Evodiamine derivatives with multi-target anti-tumor activity and their applications

Abstract

The present invention discloses a class of rutaecarpine derivatives or their pharmaceutically acceptable salts with multi-target anti-tumor activity, and the general structural formula is shown as Formula I or Formula II: #imgabs0# Wherein: R1 is selected from C1-C5 alkoxy, hydroxyl, hydrogen; R2 is selected from hydrogen, halogen; R3 is selected from hydroxyl, #imgabs1# Through enzyme inhibition activity and in vitro anti-tumor activity tests, it was found that the compounds of the present invention have strong inhibitory activities against topoisomerase 1, topoisomerase 2 and c-MYC oncogene, opening up a new way and providing a new strategy for in-depth research and development of anti-tumor drugs with new structural types.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and specifically relates to a class of rutaecarpine derivatives with multi-target anti-tumor activity and their applications. Background Art

[0002] 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-stranded or double-stranded DNA by cleaving single DNA.

[0003] In the field of DNA-targeted drugs, small molecules that stabilize c-MYC G-quadruplex (c-MYC G4) DNA are also considered a promising anti-cancer 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, nuclease hypersensitive element (NHE III1) controls 85-90% of c-MYC transcriptional activity and folds into DNA G-quadruplex (G4) under transcription-related negative supercoiling, thus 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, simultaneously targeting Top and c-MYC G4 will be a promising strategy for anti-tumor drug discovery.

[0004] Previous researchers have conducted in-depth studies on the structure-activity relationship of rutaecarpine derivatives. Through the study of the anti-tumor mechanism of action, it was found that rutaecarpine derivatives are dual inhibitors of Top1 and Top2 and can effectively induce apoptosis of tumor cells. The patent application with the publication number CN101787025A discloses a substituted rutaecarpine anti-tumor and anti-fungal compound and its preparation method; the patent application with the publication number CN1012311434A discloses a rutaecarpine compound and its preparation method and application; the patent application with the publication number CN103992336A discloses an oxa- or thia-rutaecarpine anti-tumor derivative and its preparation method.

[0005] In view of this, there is an urgent need for a class of compounds that target Top and c-MYC G4 inhibitors to achieve a synergistic anti-tumor effect. Summary of the Invention

[0006] The first object of the present invention is to provide a class of rutaecarpine derivatives with multi-target anti-tumor activity.

[0007] The second object of the present invention is to provide an application of the rutaecarpine derivative with multi-target anti-tumor activity in the preparation of anti-tumor drugs.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] The first aspect of the present invention provides a class of rutaecarpine derivatives with multi-target anti-tumor activity or their medicinal salts, and the general structural formula is shown as Formula I or Formula II:

[0010]

[0011] Wherein:

[0012] R1 is selected from C1-C5 alkoxy, hydroxyl, hydrogen;

[0013] R2 is selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine);

[0014] R3 is selected from hydroxyl,

[0015]

[0016] Preferably, in the rutaecarpine derivative with multi-target anti-tumor activity,

[0017] R1 is selected from methoxy, ethoxy, hydroxyl, hydrogen;

[0018] R2 is selected from hydrogen, fluorine;

[0019] R3 is selected from hydroxyl,

[0020]

[0021] Most preferably, the rutaecarpine derivative with multi-target anti-tumor activity is selected from one of the following structures:

[0022]

[0023]

[0024]

[0025]

[0026] The medicinal salt is its organic acid salt or inorganic acid salt.

[0027] 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.

[0028] The second aspect of the present invention provides an application of the rutaecarpine derivative with multi-target anti-tumor activity or its pharmaceutically acceptable salt in the preparation of anti-tumor drugs.

[0029] The tumors include breast cancer, colon cancer, cervical cancer, lung cancer, etc.

[0030] The third aspect of the present invention provides an application of the rutaecarpine derivative with multi-target anti-tumor activity or its pharmaceutically acceptable salt in the preparation of Top1 topoisomerase inhibitor, Top2 topoisomerase inhibitor and / or c-MYC oncogene inhibitor.

[0031] The rutaecarpine derivative with multi-target anti-tumor activity or its pharmaceutically acceptable salt can be used as a triple-target inhibitor of Top1, Top2 and c-MYC for the treatment of malignant tumors or diseases related to differentiation and proliferation.

[0032] The tumors include breast cancer, colon cancer, cervical cancer, etc.

[0033] The fourth aspect of the present invention provides an application of the rutaecarpine derivative with multi-target anti-tumor activity or its pharmaceutically acceptable salt in the preparation of drugs for treating diseases caused by abnormal gene expression.

[0034] The diseases caused by abnormal gene expression are tumors, endocrine disorders, immune system diseases, genetic diseases or nervous system diseases.

[0035] Due to the adoption of the above technical solutions, the present invention has the following advantages and beneficial effects:

[0036] The present invention provides a class of rutaecarpine derivatives with multi-target anti-tumor activity. This class of rutaecarpine derivatives has multi-target anti-tumor activity, and the multi-target is a triple-target inhibitor of Top1 / Top2 / c-MYC. Through enzyme inhibition activity and in vitro anti-tumor activity tests, it is found that the compounds of the present invention have strong inhibitory activities against topoisomerase 1, topoisomerase 2 and c-MYC oncogene, opening up a new way and providing a new strategy for the in-depth research and development of new structural types of anti-tumor drugs.

[0037] The present invention provides a class of rutaecarpine derivatives with multi-target anti-tumor activity, which are small molecule anti-cancer drugs based on multi-targets of topoisomerase 1 (Top1), topoisomerase 2 (Top2) and c-MYC oncogene. The compounds of the present invention show good inhibitory activities against Top1, Top2 and c-MYC oncogene, and have a certain broad-spectrum anti-tumor activity, and can be used as anti-tumor drugs. Description of the Drawings

[0038] Figure 1 It is a schematic diagram of the results of the induction and stabilization effect of the rutaecarpine derivative on c-MYC.

[0039] Figure 2 It is a schematic diagram of the experimental results of the inhibition of Top1 / 2 by rutaecarpine derivatives.

[0040] Figure 3 It is a schematic diagram of the results of the transcriptional and expression effects of compound II-13 and rutaecarpine on the c-MYC oncogene. Detailed implementation manners

[0041] To illustrate the present invention more clearly, the present invention will be further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0042] For the experimental methods without specific conditions indicated in the following examples, they are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0043] Synthesis of compounds I-1 to I-15 and I-25 to I-34

[0044]

[0045]

[0046] (a) HCOOCH2CH3, 70 °C, 12 h, yield 80 - 86%; (b) POCl3, 0 °C, 10 h, yield 78 - 85%; (c) THF, BTC, 70 °C, 12 h, yield 80 - 87%; (d) CH3I, NaH, DMF, 2.5 h, yield 90 - 95%; (e) DCM, rt, 6 h, yield 50 - 55%; (f) DMF, NaH, 65 °C, 12 h, yield 43 - 78%; (g) DCM, BBr3, under N2, -78 °C, 2 h, yield 24 - 85%.

[0047] Example 1

[0048] Tryptamine (compound 1a, R1 = H, 0.062 mol, 10 g) was added to the ethyl formate solution (148.8 mL, 1.872 mol), and the reaction was carried out at 70 °C overnight. After the reaction was completed, the reaction solution was evaporated to dryness, and according to the theoretical product weight, that is, compound 2a (R1 = H) (11.7 g, 0.062 mol), it was directly used for the next step of the reaction.

[0049] The crude product of the above 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 the mixture was stirred overnight. After the reaction, the reaction solution was evaporated to dryness, dissolved in 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. The impurities were removed by suction filtration under reduced pressure. The filtrate was adjusted to pH = about 11.0 with ammonia water, and a solid precipitated. The solid was filtered by suction to obtain 9.46 g of a yellow solid, namely compound 3a (R1 = H), with a yield of 89%.

[0050] Anthranilic acid 4a (5.0 g, 0.032 mol) was dissolved in dry tetrahydrofuran (100 mL), and triphosgene (3.8 g, 0.0129 mol) was added. The mixture was heated under reflux at 70 °C for 16 h. After the reaction, the reaction solution was poured into 400 mL of ice-cold saturated brine. After the white solid had completely precipitated, it was filtered by suction, washed twice with water, and the filter cake was dried in an oven at 45 °C. 5.0 g of a white solid, namely compound 5a (R2 = H), was obtained, with a yield of 86%.

[0051] 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. After half an hour, methyl iodide (1.1 mL, 0.016 mol) was added, and the reaction was carried out at room temperature for 4 h. After the reaction, the reaction solution was poured into 400 mL of ice-cold saturated brine, stirred, and filtered by suction. The filter cake was dried in an oven at 45 °C to obtain 2.5 g of a white solid, namely compound 6a (R2 = H), with a yield of 90%.

[0052] 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 the reaction was carried out at room temperature for 6 h. After the reaction, it was filtered by suction, washed twice with dichloromethane, and 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%.

[0053] Dissolve compound 7a (200 mg, 0.660 mmol) in dry DMF (5 mL). Slowly add sodium hydride (purity 60%, 53 mg, 1.320 mmol) under ice bath conditions. After half an hour, slowly add 3-bromopropanol (95 mg, 0.684 mmol) dropwise. Then transfer it to an oil bath at 65 °C and heat for 12 h. After the reaction is complete, pour the reaction solution into a 250 mL separatory funnel, add water (100 mL), and extract with ethyl acetate (40 mL × 3). Combine the organic phases, wash with saturated brine (100 mL), collect the organic phase, and separate and purify by column chromatography (petroleum ether:ethyl acetate = 2:1) after distillation under reduced pressure to obtain 120 mg of yellow solid compound I-1 with a yield of 33%.

[0054] Example 2

[0055] In the preparation of compound I-2, replace 3-bromopropanol with (3-chloropropyl)pyrrolidine (0.684 mmol), and the others are the same as in Example 1. The yield of compound I-2 is 48%.

[0056] Example 3

[0057] In the preparation of compound I-3, replace 3-bromopropanol with 4-(5-chloropropyl)morpholine (0.684 mmol), and the others are the same as in Example 1. The yield of compound I-3 is 7%.

[0058] Example 4

[0059] In the preparation of compound I-4, replace 3-bromopropanol with 3-chloro-N-methylpropan-1-amine hydrochloride (0.684 mmol), and the others are the same as in Example 1. The yield of compound I-4 is 28%.

[0060] Example 5

[0061] In the preparation of compound I-5, replace 3-bromopropanol with 3-chloro-1-(N,N-dimethyl)propylamine (0.684 mmol), and the others are the same as in Example 1. The yield of compound I-5 is 27%.

[0062] Example 6

[0063] Preparation of 3-fluoro-13-(3-hydroxypropyl)-10-methoxy-14-methyl-8,13,13b,14-tetrahydroindolo[2’,3’:3,4]pyrido[2,1-b]quinazolin-5(7H)-one (Compound I-6)

[0064] (1) Preparation of intermediate 3b (R1 = OCH3): 6-methoxycarbazole

[0065] 5-Methoxytryptamine compound 1b (R1 = OCH3) (10.0 g, 0.053 mol) was added to ethyl formate solution (125.3 mL, 1.576 mol), and the reaction was carried out at 70 °C overnight. After the reaction, the reaction solution was evaporated to dryness. According to the theoretical product weight (11.6 g, 0.053 mol), it was directly used for the next reaction. The crude product was dissolved in dry dichloromethane (200 mL), and POCl3 (6.9 mL, 0.074 mol) was slowly added dropwise under ice bath conditions, and the mixture was stirred overnight. After the reaction, the reaction solution was evaporated to dryness, dissolved in 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. The impurities were removed by vacuum filtration. The filtrate was adjusted to pH = about 11.0 with ammonia water, and a solid precipitated. The solid was filtered by suction to obtain 9.46 g of yellow solid compound 3b, with a yield of 89%. 1 H NMR (600 MHz, DMSO-d6) δ: 11.21 (s, 1H), 8.35 (s, 1H), 7.31 (d, J = 8.8 Hz, 1H), 7.02 (d, J = 2.5 Hz, 1H), 6.85 (dd, J = 8.9, 2.5 Hz, 1H), 3.78 (dd, J = 8.4, 1.8 Hz, 2H), 3.77 (s, 3H), 2.80 (t, J = 8.5 Hz, 2H).

[0066] (2) Preparation of intermediate 5b: 6-Fluoro isatoic anhydride

[0067] o-Aminobenzoic acid 4b (R2 = F) (5.0 g, 0.032 mol) was dissolved in dry tetrahydrofuran (100 mL), and triphosgene (3.8 g, 0.0129 mol) was added. The mixture was heated under reflux at 70 °C for 16 h. After the reaction, the reaction solution was poured into 400 mL of ice-cold saturated brine. After the white solid precipitated completely, it was filtered by suction and washed twice with water. The filter cake was dried in an oven at 45 °C. 5.0 g of white solid compound 5b was obtained, with a yield of 86%. 1 H NMR (600 MHz, DMSO-d6) δ: 11.79 (s, 1H), 7.69 - 7.61 (m, 2H), 7.19 (q, J = 4.8 Hz, 1H).

[0068] (3) Preparation of intermediate 6b: 6-Fluoro-N-methyl isatoic anhydride

[0069] Intermediate 5b (R2 = F) (5.0 g, 0.028 mol) was dissolved in dry DMF (60 mL). Sodium hydride (purity 60%, 1.3 g, 0.033 mol) was slowly added under an ice bath condition. After half an hour, methyl iodide (2.1 mL, 0.033 mol) was added, and the reaction was carried out at room temperature for 4 h. After the reaction was completed, the reaction solution was poured into 400 mL of ice-cold saturated brine, stirred, filtered by suction, and the filter cake was dried in an oven at 45 °C to obtain 4.9 g of white solid compound 6b, with a yield of 90%. 1H NMR (600 MHz, DMSO-d6) δ: 7.78 - 7.73 (m, 2H), 7.52 - 7.48 (m, 1H), 3.47 (s, 3H).

[0070] (4) Preparation of intermediate 7b: 3-fluoro-10-methoxydictamnine

[0071] 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 the reaction was carried out at room temperature for 6 h. After the reaction was completed, filtration by suction was carried out, and it was rinsed twice with dichloromethane. The filter cake was freeze-dried. 5.2 g of yellow solid compound 7b (R1 = OCH3, R2 = F) was obtained, with a yield of 59%. 1H NMR (600 MHz, DMSO-d6) δ: 11.01 (s, 1H), 7.55 (dd, J = 8.9, 3.1 Hz, 1H), 7.39 (td, J = 8.6, 3.1 Hz, 1H), 7.27 (d, J = 8.7 Hz, 1H), 7.19 (dd, J = 8.9, 4.5 Hz, 1H), 7.01 (d, J = 2.4 Hz, 1H), 6.78 (dd, J = 8.7, 2.4 Hz, 1H), 6.08 (s, 1H), 4.63 (dt, J = 11.4, 3.6 Hz, 1H), 3.77 (s, 3H), 3.21 (m, 1H), 2.81 (t, J = 11.4 Hz, 2H), 2.69 (s, 3H).

[0072] (5) Preparation of the target compound I-6:

[0073] The intermediate 7b (200 mg, 0.570 mmol) was dissolved in dry DMF (5 mL). Sodium hydride (purity 60%, 34 mg, 0.855 mmol) was slowly added under an ice bath condition. After half an hour, 3-bromopropanol (95 mg, 0.684 mmol) was slowly added dropwise. Then it was transferred to an oil bath at 65 °C 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, washed with saturated brine (100 mL), the organic phase was collected, and after reduced pressure distillation, it was separated and purified by column chromatography (petroleum ether:ethyl acetate = 2:1) to obtain 120 mg of yellow solid compound I-6, with a yield of 52%.

[0074] Example 7

[0075] In the preparation of compound I-7, 3-bromopropanol was replaced with (3-chloropropyl)pyrrolidine (0.684 mmol), and the others were the same as in Example 6. The yield of compound I-7 was 70%.

[0076] Example 8

[0077] In the preparation of compound I-8, 3-bromopropanol was replaced with 4-(5-chloropropyl)morpholine (0.684 mmol), and the others were the same as in Example 6. The yield of compound I-8 was 48%.

[0078] Example 9

[0079] In the preparation of compound I-9, 3-bromopropanol was replaced with 3-chloro-N-methylpropan-1-amine hydrochloride (0.684 mmol), and the others were the same as in Example 6. The yield of compound I-9 was 33%.

[0080] Example 10

[0081] In the preparation of compound I-10, 3-bromopropanol was replaced with 3-chloro-1-(N,N-dimethyl)propylamine (0.684 mmol), and the others were the same as in Example 6. The yield of compound I-10 was 61%.

[0082] Example 11

[0083] The intermediate 3b (5.0 g, 0.025 mol) and the intermediate 6a (4.4 g, 0.025 mol) were dissolved in dry dichloromethane (100 mL), and the reaction was carried out at room temperature for 6 h. After the reaction was completed, filtration was carried out, and it was rinsed twice with dichloromethane. The filter cake was freeze-dried. 5.0 g of yellow solid compound 7c (R1 = OCH3, R2 = H) was obtained, with a yield of 60%.

[0084] In the preparation of compound I-11, compound 7c (0.660 mmol) and 3-bromopropanol (0.684 mmol) were used to prepare compound I-11 according to the method steps in Example 1, with a yield of 47%.

[0085] Example 12

[0086] In the preparation of compound I-12, compound 7c (0.660 mmol) and (3-chloropropyl)pyrrolidine (0.684 mmol) were used to prepare compound I-12 according to the method steps in Example 1, with a yield of 63%.

[0087] Example 13

[0088] In the preparation of compound I-13, compound 7c (0.660 mmol) and 4-(5-chloropropyl)morpholine (0.684 mmol) were used to prepare compound I-13 according to the method steps in Example 1, with a yield of 51%.

[0089] Example 14

[0090] In the preparation of compound I-14, compound 7c (0.660 mmol) and 3-chloro-N-methylpropan-1-amine hydrochloride (0.684 mmol) were used to prepare compound I-14 according to the method steps in Example 1, with a yield of 68%.

[0091] Example 15

[0092] In the preparation of compound I-15, compound 7c (0.660 mmol) and 3-chloro-1-(N,N-dimethyl)propylamine (0.684 mmol) were used to prepare compound I-15 according to the method steps in Example 1, with a yield of 56%.

[0093] Synthesis of Compounds I-16 to I-24 and I-35 to I-40

[0094]

[0095] (a) DMF, KOH, 1,3-dibromo propane, rt, 12 h, yield 35 - 80%; (b) I-16 - I-17: DMF, K2CO3, 65 °C, 12 h; yield 32 - 60%; (c) I-18: MeCN, rt, 72 h, yield 44 - 81%; (d) DCM, BBr3, under N2, -78 °C, 2 h, yield 34 - 72%.

[0096] Example 16

[0097] Preparation of 13-(3-((4,5-thiazolin-2-yl)amino)propyl)-14-methyl-8,13,13b,14-tetrahydroindolo[2’,3’:3,4]pyrido[2,1-b]quinazolin-5(7H)-one (Compound I-16)

[0098] (1) Preparation of Intermediate 12a:

[0099] Dissolve Intermediate 7a (1.0 g, 3.30 mmol) in dry DMF (10 mL), add potassium hydroxide (370.0 mg, 6.60 mmol), and then add 1,3-dibromopropane (2.0 g, 9.90 mmol). React at room temperature overnight. After the reaction is complete, pour the reaction solution into a 250 mL separatory funnel, add water (100 mL) and ethyl acetate (40 mL × 3) for extraction. Combine the organic phases, wash with saturated brine (100 mL), collect the organic phase, and separate and purify by column chromatography (petroleum ether:ethyl acetate = 6:1) after vacuum distillation to obtain 360 mg of yellow oil Compound 12a with a yield of 26%.

[0100] (2) Preparation of Target Compound I-16:

[0101] Dissolve Intermediate 12a (120 mg, 0.284 mmol) in dry DMF (5 mL), add potassium carbonate (59 mg, 0.43 mmol), then add 2-amino-2-thiazoline (87 mg, 0.85 mmol), and then transfer to an oil bath at 65 °C and heat for 12 h. After the reaction is complete, pour the reaction solution into a 250 mL separatory funnel, add water (100 mL) and ethyl acetate (40 mL × 3) for extraction. Combine the organic phases, wash with saturated brine (100 mL), collect the organic phase, and separate and purify by column chromatography (dichloromethane:methanol = 14:1) after vacuum distillation to obtain 50 mg of yellow solid Compound I-16 with a yield of 40%.

[0102] Example 17

[0103] During the preparation of Compound I-17, Compound 12a (0.284 mmol) and isopropylamine (0.85 mmol) were prepared to obtain Compound I-17 according to the method steps in Example 16 with a yield of 44%.

[0104] Example 18

[0105] During the preparation of Compound I-18, Compound 12a (0.284 mmol) and ethanolamine (0.85 mmol) were prepared to obtain Compound I-18 according to the method steps in Example 16 with a yield of 44%.

[0106] Example 19

[0107] Preparation of 13-(3-((4,5-thiazolin-2-yl)amino)propyl)-3-fluoro-10-methoxy-14-methyl-8,13,13b,14-tetrahydroindolo[2’,3’:3,4]pyrido[2,1-b]quinazolin-5(7H)-one (Compound I-19)

[0108] (1) Preparation of Intermediate 12b:

[0109] Dissolve Intermediate 7b (200 mg, 0.57 mmol) in dry DMF (5 mL), add potassium hydroxide (64 mg, 1.14 mmol), then add 1,3-dibromopropane (345 mg, 1.71 mmol), and react at room temperature overnight. After the reaction is complete, pour the reaction solution into a 250 mL separatory funnel, add water (100 mL) and ethyl acetate (40 mL×3) for extraction. Combine the organic phases, wash with saturated brine (100 mL), collect the organic phase, and separate and purify by column chromatography (petroleum ether:ethyl acetate = 6:1) after distillation under reduced pressure to obtain 85 mg of yellow oil of Compound 12b with a yield of 32%. 1 H NMR (600 MHz, DMSO-d6) δ: 7.63 (dd, J = 9.0, 3.1 Hz, 1H), 7.49 - 7.43 (m, 2H), 7.39 (dd, J = 8.8, 4.7 Hz, 1H), 7.11 (d, J = 2.5 Hz, 1H), 6.89 (dd, J = 8.9, 2.5 Hz, 1H), 6.16 (s, 1H), 4.67 (ddd, J = 12.6, 5.0, 1.6 Hz, 1H), 4.43 - 4.36 (m, 1H), 4.35 - 4.28 (m, 1H), 3.80 (s, 3H), 3.59 (td, J = 6.2, 1.3 Hz, 2H), 3.12 (td, J = 12.2, 3.9 Hz, 1H), 2.97 (d, J = 15.0 Hz, 1H), 2.76 (t, J = 12.1 Hz, 1H), 2.38 - 2.23 (m, 5H).

[0110] (2) Preparation of the target compound I-19:

[0111] Intermediate 12b (120 mg, 0.28 mmol) was dissolved in dry DMF (5 mL), potassium carbonate (59 mg, 0.43 mmol) was added, followed by 2-amino-2-thiazoline (87 mg, 0.85 mmol). Then it was transferred to an oil bath at 65 °C and heated for 12 h. After the reaction, the reaction solution was poured into a 250 mL separatory funnel, extracted with water (100 mL) and ethyl acetate (40 mL × 3). The organic phases were combined, washed with saturated brine (100 mL), the organic phase was collected, and after distillation under reduced pressure, it was separated and purified by column chromatography (methylene chloride:methanol = 14:1) to obtain 60 mg of yellow solid Compound I-19 with a yield of 48%.

[0112] Example 20

[0113] In the preparation of Compound I-20, Compound 12b (0.28 mmol) and isopropylamine (0.85 mmol) were prepared to obtain Compound I-20 according to the method steps in Example 19 with a yield of 32%.

[0114] Example 21

[0115] In the preparation of Compound I-21, Compound 12b (0.28 mmol) and ethanolamine (0.85 mmol) were prepared to obtain Compound I-21 according to the method steps in Example 19 with a yield of 81%.

[0116] Example 22

[0117] Preparation of 13-(3-((4,5-thiazolin-2-yl)amino)propyl)-10-methoxy-14-methyl-8,13,13b,14-tetrahydroindolo[2’,3’:3,4]pyrido[2,1-b]quinazolin-5(7H)-one (Compound I-22)

[0118] Intermediate 7c (200 mg, 0.60 mmol) was dissolved in dry DMF (5 mL), potassium hydroxide (67 mg, 1.2 mmol) was added, followed by 1,3-dibromopropane (363 mg, 1.80 mmol). The reaction was carried out overnight at room temperature. After the reaction, the reaction solution was poured into a 250 mL separatory funnel, extracted with water (100 mL) and ethyl acetate (40 mL × 3). The organic phases were combined, washed with saturated brine (100 mL), the organic phase was collected, and after distillation under reduced pressure, it was separated and purified by column chromatography (petroleum ether:ethyl acetate = 6:1) to obtain 68 mg of yellow oil Compound 12c with a yield of 25%.

[0119] In the preparation of Compound I-22, Compound 12c (0.28 mmol) and 2-amino-2-thiazoline (0.85 mmol) were used to prepare Compound I-22 according to the method steps in Example 19, with a yield of 53%.

[0120] Example 23

[0121] In the preparation of Compound I-23, Compound 12c (0.28 mmol) and isopropylamine (0.85 mmol) were used to prepare Compound I-23 according to the method steps in Example 19, with a yield of 60%.

[0122] Example 24

[0123] In the preparation of Compound I-24, Compound 12c (0.28 mmol) and ethanolamine (0.85 mmol) were used to prepare Compound I-24 according to the method steps in Example 19, with a yield of 63%.

[0124] Example 25

[0125] Preparation of 3-fluoro-10-hydroxy-13-(3-hydroxypropyl)-14-methyl-8,13,13b,14-tetrahydroindolo[2’,3’:3,4]pyrido[2,1-b]quinazolin-5(7H)-one Compound I-25

[0126] Compound I-6 (270 mg, 0.66 mmol) was dissolved in dry dichloromethane (10 mL). Under N2 protection, boron tribromide (495 mg, 1.98 mmol) was added at -78 °C and reacted for 2 h, and then transferred to room temperature and reacted overnight. After the reaction, 25 mL of ice-cold aqueous NaHCO3 solution was added, stirred for 30 minutes, extracted with dichloromethane (40 mL × 3), the organic phase was collected, and 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%.

[0127] Example 26

[0128] The preparation of Compound I-26 was carried out according to the method of Example 25, using Compound I-7 (0.66 mmol) as the raw material, and the others were the same as in Example 25, with a yield of 30%.

[0129] Example 27

[0130] The preparation of Compound I-27 was carried out according to the method of Example 25, using Compound I-8 (0.66 mmol) as the raw material, and the others were the same as in Example 25, with a yield of 12%.

[0131] Example 28

[0132] The preparation of Compound I-28 was carried out according to the method of Example 25, using Compound I-9 (0.66 mmol) as the raw material. Other conditions were the same as those in Example 25, and the yield was 81%.

[0133] Example 29

[0134] The preparation of Compound I-29 was carried out according to the method of Example 25, using Compound I-10 (0.66 mmol) as the raw material. Other conditions were the same as those in Example 25, and the yield was 36%.

[0135] Example 30

[0136] The preparation of Compound I-30 was carried out according to the method of Example 25, using Compound I-11 (0.66 mmol) as the raw material. Other conditions were the same as those in Example 25, and the yield was 47%.

[0137] Example 31

[0138] The preparation of Compound I-31 was carried out according to the method of Example 25, using Compound I-12 (0.66 mmol) as the raw material. Other conditions were the same as those in Example 25, and the yield was 63%.

[0139] Example 32

[0140] The preparation of Compound I-32 was carried out according to the method of Example 25, using Compound I-13 (0.66 mmol) as the raw material. Other conditions were the same as those in Example 25, and the yield was 51%.

[0141] Example 33

[0142] The preparation of Compound I-33 was carried out according to the method of Example 25, using Compound I-14 (0.66 mmol) as the raw material. Other conditions were the same as those in Example 25, and the yield was 68%.

[0143] Example 34

[0144] The preparation of Compound I-34 was carried out according to the method of Example 25, using Compound I-15 (0.66 mmol) as the raw material. Other conditions were the same as those in Example 25, and the yield was 56%.

[0145] Example 35

[0146] The preparation of Compound I-35 was carried out according to the method of Example 25, using Compound I-19 (0.66 mmol) as the raw material. Other conditions were the same as those in Example 25, and the yield was 71%.

[0147] Example 36

[0148] The preparation of compound I-36 was carried out according to the method of Example 25, using compound I-20 (0.66 mmol) as the raw material. Other conditions were the same as those in Example 25, and the yield was 50%.

[0149] Example 37

[0150] The preparation of compound I-37 was carried out according to the method of Example 25, using compound I-21 (0.66 mmol) as the raw material. Other conditions were the same as those in Example 25, and the yield was 34%.

[0151] Example 38

[0152] The preparation of compound I-38 was carried out according to the method of Example 25, using compound I-22 (0.66 mmol) as the raw material. Other conditions were the same as those in Example 25, and the yield was 66%.

[0153] Example 39

[0154] The preparation of compound I-39 was carried out according to the method of Example 25, using compound I-23 (0.66 mmol) as the raw material. Other conditions were the same as those in Example 25, and the yield was 72%.

[0155] Example 40

[0156] The preparation of compound I-40 was carried out according to the method of Example 25, using compound I-24 (0.66 mmol) as the raw material. Other conditions were the same as those in Example 25, and the yield was 65%.

[0157] Example 41

[0158] Preparation of 13-(3-(pyrrolidin-1-yl)propyl)-8,13-tetrahydroindolo[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one compound II-1

[0159]

[0160] (a) DCM, rt, 6 h, yield 47%; (b) DMF, Cs2CO3, 65 °C, 12 h, yield 25 - 65%.

[0161] (1) Preparation of intermediate m7:

[0162] Intermediate 3a (200 mg, 1.18 mmol) and intermediate 5a (192 mg, 1.18 mmol) were dissolved in dry dichloromethane (10 mL), and the reaction was carried out at room temperature for 6 h. After the reaction was completed, suction filtration was carried out, and the filter cake was washed twice with dichloromethane and then freeze-dried. 34 mg of yellow solid compound m7 was obtained, and the yield was 10%. 11H NMR (600 MHz, DMSO-d6) δ: 10.97 (s, 1H), 7.80 (dd, J = 7.8, 1.6 Hz, 1H), 7.53 (d, J = 7.8 Hz, 1H), 7.46 (d, J = 7.8 Hz, 1H), 7.38 (td, J = 8.4, 1.8 Hz, 1H), 7.20 - 7.15 (m, 1H), 7.07 (td, J = 8.4, 1.8 Hz, 1H), 6.97 (s, 1H), 6.90 (d, J = 1.8 Hz, 1H), 6.86 (td, J = 7.8, 0.6 Hz, 1H), 6.08 (d, J = 0.6 Hz, 1H), 4.84 (ddd, J = 12.9, 5.2, 1.6 Hz, 1H), 3.05 (td, J = 15.0, 7.2 Hz, 1H), 2.89 - 2.76 (m, 2H).

[0163] (2) Preparation of the target compound II-1:

[0164] Dissolve intermediate m7 (200 mg, 0.69 mmol) in dry DMF (10 mL), add cesium carbonate (670 mg, 2.08 mmol), then add (3-chloropropyl)pyrrolidine (120 mg, 0.83 mmol), and then transfer to an oil bath at 65 °C and heat for 12 h. After the reaction is completed, pour the reaction solution into a 250 mL separatory funnel, add water (100 mL) and ethyl acetate (40 mL × 3) for extraction. Combine the organic phases, wash with saturated brine (100 mL), collect the organic phase, and after distillation under reduced pressure, separate and purify by column chromatography (methylene chloride:methanol = 19:1) to obtain 70 mg of yellow solid compound II-1, with a yield of 25%.

[0165] Example 42

[0166] In the preparation of compound II-2, compound m7 (0.69 mmol) and 3-chloro-1-(N,N-dimethyl)propylamine (0.83 mmol) were prepared to obtain compound II-2 according to the method steps in Example 41, with a yield of 65%.

[0167] Example 43

[0168]

[0169] (a) DCM, DDQ, rt, 8 h, yield 45 - 55%; (b) DMF, NaH, 65 °C, 12 h, yield 8%.

[0170] (1) Preparation of intermediate 8a:

[0171] Intermediate m7 (200 mg, 0.69 mmol) was dissolved in dry DCM (10 mL), and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (157 mg, 0.69 mmol) was added. The reaction was carried out at room temperature for 8 h. After the reaction was completed, it was separated and purified by column chromatography (dichloromethane:methanol = 100:1) under reduced pressure distillation to obtain 90 mg of yellow solid compound 8a with a yield of 45%.

[0172] (2) Preparation of target compound II-3:

[0173] Compound 8a (200 mg, 0.70 mmol) was dissolved in dry DMF (5 mL). Under ice bath conditions, sodium hydride (purity 60%, 111 mg, 2.79 mmol) was slowly added. After half an hour, 3-chloro-N-methylpropan-1-amine hydrochloride (1.39 mmol) was slowly added dropwise, and then it was transferred to an oil bath at 65 °C 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, washed with saturated brine (100 mL), the organic phase was collected, and after reduced pressure distillation, it was separated and purified by column chromatography (dichloromethane:methanol = 100:6) to obtain 19 mg of yellow solid compound II-3 with a yield of 8%.

[0174] Synthesis of compounds II-4 to II-15

[0175]

[0176]

[0177] (a) DMF, KOH, 1-bromo-3-chloropropane, rt, 12 h, yield 19 - 22%; (b) DMF, Cs2CO3, 65 °C, 12 h, yield 10 - 37%; (c) DCM, BBr3, under N2, -78 °C, 2 h, yield 44 - 98%.

[0178] Example 44

[0179] Preparation of 13-(3-((4,5-dihydrothiazol-2-yl)amino)propyl)-8,13-dihydroindolo[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one (compound II-4)

[0180] (1) Preparation of intermediate 14a:

[0181] 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. The reaction was carried out 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, washed with saturated brine (100 mL), the organic phase was collected, and after distillation under reduced pressure, it was separated and purified by column chromatography (petroleum ether:ethyl acetate = 6:1) to obtain 54 mg of a yellow oil, compound 14a, with a yield of 21%.

[0182] (2) Preparation of the target compound II-4:

[0183] 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. Then it was transferred to an oil bath at 65 °C 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, washed with saturated brine (100 mL), the organic phase was collected, and after distillation under reduced pressure, it was separated and purified by column chromatography (methylene chloride:methanol = 14:1) to obtain 23 mg of a yellow solid, compound II-4, with a yield of 10%.

[0184] Example 45

[0185] In the preparation of compound II-5, compound 14a (0.55 mmol) and isopropylamine (1.65 mmol) were prepared into compound II-5 according to the method steps in Example 44, with a yield of 14%.

[0186] Example 46

[0187] Preparation of 13-(3-((4,5-dihydrothiazol-2-yl)amino)propyl)-3-fluoro-10-methoxy-8,13-dihydroindolo[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one (Compound II-6)

[0188] Compound 3b and compound 5b were prepared into compound m8 according to the method in Example 41, and then into compound 8b according to the method in Example 43.

[0189] (1) Preparation of intermediate 14b:

[0190] 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. The reaction was carried out at room temperature overnight. After the reaction, 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, washed with saturated brine (100 mL), the organic phase was collected, and after distillation under reduced pressure, it was separated and purified by column chromatography (petroleum ether: ethyl acetate = 6:1) to obtain 81 mg of yellow oil of compound 14b, with a yield of 20%.

[0191] (2) Preparation of target compound II-6:

[0192] 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. Then it was transferred to an oil bath at 65 °C and heated for 12 h. After the reaction, 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, washed with saturated brine (100 mL), the organic phase was collected, and after distillation under reduced pressure, it was separated and purified by column chromatography (methylene chloride: methanol = 14:1) to obtain 90 mg of yellow solid of compound II-6, with a yield of 22%.

[0193] Example 47

[0194] In the preparation process of compound II-7, compound 14b (0.88 mmol) and isopropylamine (2.63 mmol) were prepared into compound II-7 according to the method steps in Example 46, with a yield of 29%.

[0195] Example 48

[0196] 13-(3-((4,5-Dihydrothiazol-2-yl)amino)propyl)-10-methoxy-8,13-dihydroindolo[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one

[0197] Compound 3b and compound 5a were prepared into compound m9 according to the method of Example 41, and then into compound 8c according to the method of Example 43.

[0198] (1) Preparation of intermediate 14c:

[0199] 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. The reaction was carried out overnight at room temperature. After the reaction, 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, washed with saturated brine (100 mL), the organic phase was collected, and after distillation under reduced pressure, it was separated and purified 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%.

[0200] (2) Preparation of the target compound II-8:

[0201] During the preparation of compound II-8, compound 14c (0.88 mmol) and 2-amino-2-thiazoline (2.63 mmol) were prepared into compound II-8 according to the method steps in Example 46, with a yield of 20%.

[0202] Example 49

[0203] During the preparation of compound II-9, compound 14c (0.88 mmol) and isopropylamine (2.63 mmol) were prepared into compound II-9 according to the method steps in Example 46, with a yield of 29%.

[0204] Example 50

[0205] Preparation of 13-(3-((4,5-dihydrothiazol-2-yl)amino)propyl)-3-fluoro-8,13-dihydroindolo[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one (Compound II-10)

[0206] Compound 3a and compound 5b were prepared into compound m10 according to the method of Example 41, and then into compound 8d according to the method of Example 43.

[0207] (1) Preparation of intermediate 14d:

[0208] 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. The reaction was carried out overnight at room temperature. After the reaction, 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, washed with saturated brine (100 mL), the organic phase was collected, and after distillation under reduced pressure, it was separated and purified by column chromatography (petroleum ether: ethyl acetate = 6:1) to obtain 55 mg of a yellow oil of compound 14d, with a yield of 22%.

[0209] (2) Preparation of the target compound II-10:

[0210] During the preparation of compound II-10, compound 14d (0.88 mmol) and 2-amino-2-thiazoline (2.63 mmol) were used to prepare compound II-10 according to the method steps in Example 46, with a yield of 7%.

[0211] Example 51

[0212] During the preparation of compound II-11, compound 14d (0.88 mmol) and isopropylamine (2.63 mmol) were used to prepare compound II-11 according to the method steps in Example 46, with a yield of 27%.

[0213] Example 52

[0214] The preparation of compound II-12 was carried out according to the method of Example 25, using compound II-6 (0.66 mmol) as the raw material, and the others were the same as in Example 25, with a yield of 80%.

[0215] Example 53

[0216] The preparation of compound II-13 was carried out according to the method of Example 25, using compound II-7 (0.66 mmol) as the raw material, and the others were the same as in Example 25, with a yield of 98%.

[0217] Example 54

[0218] The preparation of compound II-14 was carried out according to the method of Example 25, using compound II-8 (0.66 mmol) as the raw material, and the others were the same as in Example 25, with a yield of 44%.

[0219] Example 55

[0220] The preparation of compound II-15 was carried out according to the method of Example 25, using compound II-9 (0.66 mmol) as the raw material, and the others were the same as in Example 25, with a yield of 64%.

[0221] The 1 H NMR, 13 C NMR and MS data of the compounds prepared by the present invention are shown in Table 1 in detail.

[0222] Table 1 Preferred compounds of the present invention 1 H NMR, 13 C NMR and MS data

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232] Example 56

[0233] Induction and stabilization effects of the target compound on c-MYC

[0234] 1. Induction effect of the target compound on c-MYC

[0235] (1) Experimental materials: probe 3’-FAM-c-MYC Pu28-BHQ1-5’, 200 mM KCl, 50 mM Tris acetate buffer, pH = 7.0 and black 96-well plates.

[0236] (2) Experimental instruments: shaker and Tecan microplate reader.

[0237] (3) Experimental method: Take 1 μL of the probe of 3’-FAM-c-MYC Pu28-BHQ1-5’ with a concentration of 100 μM and place it in a black 96-well plate. Add 49 μL of 50 mM Tris acetate buffer, pH = 7.0, to prepare a 2 μM probe. Let it stand on a shaker for 1 h, and then add 50 μL to each well. Then add 50 μL of 20 μM compound or 200 mM KCl to the 2 μM probe to prepare a 1 μM probe. Let it stand on a shaker for 1 h, and each well totals 100 μL. DMSO and KCl are used as control groups. The excitation wavelength of the Tecan microplate reader is set at 490 nm, the emission wavelength is set at 520 nm, and the bandwidth is 20 nm.

[0238] Calculate the final fluorescence intensity percentage (%) using Excel software based on the fluorescence values obtained by the above method.

[0239] (4) Experimental results: As Figure 1 shown, Figure 1 is a schematic diagram of the induction and stabilization effects of rutaecarpine derivatives on c-MYC. Among them, A is a schematic diagram of the induction effect of rutaecarpine derivatives on c-MYC, and C is a schematic diagram of the induction effect of rutaecarpine derivatives on c-MYC. The experimental results show that the tested compounds exhibit a relatively high c-MYC induction effect, and among them, compounds II-7, II-11, and II-13 show the best induction effect.

[0240] 2. Stabilization effect of the target compound on c-MYC

[0241] (1) Experimental materials: Probe 5’-FAM-c-MYC Pu22-TAMRA-3’, 7.5 mM KCl / 2.5 mM PBS, pH = 7.0, and DMSO.

[0242] (2) Experimental instrument: Thermo Fisher quantstudio 3 q-PCR instrument.

[0243] (3) Experimental method: Dilute 100 μM 5’-FAM-c-MYC Pu22-TAMRA-3’ to 400 nM in a buffer containing 7.5 mM KCl / 2.5 mM PBS, pH = 7.0, heat at 95 °C for 5 minutes, and then slowly cool to room temperature. Incubate 10 μL of the annealed 400 nM probe and 10 μL of 20 μM compound overnight at 4 °C. Measure at a rate of 0.9 °C / min from 25 °C to 95 °C using the Thermo Fisher quantstudio 3 q-PCR instrument. Use DMSO as the control group.

[0244] The fluorescence values obtained according to the above method were used to calculate the final ΔT by GraphPad Prism 8.0 software. m (°C).

[0245] (4) Experimental results: As Figure 1 shown, Figure 1 is a schematic diagram of the induction and stabilization effects of rutaecarpine derivatives on c-MYC. Among them, B is the schematic diagram of the stabilization effect of evodiamine derivatives on c-MYC, and D is the schematic diagram of the stabilization effect of rutaecarpine derivatives on c-MYC. The experimental results show that the tested compounds exhibit a high c-MYC stabilization effect, and among them, compounds II-6, II-9, and II-13 exhibit the best stabilization effect.

[0246] Example 57

[0247] Target compound topoisomerase I / II inhibitory activity

[0248] 1. DNA unwinding experiment mediated by topoisomerase I

[0249] (1) Experimental materials: Calf thymus DNA topoisomerase I, negatively supercoiled DNA plasmid pBR322, 10×buffer buffer, 0.1% BSA, agarose, DMSO, and EtBr.

[0250] (2) Experimental instruments: Gel electrophoresis was performed using a BioRad PowerPac electrophoresis apparatus and a Sub-Cell Model electrophoresis tank, and gel scanning and quantification were performed using a BioRad Gel Doc EZ fully automatic gel system.

[0251] (3) Experimental method: First, prepare a 0.8% agarose gel with 1×TAE solution. Add 10 μL of water, 2 μL of 10×buffer buffer, 2 μL of 0.1% BSA, 0.5 U of Top1, 0.5 μL of DNA, and 0.2 μL of different drugs to a 1.5 mL sample tube in sequence, and make up the volume to 20 μL. Then place the sample in a 37°C water bath and incubate for 15 minutes, and observe the electrophoresis results with a gel imaging system.

[0252] (4) Experimental results: As Figure 2 shown, Figure 2It is a schematic diagram of the experimental results of the inhibition of Top1 / 2 by rutaecarpine derivatives. Among them, A is a schematic diagram of the inhibitory effect of rutaecarpine derivative (200 μM) on Top1 enzyme activity, C is a schematic diagram of the inhibitory effect of rutaecarpine derivative (50 μM) on Top1 enzyme activity, D is a schematic diagram of the inhibitory effect of rutaecarpine derivative (25 / 12.5 μM) on Top1 enzyme activity, and E is a schematic diagram of the inhibitory effect of rutaecarpine derivative (10 / 5 / 2.5 / 1 μM) on Top1 enzyme activity. 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, and among them, compounds II-13 and II-15 exhibit the best Top1 inhibitory activity.

[0253] 2. DNA Unwinding Experiment Mediated by Topoisomerase Ⅱ

[0254] (1) Experimental Materials: Calf thymus DNA topoisomerase Ⅱ, negatively supercoiled DNA plasmid pBR322, 30 mM ATP, dilution buffer, assay buffer, agarose, DMSO, and EtBr.

[0255] (2) Experimental Instruments: For gel electrophoresis, a PowerPac electrophoresis apparatus and a Sub-Cell Model electrophoresis tank from BioRad are used. For gel scanning and quantification, a Gel Doc EZ fully automatic gel system from BioRad is used.

[0256] (3) Experimental Method: First, prepare a 0.8% agarose gel with 1×TAE solution. Add 10 μL of water, 2 μL of dilution buffer, 2 μL of assay buffer, 0.5 U of Top2, 0.5 μL of DNA, and 0.2 μL of different drugs into a 1.5 mL sample tube in sequence, and make up the volume to 20 μL. Then place the sample in a 37°C water bath and incubate for 30 minutes, and observe the electrophoresis results with a gel imaging system.

[0257] (4) Experimental Results: As Figure 2 shown, Figure 2It is a schematic diagram of the inhibitory experiment results of rutaecarpine derivatives on Top1 / 2. Among them, B is the schematic diagram of the inhibitory effect of rutaecarpine derivative (200 μM) on Top2 enzyme activity, and F is the schematic diagram of the inhibitory effect of rutecarpine derivative (50 μM) on Top2 enzyme activity. 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.

[0258] Example 58

[0259] In vitro antitumor activity test of the target compound (IC 50 )

[0260] (1) Sample preparation: Dissolve the target compound in DMSO to make a 10 mM solution.

[0261] (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) are cryopreserved and passaged in our laboratory.

[0262] (3) Experimental method: The inhibitory ability of the target compound on the proliferation of four types of tumor cells was tested. The conventional CCK8 method was used for the test. Digest the tumor cells (MCF-7, MDA-MB-231, HCT116 and HeLa) in the logarithmic growth phase with trypsin, and then dilute and suspend the cells into a single-cell suspension with a medium (DMEM or PRMI1640 + 10% FBS + 1% double antibody). Adjust the cell density to 5×10 4 cells / mL, add 100 μL to each well and inoculate in a 96-well plate. Incubate in an incubator at 37 °C and 5% CO2 for 24 hours, then add compounds at different concentrations. Each concentration has three parallel replicates, and set experimental groups and control groups. After continuing to incubate for 72 hours, add 10 μL of CCK8 solution to each well, and then incubate in the dark at 37 °C for 1 - 4 hours. Measure the OD value at 450 nm with a Biotek-Synergy microplate reader and calculate the half-maximal inhibitory concentration IC 50 .

[0263] (4) Experimental results: The half-maximal inhibitory concentration IC 50 values of the target compound on tumor cells are shown in Table 2. The test results show that these multi-target compounds have broad-spectrum antitumor activity. Among them, compound II-13 shows the best IC 50 value against MCF-7 and HeLa cells.

[0264] Table 2 The half inhibitory concentration IC of the target compound cells against tumor cells 50 (unit: μmol / L)

[0265]

[0266]

[0267] CPT is the positive drug camptothecin, and its structure is as follows:

[0268]

[0269] Figure 3 It is a schematic diagram of the results of the transcriptional and expression effects of compound II-13 and rutaecarpine on the c-MYC oncogene. A represents the schematic diagram of the transcriptional effect of compound II-13 and rutaecarpine on the c-MYC oncogene. Compound II-13 has a transcriptional inhibitory trend on the c-MYC oncogene from 0 to 15 μM, and basically inhibits the expression of the c-MYC oncogene at 15 μM. The negative control rutaecarpine does not have a complete transcriptional inhibitory effect on the c-MYC oncogene. B represents the schematic diagram of the expression level results of compound II-13 and rutaecarpine on the c-MYC oncogene. Compound II-13 has a downward trend in the expression level of the c-MYC oncogene from 0 to 15 μM, indicating that compound II-13 has an inhibitory effect on the c-MYC oncogene. With the increase in the concentration of rutaecarpine, the negative control rutaecarpine shows no downward trend in the expression level of the c-MYC oncogene, indicating that rutaecarpine does not have an inhibitory effect on the expression level of the c-MYC oncogene.

[0270] The above description is only a preferred embodiment of the present invention and does not impose any form of limitation on the present invention. Although the present invention has been disclosed above with the preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art of this patent, without departing from the scope of the technical solution of the present invention, may make some changes or modifications using the technical content prompted above into equivalent embodiments of equivalent changes. However, as long as it does not depart from the content of the technical solution of the present invention, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention still fall within the scope of the present invention.

Claims

1. A rutaecarpine derivative or its medicinal salt with multi-target anti-tumor activity, characterized in that, The rutaecarpine derivatives with multi-target anti-tumor activity are selected from one of the following structures:

2. Use of the rutaecarpine derivative with multi-target anti-tumor activity or a pharmaceutically acceptable salt thereof as claimed in claim 1 in the preparation of an anti-tumor drug; the tumors include breast cancer, colon cancer, cervical cancer, and lung cancer.

3. Use of the rutaecarpine derivative with multi-target anti-tumor activity or a pharmaceutically acceptable salt thereof as claimed in claim 1 in the preparation of a Top1 topoisomerase inhibitor, a Top2 topoisomerase inhibitor, and / or a c-MYC oncogene inhibitor.

4. Use of the rutaecarpine derivative with multi-target anti-tumor activity or its pharmaceutically acceptable salt according to claim 1 in the preparation of a drug for treating diseases caused by abnormal gene expression, characterized in that, The disease caused by abnormal gene expression is a tumor.

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