Evodiamine derivatives with multi-target antitumor activity and use thereof

Abstract

This invention discloses a class of Evodia rutaecarpa derivatives or their pharmaceutical salts with multi-target antitumor activity, the general structural formula of which is shown in Formula I or Formula II: wherein: R1 is selected from C1-C5 alkoxy, hydroxyl, and hydrogen; R2 is selected from hydrogen and halogen; R3 is selected from hydroxyl. Enzyme inhibition activity and in vitro antitumor activity tests revealed that the compounds of this invention have strong inhibitory activity against topoisomerase 1, topoisomerase 2 and c-MYC oncogene, opening up new avenues and providing new strategies for in-depth research and development of new structural types of antitumor drugs.

Description

[0001] This application is a divisional application of patent application filed on January 11, 2022, with application number 202210025250.5 and invention title "Evodiae acetylalkaloid derivatives with multi-target antitumor activity and their applications". Technical Field

[0002] This invention belongs to the field of pharmaceutical technology, specifically, it relates to a class of Evodia rutaecarpa derivatives with multi-target antitumor activity and their applications. Background Technology

[0003] Topoisomerases (Top) are important components of ribozymes, playing a crucial role in DNA transcription, replication, and chromatin assembly. Top1 and Top2 induce transient breaks in single-stranded or double-stranded DNA by cleaving individual DNA molecules.

[0004] In the field of DNA-targeted drugs, small molecules stabilizing c-MYC G-quadruplex (c-MYC G4) DNA are considered a promising anti-cancer strategy. c-MYC is one of the most important oncogenes, overexpressed in over 80% of cancer cells and contributing 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 a DNA G-quadruplex (G4) under transcription-associated negative supercoiling, thereby silencing c-MYC transcription. Therefore, c-MYC G4 has become a promising cancer-specific molecular target for anti-tumor drug discovery. Furthermore, the synergistic anti-tumor effect between c-MYC G4 and Top inhibitors has been demonstrated. Therefore, simultaneously targeting Top and c-MYCG4 would be a promising strategy for anti-tumor drug discovery.

[0005] Previous studies have conducted in-depth research on the structure-activity relationship of evodiamine derivatives. Through research on the antitumor mechanism of action, it was found that evodiamine derivatives are dual Top1 and Top2 inhibitors, effectively inducing tumor cell apoptosis. Patent application CN101787025A discloses a substituted evodiamine antitumor and antifungal compound and its preparation method; patent application CN1012311434A discloses an evodiamine compound and its preparation method and application; patent application 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 objective of this invention is to provide a class of evodiamine derivatives with multi-target antitumor activity.

[0008] A second objective of this invention is to provide the application of the evodiamine derivative with the aforementioned multi-target antitumor activity in the preparation of antitumor drugs.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] The first aspect of this invention provides a class of evodiamine derivatives or their pharmaceutical salts with multi-target antitumor activity, 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, and hydrogen;

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

[0015] R3 is selected from hydroxyl group,

[0016]

[0017] Preferably, among the evodiamine derivatives with multi-target antitumor activity,

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

[0019] R2 is selected from hydrogen and fluorine;

[0020] R3 is selected from hydroxyl groups,

[0021]

[0022] Most preferably, the evodiamine derivative with multi-target antitumor activity is selected from one of the following structures:

[0023]

[0024]

[0025]

[0026]

[0027] The medicinal salt is its organic acid salt or inorganic acid salt.

[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] A second aspect of the present invention provides the use of the evodiamine derivative or its pharmaceutical salt with the aforementioned multi-target antitumor activity in the preparation of antitumor drugs.

[0030] The tumors are selected from breast cancer, colon cancer, cervical cancer, lung cancer, etc.

[0031] A third aspect of the present invention provides the use of the evodia alkaloid derivative or its medicinal salt with the multi-target antitumor activity in the preparation of Top1 topoisomerase inhibitors, Top2 topoisomerase inhibitors and / or c-MYC oncogene inhibitors.

[0032] The multi-target antitumor active derivatives of Evodia rutaecarpa or their medicinal salts can be used as Top1, Top2 and c-MYC three-target inhibitors to treat malignant tumors or diseases related to differentiation and proliferation.

[0033] The tumors mentioned include breast cancer, colon cancer, cervical cancer, etc.

[0034] The fourth aspect of the present invention provides the use of the evodiamine derivative or its pharmaceutical salt with the aforementioned multi-target antitumor activity in the preparation of a medicament for treating diseases caused by abnormal gene expression.

[0035] The diseases caused by abnormal gene expression include tumors, endocrine disorders, immune system diseases, genetic diseases, or nervous system diseases.

[0036] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0037] This invention provides a class of evodiamine derivatives with multi-target antitumor activity. These evodiamine derivatives possess multi-target antitumor activity, with the multi-target being a three-target inhibitor of Top1 / Top2 / c-MYC. Enzyme inhibition activity and in vitro antitumor activity tests revealed that the compounds of this invention have strong inhibitory activity against topoisomerase 1, topoisomerase 2, and the c-MYC oncogene. This opens up new avenues and provides new strategies for in-depth research and development of antitumor drugs with novel structural types.

[0038] This invention provides a class of Evodia rutaecarpa derivatives with multi-target antitumor activity. These are small molecule anticancer drugs targeting multiple targets, including topoisomerase 1 (Top1), topoisomerase 2 (Top2), and the c-MYC oncogene. The compounds of this invention exhibit good inhibitory activity against Top1, Top2, and c-MYC oncogenes, and also have a certain broad-spectrum antitumor activity, making them suitable for use as antitumor drugs. Attached Figure Description

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

[0040] Figure 2 This is a schematic diagram of the experimental results of the inhibition of Top1 / 2 by the Evodia rutaecarpa alkaloid derivative.

[0041] Figure 3 This is a schematic diagram showing the effects of compound II-13 and evodiamine on the transcription and expression of the c-MYC oncogene. Detailed Implementation

[0042] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0043] Unless otherwise specified, the experimental methods described in the following examples are generally performed under standard conditions or as recommended by the manufacturer.

[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 the reaction was carried out overnight at 70 °C. After the reaction was complete, 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 reaction.

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

[0051] 5.0 g (0.032 mol) of anthranilic acid 4a was dissolved in 100 mL of dry tetrahydrofuran, 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 was complete, the reaction solution was poured into 400 mL of ice-cold saturated brine. Once a white solid had completely precipitated, the mixture was filtered, washed twice with water, and the filter cake was dried in an oven at 45 °C. 5.0 g of a white solid, compound 5a (R₂ = H), was obtained, with a yield of 86%.

[0052] Compound 5a (5.0 g, 0.03 mol) was dissolved in 60 mL of dry DMF. Sodium hydride (60% purity, 0.65 g, 0.016 mol) was slowly added under ice bath conditions. After half an hour, 1.1 mL of iodomethane (0.016 mol) was added, and the reaction was allowed to proceed at room temperature for 4 hours. After the reaction was complete, the reaction solution was poured into 400 mL of ice-cold saturated brine, stirred, filtered, and the filter cake was dried in an oven at 45 °C to obtain 2.5 g of white solid, which is 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 100 mL of dry dichloromethane and reacted at room temperature for 6 h. After the reaction was complete, the mixture was filtered, washed twice with dichloromethane, and the filter cake was freeze-dried to give 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). Sodium hydride (60% purity, 53 mg, 1.320 mmol) was slowly added under ice bath conditions. After half an hour, 3-bromopropanol (95 mg, 0.684 mmol) was slowly added dropwise. The mixture was then 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 extracted with water (100 mL) and ethyl acetate (40 mL × 3). The organic phases were combined, washed with saturated brine (100 mL), and collected. The organic phase was then purified by column chromatography (petroleum ether: ethyl acetate = 2:1) after vacuum distillation to obtain 120 mg of compound I-1 as a yellow solid, with a yield of 33%.

[0055] Example 2

[0056] In 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] In 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] In the preparation of compound I-4, 3-bromopropanol was replaced with 3-chloro-N-methylprop-1-amine hydrochloride (0.684 mmol), otherwise it was the same as in Example 1, and the yield of compound I-4 was 28%.

[0061] Example 5

[0062] In the preparation of compound I-5, 3-bromopropanol was replaced with 3-chloro-1-(N,N-dimethyl)propane (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]pyridine[2,1-b]quinazolin-5(7H)-one (compound I-6)

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

[0066] 10.0 g (0.053 mol) of 5-methoxytryptamine compound 1b (R1 = OCH3) was added to ethyl formate solution (125.3 mL, 1.576 mol) and reacted overnight at 70 °C. After the reaction was complete, the reaction solution was evaporated to dryness, and the product weight (11.6 g, 0.053 mol) was used directly in 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, stirring overnight. After the reaction was complete, 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 under ice bath conditions. Impurities were removed by vacuum filtration, and the filtrate was adjusted to pH = 11.0 with ammonia. A solid precipitated out, and was filtered to give 9.46 g 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-fluoroindosan anhydride

[0068] 5.0 g (0.032 mol) of anthranilic acid 4b (R2=F) was dissolved in 100 mL of dry tetrahydrofuran, 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 was complete, the reaction solution was poured into 400 mL of ice-cold saturated brine. Once a white solid had completely precipitated, the mixture was filtered, washed twice with water, and the filter cake was dried in an oven at 45 °C. 5.0 g of compound 5b was 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-methylindigo anhydride

[0070] Intermediate 5b (R2=F) (5.0 g, 0.028 mol) was dissolved in 60 mL of dry DMF. Sodium hydride (60% purity, 1.3 g, 0.033 mol) was slowly added under ice bath conditions. After half an hour, iodomethane (2.1 mL, 0.033 mol) was added, and the reaction was carried out at room temperature for 4 h. After the reaction was complete, the reaction solution was poured into 400 mL of ice-cold saturated brine, stirred, filtered, and the filter cake was dried in an oven at 45 °C to give 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).

[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 100 mL of dry dichloromethane and reacted at room temperature for 6 h. After the reaction was complete, the mixture was filtered, washed twice with dichloromethane, and the filter cake was freeze-dried. 5.2 g of a yellow solid, compound 7b (R1 = OCH3, R2 = F), was obtained, 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] Intermediate 7b (200 mg, 0.570 mmol) was dissolved in dry DMF (5 mL). Sodium hydride (60% purity, 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. The mixture was then 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 extracted with water (100 mL) and ethyl acetate (40 mL × 3). The organic phases were combined, washed with saturated brine (100 mL), and collected. The organic phase was then purified by column chromatography (petroleum ether: ethyl acetate = 2:1) after vacuum distillation to obtain 120 mg of compound I-6 as a yellow solid, with a yield of 52%.

[0075] Example 7

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

[0077] Example 8

[0078] In 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] In the preparation of compound I-9, 3-bromopropanol was replaced with 3-chloro-N-methylprop-1-amine hydrochloride (0.684 mmol), otherwise it was the same as in Example 6, and the yield of compound I-9 was 33%.

[0081] Example 10

[0082] In 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] Example 11

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

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

[0086] Example 12

[0087] In 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] In the preparation of compound I-13, compound 7c (0.660 mmol) and 4-(5-chloropropyl)morpholine (0.684 mmol) were prepared according to the method steps in Example 1 to obtain compound I-13, with a yield of 51%.

[0090] Example 14

[0091] In the preparation of compound I-14, compound 7c (0.660 mmol) and 3-chloro-N-methylprop-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] Example 15

[0093] In 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~I-24 and I-35~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] 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, followed by 1,3-dibromopropane (2.0 g, 9.90 mmol), and the mixture was reacted overnight at room temperature. After the reaction was complete, the reaction mixture was poured into a 250 mL separatory funnel, and extracted with water (100 mL) and ethyl acetate (40 mL × 3). The organic phases were combined, washed with saturated brine (100 mL), and collected. The organic phase was then purified by column chromatography (petroleum ether: ethyl acetate = 6:1) after vacuum distillation to obtain 360 mg of a yellow oily compound 12a, in 26% yield.

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

[0102] Intermediate 12a (120 mg, 0.284 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). The mixture was then transferred to a 65 °C oil bath and heated for 12 h. After the reaction was complete, the reaction mixture was poured into a 250 mL separatory funnel, extracted with water (100 mL) and ethyl acetate (40 mL × 3), and the organic phases were combined. The organic phase was washed with saturated brine (100 mL), collected, and purified by column chromatography (dichloromethane:methanol = 14:1) after vacuum distillation to obtain 50 mg of a yellow solid, compound I-16, in 40% yield.

[0103] Example 17

[0104] In 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] Example 18

[0106] In 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] Example 19

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

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

[0110] Intermediate 7b (200 mg, 0.57 mmol) was dissolved in dry DMF (5 mL), potassium hydroxide (64 mg, 1.14 mmol) was added, followed by 1,3-dibromopropane (345 mg, 1.71 mmol), and the mixture was reacted overnight at room temperature. After the reaction was complete, the reaction mixture was poured into a 250 mL separatory funnel, and extracted with water (100 mL) and ethyl acetate (40 mL × 3). The organic phases were combined, washed with saturated brine (100 mL), and collected. The organic phase was then purified by column chromatography (petroleum ether: ethyl acetate = 6:1) after vacuum distillation to obtain 85 mg of a yellow oily substance, compound 12b, in 32% yield. 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, followed by 2-amino-2-thiazoline (87 mg, 0.85 mmol). The mixture was then 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 extracted with water (100 mL) and ethyl acetate (40 mL × 3). The organic phases were combined, washed with saturated brine (100 mL), and collected. The organic phase was then purified by column chromatography (dichloromethane:methanol = 14:1) under reduced pressure to obtain 60 mg of a yellow solid, compound I-19, in 48% yield.

[0113] Example 20

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

[0115] Example 21

[0116] In 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] Example 22

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

[0119] 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), and the mixture was reacted overnight at room temperature. After the reaction was complete, the reaction mixture was poured into a 250 mL separatory funnel, and extracted with water (100 mL) and ethyl acetate (40 mL × 3). The organic phases were combined, washed with saturated brine (100 mL), and collected. The organic phase was then purified by column chromatography (petroleum ether: ethyl acetate = 6:1) after vacuum distillation to obtain 68 mg of a yellow oily compound 12c, in 25% yield.

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

[0121] Example 23

[0122] In 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] Example 24

[0124] In 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] Example 25

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

[0127] 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 and the reaction proceeded at -78 °C for 2 h, followed by overnight reaction at room temperature. After the reaction was complete, 25 mL of ice-cold NaHCO3 aqueous solution was added, and the mixture was stirred for 30 min. The mixture was extracted with dichloromethane (40 mL × 3), and the organic phase was collected. After vacuum distillation, the organic phase was purified by column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain 150 mg of a yellow solid, compound I-25, in 58% yield.

[0128] Example 26

[0129] Compound I-26 was prepared according to the method of Example 25, using compound I-7 (0.66 mmol) as the starting material, and otherwise consistent with Example 25, with a yield of 30%.

[0130] Example 27

[0131] Compound I-27 was prepared according to the method of Example 25, using compound I-8 (0.66 mmol) as the starting material, and otherwise consistent with Example 25, with a yield of 12%.

[0132] Example 28

[0133] Compound I-28 was prepared according to the method of Example 25, using compound I-9 (0.66 mmol) as the starting material, and otherwise consistent with Example 25, with a yield of 81%.

[0134] Example 29

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

[0136] Example 30

[0137] Compound I-30 was prepared according to the method of Example 25, using compound I-11 (0.66 mmol) as the starting material, and otherwise consistent with Example 25, with a yield of 47%.

[0138] Example 31

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

[0140] Example 32

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

[0142] Example 33

[0143] Compound I-33 was prepared according to the method of Example 25, using compound I-14 (0.66 mmol) as the starting material, and otherwise consistent with Example 25, with a yield of 68%.

[0144] Example 34

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

[0146] Example 35

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

[0148] Example 36

[0149] Compound I-36 was prepared according to the method of Example 25, using compound I-20 (0.66 mmol) as the starting material, and otherwise consistent with Example 25, with a yield of 50%.

[0150] Example 37

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

[0152] Example 38

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

[0154] Example 39

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

[0156] Example 40

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

[0158] Example 41

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

[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 10 mL of dry dichloromethane and reacted at room temperature for 6 h. After the reaction was complete, the mixture was filtered, washed twice with dichloromethane, and the filter cake was freeze-dried. 34 mg of a yellow solid, compound m7, was given, in 10% yield. 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] Intermediate m7 (200 mg, 0.69 mmol) was dissolved in dry DMF (10 mL), cesium carbonate (670 mg, 2.08 mmol) was added, followed by (3-chloropropyl)pyrrolidine (120 mg, 0.83 mmol). The mixture was then transferred to a 65 °C oil bath and heated for 12 h. After the reaction was complete, the reaction mixture was poured into a 250 mL separatory funnel, extracted with water (100 mL) and ethyl acetate (40 mL × 3), and the organic phases were combined. The organic phase was washed with saturated brine (100 mL), collected, and purified by column chromatography (dichloromethane:methanol = 19:1) after vacuum distillation to obtain 70 mg of yellow solid compound II-1, with a yield of 25%.

[0166] Example 42

[0167] 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 according to the method steps in Example 41 to obtain compound II-2, with a yield of 65%.

[0168] Example 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] Intermediate m7 (200 mg, 0.69 mmol) was dissolved in dry DCM (10 mL), and 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) (157 mg, 0.69 mmol) was added. The reaction was carried out at room temperature for 8 h. After the reaction was complete, the mixture was purified by column chromatography (dichloromethane:methanol = 100:1) under reduced pressure to give 90 mg of a yellow solid, compound 8a, in 45% yield.

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

[0174] Compound 8a (200 mg, 0.70 mmol) was dissolved in dry DMF (5 mL). Sodium hydride (60% purity, 111 mg, 2.79 mmol) was slowly added under ice bath conditions. After half an hour, 3-chloro-N-methylpropyl-1-amine hydrochloride (1.39 mmol) was slowly added dropwise. The mixture was then 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 extracted with water (100 mL) and ethyl acetate (40 mL × 3). The organic phases were combined, washed with saturated brine (100 mL), and collected. The organic phase was then purified by column chromatography (dichloromethane:methanol = 100:6) after vacuum distillation to obtain 19 mg of compound II-3 as a yellow solid, 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] Example 44

[0180] Preparation of 13-(3-((4,5-dihydrothiazolyl-2-yl)amino)propyl)-8,13-dihydroindolo[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one (compound II-4)

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

[0182] Intermediate 8a (200 mg, 0.70 mmol) was dissolved in dry DMF (5 mL), potassium hydroxide (78 mg, 1.39 mmol) was added, followed by 1-bromo-3-chloropropane (329 mg, 2.09 mmol), and the mixture was reacted overnight at room temperature. After the reaction was complete, the reaction mixture was poured into a 250 mL separatory funnel, and extracted with water (100 mL) and ethyl acetate (40 mL × 3). The organic phases were combined, washed with saturated brine (100 mL), and collected. The organic phase was then purified by column chromatography (petroleum ether: ethyl acetate = 6:1) after vacuum distillation to obtain 54 mg of a yellow oily compound 14a, with a yield of 21%.

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

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

[0185] Example 45

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

[0187] Example 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] Compounds 3b and 5b were prepared into compound m8 according to the method of Example 41, and then compound 8b was prepared according to the method of Example 43.

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

[0191] Intermediate 8b (200 mg, 0.60 mmol) was dissolved in dry DMF (5 mL), potassium hydroxide (67 mg, 1.19 mmol) was added, followed by 1-bromo-3-chloropropane (282 mg, 1.79 mmol), and the mixture was reacted overnight at room temperature. After the reaction was complete, the reaction mixture was poured into a 250 mL separatory funnel, and extracted with water (100 mL) and ethyl acetate (40 mL × 3). The organic phases were combined, washed with saturated brine (100 mL), and collected. The organic phase was purified by column chromatography (petroleum ether: ethyl acetate = 6:1) after vacuum distillation 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, followed by 2-amino-2-thiazoline (268 mg, 2.63 mmol). The mixture was then transferred to a 65°C oil bath and heated for 12 h. After the reaction was complete, the reaction mixture was poured into a 250 mL separatory funnel, extracted with water (100 mL) and ethyl acetate (40 mL × 3), and the organic phases were combined. The organic phase was washed with saturated brine (100 mL), collected, and purified by column chromatography (dichloromethane:methanol = 14:1) after vacuum distillation to give 90 mg of compound II-6 as a yellow solid, with a yield of 22%.

[0194] Example 47

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

[0196] Example 48

[0197] 13-(3-((4,5-dihydrothiazolyl-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 compound 8c was prepared according to the method of Example 43.

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

[0200] Intermediate 8c (200 mg, 0.63 mmol) was dissolved in dry DMF (5 mL), potassium hydroxide (71 mg, 1.26 mmol) was added, followed by 1-bromo-3-chloropropane (298 mg, 1.89 mmol), and the mixture was reacted overnight at room temperature. After the reaction was complete, the reaction solution was poured into a 250 mL separatory funnel, and extracted with water (100 mL) and ethyl acetate (40 mL × 3). The organic phases were combined, washed with saturated brine (100 mL), and collected. The organic phase was then purified by column chromatography (petroleum ether: ethyl acetate = 6:1) after vacuum distillation to obtain 48 mg of a yellow oily substance, compound 14c, in 19% yield.

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

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

[0203] Example 49

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

[0205] Example 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] Compounds 3a and 5b were prepared into compound m10 according to the method of Example 41, and then compound 8d was prepared according to the method of Example 43.

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

[0209] Intermediate 8d (200 mg, 0.66 mmol) was dissolved in dry DMF (5 mL), potassium hydroxide (74 mg, 1.31 mmol) was added, followed by 1-bromo-3-chloropropane (310 mg, 1.97 mmol), and the reaction was allowed to proceed overnight at room temperature. After the reaction was complete, the reaction mixture was poured into a 250 mL separatory funnel, and extracted with water (100 mL) and ethyl acetate (40 mL × 3). The organic phases were combined, washed with saturated brine (100 mL), and collected. The organic phase was then purified by column chromatography (petroleum ether: ethyl acetate = 6:1) after vacuum distillation to obtain 55 mg of a yellow oily substance, compound 14d, in 22% yield.

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

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

[0212] Example 51

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

[0214] Example 52

[0215] Compound II-12 was prepared according to the method of Example 25, using compound II-6 (0.66 mmol) as the starting material, and otherwise consistent with Example 25, with a yield of 80%.

[0216] Example 53

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

[0218] Example 54

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

[0220] Example 55

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

[0222] The compounds prepared in this invention 1 H NMR, 13 Detailed C NMR and MS data are shown 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] Example 56

[0234] Inducing and stabilizing effects of the target compound on c-MYC

[0235] 1. The inducing effect of the target compound on c-MYC

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

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

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

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

[0240] (4) Experimental results: such as Figure 1 As shown, Figure 1 This is a schematic diagram illustrating the induction and stabilization effects of evodiamine derivatives on c-MYC. In the diagram, A represents the induction effect of evodiamine derivatives on c-MYC, and C represents the induction effect of evodiamine derivatives on c-MYC. The experimental results show that the tested compounds exhibit high c-MYC induction activity, with compounds II-7, II-11, and II-13 showing the best induction.

[0241] 2. Stabilizing effect of the target compound on c-MYC

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

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

[0244] (3) Experimental Methods: 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 to 95 °C for 5 minutes, and then slowly cooled to room temperature. A mixture containing 10 μL of the annealed 400 nM probe and 10 μL of the 20 μM compound was incubated overnight at 4 °C. The temperature was increased from 25 °C to 95 °C at a rate of 0.9 °C / min, and the results were measured using a Thermo Fisher Quantstudio 3q-PCR instrument. DMSO was used as a control group.

[0245] Based on the fluorescence values ​​obtained using the above method, the final ΔT was calculated using GraphPad Prism 8.0 software. m (°C).

[0246] (4) Experimental results: such as Figure 1 As shown, Figure 1 This diagram illustrates the induction and stabilization effects of evodiamine derivatives on c-MYC. In diagram B, evodiamine derivatives stabilize c-MYC, and in diagram D, they stabilize c-MYC. The experimental results show that the tested compounds exhibit high c-MYC stabilization effects, with compounds II-6, II-9, and II-13 showing the best stabilizing effects.

[0247] Example 57

[0248] The target compound's topoisomerase I / II inhibitory activity

[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 apparatus: Gel electrophoresis was performed using a BioRad PowerPac electrophoresis apparatus and a Sub-Cell Model electrophoresis tank, and gel scanning quantification was performed using a BioRad Gel Doc EZ fully automated gel system.

[0252] (3) Experimental method: First, prepare a 0.8% agarose gel with 1×TAE solution. Then, add 10 μL of water, 2 μL of 10× 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, and bring the volume to 20 μL. Then, place the sample in a 37℃ water bath and incubate for 15 minutes. Observe the electrophoresis results using a gel imaging system.

[0253] (4) Experimental results: such as Figure 2 As shown, Figure 2This diagram illustrates the results of Top1 / 2 inhibition experiments using Evodia rutaecarpa derivatives. A represents the inhibitory effect of Evodia rutaecarpa derivative (200 μM) on Top1 enzyme activity; C represents the inhibitory effect of Evodia rutaecarpa derivative (50 μM) on Top1 enzyme activity; D represents the inhibitory effect of Evodia rutaecarpa derivative (25 / 12.5 μM) on Top1 enzyme activity; and E represents the inhibitory effect of Evodia rutaecarpa derivative (10 / 5 / 2.5 / 1 μM) on Top1 enzyme activity. The results show that compounds II-5, II-13, II-11, II-8, II-9, II-14, and II-15 exhibited strong Top1 inhibitory activity at 50 μM, with compounds II-13 and II-15 showing 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, 30mM ATP, dilution buffer, assay buffer, agarose, DMSO and EtBr.

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

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

[0258] (4) Experimental results: such as Figure 2 As shown, Figure 2This is a schematic diagram of the inhibitory effect of evodiamine derivatives on Top1 / 2 enzyme activity. In the diagram, B is the result of the inhibitory effect of evodiamine derivative (200 μM) on Top2 enzyme activity, and F is the result of the inhibitory effect of evodiamine 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.

[0259] Example 58

[0260] In vitro antitumor activity assay of the target compound (IC) 50 )

[0261] (1) Sample preparation: The target compound was prepared into 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 methods: The inhibitory effect of the target compound on tumor cell proliferation was tested using four methods, employing the conventional CCK8 assay. Tumor cells in logarithmic growth phase (MCF-7, MDA-MB-231, HCT116, and HeLa) were digested with trypsin, then diluted and resuspended in culture medium (DMEM or PRMI1640 + 10% FBS + 1% penicillin antibiotics) to form a single-cell suspension, adjusting the cell density to 5 × 10⁶ cells / year. 4 100 μL of the compound was seeded per well in a 96-well plate at a concentration of 1 / mL. The plates were incubated at 37°C with 5% CO2 for 24 hours. Different concentrations of the compound were then added, with three replicates for each concentration. Experimental and control groups were also established. After incubation for another 72 hours, 10 μL of CCK8 solution was added to each well. The plates were then incubated at 37°C in the dark for 1-4 hours. The OD value at 450 nm was measured using a Biotek-Synergy microplate reader, and the half-maximal inhibitory concentration (IC50) was calculated. 50 .

[0264] (4) Experimental results: The half-maximal inhibitory concentration (IC50) of the target compound on tumor cells 50 The values ​​are shown in Table 2. The test results show that these multi-target compounds have broad-spectrum antitumor activity, among which compound II-13 showed the best inhibitory activity against MCF-7 and HeLa cells with an IC50 value. 50 value.

[0265] Table 2. Half-maximal inhibitory concentration (IC50) of the target compound against tumor cells. 50 (Unit: μmol / L)

[0266]

[0267]

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

[0269]

[0270] Figure 3 This diagram illustrates the effects of compound II-13 and rutaecarpine on the transcription and expression of the c-MYC oncogene. A shows the transcriptional effects of these two compounds on the c-MYC oncogene. Compound II-13 showed a trend of transcriptional repression against the c-MYC oncogene from 0-15 μM, essentially inhibiting its expression at 15 μM. The negative control, rutaecarpine, did not completely repress the c-MYC oncogene. B shows the expression levels of compound II-13 and rutaecarpine on the c-MYC oncogene. Compound II-13 showed a decreasing trend in c-MYC oncogene expression from 0-15 μM, indicating that it has an inhibitory effect on the c-MYC oncogene. As the concentration of rutaecarpine increased, the negative control rutaecarpine did not show a decreasing 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.

[0271] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A class of evodiamine derivatives or their pharmaceutical salts with multi-target antitumor activity, characterized in that, The evodiamine derivative with multi-target antitumor activity is selected from one of the following structures: 。 2. The use of a multi-target antitumor activity evodiamine derivative or its pharmaceutical salt as described in claim 1 in the preparation of antitumor drugs, characterized in that, The tumor was selected from breast cancer and colon cancer.

3. The application of a multi-target antitumor alkaloid derivative or its pharmaceutical salt in the preparation of antitumor drugs, characterized in that, The tumor was selected from cervical cancer. The evodiamine derivative with multi-target antitumor activity is selected from one of the following structures: 。

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