Multi-target antineoplastic active rutaecarpin derivative and application thereof

Abstract

The invention discloses a multi-target anti-tumor active rutaecarpin derivative or a pharmaceutical salt thereof. The structural general formula of the rutaecarpin derivative or the pharmaceutical salt thereof is shown as a formula I or a formula II: # imgabs0 #, wherein R1 is selected from C1-C5 alkoxy, hydroxyl and hydrogen; r2 is selected from hydrogen and halogen; according to the present invention, the enzyme inhibition activity and in vitro anti-tumor activity test results show that the compound of the present invention has strong inhibition activity on topoisomerase 1, topoisomerase 2 and c-MYC oncogenes, such that the new approach is developed for the deep research and the development of the new structure type anti-tumor drug, and the new strategy is provided.

Description

[0001] This application is a divisional application of the patent application filed on January 11, 2022, with application number 202210025250.5 and invention name “Evodia rutaecarpine derivatives with multi-target anti-tumor activity and their applications”. Technical Field

[0002] The invention belongs to the field of medical technology, and in particular relates to a class of evodia rutaecarpine derivatives with multi-target anti-tumor activity and applications thereof. Background Art

[0003] Topoisomerase (Top) is an important component of ribozymes and plays an important role in DNA transcription, replication and chromatin assembly. Top1 and Top2 induce transient breaks in single or double strands of DNA by cutting a single DNA strand.

[0004] In the field of DNA-targeted drugs, small molecules that stabilize c-MYC G-quadruplex (c-MYC G4) DNA are also considered a promising anticancer strategy. c-MYC is one of the most important oncogenes, which is overexpressed in more than 80% of cancer cells and contributes to cell proliferation, differentiation, and apoptosis. However, the transcriptional control of c-MYC is quite complex. c-MYC is difficult to target due to its short half-life and lack of binding pockets. Among various transcription factors, the nuclease hypersensitive element (NHE III1) controls 85-90% of c-MYC transcriptional activity, folding into DNA G-quadruplex (G4) under transcription-related negative supercoiling, thereby silencing c-MYC transcription. Therefore, c-MYC G4 has become a promising cancer-specific molecular target for anti-tumor drug discovery. In addition, the synergistic anti-tumor effect between c-MYC G4 and Top inhibitors has been confirmed. Therefore, simultaneous targeting of Top and c-MYCG4 will be a promising strategy for anti-tumor drug discovery.

[0005] In the early stage, researchers conducted in-depth research on the structure-activity relationship of evodiamine derivatives. Through the study of the anti-tumor mechanism, it was found that evodiamine derivatives are dual inhibitors of Top1 and Top2, which can effectively induce tumor cell apoptosis. The patent application with publication number CN101787025A discloses a substituted evodiamine antitumor and antifungal compound and its preparation method; the patent application with publication number CN1012311434A discloses an evodiamine compound and its preparation method and application; the patent application with publication number CN103992336A discloses an oxa- or thia-evodiamine antitumor derivative and its preparation method.

[0006] In view of this, there is an urgent need for a class of compounds that target Top and c-MYC G4 inhibitors to achieve synergistic anti-tumor effects. Summary of the invention

[0007] The first object of the present invention is to provide a class of Evodia rutaecarpine derivatives with multi-target anti-tumor activity.

[0008] The second object of the present invention is to provide an application of the evodia rutaecarpine derivative with multi-target anti-tumor activity in the preparation of anti-tumor drugs.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] The first aspect of the present invention provides a class of multi-target anti-tumor activity of evodia rutaecarpine derivatives or pharmaceutically acceptable salts thereof, the general structural formula of which is shown in Formula I or Formula II:

[0011]

[0012] in:

[0013] R1 is selected from C1-C5 alkoxy, hydroxyl, hydrogen;

[0014] R2 is selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine);

[0015] R3 is selected from hydroxyl,

[0016]

[0017] More preferably, among the evodiamine derivatives having multi-target anti-tumor activity,

[0018] R1 is selected from methoxy, ethoxy, hydroxyl, hydrogen;

[0019] R2 is selected from hydrogen, fluorine;

[0020] R3 is selected from hydroxyl,

[0021]

[0022] Most preferably, the multi-target anti-tumor activity of the evodia rutaecarpine derivative is selected from one of the following structures:

[0023]

[0024]

[0025]

[0026]

[0027] The pharmaceutically acceptable salt is an organic acid salt or an inorganic acid salt thereof.

[0028] The organic acid is hexanoic acid, maleic acid, fumaric acid, tartaric acid, succinic acid, lactic acid, p-toluenesulfonic acid, salicylic acid, oxalic acid, tannic acid, citric acid, trifluoroacetic acid, malic acid or benzenesulfonate; the inorganic acid is hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid or nitric acid.

[0029] The second aspect of the present invention provides a use of the evodia rutaecarpine derivative or a pharmaceutically acceptable salt thereof having multi-target anti-tumor activity in the preparation of an anti-tumor drug.

[0030] The tumor is selected from breast cancer, colon cancer, cervical cancer, lung cancer and the like.

[0031] The third aspect of the present invention provides a use of the multi-target anti-tumor activity evodiamine derivative or a pharmaceutically acceptable salt thereof in the preparation of a Top1 topoisomerase inhibitor, a Top2 topoisomerase inhibitor and / or a c-MYC oncogene inhibitor.

[0032] The multi-target anti-tumor activity evodia rutaecarpine derivative or its pharmaceutically acceptable salt can be used as a triple-target inhibitor of Top1, Top2 and c-MYC to treat malignant tumors or diseases related to differentiation and proliferation.

[0033] The tumors include breast cancer, colon cancer, cervical cancer and the like.

[0034] The fourth aspect of the present invention provides a use of the evodia rutaecarpine derivative or a pharmaceutically acceptable salt thereof having multi-target anti-tumor activity in the preparation of a drug for treating diseases caused by abnormal gene expression.

[0035] The disease caused by abnormal gene expression is tumor, endocrine disorder, immune system disease, genetic disease or nervous system disease.

[0036] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0037] The present invention provides a class of evodia rutaecarpine derivatives with multi-target anti-tumor activity. The class of evodia rutaecarpine derivatives have multi-target anti-tumor activity, and the multi-targets are Top1 / Top2 / c-MYC triple-target inhibitors. Enzyme inhibition activity and in vitro anti-tumor activity tests show that the compounds of the present invention have strong inhibitory activity against topoisomerase 1, topoisomerase 2 and c-MYC oncogenes, which opens up a new path and provides a new strategy for in-depth research and development of new structural types of anti-tumor drugs.

[0038] The present invention provides a class of evodia rutaecarpine derivatives with multi-target anti-tumor activity, which are a class of small molecule anticancer drugs based on multi-targets of topoisomerase 1 (Top1), topoisomerase 2 (Top2) and c-MYC oncogenes. The compounds of the present invention exhibit good Top1, Top2 and c-MYC oncogene inhibitory activity and have a certain broad-spectrum anti-tumor activity, and can be used as anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a schematic diagram of the induction and stabilization effects of Evodia rutaecarpine derivatives on c-MYC.

[0040] Figure 2 This is a schematic diagram of the results of the inhibition experiment of Evodia rutaecarpa derivatives on Top1 / 2.

[0041] Figure 3 This is a schematic diagram of the effects of compound II-13 and evodia rutaecarpine on the transcription and expression of c-MYC oncogene. DETAILED DESCRIPTION

[0042] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0043] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers.

[0044] Synthesis of compounds I-1 to I-15 and I-25 to I-34

[0045]

[0046]

[0047] (a)HCOOCH2CH3, 70℃, 12h, yield 80-86%; (b) POCl3, 0℃, 10h, yield 78-85%; (c) THF, BTC, 70℃, 12h, yield 80-87%; (d) CH3I, NaH, DMF, 2.5h, yield 90-95%; (e) DCM, rt, 6h, yield 50-55%; (f) DMF, NaH, 65℃, 12h, yield 43-78%; (g) DCM, BBr3, under N2, -78℃, 2h, yield 24-85%.

[0048] Example 1

[0049] Tryptamine (compound 1a, R1 = H, 0.062 mol, 10 g) was added to ethyl formate solution (148.8 mL, 1.872 mol) and reacted overnight at 70° C. After the reaction, the reaction solution was evaporated to dryness, and the theoretical product weight, compound 2a (R1 = H) (11.7 g, 0.062 mol), was directly used in the next step.

[0050] The crude product of the above-mentioned compound 2a (R1 = H) was dissolved in dry dichloromethane (200 mL), and POCl3 (8.1 mL, 0.087 mol) was slowly added dropwise under ice bath conditions, and stirred overnight. After the reaction was completed, the reaction solution was evaporated to dryness, dissolved with a mixed solution of acetic acid and water (v / v = 1:1, 400 mL), and the solution was adjusted to neutral with ammonia water under ice bath conditions, and impurities were removed by vacuum filtration. The filtrate was adjusted to pH = 11.0 with ammonia water, and solids precipitated and were filtered to obtain 9.46 g of yellow solid, namely compound 3a (R1 = H), with a yield of 89%.

[0051] Dissolve anthranilic acid 4a (5.0 g, 0.032 mol) in dry tetrahydrofuran (100 mL), add triphosgene (3.8 g, 0.0129 mol), and heat under reflux at 70°C for 16 h. After the reaction is complete, pour the reaction solution into 400 mL of ice-cold saturated brine, wait for the white solid to precipitate completely, filter it, wash it twice with water, and dry the filter cake in an oven at 45°C. 5.0 g of white solid, i.e., compound 5a (R2=H), is obtained, with a yield of 86%.

[0052] Compound 5a (5.0 g, 0.03 mol) was dissolved in dry DMF (60 mL), and sodium hydride (purity 60%, 0.65 g, 0.016 mol) was slowly added under ice bath conditions, and iodomethane (1.1 mL, 0.016 mol) was added half an hour later, and the reaction was carried out at room temperature for 4 hours. After the reaction, the reaction solution was poured into 400 mL of ice saturated brine, stirred, and filtered, and the filter cake was dried in an oven at 45°C to obtain 2.5 g of white solid, namely compound 6a (R2=H), with a yield of 90%.

[0053] Compound 3a (R1 = H) (5.0 g, 0.025 mol) and compound 6a (R2 = H) (4.5 g, 0.025 mol) were dissolved in dry dichloromethane (100 mL) and reacted at room temperature for 6 h. After the reaction, the mixture was filtered and rinsed twice with dichloromethane. The filter cake was freeze-dried to obtain 5.2 g of a yellow solid, namely compound 7a (R1 = H, R2 = H), with a yield of 59%.

[0054] Compound 7a (200 mg, 0.660 mmol) was dissolved in dry DMF (5 mL), and sodium hydride (purity of 60%, 53 mg, 1.320 mmol) was slowly added under ice bath conditions. 3-bromopropanol (95 mg, 0.684 mmol) was slowly added dropwise after half an hour, and then transferred to a 65°C oil bath and heated for 12 h. After the reaction was complete, the reaction solution was poured into a 250 mL separatory funnel, and water (100 mL) and ethyl acetate (40 mL×3) were added for extraction. The organic phases were combined and washed with saturated brine (100 mL). The organic phases were collected, and after reduced pressure distillation, separation and purification were performed by column chromatography (petroleum ether: ethyl acetate = 2:1) to obtain 120 mg of yellow solid compound I-1, with a yield of 33%.

[0055] Example 2

[0056] During the preparation of compound I-2, 3-bromopropanol was replaced with (3-chloropropyl)pyrrolidine (0.684 mmol), and the rest was the same as in Example 1. The yield of compound I-2 was 48%.

[0057] Example 3

[0058] During the preparation of compound I-3, 3-bromopropanol was replaced with 4-(5-chloropropyl)morpholine (0.684 mmol), and the rest was the same as in Example 1. The yield of compound I-3 was 7%.

[0059] Example 4

[0060] During the preparation of compound I-4, 3-bromopropanol was replaced with 3-chloro-N-methylpropan-1-amine hydrochloride (0.684 mmol), and the rest was the same as in Example 1. The yield of compound I-4 was 28%.

[0061] Example 5

[0062] During the preparation of compound I-5, 3-bromopropanol was replaced with 3-chloro-1-(N,N-dimethyl)propylamine (0.684 mmol), and the rest was the same as in Example 1. The yield of compound I-5 was 27%.

[0063] Example 6

[0064] Preparation of 3-fluoro-13-(3-hydroxypropyl)-10-methoxy-14-methyl-8,13,13b,14-tetrahydroindole[2',3':3,4]pyridin[2,1-b]quinazolin-5(7H)-one (Compound I-6)

[0065] (1) Preparation of intermediate 3b (R1 = OCH3): 6-methoxycarboline

[0066] 5-Methoxytryptamine compound 1b (R1 = OCH3) (10.0g, 0.053mol) was added to ethyl formate solution (125.3mL, 1.576mol) and reacted at 70℃ overnight. After the reaction, the reaction solution was evaporated to dryness and used directly in the next step according to the theoretical product weight (11.6g, 0.053mol). The crude product was dissolved in dry dichloromethane (200mL), and POCl3 (6.9mL, 0.074mol) was slowly added dropwise under ice bath conditions and stirred overnight. After the reaction, the reaction solution was evaporated to dryness and dissolved with a mixed solution of acetic acid and water (v / v = 1:1, 400mL). The solution was adjusted to neutral with ammonia water under ice bath conditions, and impurities were removed by vacuum filtration. The filtrate was adjusted to pH = about 11.0 with ammonia water, and solids were precipitated and filtered to obtain 9.46g of yellow solid compound 3b with a yield of 89%. 1 H NMR(600MHz,DMSO-d6)δ:11.21(s,1H),8.35(s,1H),7.31(d,J=8.8Hz,1H),7.02(d,J=2.5Hz,1H ),6.85(dd,J=8.9,2.5Hz,1H),3.78(dd,J=8.4,1.8Hz,2H),3.77(s,3H),2.80(t,J=8.5Hz,2H).

[0067] (2) Preparation of intermediate 5b: 6-fluoroindigo carboxylic anhydride

[0068] Dissolve anthranilic acid 4b (R2 = F) (5.0 g, 0.032 mol) in dry tetrahydrofuran (100 mL), add triphosgene (3.8 g, 0.0129 mol), and heat under reflux at 70°C for 16 h. After the reaction is complete, pour the reaction solution into 400 mL of ice-saturated saline water, wait for the white solid to precipitate completely, filter it, wash it twice with water, and dry the filter cake in an oven at 45°C. 5.0 g of compound 5b is obtained as a white solid, with a yield of 86%. 1 H NMR (600MHz, DMSO-d6) δ: 11.79 (s, 1H), 7.69-7.61 (m, 2H), 7.19 (q, J = 4.8Hz, 1H).

[0069] (3) Preparation of intermediate 6b: 6-fluoro-N-methylisatoic anhydride

[0070] The intermediate 5b (R2 = F) (5.0g, 0.028mol) was dissolved in dry DMF (60mL), and sodium hydride (purity 60%, 1.3g, 0.033mol) was slowly added under ice bath conditions. After half an hour, methyl iodide (2.1mL, 0.033mol) was added and reacted at room temperature for 4h. After the reaction, the reaction solution was poured into 400mL of ice saturated salt water, stirred, filtered, and the filter cake was dried in an oven at 45℃ to obtain 4.9g of white solid compound 6b, with a yield of 90%. 1H NMR (600MHz, DMSO-d6) δ: 7.78-7.73 (m, 2H), 7.52-7.48 (m, 1H), 3.47 (s, 3H).

[0071] (4) Preparation of intermediate 7b: 3-fluoro-10-methoxyevodiamine

[0072] Intermediate 3b (5.0 g, 0.025 mol) and intermediate 6b (4.5 g, 0.025 mol) were dissolved in dry dichloromethane (100 mL) and reacted at room temperature for 6 h. After the reaction, the mixture was filtered and rinsed twice with dichloromethane. The filter cake was freeze-dried to obtain 5.2 g of yellow solid compound 7b (R1 = OCH3, R2 = F), with a yield of 59%. 1H NMR (600MHz, DMSO-d6) δ: 11.01 (s, 1H), 7.55 (dd, J = 8.9, 3.1Hz, 1H), 7.39 (td ,J=8.6,3.1Hz,1H),7.27(d,J=8.7Hz,1H),7.19(dd,J=8.9,4.5Hz,1H),7.01 (d,J=2.4Hz,1H),6.78(dd,J=8.7,2.4Hz,1H),6.08(s,1H),4.63(dt,J=11.4 ,3.6Hz,1H),3.77(s,3H),3.21(m,1H),2.81(t,J=11.4Hz,2H),2.69(s,3H).

[0073] (4) Preparation of target compound I-6:

[0074] The intermediate 7b (200 mg, 0.570 mmol) was dissolved in dry DMF (5 mL), and sodium hydride (purity of 60%, 34 mg, 0.855 mmol) was slowly added under ice bath conditions. After half an hour, 3-bromopropanol (95 mg, 0.684 mmol) was slowly added dropwise, and then the mixture was transferred to a 65°C oil bath and heated for 12 h. After the reaction was complete, the reaction solution was poured into a 250 mL separatory funnel, and water (100 mL) and ethyl acetate (40 mL×3) were added for extraction. The organic phases were combined and washed with saturated brine (100 mL). The organic phases were collected, and after reduced pressure distillation, separation and purification were performed by column chromatography (petroleum ether: ethyl acetate = 2:1) to obtain 120 mg of yellow solid compound I-6, with a yield of 52%.

[0075] Example 7

[0076] During the preparation of compound I-7, 3-bromopropanol was replaced with (3-chloropropyl)pyrrolidine (0.684 mmol), and the rest was the same as in Example 6. The yield of compound I-7 was 70%.

[0077] Example 8

[0078] During the preparation of compound I-8, 3-bromopropanol was replaced with 4-(5-chloropropyl)morpholine (0.684 mmol), and the rest was the same as in Example 6. The yield of compound I-8 was 48%.

[0079] Example 9

[0080] During the preparation of compound I-9, 3-bromopropanol was replaced with 3-chloro-N-methylpropan-1-amine hydrochloride (0.684 mmol), and the rest was the same as in Example 6. The yield of compound I-9 was 33%.

[0081] Example 10

[0082] During the preparation of compound I-10, 3-bromopropanol was replaced with 3-chloro-1-(N,N-dimethyl)propylamine (0.684 mmol), and the rest was the same as in Example 6. The yield of compound I-10 was 61%.

[0083] Embodiment 11

[0084] Intermediate 3b (5.0 g, 0.025 mol) and intermediate 6a (4.4 g, 0.025 mol) were dissolved in dry dichloromethane (100 mL) and reacted at room temperature for 6 h. After the reaction, the mixture was filtered and rinsed twice with dichloromethane. The filter cake was freeze-dried to obtain 5.0 g of compound 7c (R1 = OCH3, R2 = H) as a yellow solid with a yield of 60%.

[0085] During the preparation of compound I-11, compound 7c (0.660 mmol) and 3-bromopropanol (0.684 mmol) were reacted according to the method steps in Example 1 to obtain compound I-11 with a yield of 47%.

[0086] Example 12

[0087] During the preparation of compound I-12, compound 7c (0.660 mmol) and (3-chloropropyl)pyrrolidine (0.684 mmol) were prepared according to the method steps in Example 1 to obtain compound I-12 with a yield of 63%.

[0088] Example 13

[0089] During the preparation of compound I-13, compound 7c (0.660 mmol) and 4-(5-chloropropyl)morpholine (0.684 mmol) were reacted according to the method steps in Example 1 to obtain compound I-13 with a yield of 51%.

[0090] Embodiment 14

[0091] During the preparation of compound I-14, compound 7c (0.660 mmol) and 3-chloro-N-methylpropan-1-amine hydrochloride (0.684 mmol) were prepared according to the method steps in Example 1 to obtain compound I-14 with a yield of 68%.

[0092] Embodiment 15

[0093] During the preparation of compound I-15, compound 7c (0.660 mmol) and 3-chloro-1-(N,N-dimethyl)propylamine (0.684 mmol) were prepared according to the method steps in Example 1 to obtain compound I-15 with a yield of 56%.

[0094] Synthesis of compounds I-16 to I-24 and I-35 to I-40

[0095]

[0096] (a) DMF, KOH, 1,3-dibromo propane, rt, 12h, yield 35-80%; (b) I-16~I-17: DMF, K2CO3, 65℃, 12h; yield 32-60%; (c) I-18: MeCN, rt, 72h, yield 44-81%; (d) DCM, BBr3, under N2, -78℃, 2h, yield 34-72%.

[0097] Example 16

[0098] Preparation of 13-(3-((4,5-thiazolin-2-yl)amino)propyl)-14-methyl-8,13,13b,14-tetrahydroindole[2',3':3,4]pyridin[2,1-b]quinazolin-5(7H)-one (Compound Ⅰ-16)

[0099] (1) Preparation of intermediate 12a:

[0100] The intermediate 7a (1.0 g, 3.30 mmol) was dissolved in dry DMF (10 mL), potassium hydroxide (370.0 mg, 6.60 mmol) was added, and then 1,3-dibromopropane (2.0 g, 9.90 mmol) was added, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the reaction solution was poured into a 250 mL separatory funnel, and water (100 mL) and ethyl acetate (40 mL×3) were added for extraction, the organic phases were combined, and washed with saturated brine (100 mL), the organic phases were collected, and after reduced pressure distillation, separation and purification by column chromatography (petroleum ether: ethyl acetate = 6:1) were performed to obtain 360 mg of compound 12a as a yellow oil, with a yield of 26%.

[0101] (2) Preparation of target compound I-16:

[0102] The intermediate 12a (120 mg, 0.284 mmol) was dissolved in dry DMF (5 mL), potassium carbonate (59 mg, 0.43 mmol) was added, and then 2-amino-2-thiazoline (87 mg, 0.85 mmol) was added, and then the mixture was transferred to a 65°C oil bath and heated for 12 h. After the reaction was completed, the reaction solution was poured into a 250 mL separatory funnel, and water (100 mL) and ethyl acetate (40 mL×3) were added for extraction. The organic phases were combined and washed with saturated brine (100 mL). The organic phases were collected, and after reduced pressure distillation, they were separated and purified by column chromatography (dichloromethane: methanol = 14: 1) to obtain 50 mg of compound I-16 as a yellow solid, with a yield of 40%.

[0103] Embodiment 17

[0104] During the preparation of compound I-17, compound 12a (0.284 mmol) and isopropylamine (0.85 mmol) were prepared according to the method steps in Example 16 to obtain compound I-17 with a yield of 44%.

[0105] Embodiment 18

[0106] During the preparation of compound I-18, compound 12a (0.284 mmol) and ethanolamine (0.85 mmol) were prepared according to the method steps in Example 16 to obtain compound I-18 with a yield of 44%.

[0107] Embodiment 19

[0108] Preparation of 13-(3-((4,5-thiazolin-2-yl)amino)propyl)-3-fluoro-10-methoxy-14-methyl-8,13,13b,14-tetrahydroindole[2',3':3,4]pyridin[2,1-b]quinazolin-5(7H)-one (Compound I-19)

[0109] (1) Preparation of intermediate 12b:

[0110] The intermediate 7b (200 mg, 0.57 mmol) was dissolved in dry DMF (5 mL), potassium hydroxide (64 mg, 1.14 mmol) was added, and then 1,3-dibromopropane (345 mg, 1.71 mmol) was added, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the reaction solution was poured into a 250 mL separatory funnel, and water (100 mL) and ethyl acetate (40 mL×3) were added for extraction, the organic phases were combined, and washed with saturated brine (100 mL), the organic phases were collected, and after reduced pressure distillation, separation and purification by column chromatography (petroleum ether: ethyl acetate = 6:1) were performed to obtain 85 mg of compound 12b as a yellow oil, with a yield of 32%. 1 H NMR(600MHz, DMSO-d6)δ:7.63(dd,J=9.0,3.1Hz,1H),7.49-7.43(m,2H),7.39(dd,J=8.8,4.7H z,1H),7.11(d,J=2.5Hz,1H),6.89(dd,J=8.9,2.5Hz,1H),6.16(s,1H),4.67(ddd,J=12.6,5.0 ,1.6Hz,1H),4.43-4.36(m,1H),4.35-4.28(m,1H),3.80(s,3H),3.59(td,J=6.2,1.3Hz,2H),3 .12(td,J=12.2,3.9Hz,1H),2.97(d,J=15.0Hz,1H),2.76(t,J=12.1Hz,1H),2.38-2.23(m,5H).

[0111] (2) Preparation of target compound I-19:

[0112] Intermediate 12b (120 mg, 0.28 mmol) was dissolved in dry DMF (5 mL), potassium carbonate (59 mg, 0.043 mmol) was added, and then 2-amino-2-thiazoline (87 mg, 0.85 mmol) was added, and then the mixture was transferred to a 65°C oil bath and heated for 12 h. After the reaction was completed, the reaction solution was poured into a 250 mL separatory funnel, and water (100 mL) and ethyl acetate (40 mL×3) were added for extraction. The organic phases were combined and washed with saturated brine (100 mL). The organic phases were collected, and after reduced pressure distillation, they were separated and purified by column chromatography (dichloromethane: methanol = 14: 1) to obtain 60 mg of compound I-19 as a yellow solid with a yield of 48%.

[0113] Embodiment 20

[0114] During the preparation of compound I-20, compound 12b (0.28 mmol) and isopropylamine (0.85 mmol) were reacted according to the method steps in Example 19 to obtain compound I-20 with a yield of 32%.

[0115] Embodiment 21

[0116] During the preparation of compound I-21, compound 12b (0.28 mmol) and ethanolamine (0.85 mmol) were prepared according to the method steps in Example 19 to obtain compound I-21 with a yield of 81%.

[0117] Embodiment 22

[0118] Preparation of 13-(3-((4,5-thiazolin-2-yl)amino)propyl)-10-methoxy-14-methyl-8,13,13b,14-tetrahydroindole[2',3':3,4]pyridin[2,1-b]quinazolin-5(7H)-one (Compound I-22)

[0119] The intermediate 7c (200 mg, 0.60 mmol) was dissolved in dry DMF (5 mL), potassium hydroxide (67 mg, 1.2 mmol) was added, and then 1,3-dibromopropane (363 mg, 1.80 mmol) was added, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the reaction solution was poured into a 250 mL separatory funnel, and water (100 mL) and ethyl acetate (40 mL×3) were added for extraction, the organic phases were combined, and washed with saturated brine (100 mL), the organic phases were collected, and after reduced pressure distillation, separation and purification by column chromatography (petroleum ether: ethyl acetate = 6:1) were performed to obtain 68 mg of compound 12c as a yellow oil, with a yield of 25%.

[0120] During the preparation of compound I-22, compound 12c (0.28 mmol) and 2-amino-2-thiazoline (0.85 mmol) were reacted according to the method steps in Example 19 to obtain compound I-22 with a yield of 53%.

[0121] Embodiment 23

[0122] During the preparation of compound I-23, compound 12c (0.28 mmol) and isopropylamine (0.85 mmol) were prepared according to the method steps in Example 19 to obtain compound I-23 with a yield of 60%.

[0123] Embodiment 24

[0124] During the preparation of compound I-24, compound 12c (0.28 mmol) and ethanolamine (0.85 mmol) were prepared according to the method steps in Example 19 to obtain compound I-24 with a yield of 63%.

[0125] Embodiment 25

[0126] Preparation of 3-fluoro-10-hydroxy-13-(3-hydroxypropyl)-14-methyl-8,13,13b,14-tetrahydroindole[2',3':3,4]pyridin[2,1-b]quinazolin-5(7H)-one compound I-25

[0127] Compound I-6 (270 mg, 0.66 mmol) was dissolved in dry dichloromethane (10 mL), and boron tribromide (495 mg, 1.98 mmol) was added at -78 ° C under N2 protection for 2 h, and then transferred to room temperature for overnight reaction. After the reaction, 25 mL of icy NaHCO3 aqueous solution was added, stirred for 30 minutes, extracted with dichloromethane (40 mL×3), and the organic phase was collected. After reduced pressure distillation, it was separated and purified by column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain 150 mg of yellow solid compound I-25, with a yield of 58%.

[0128] Embodiment 26

[0129] Compound I-26 was prepared according to the method of Example 25, using compound I-7 (0.66 mmol) as the raw material. Other steps were the same as those of Example 25, and the yield was 30%.

[0130] Embodiment 27

[0131] Compound I-27 was prepared according to the method of Example 25, using compound I-8 (0.66 mmol) as the raw material. Other steps were the same as those of Example 25, and the yield was 12%.

[0132] Embodiment 28

[0133] Compound I-28 was prepared according to the method of Example 25, using compound I-9 (0.66 mmol) as the raw material. Other steps were the same as those of Example 25, and the yield was 81%.

[0134] Embodiment 29

[0135] Compound I-29 was prepared according to the method of Example 25, using compound I-10 (0.66 mmol) as the raw material. Other steps were consistent with Example 25, and the yield was 36%.

[0136] Embodiment 30

[0137] Compound I-30 was prepared according to the method of Example 25, using compound I-11 (0.66 mmol) as the raw material. Other steps were the same as those of Example 25, and the yield was 47%.

[0138] Embodiment 31

[0139] Compound I-31 was prepared according to the method of Example 25, using compound I-12 (0.66 mmol) as the raw material. Other steps were consistent with Example 25, and the yield was 63%.

[0140] Embodiment 32

[0141] Compound I-32 was prepared according to the method of Example 25, using compound I-13 (0.66 mmol) as the raw material. Other steps were consistent with Example 25, and the yield was 51%.

[0142] Embodiment 33

[0143] Compound I-33 was prepared according to the method of Example 25, using compound I-14 (0.66 mmol) as the raw material. Other steps were the same as Example 25, and the yield was 68%.

[0144] Embodiment 34

[0145] Compound I-34 was prepared according to the method of Example 25, using compound I-15 (0.66 mmol) as raw material, and the other steps were consistent with Example 25, with a yield of 56%.

[0146] Embodiment 35

[0147] Compound I-35 was prepared according to the method of Example 25, using compound I-19 (0.66 mmol) as raw material, and the other steps were consistent with Example 25, with a yield of 71%.

[0148] Embodiment 36

[0149] Compound I-36 was prepared according to the method of Example 25, using compound I-20 (0.66 mmol) as the raw material. Other steps were the same as Example 25, and the yield was 50%.

[0150] Embodiment 37

[0151] Compound I-37 was prepared according to the method of Example 25, using compound I-21 (0.66 mmol) as the raw material. Other steps were consistent with Example 25, and the yield was 34%.

[0152] Embodiment 38

[0153] Compound I-38 was prepared according to the method of Example 25, using compound I-22 (0.66 mmol) as the raw material. Other steps were consistent with Example 25, and the yield was 66%.

[0154] Embodiment 39

[0155] Compound I-39 was prepared according to the method of Example 25, using compound I-23 (0.66 mmol) as the raw material. Other steps were consistent with Example 25, and the yield was 72%.

[0156] Embodiment 40

[0157] Compound I-40 was prepared according to the method of Example 25, using compound I-24 (0.66 mmol) as the raw material, and the other steps were consistent with Example 25, with a yield of 65%.

[0158] Embodiment 41

[0159] Preparation of 13-(3-(pyrrolidin-1-yl)propyl)-8,13-tetrahydroindole[2',3':3,4]pyridin[2,1-b]quinazolin-5(7H)-one Compound Ⅱ-1

[0160]

[0161] (a) DCM, rt, 6h, yield 47%; (b) DMF, Cs2CO3, 65℃, 12h, yield 25-65%.

[0162] (1) Preparation of intermediate m7:

[0163] Intermediate 3a (200 mg, 1.18 mmol) and intermediate 5a (192 mg, 1.18 mmol) were dissolved in dry dichloromethane (10 mL) and reacted at room temperature for 6 h. After the reaction, the mixture was filtered and rinsed twice with dichloromethane. The filter cake was freeze-dried to obtain 34 mg of compound m7 as a yellow solid with a yield of 10%. 1H NMR(600MHz,DMSO-d6)δ:10.97(s,1H),7.80(dd,J=7.8,1.6Hz,1H),7.53(d,J=7.8Hz,1H) ,7.46(d,J=7.8Hz,1H),7.38(td,J=8.4,1.8Hz,1H),7.20-7.15(m,1H),7.07(td,J=8.4,1. 8Hz,1H),6.97(s,1H),6.90(d,J=1.8Hz,1H),6.86(td,J=7.8,0.6Hz,1H),6.08(d,J=0.6Hz ,1H),4.84(ddd,J=12.9,5.2,1.6Hz,1H),3.05(td,J=15.0,7.2Hz,1H),2.89-2.76(m,2H).

[0164] (2) Preparation of target compound II-1:

[0165] The intermediate m7 (200 mg, 0.69 mmol) was dissolved in dry DMF (10 mL), cesium carbonate (670 mg, 2.08 mmol) was added, and then (3-chloropropyl) pyrrolidine (120 mg, 0.83 mmol) was added, and then transferred to a 65°C oil bath and heated for 12 h. After the reaction was completed, the reaction solution was poured into a 250 mL separatory funnel, and water (100 mL) and ethyl acetate (40 mL×3) were added for extraction. The organic phases were combined and washed with saturated brine (100 mL). The organic phases were collected, and after reduced pressure distillation, separation and purification were performed by column chromatography (dichloromethane: methanol = 19: 1) to obtain 70 mg of yellow solid compound II-1, with a yield of 25%.

[0166] Embodiment 42

[0167] During the preparation of compound II-2, compound m7 (0.69 mmol) and 3-chloro-1-(N,N-dimethyl)propylamine (0.83 mmol) were reacted according to the method steps in Example 41 to obtain compound II-2 with a yield of 65%.

[0168] Embodiment 43

[0169]

[0170] (a) DCM, DDQ, rt, 8h, yield 45-55%; (b) DMF, NaH, 65°C, 12h, yield 8%.

[0171] (1) Preparation of intermediate 8a:

[0172] The intermediate m7 (200 mg, 0.69 mmol) was dissolved in dry DCM (10 mL), and 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) (157 mg, 0.69 mmol) was added, and the mixture was reacted at room temperature for 8 h. After the reaction, the mixture was separated and purified by column chromatography (dichloromethane: methanol = 100: 1) under reduced pressure to obtain 90 mg of compound 8a as a yellow solid with a yield of 45%.

[0173] (2) Preparation of target compound II-3:

[0174] Compound 8a (200 mg, 0.70 mmol) was dissolved in dry DMF (5 mL), and sodium hydride (purity of 60%, 111 mg, 2.79 mmol) was slowly added under ice bath conditions. 3-chloro-N-methylpropan-1-amine hydrochloride (1.39 mmol) was slowly added dropwise after half an hour, and then the mixture was transferred to a 65°C oil bath and heated for 12 h. After the reaction was complete, the reaction solution was poured into a 250 mL separatory funnel, and water (100 mL) and ethyl acetate (40 mL×3) were added for extraction. The organic phases were combined and washed with saturated brine (100 mL). The organic phases were collected, and after reduced pressure distillation, separation and purification were performed by column chromatography (dichloromethane: methanol = 100: 6) to obtain 19 mg of yellow solid compound II-3, with a yield of 8%.

[0175] Synthesis of compounds Ⅱ-4 to Ⅱ-15

[0176]

[0177]

[0178] (a) DMF, KOH, 1-bromo-3-chloro propane, rt, 12h, yield 19-22%; (b) DMF, Cs2CO3, 65℃, 12h, yield 10-37%; (c) DCM, BBr3, under N2, -78℃, 2h, yield 44-98%.

[0179] Embodiment 44

[0180] Preparation of 13-(3-((4,5-dihydrothiazol-2-yl)amino)propyl)-8,13-dihydroindole[2',3':3,4]pyrido[2,1-b]quinazoline-5(7H)-one (Compound II-4)

[0181] (1) Preparation of intermediate 14a:

[0182] The intermediate 8a (200 mg, 0.70 mmol) was dissolved in dry DMF (5 mL), potassium hydroxide (78 mg, 1.39 mmol) was added, and then 1-bromo-3-chloropropane (329 mg, 2.09 mmol) was added, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the reaction solution was poured into a 250 mL separatory funnel, and water (100 mL) and ethyl acetate (40 mL×3) were added for extraction, the organic phases were combined, and washed with saturated brine (100 mL), the organic phases were collected, and after reduced pressure distillation, separation and purification by column chromatography (petroleum ether: ethyl acetate = 6:1) were performed to obtain 54 mg of compound 14a as a yellow oil, with a yield of 21%.

[0183] (2) Preparation of target compound II-4:

[0184] The intermediate 14a (200 mg, 0.55 mmol) was dissolved in dry DMF (5 mL), cesium carbonate (269 mg, 0.83 mmol) was added, and then 2-amino-2-thiazoline (169 mg, 1.65 mmol) was added, and then the mixture was transferred to a 65°C oil bath and heated for 12 h. After the reaction was completed, the reaction solution was poured into a 250 mL separatory funnel, and water (100 mL) and ethyl acetate (40 mL×3) were added for extraction. The organic phases were combined and washed with saturated brine (100 mL). The organic phases were collected, and after reduced pressure distillation, separation and purification were performed by column chromatography (dichloromethane: methanol = 14: 1) to obtain 23 mg of yellow solid compound II-4, with a yield of 10%.

[0185] Embodiment 45

[0186] During the preparation of compound II-5, compound 14a (0.55 mmol) and isopropylamine (1.65 mmol) were reacted according to the method steps in Example 44 to obtain compound II-5 with a yield of 14%.

[0187] Embodiment 46

[0188] Preparation of 13-(3-((4,5-dihydrothiazol-2-yl)amino)propyl)-3-fluoro-10-methoxy-8,13-dihydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one (Compound II-6)

[0189] Compound 3b and compound 5b were prepared into compound m8 according to the method of Example 41, and then prepared into compound 8b according to the method of Example 43.

[0190] (1) Preparation of intermediate 14b:

[0191] The intermediate 8b (200 mg, 0.60 mmol) was dissolved in dry DMF (5 mL), potassium hydroxide (67 mg, 1.19 mmol) was added, and then 1-bromo-3-chloropropane (282 mg, 1.79 mmol) was added, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the reaction solution was poured into a 250 mL separatory funnel, and water (100 mL) and ethyl acetate (40 mL×3) were added for extraction, the organic phases were combined, and washed with saturated brine (100 mL), the organic phases were collected, and after reduced pressure distillation, separation and purification were performed by column chromatography (petroleum ether: ethyl acetate = 6:1) to obtain 81 mg of compound 14b as a yellow oil, with a yield of 20%.

[0192] (2) Preparation of target compound II-6:

[0193] Intermediate 14b (360 mg, 0.88 mmol) was dissolved in dry DMF (5 mL), cesium carbonate (428 mg, 1.31 mmol) was added, and then 2-amino-2-thiazoline (268 mg, 2.63 mmol) was added, and then the mixture was transferred to a 65°C oil bath and heated for 12 h. After the reaction was completed, the reaction solution was poured into a 250 mL separatory funnel, and water (100 mL) and ethyl acetate (40 mL×3) were added for extraction. The organic phases were combined and washed with saturated brine (100 mL). The organic phases were collected, and after reduced pressure distillation, they were separated and purified by column chromatography (dichloromethane: methanol = 14: 1) to obtain 90 mg of yellow solid compound II-6 with a yield of 22%.

[0194] Embodiment 47

[0195] During the preparation of compound II-7, compound 14b (0.88 mmol) and isopropylamine (2.63 mmol) were reacted according to the method steps in Example 46 to obtain compound II-7 with a yield of 29%.

[0196] Embodiment 48

[0197] 13-(3-((4,5-dihydrothiazol-2-yl)amino)propyl)-10-methoxy-8,13-dihydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one

[0198] Compound 3b and compound 5a were prepared into compound m9 according to the method of Example 41, and then prepared into compound 8c according to the method of Example 43.

[0199] (1) Preparation of intermediate 14c:

[0200] The intermediate 8c (200 mg, 0.63 mmol) was dissolved in dry DMF (5 mL), potassium hydroxide (71 mg, 1.26 mmol) was added, and then 1-bromo-3-chloropropane (298 mg, 1.89 mmol) was added, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the reaction solution was poured into a 250 mL separatory funnel, and water (100 mL) and ethyl acetate (40 mL×3) were added for extraction, the organic phases were combined, and washed with saturated brine (100 mL), the organic phases were collected, and after reduced pressure distillation, separation and purification were performed by column chromatography (petroleum ether: ethyl acetate = 6:1) to obtain 48 mg of compound 14c as a yellow oil, with a yield of 19%.

[0201] (2) Preparation of target compound II-8:

[0202] During the preparation of compound II-8, compound 14c (0.88 mmol) and 2-amino-2-thiazoline (2.63 mmol) were reacted according to the method steps in Example 46 to obtain compound II-8 with a yield of 20%.

[0203] Embodiment 49

[0204] During the preparation of compound II-9, compound 14c (0.88 mmol) and isopropylamine (2.63 mmol) were reacted according to the method steps in Example 46 to obtain compound II-9 with a yield of 29%.

[0205] Embodiment 50

[0206] Preparation of 13-(3-((4,5-dihydrothiazol-2-yl)amino)propyl)-3-fluoro-8,13-dihydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one (Compound II-10)

[0207] Compound 3a and compound 5b were prepared into compound m10 according to the method of Example 41, and then prepared into compound 8d according to the method of Example 43.

[0208] (1) Preparation of intermediate 14d:

[0209] The intermediate 8d (200 mg, 0.66 mmol) was dissolved in dry DMF (5 mL), potassium hydroxide (74 mg, 1.31 mmol) was added, and then 1-bromo-3-chloropropane (310 mg, 1.97 mmol) was added, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the reaction solution was poured into a 250 mL separatory funnel, and water (100 mL) and ethyl acetate (40 mL×3) were added for extraction, the organic phases were combined, and washed with saturated brine (100 mL), the organic phases were collected, and after reduced pressure distillation, separation and purification were performed by column chromatography (petroleum ether: ethyl acetate = 6:1) to obtain 55 mg of compound 14d as a yellow oil, with a yield of 22%.

[0210] (2) Preparation of target compound II-10:

[0211] During the preparation of compound II-10, compound 14d (0.88 mmol) and 2-amino-2-thiazoline (2.63 mmol) were reacted according to the method steps in Example 46 to obtain compound II-10 with a yield of 7%.

[0212] Embodiment 51

[0213] During the preparation of compound II-11, compound 14d (0.88 mmol) and isopropylamine (2.63 mmol) were reacted according to the method steps in Example 46 to obtain compound II-11 with a yield of 27%.

[0214] Embodiment 52

[0215] Compound II-12 was prepared according to the method of Example 25, using compound II-6 (0.66 mmol) as the raw material. Other steps were the same as those of Example 25, and the yield was 80%.

[0216] Embodiment 53

[0217] Compound II-13 was prepared according to the method of Example 25, using compound II-7 (0.66 mmol) as raw material. Other steps were consistent with Example 25, and the yield was 98%.

[0218] Embodiment 54

[0219] Compound II-14 was prepared according to the method of Example 25, using compound II-8 (0.66 mmol) as raw material. Other steps were consistent with Example 25, and the yield was 44%.

[0220] Embodiment 55

[0221] Compound II-15 was prepared according to the method of Example 25, using compound II-9 (0.66 mmol) as raw material. Other steps were consistent with Example 25, and the yield was 64%.

[0222] The compounds prepared by the present invention 1 H NMR, 13 C NMR and MS data are detailed in Table 1.

[0223] Table 1 Preferred compounds of the present invention 1 H NMR, 13 C NMR and MS data

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233] Embodiment 56

[0234] Induction and stabilization of c-MYC by target compounds

[0235] 1. Induction of c-MYC by target compounds

[0236] (1) Experimental materials: probe 3'-FAM-c-MYC Pu28-BHQ1-5', 200 mM KCl, 50 mM Tris acetate buffer (Tris acetate buffer), pH = 7.0, and black 96-well plate.

[0237] (2) Experimental instruments: shaker and Tecan microplate reader.

[0238] (3) Experimental method: Take 1 μL of 100 μM 3'-FAM-c-MYC Pu28-BHQ1-5' probe in a black 96-well plate, add 49 μL 50 mM Tris acetate buffer, pH = 7.0, to make a 2 μM probe, let it stand on a shaker for 1 hour, and add 50 μL to each well. Add 50 μL of 20 μM compound or 200 mM KCl to the 2 μM probe to make a 1 μM probe, let it stand on a shaker for 1 hour, a total of 100 μL per well. DMSO and KCl were used as control groups. The excitation wavelength of the Tecan microplate reader was set to 490 nm, the emission wavelength was set to 520 nm, and the bandwidth was 20 nm.

[0239] Based on the fluorescence values ​​obtained by the above method, the final fluorescence intensity percentage (%) was calculated using Excel software.

[0240] (4) Experimental results: Figure 1 As shown, Figure 1 It is a schematic diagram of the induction and stabilization effect of evodiamine derivatives on c-MYC. Among them, A is a schematic diagram of the induction effect of evodiamine derivatives on c-MYC, and C is a schematic diagram of the induction effect of evodiamine derivatives on c-MYC. The experimental results show that the tested compounds show a high c-MYC induction effect, among which compounds II-7, II-11 and II-13 show the best induction effect.

[0241] 2. Stabilization effect of target compounds on c-MYC

[0242] (1) Experimental materials: probe 5'-FAM-c-MYC Pu22-TAMRA-3', 7.5 mM KCl / 2.5 mM PBS, pH = 7.0, and DMSO.

[0243] (2) Experimental instrument: Thermo Fisher quantstudio 3q-PCR instrument.

[0244] (3) Experimental method: 100 μM 5'-FAM-c-MYC Pu22-TAMRA-3' was diluted to 400 nM in a buffer containing 7.5 mM KCl / 2.5 mM PBS, pH = 7.0, heated at 95 ° C for 5 minutes, and then slowly cooled to room temperature. 10 μL of annealed 400 nM probe and 10 μL of 20 μM compound were incubated at 4 ° C overnight. The temperature was increased from 25 ° C to 95 ° C at a rate of 0.9 ° C / min, and the results were measured using the Thermo Fisher quantstudio 3q-PCR instrument. DMSO was used as the control group.

[0245] According to the fluorescence values ​​obtained by the above method, the final ΔT was calculated using GraphPad Prism 8.0 software. m (℃).

[0246] (4) Experimental results: Figure 1 As shown, Figure 1 Schematic diagram of the induction and stabilization of c-MYC by evodiamine derivatives. B is a schematic diagram of the stabilization of c-MYC by evodiamine derivatives, and D is a schematic diagram of the stabilization of c-MYC by evodiamine derivatives. The experimental results show that the tested compounds exhibit a high c-MYC stabilization effect, among which compounds II-6, II-9 and II-13 exhibit the best stabilization effect.

[0247] Embodiment 57

[0248] Topoisomerase Ⅰ / Ⅱ inhibitory activity of target compounds

[0249] 1. Topoisomerase I-mediated DNA unwinding experiment

[0250] (1) Experimental materials: calf thymus DNA topoisomerase I, negative supercoiled DNA plasmid pBR322, 10× buffer, 0.1% BSA, agarose, DMSO and EtBr.

[0251] (2) Experimental instruments: Gel electrophoresis was performed using a BioRad PowerPac electrophoresis instrument and a Sub-Cell Model electrophoresis tank. Gel scanning and quantification were performed using a BioRad Gel Doc EZ fully automated gel system.

[0252] (3) Experimental method: First, prepare 1×TAE solution into 0.8% agarose gel. Then, add 10 μL water, 2 μL 10× buffer, 2 μL 0.1% BSA, 0.5U Top1, 0.5 μL DNA, and 0.2 μL of different drugs to a 1.5 mL sample tube, and adjust the volume to 20 μL. Then, place the sample in a 37°C water bath and incubate for 15 minutes. The electrophoresis results are observed using a gel imaging system.

[0253] (4) Experimental results: Figure 2 As shown, Figure 2Figure 1 is a schematic diagram of the results of the inhibition experiment of evodiamine derivatives on Top1 / 2, wherein A is a schematic diagram of the results of the inhibition of Top1 enzyme activity by evodiamine derivatives (200 μM), C is a schematic diagram of the results of the inhibition of Top1 enzyme activity by evodiamine derivatives (50 μM), D is a schematic diagram of the results of the inhibition of Top1 enzyme activity by evodiamine derivatives (25 / 12.5 μM), and E is a schematic diagram of the results of the inhibition of Top1 enzyme activity by evodiamine derivatives (10 / 5 / 2.5 / 1 μM). The experimental results show that compounds II-5, II-13, II-11, II-8, II-9, II-14 and II-15 exhibit strong Top1 inhibitory activity at 50 μM, among which compounds II-13 and II-15 exhibit the best Top1 inhibitory activity.

[0254] 2. Topoisomerase II-mediated DNA unwinding experiment

[0255] (1) Experimental materials: calf thymus DNA topoisomerase II, negative supercoiled DNA plasmid pBR322, 30 mM ATP, dilution buffer, assay buffer, agarose, DMSO and EtBr.

[0256] (2) Experimental instruments: Gel electrophoresis was performed using a BioRad PowerPac electrophoresis instrument and a Sub-Cell Model electrophoresis tank. Gel scanning and quantification were performed using a BioRad Gel Doc EZ fully automated gel system.

[0257] (3) Experimental method: First, prepare 1×TAE solution into a 0.8% agarose gel. Then, add 10 μL of water, 2 μL of dilution buffer, 2 μL of assay buffer, 0.5U of Top2, 0.5 μL of DNA, and 0.2 μL of different drugs to a 1.5 mL sample tube, and adjust the volume to 20 μL. Then, place the sample in a 37°C water bath and incubate for 30 minutes. Observe the electrophoresis results using a gel imaging system.

[0258] (4) Experimental results: Figure 2 As shown, Figure 2Figure 1 is a schematic diagram of the results of the inhibition experiment of evodiamine derivatives on Top1 / 2, wherein B is a schematic diagram of the results of the inhibition of Top2 enzyme activity by evodiamine derivatives (200 μM), and F is a schematic diagram of the results of the inhibition of Top2 enzyme activity by evodiamine derivatives (50 μM). The experimental results show that compounds II-3, II-4, II-5, II-6, II-7, II-13, II-11, II-8, II-9, II-14 and II-15 exhibit strong Top2 inhibitory activity at 50 μM.

[0259] Embodiment 58

[0260] In vitro antitumor activity test of target compounds (IC 50 )

[0261] (1) Sample preparation: The target compound was prepared at 10 mM using DMSO.

[0262] (2) Cell lines: MCF-7 (human breast cancer cells), MDA-MB-231 (human breast cancer cells), HCT116 (human colon cancer cells), and HeLa (human cervical cancer cells) were all cryopreserved and passaged in our laboratory.

[0263] (3) Experimental method: The target compounds were tested for their ability to inhibit the proliferation of four types of tumor cells using the conventional CCK8 method. Tumor cells (MCF-7, MDA-MB-231, HCT116 and HeLa) in the logarithmic growth phase were digested with trypsin, and then the cells were diluted and suspended into a single cell suspension using culture medium (DMEM or PRMI1640 + 10% FBS + 1% double antibody). The cell density was adjusted to 5×10 4 / mL, 100 μL was added to each well and inoculated into a 96-well plate, and cultured in a 37°C, 5% CO2 incubator for 24 hours. Then, different concentrations of compounds were added, and three replicates were made for each concentration. Experimental and control groups were set up. After incubation for 72 hours, 10 μL CCK8 solution was added to each well, and then incubated at 37°C in the dark for 1-4 hours. The OD value at 450 nm was measured with a Biotek-Synergy microplate reader to calculate the half inhibitory concentration IC 50 .

[0264] (4) Experimental results: IC50 inhibitory concentration of target compound on tumor cells 50 The values ​​are shown in Table 2. The test results show that these multi-target compounds have a wide range of anti-tumor activities, among which compound II-13 exhibits the best IC for the inhibitory activity against MCF-7 and HeLa cells. 50 value.

[0265] Table 2 IC50 inhibitory concentration of target compounds on tumor cells 50 (Unit: μmol / L)

[0266]

[0267]

[0268] CPT is a positive drug camptothecin, with the following structure:

[0269]

[0270] Figure 3 The figure is a schematic diagram of the transcription and expression effects of compound II-13 and Rutaecarpine on c-MYC oncogene. A is a schematic diagram of the transcription effects of compound II-13 and Rutaecarpine on c-MYC oncogene. Compound II-13 has a transcriptional inhibitory trend on c-MYC oncogene from 0-15 μM, and basically inhibits the expression of c-MYC oncogene at 15 μM. The negative control Rutaecarpine does not have a complete transcriptional inhibitory effect on c-MYC oncogene. B is a schematic diagram of the expression level of compound II-13 and Rutaecarpine on c-MYC oncogene. Compound II-13 has a decreasing expression level trend on c-MYC oncogene from 0-15 μM, indicating that compound II-13 has an inhibitory effect on c-MYC oncogene. As the concentration of Rutaecarpine increased, the negative control Rutaecarpine showed no downward trend in the expression level of c-MYC oncogene, indicating that Rutaecarpine has no inhibitory effect on the expression level of c-MYC oncogene.

[0271] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. A multi-target anti-tumor activity evodiamine derivative or a pharmaceutically acceptable salt thereof, characterized in that: The general structural formula is shown in Formula I: in: R1 is selected from C1-C5 alkoxy, hydroxyl, hydrogen; R2 is selected from hydrogen, halogen; R3 is selected from hydroxyl, 2. The multi-target anti-tumor activity evodiamine derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Among the evodiamine derivatives with multi-target anti-tumor activity, R1 is selected from methoxy, ethoxy, hydroxyl, hydrogen; R2 is selected from hydrogen, fluorine; R3 is selected from hydroxyl, 3. The multi-target anti-tumor activity evodiamine derivative or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: The multi-target anti-tumor activity of the evodia rutaecarpine derivative is selected from one of the following structures:

4. Use of the Evodia rutaecarpine derivative or a pharmaceutically acceptable salt thereof having multi-target anti-tumor activity as claimed in any one of claims 1 to 3 in the preparation of anti-tumor drugs.

5. The use according to claim 4, characterized in that: The tumor is selected from breast cancer, colon cancer, cervical cancer, and lung cancer.

6. Use of the multi-target anti-tumor activity evodiamine derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 in the preparation of a Top1 topoisomerase inhibitor, a Top2 topoisomerase inhibitor and / or a c-MYC oncogene inhibitor.

7. Use of the multi-target anti-tumor activity evodiamine derivative or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 3 in the preparation of a drug for treating diseases caused by abnormal gene expression.

8. The use according to claim 7, characterized in that: The disease caused by abnormal gene expression is tumor, endocrine disorder, immune system disease, genetic disease or nervous system disease.

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