 2.18 (s, 3H), 2.04 – 1.97 (m, 3H), 1.54 (d, J = 6.8Hz, 6H), 1.25 (s, 2H).

[0192] Example 26 3-(N-ethylcyclopropanecarbamate)-N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-5-(1-(2-morpholinoacetyl)indoline-5-yl)benzamide (Compound 26)

[0193]

[0194] MS (ESI), m / z: 641.32[M+H]+; 1 H NMR (400 MHz, CDCl3) δ 13.11 (s, 1H),8.24 – 8.16 (m, 2H), 7.77 (s, 1H), 7.60 (s, 1H), 7.49 – 7.45 (m, 2H), 7.28(d, J = 3.2 Hz, 1H), 6.97 (d, J = 3.2 Hz, 1H), 5.85 (s, 1H), 4.78 – 4.69 (m,3H), 4.13 – 4.09 (m, 2H), 3.87 (s, 3H), 3.78 – 3.75 (m, 3H), 3.26 (s, 1H),3.15 –3.10 (m, 2H), 2.63 (s, 3H), 2.11 (s, 3H), 1.54 (d, J = 6.4 Hz, 6H), 1.33 – 1.25 (s, 3H).

[0195] Example 27 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-isopropyl-6-(1-(2-(4-methylpiperazin-1-yl)acetyl)indoline-5-yl)-1H-indo-4-carboxamide (Compound 27)

[0196]

[0197] MS (ESI), m / z: 594.33[M+H]+; 1 H NMR (400 MHz, CDCl3) δ 12.22 (s, 1H),8.24 – 8.16 (m, 1H), 7.83 – 7.79(m, 2H), 7.60 (s, 1H), 7.49 – 7.47 (m, 2H),7.28 – 7.27 (m, 1H), 6.92 (d, J = 3.2 Hz, 1H), 5.90 (s, 1H), 4.64 (d, J =6.0 Hz, 2H), 4.14 (t, J = 8.4 Hz, 2H), 3.26 (s, 1H), 3.16 (t, J = 8.4 Hz, 2H), 2.70 – 2.61 (m, 6H), 2.43 – 2.37 (m, 6H), 2.13 (s, 3H), 1.53 (d, J = 6.8Hz, 6H), 1.25 (s, 4H).

[0198] Example 28 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(N-ethylcyclopropanecarbamate)-2-methyl-5-(1-(3-morpholinopropionyl)-1,2,3,4-tetrahydroquinoline-6-yl)benzamide (Compound 28)

[0199]

[0200] MS (ESI), m / z: 653.36[M+H]+; 1H NMR (400 MHz, CDCl3) δ 11.41 (s, 1H),7.55 – 7.29 (m, 5H), 5.95 (s, 1H), 4.55 (d, J = 5.6 Hz, 2H), 3.81–3.73 (m,5H), 2.86 – 2.76(m, 8H), 2.40 – 2.31 (s, 6H), 2.19 (s, 3H), 2.03 – 1.95 (m, 2H), 1.42 – 0.87 (m, 12H), 0.62 – 0.53 (m, 2H).

[0201] Example 29 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-isopropyl-6-(1-(2-(4-methylpiperazin-1-yl)acetyl)-1,2,3,4-tetrahydroquinoline-6-yl)-1H-indazole-4-carboxamide (Compound 29)

[0202]

[0203] MS (ESI), m / z: 609.34[M+H]+; 1 H NMR (400 MHz, CDCl3) δ 12.29 (s, 1H), 8.44 (s, 1H), 7.92 – 7.89 (m, 1H), 7.81 (s, 1H), 7.69 (s, 1H), 7.51 – 7.49(m, 2H), 6.00 (s, 1H), 4.98 – 4.91 (m, 1H), 4.67 (d, J = 6.0 Hz, 2H), 3.87 –3.83(m, 2H), 3.40 (s, 2H), 2.84 – 2.74 (s, 9H), 2.45 (d, J = 6.8 Hz, 6H),2.23 (s, 3H), 2.06 – 2.00 (m, 4H), 1.63 (d, J = 6.8 Hz, 6H).

[0204] Example 30 6-(1-((2S,6R)-2,6-dimethylmorpholino)diazanaphth-6-yl)-1-isopropyl-N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1H-indazole-4-carboxamide (Compound 30)

[0205]

[0206] MS (ESI), m / z: 595.29[M+H]+; 1 H NMR (400 MHz, CDCl3) δ 12.18 (s, 1H),8.64 (s, 1H), 7.99 (s, 1H), 7.83 (s, 1H), 7.69 – 7.46 (m, 4H), 6.72 (s, 1H),5.32 (s, 1H), 4.17 (d, J = 5.6 Hz, 2H), 3.48 – 3.40 (m, 2H), 3.34 (s, 2H), 3.23 (d, J = 12.4Hz, 2H), 2.43 – 2.31 (m, 2H), 1.65 (s, 3H), 1.09 (d, J = 6.4Hz, 6H), 0.70 (d, J = 6.4 Hz, 8H).

[0207] Example 31 5-(1-((2S,6R)-2,6-dimethylmorpholino)phthalazin-6-yl)-3-(N-ethylcyclopropanecarbamate)-N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methylbenzamide (Compound 31)

[0208]

[0209] MS (ESI), m / z: 638.32[M+H]+; 1 H NMR (400 MHz, CDCl3) δ 11.80 (s, 1H),9.16 (s, 1H), 8.08 – 8.01 (m, 2H), 7.78 (s, 1H), 7.60 (s, 1H), 7.53 (s, 1H),5.91 (s, 1H), 4.62 (d, J = 6.0 Hz, 2H), 4.00 – 3.94(m, 2H), 3.89 (s, 2H), 3.77 (d, J = 12.4 Hz, 2H), 2.93 – 2.87 (m, 2H), 2.40 (s, 3H), 2.23 (s, 3H),1.27 – 1.13 (m, 14H), 1.03 – 0.95 m, 2H), 0.60 – 0.56 (m, 2H).

[0210] Example 32 3-(N-ethylcyclopropanecarbamate)-N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-5-(1-morpholinophthalazine-6-yl)benzamide (Compound 32)

[0211]

[0212] MS (ESI), m / z: 610.29[M+H]+; 1 H NMR (400 MHz, CDCl3) δ 12.10 (s, 1H),9.17 (s, 1H), 8.09 – 8.00 (m, 3H), 7.79 (s, 1H), 7.66 – 7.63 (m, 1H), 7.53(d, J = 2.8 Hz, 1H), 5.88 (s, 1H), 4.62 (d, J = 5.6 Hz, 2H),, 3.97 – 3.88 (s,6H), 3.54 – 3.51 (m, 4H), 2.42 (s, 3H), 2.22 (s, 3H), 1.33 – 1.14 (m, 7H),1.02 – 0.97 (m, 2H), 0.61– 0.58 (m, 2H).

[0213] Example 33 3-(N-ethylcyclopropanecarbamate)-N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-5-(1-(2-morpholinoacetyl)-1,2,3,4-tetrahydroquinoline-6-yl)benzamide (Compound 33)

[0214]

[0215] MS (ESI), m / z: 655.34[M+H]+; 1H NMR (400 MHz, CDCl3) δ 12.08 (s, 1H),7.71 – 7.53 (m, 2H), 7.39 – 7.32 (m, 3H), 5.94 (s, 1H), 4.60 (s, 2H), 3.89(s, 3H), 3.81 – 3.78 (m, 5H), 3.53 (s, 2H), 2.77 (d, J = 7.4 Hz, 5H), 2.34(s, 3H), 2.23 (s, 3H), 2.00 – 1.97 (m, 2H), 1.40 – 1.17 (m, 5H), 1.14 – 1.10(m, 2H), 0.97 – 0.85 (m, 4H), 0.59 – 0.54 (m, 2H).

[0216] Example 34 3-(N-ethylcyclopropanecarbamate)-N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-5-(1-(2-morpholinoacetyl)-1,2,3,4-tetrahydroquinoline-6-yl)benzamide (Compound 34)

[0217]

[0218] MS (ESI), m / z: 578.30[M+H]+; 1 H NMR (400 MHz, CDCl3) δ 11.68 (s, 1H), 9.16 (s, 1H), 8.14 – 7.97 (m, 2H), 7.85 – 7.75 (m, 1H), 7.37 (s, 1H), 6.98 (s,1H), 5.93 (s, 1H), 4.64 (s, 1H), 3.83 (s, 1H), 3.43 (s, 2H), 3.29 (s, 1H), 2.44 (s, 2H), 2.18 (s, 3H), 1.86 (s, 2H), 1.57 (d, J = 6.4 Hz, 4H), 1.42 (s,1H), 1.25 (s, 9H), 0.87 – 0.86 (m, 4H).

[0219] Example 35 5-(1-(4-acetylpiperidin-1-yl)diazanaphth-6-yl)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(N-ethylcyclopropanecarbamate)-2-methylbenzamide (Compound 35)

[0220]

[0221] MS (ESI), m / z: 635.32[M+H]+; 1 H NMR (400 MHz, CDCl3) δ 11.63 (s, 1H),9.25 (s, 1H), 8.11– 8.04 (m, 3H), 7.76 (s, 1H), 7.55 (s, 2H), 5.91 (s, 1H),4.58 (d, J = 5.6Hz, 2H), 3.86 – 3.75 (m, 2H), 3.60 – 3.48 (m, 4H), 2.40 –2.38 (m, 6H), 2.19 – 2.17 (m, 6H), 1.39 – 1.21 (m, 6H), 0.99 (s, 2H), 0.60 –0.54 (m, 2H).

[0222] Example 36 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(N-ethylcyclopropanecarbamate)-2-methyl-5-(1-morpholinophthalazine-6-yl)benzamide (Compound 36)

[0223]

[0224] MS (ESI), m / z: 594.30[M+H]+; 1 H NMR (400 MHz, CDCl3) δ 11.73 (s, 1H),9.22 (s, 1H), 8.11– 8.00 (m, 3H), 7.73 (s, 1H), 7.55 (s, 1H), 7.42 (s, 1H),6.00 (s, 1H), 4.57 (d, J = 6.0 Hz, 2H), 3.99 – 3.97 (m, 3H), 3.57 – 3.54 (m,3H), 2.44 – 2.37 (m, 6H), 2.23 (s, 3H), 2.06 (s, 2H), 1.24 – 1.14(m, 6H),1.01 (s, 2H), 0.63–0.58 (m, 2H).

[0225] Example 37 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(N-ethylcyclopropanecarbamate)-2-methyl-5-(1-(4-morpholinopiperidin-1-yl)phthalazin-6-yl)benzamide (Compound 37)

[0226]

[0227] MS (ESI), m / z: 677.38[M+H]+; 1 H NMR (400 MHz, CDCl3) δ 11.80 (s, 1H), 9.09 (s, 1H), 8.05 – 7.98 (m, 3H), 7.77 (d, J = 2.0 Hz, 1H), 7.63 (t, J = 5.6Hz, 1H), 7.54 – 7.53(m, 1H), 5.87 (s, 1H), 4.58 (d, J = 6.0Hz, 2H), 3.98 (d,J = 13.2Hz, 2H), 3.81 – 3.78 (m, 4H), 3.10 – 3.03 (m, 2H), 2.68 – 2.66(m,4H), 2.38 (d, J=4.4Hz, 6H), 2.15 (s, 3H), 2.07 – 2.05 (m, 2H), 1.24 – 1.12 (m, 9H), 1.02 – 0.97 (m, 2H), 0.62 – 0.53 (m, 2H).

[0228] Example 38 N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(1-(4-(dimethylamino)piperidin-1-yl)phthalazin-6-yl)-3-(N-ethylcyclopropanecarbamate)-2-methylbenzamide (Compound 38)

[0229]

[0230] MS (ESI), m / z: 635.36[M+H]+; 1H NMR (400 MHz, CDCl3) δ 11.78 (s, 1H), 9.09 (s, 1H), 8.06 – 8.00 (m, 3H), 7.78 (d, J = 2.0Hz, 1H), 7.65 – 7.62 (m,1H), 7.53 (d, J = 2.0 Hz, 1H), 5.87 (s, 1H), 4.58 (d, J = 6.0 Hz, 2H), 3.97(d, J = 13.2 Hz, 2H), 3.08 – 3.02 (m, 2H), 2.80 (s, 2H), 2.40 – 2.38 (m,12H), 2.15 (s, 3H), 2.06 – 2.01 (m, 2H), 1.86 – 1.77 (m, 2H), 1.24 – 1.12 (m,5H), 1.02 – 0.95 (m, 2H), 0.61 – 0.52 (m, 2H).

[0231] Example 39 In vitro antitumor cell activity

[0232] The novel compounds substituted with benzoylpyridinones were screened for their in vitro inhibitory activity against human B lymphoma cells (SU-DHL-6, WSU-DLCL-2).

[0233] 1) Cell Plating: Centrifuge SU-DHL-6 and WSU-DLCL-2 cell suspensions in logarithmic growth phase (800 r, 5 min), discard the supernatant, and resuspend the cells in 1 mL of fresh culture medium. Prepare a 1:10 dilution by mixing 100 μL of cell suspension with 900 μL of culture medium and quantify the cells using a hemocytometer. Adjust the concentration based on the counting results, and seed each well of a 96-well plate with 100 μL of a solution containing 1×10⁻⁶ cells / mL. 4 A suspension of cells was prepared. After inoculation, the culture plate was pre-cultured in a 37 ℃, 5% CO2 incubator for 24 h.

[0234] 2) Drug preparation: Accurately weigh the test compound and the positive control drug Tazematostat using an analytical balance. For initial dissolution, dissolve the sample in 100 μL DMSO, add 50 μL Tween 80 to aid dissolution, and then bring the volume to 2 mL with RPMI-1640 medium. Vortex to obtain a clear stock solution (1 mmol / L). All solution preparation processes must be performed under aseptic conditions.

[0235] 3) Gradual dosing: The stock solution was serially diluted to obtain five concentration gradients: 100 μmol / L, 10 μmol / L, 1 μmol / L, 0.1 μmol / L, and 0.01 μmol / L. 100 μL of the corresponding concentration was added to each well of the pre-cultured 96-well plate (the actual concentration in the well was 50% of the labeled concentration). After dosing, the plate was incubated for another 72 h for efficacy observation.

[0236] 4) MTT assay: Add 20 μL of 0.5% MTT solution (final concentration 0.1%) to each well under light-protected conditions, incubate at 37 ℃ for 4 h, then centrifuge (1500 rpm, 5 min) to remove the supernatant. Add 150 μL of DMSO to each well and vortex until the formazan crystals are completely dissolved. Measure the absorbance at 490 nm using a microplate reader. Plot the dose-response curve by calculating the cell viability of different concentration groups, and calculate the IC50 using GraphPad Prism software. 50 The values ​​are shown in Table 1.

[0237] Table 1 Results of the in vitro antitumor cell activity of the compounds

[0238]

[0239] Example 40 EZH2 kinase inhibition experiment

[0240] 1) Reagent preparation: Prepare the reaction buffer system, enzyme working solution, substrate working solution, and radiolabeled […] in advance. 3 H]-S-adenosylmethionine ([ 3 H]-SAM) solution.

[0241] 2) Enzymatic reaction: Dispense 10 μL of enzyme reaction solution into each well of a 96-well microplate and incubate at room temperature for 15 min; add 10 μL of substrate working solution and 10 μL of […] to each well sequentially. 3 The reaction was initiated with H-SAM solution, and then the reaction system was placed in a constant temperature environment of 25 °C for 90 min. The reaction was terminated by adding 5 μL of termination buffer, and then 25 μL of the reaction mixture was quantitatively transferred to the flashplate using a pipette.

[0242] 3) Results Analysis: The radioactive signal of the scintillator was collected using a liquid scintillator, and the suppression rate was calculated according to the formula:

[0243] Inhibition rate (%) = Number of experimental groups × 100%

[0244] The inhibitory effect on enzyme activity was assessed by comparing the differences in radioactivity intensity among different treatment groups.

[0245] The results of the compounds inhibiting EZH2 kinase activity at concentrations of 100 nM and 10 nM are shown in Table 2.

[0246] Table 2 Results of compound kinase inhibition experiments

[0247]

Claims

1. Novel substituted benzoylpyridinone compounds and their pharmaceutically acceptable salts, characterized in that, The novel substituted benzoylpyridinone compounds and their pharmaceutically acceptable salts have the general structural formulas shown as (Ⅰ), (Ⅱ), (Ⅲ), (Ⅳ), (Ⅴ), (Ⅵ), (Ⅶ), (Ⅷ) or (Ⅸ): ； in, A is selected from primary amine compounds; R1 or R2 is selected from secondary amines or structures containing secondary amine substitutions.

2. The novel substituted benzoylpyridinone compound and its pharmaceutically acceptable salt according to claim 1, characterized in that, A is selected from ； R1 is selected from ； R2 is selected from 。 3. The novel substituted benzoylpyridinone compound and its pharmaceutically acceptable salt according to claim 2, characterized in that, The novel substituted benzoylpyridinone compounds have the following structural formula: 。 4. A pharmaceutical composition, characterized in that, It is prepared by combining the novel substituted benzoylpyridinone compound as described in any one of claims 1-3 and its pharmaceutically acceptable salt as the active ingredient with a pharmaceutically acceptable carrier.

5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutically acceptable carrier is one or more of solid dosage form carriers and liquid dosage form carriers.

6. The pharmaceutical composition according to claim 4, characterized in that, Formulating a pharmaceutical composition into a dosage form such as tablets, capsules, granules, sprays, or injections.

7. The use of the novel substituted benzoylpyridinone compound of any one of claims 1-3 and its pharmaceutically acceptable salt, or the pharmaceutical composition of any one of claims 4-6, in the preparation of a medicament for treating EZH2, PRC2, or EZH2 / PRC2-mediated diseases.

8. The use of the novel substituted benzoylpyridinone compound of any one of claims 1-3 and its pharmaceutically acceptable salt, or the pharmaceutical composition of any one of claims 4-6, in the preparation of a medicament targeting EZH2.

9. The use of the novel substituted benzoylpyridinone compound of any one of claims 1-3 and its pharmaceutically acceptable salt, or the pharmaceutical composition of any one of claims 4-6, in the preparation of an antitumor drug.

10. The application according to claim 9, characterized in that, The tumors mentioned are: liver cancer, nasopharyngeal carcinoma, colorectal cancer, melanoma, bladder cancer, leukemia, esophageal cancer, breast cancer, stomach cancer, prostate cancer, pancreatic cancer, lung cancer, ovarian cancer, epithelioid sarcoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, and follicular lymphoma.

Images