Water-soluble evodiamine derivatives and uses thereof

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to a water-soluble evodiamine derivative and its application, with the following structural formula: wherein X is a C1-6 alkylene group or X together with R forms R1 and R2 independently selected from hydroxyl or C1-20 alkoxy groups; R is a hydroxyl group with or without alkylene group, a carboxyl group with or without alkylene group, a C1-20 oxane group, an amino group, a substituted amino group with or without alkylene group, or a heterocyclic amino group with or without alkylene group. This invention can improve its water solubility and antitumor effect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a water-soluble evodiamine derivative and its application. Background Technology

[0002] Liver cancer is the fourth most common cancer and the second most deadly cancer in my country, accounting for half of all new cases and deaths worldwide. Given this regional disparity, it is imperative for my country to independently explore multiple effective clinical treatment protocols for liver cancer.

[0003] Evodiamine is an alkaloid isolated from the fruit of Evodia rutaecarpa with a novel quinazoline-carbamoline skeleton and possesses broad-spectrum antitumor activity. However, studies have revealed that evodiamine suffers from poor water solubility, weak target inhibitory activity, low antitumor activity, and poor metabolic stability, which significantly limit its further clinical development.

[0004] Therefore, in recent years, a series of evodiamine derivatives have been developed using evodiamine as the lead structure of antitumor drugs. For example, CN113683615A, CN113563336A, CN111620871A, and CN110066281A have disclosed evodiamine derivatives with different structural types.

[0005] In previous studies, a class of N(14)-aryl-substituted evodiamine derivatives were discovered that can serve as dual-target inhibitors of topoisomerases 1 and 2 (J.Med.Chem.2022, 65, 7975-7992, https: / / doi.org / 10.1021 / acs.jmedchem.2c00520). Comparative compounds A and B significantly inhibited the proliferation, invasion, and migration of human hepatocellular carcinoma cells Huh7 and SK-Hep-1, blocked the cell cycle to the G2 / M phase, and induced apoptosis. Furthermore, in vivo animal models showed that comparative compound B significantly inhibited tumor growth more than sorafenib, a first-line clinical anti-hepatocellular carcinoma drug.

[0006]

[0007] However, in the existing technology, there is still huge room for improvement in the antitumor activity (especially anti-liver cancer activity), drug-likeness, and especially water solubility of evodiamine derivatives. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a water-soluble evodiamine derivative and its application, which significantly improves its water solubility while ensuring good anti-tumor effect.

[0009] This invention relates to a water-soluble erythropoietin derivative, with the following structural formula:

[0010]

[0011] Where X is a C1-6 alkylene group or Or X and R together form

[0012] R1 and R2 are independently selected from hydroxyl or C1-20 alkoxy groups;

[0013] R is a hydroxyl group with or without alkylene, a carboxyl group with or without alkylene, a C1-20 oxane group, an amino group, a substituted amino group with or without alkylene, or a heterocyclic amino group with or without alkylene.

[0014] The alkylene group is an alkyl group in which the C atom has been substituted, such as -CH2-, -(CH2)2-, or -C(CH3). 2- etc., including straight-chain alkylene or branched alkylene.

[0015] The oxaalkyl group is an alkyl group in which one or more -CH2- groups are replaced by -O- groups, including alkoxy groups, such as methoxy, CH3O(CH2CH2O)2CH2- or CH3O(CH2CH2O)3CH2-.

[0016] Preferably, R1 and R2 are independently selected from hydroxyl or C1-6 alkoxy groups.

[0017] Preferably, R1 and R2 are both hydroxyl or methoxy.

[0018] Preferably, R is a hydroxyl group with or without C1-6 alkylene groups, a carboxyl group with or without C1-6 alkylene groups, a C1-6 oxane group, an amino group, a substituted amino group with or without C1-6 alkylene groups, or a heterocyclic amino group with or without C1-6 alkylene groups.

[0019] Preferably, the substituted amino group is an amino group that is simultaneously substituted by the same substituent; the heterocyclic amino group is a five- or six-membered heterocycle containing N or simultaneously containing N and O.

[0020] Preferably, the carbon or nitrogen atom on the five- or six-membered heterocycle is replaced by an alkyl group or a five- or six-membered heterocycle containing nitrogen, wherein the five- or six-membered heterocycle is preferably...

[0021] Preferably, it also includes salts of the water-soluble evodiamine derivative.

[0022] Preferably, it is any one of the following compounds:

[0023]

[0024]

[0025]

[0026] This invention provides an application of a water-soluble evodiamine derivative, which is used to prepare a drug for treating antitumor diseases, preferably liver cancer.

[0027] The beneficial effects of this invention are that, based on the evodiamine derivative, the substituents have been adjusted, and the adjusted evodiamine derivative has a significant improvement in antitumor activity, especially in water solubility, which can greatly improve the speed and extent of oral absorption of the drug and improve bioavailability. Attached Figure Description

[0028] Figure 1 The inhibitory activity of compound 19 against topoisomerase I.

[0029] Figure 2 The figure shows the effect of compound 19 on the cell cycle of Huh7 cells.

[0030] Figure 3 The figure shows the effect of compound 19 on apoptosis in Huh7 cells.

[0031] Figure 4 The antitumor effect of compound 19 in a human hepatocellular carcinoma Huh7 xenograft model (administration route: intraperitoneal injection; dosing frequency: once a day; MPK means: mg / kg). Detailed Implementation

[0032] Example 1

[0033] Preparation of 1,3-difluoro-14-(3-fluorophenyl)-10-(2-hydroxyethoxy)-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one

[0034]

[0035] Step 1: Place 90 mmol of 2-bromo-3,5-difluorobenzoic acid, 9 mmol of copper powder, and 180 mmol of potassium carbonate in a dry 500 mL round-bottom flask. Add 100 mL of water and stir magnetically. Add 110 mmol of 3-fluoroaniline and heat at 105 °C for 5 h. Cool the reaction solution to approximately 60 °C, add activated carbon, and stir for 2 h. Filter the solution while hot to obtain the filtrate. Adjust the pH to 3, filter again to obtain the solid, and dry to obtain a white solid with a yield of 79%.

[0036] Step 2: 3,5-Difluoro-2-((3-fluorophenyl)amino)benzoic acid (50 mmol), 5-methoxytryptamine (50 mmol), EDCI (100 mmol), and HOBt (100 mmol) were added sequentially to a round-bottom flask, and DMF (60 mL) was added. The mixture was magnetically stirred at room temperature. After 12 h of reaction, water was slowly added to the reaction solution, and a solid precipitated. The mixture was stirred for 2 h, filtered, and the solid was slurried with ethyl acetate to give a pale yellow solid product with a yield of 80%.

[0037] Step 3: Step 2: Dissolve 50 mmol of 3,5-difluoro-2-((3-fluorophenyl)amino)-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)benzamide in anhydrous DCM, under argon protection, and stir at -40°C for 30 min. Add boron tribromide (150 mmol), slowly raise the temperature to room temperature, and continue the reaction for 2 h. Quench with water in an ice bath, extract and column chromatography to obtain a yellow solid with a yield of 67%.

[0038] Step 4: Weigh 70 mmol of 3,5-difluoro-2-((3-fluorophenyl)amino)-N-(2-(5-hydroxy-1H-indol-3-yl)ethyl)benzamide into a 500 mL round-bottom flask, add 100 mL of DMF, 210 mmol of triethyl orthoformate, and 70 mmol of boron trifluoride ether. Under evacuation and argon protection, heat under reflux at 130 °C for 12 h. After cooling, slowly add water and saturated sodium bicarbonate aqueous solution until no solid precipitates. Stir for 4 h, filter, dry the filter cake, and then beat with acetonitrile to obtain a pale yellow solid with a yield of 85%.

[0039] Step 5: 1,3-Difluoro-14-(3-fluorophenyl)-10-hydroxy-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one (1 mmol), potassium carbonate (3 mmol), and DMF (20 mL) were added sequentially to a round-bottom flask and stirred at room temperature for 30 minutes. Then, 2-iodoethanol (2 mmol) was added, and the mixture was heated to 60 °C and reacted for another 5 hours. The reaction was monitored by TLC and found to be complete. The reaction mixture was then transferred to a separatory funnel containing 200 mL of ethyl acetate, washed three times with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain a pale yellow solid in 89% yield.

[0040] 1H NMR(500MHz, CDCl3)δ8.5(s,1H),7.7(dd,J=8.5,2.8Hz,1H),7.3-7.3(m,1H),7.2(q,J=7.7Hz,1H),7.0( ddd,J=10.6,8.0,2.9Hz,1H),6.9(d,J=6.8Hz,2H),6.8(td,J=8.3,2.5Hz,1H),6.8(dd,J=8.1,2.1Hz,1H ),6.7(dt,J=10.2,2.4Hz,1H),6.3(s,1H),4.8(dd,J=13.2,5.5Hz,1H),4.1(t,J=4.5Hz,2H),4.0(t,J=4 .4Hz, 2H), 3.2 (td, J=12.3, 4.8Hz, 1H), 2.8 (ddd, J=16.7, 11.3, 5.6Hz, 1H), 2.7 (dd, J=15.6, 4.6Hz, 1H).

[0041] Example 2

[0042] Preparation of 1,3-difluoro-14-(3-fluorophenyl)-10-(2-methoxyethoxy)-8,13,13b-14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one

[0043]

[0044] The method was similar to that in Example 1, except that 2-iodoethanol was replaced with 1-iodo-2-methoxyethane.

[0045] 1H NMR (500MHz, CDCl3) δ8.4(s,1H),7.7(dd,J=8.3,2.9Hz,1H),7.3(d,J=8.1Hz,1H),7.2(q,J=7.9Hz,1H),7.0(ddd ,J=10.5,8.0,2.9Hz,1H),6.9(dd,J=8.8,2.4Hz,1H),6.9(d,J=2.5Hz,1H),6.8(td,J=8.2,2.4Hz,1H),6.7(dd,J =8.2,2.2Hz,1H),6.7(dt,J=10.3,2.5Hz,1H),6.3(s,1H),4.8(dd,J=13.1,5.5Hz,1H),4.2-4.1(m,2H),3.8-3.7 (m,2H),3.5(s,3H),3.2(td,J=12.3,4.8Hz,1H),2.8(td,J=13.1,11.7,5.0Hz,1H),2.7(dd,J=15.6,4.6Hz,1H).

[0046] Example 3

[0047] Preparation of 1,3-difluoro-14-(3-fluorophenyl)-10-(2-hydroxy-2-methylpropoxy)-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one

[0048]

[0049] The method was similar to that in Example 1, except that 2-iodoethanol was replaced with methyl propylene oxide.

[0050] 1 H NMR (500MHz, CDCl3) δ8.3(s,1H),7.7-7.6(m,1H),7.3(d,J=8.6Hz,1H),7.2(q,J=7.7Hz,1H),7.0(d dd,J=10.5,7.9,2.9Hz,1H),7.0-6.9(m,2H),6.8(td,J=8.3,2.5Hz,1H),6.7(dd,J=8.1,2.2Hz,1H) ,6.7(dt,J=10.2,2.4Hz,1H),6.3(s,1H),4.8(dd,J=13.1,5.5Hz,1H),3.8(s,2H),3.3(td,J=12.3, 4.8Hz, 1H), 2.8 (td, J=12.6, 10.8, 4.6Hz, 1H), 2.7 (dd, J=15.6, 4.6Hz, 1H), 2.4 (s, 1H), 1.4 (s, 6H).

[0051] Example 4

[0052] Preparation of 2-((1,3-difluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl)oxy)acetic acid

[0053]

[0054] The method was similar to that in Example 1, except that 2-iodoethanol was replaced with bromoacetic acid.

[0055] 13 C NMR (126MHz, CDCl3) δ172.3, 163.2, 163.0 (d, J = 247.2Hz), 159.3 (dd, J = 247.3, 11.2H z),155.1(dd,J=255.3,11.7Hz),152.5,147.3,132.0,130.3(d,J=9.2Hz),130.1,129 .3,127.1,126.7,119.5,113.8,113.8,112.8(d,J=20.8Hz),112.6,111.4(d,J=23.7 Hz), 110.9 (d, J = 22.7Hz), 109.1 (dd, J = 26.6, 22.8Hz), 102.4, 73.4, 66.2, 43.0, 19.8.

[0056] Example 5

[0057] Preparation of 10-(2-(dimethylamino)ethoxy)-1,3-difluoro-14-(3-fluorophenyl)-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one

[0058]

[0059] The method was similar to that in Example 1, except that 2-iodoethanol was replaced with (2-bromomethyl)dimethylamine.

[0060] 1H NMR(500MHz, CDCl3)δ8.6(s,1H),7.6(d,J=8.4Hz,1H),7.3(s,1H),7.1(q,J=7.7Hz,1 H),7.0(d,J=8.0Hz,1H),6.9(s,2H),6.8-6.8(m,1H),6.7(d,J=8.2Hz,1H),6.7(d,J= 10.3Hz,1H),6.3(s,1H),4.8(dd,J=13.3,5.4Hz,1H),4.1(t,J=5.7Hz,2H),3.2(td,J =12.4, 4.8Hz, 1H), 2.8 (q, J = 8.8, 5.8Hz, 3H), 2.7 (dd, J = 15.5, 4.7Hz, 1H), 2.4 (s, 6H).

[0061] Example 6

[0062] Preparation of 1,3-difluoro-14-(3-fluorophenyl)-10-(2-(piperidin-1-yl)ethoxy)-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one

[0063]

[0064] The method was similar to that in Example 1, except that 2-iodoethanol was replaced with 1-(2-chloroethyl)piperidine.

[0065] 1 H NMR (500MHz, CDCl3) δ8.35(s,1H),7.65(dd,J=8.6,2.8Hz,1H),7.27(s,1H),7.15(td,J=8.2,6.4Hz,1H),6.97(ddd,J =10.5,8.0,2.9Hz,1H),6.88(d,J=7.9Hz,2H),6.81(td,J=8.2,2.4Hz,1H),6.74(dd,J=8.2,2.1Hz,1H),6.66(dt,J=10 .2,2.4Hz,1H),6.33(s,1H),4.80(dd,J=13.2,5.5Hz,1H),4.15(t,J=6.0Hz,2H),3.28-3.21(m,1H),2.84(d,J=6.0Hz ,2H),2.81(d,J=10.0Hz,1H),2.67(dd,J=15.7,4.7Hz,1H),2.57(s,4H),1.64(p,J=5.7Hz,4H),1.46(t,J=6.0Hz,2H).

[0066] Example 7

[0067] Preparation of 1,3-difluoro-14-(3-fluorophenyl)-10-(2-(4-methylpiperazin-1-yl)ethoxy)-8,13,13b-14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one

[0068]

[0069] The method was similar to that in Example 1, except that 2-iodoethanol was replaced with 1-(2-bromoethyl)-4-methylpiperazine.

[0070] 1 H NMR(500MHz, CDCl3)δ8.6(s,1H),7.7(dd,J=8.3,2.9Hz,1H),7.3(s,1H),7.13(q,J=7.7Hz,1H) ,7.0(ddd,J=10.6,7.9,2.9Hz,1H),6.9(d,J=7.2Hz,2H),6.8(td,J=8.2,2.5Hz,1H),6.7(dd,J =8.1,2.2Hz,1H),6.7(dt,J=10.3,2.4Hz,1H),6.3(s,1H),4.8(dd,J=13.1,5.5Hz,1H),4.1(t, J=5.8Hz,2H),3.2(td,J=12.3,4.8Hz,1H),2.8(t,J=5.8Hz,2H),2.8-2.4(m,10H),2.3(s,3H).

[0071] Example 8

[0072] Preparation of 1,3-difluoro-14-(3-fluorophenyl)-10-(2-(piperazin-1-yl)ethoxy)-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one

[0073]

[0074] The method was similar to that in Example 1, except that 2-iodoethanol was replaced with 1-(2-chloroethyl)piperazine.

[0075] 1H NMR (500MHz, CDCl3) δ8.7(s,1H),7.6(dd,J=8.4,2.9Hz,1H),7.3(s,1H),7.1(q,J=7.7Hz,1H),7.0(ddd,J=1 0.5,8.0,2.9Hz,1H),6.9(d,J=8.0Hz,2H),6.8(td,J=8.3,2.5Hz,1H),6.7(dd,J=8.0,2.1Hz,1H),6.7(dd,J= 10.2,2.5Hz,1H),6.3(s,1H),4.8(dd,J=13.1,5.4Hz,1H),4.1(t,J=5.6Hz,2H),3.2(td,J=12.4,4.8Hz,1H), 3.0(d,J=4.1Hz,3H),2.8(t,J=5.7Hz,4H),2.8-2.8(m,1H),2.7(d,J=4.6Hz,1H),2.6(dt,J=9.5,4.7Hz,4H).

[0076] Example 9

[0077] Preparation of 1,3-difluoro-14-(3-fluorophenyl)-10-(2-morpholinethoxy)-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one

[0078]

[0079] The method was similar to that in Example 1, except that 2-iodoethanol was replaced with 2-(4-morpholine)ethyl bromide. 1 H NMR (500MHz, CDCl3) δ8.5 (s, 1H), 7.65 (d, J = 8.3Hz, 1H), 7.3 (s, 1H), 7.2 (q, J = 7.8Hz, 1H), 7. 0-6.9(m,1H),6.9(d,J=5.3Hz,2H),6.8(t,J=8.3Hz,1H),6.7(d,J=8.2Hz,1H),6.7(d,J=10.2 Hz,1H),6.3(s,1H),4.8(dd,J=13.2,5.5Hz,1H),4.1(t,J=5.7Hz,2H),3.8(t,J=4.7Hz,4H), 3.2(td,J=12.4,4.8Hz,1H),2.8(h,J=6.6,5.5Hz,3H),2.72-2.65(m,1H),2.65-2.52(m,4H).

[0080] Example 10

[0081] Preparation of 1,3-difluoro-14-(3-fluorophenyl)-10-(3-methoxypropoxy)-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one

[0082]

[0083] The method was similar to that in Example 1, except that 2-iodoethanol was replaced with 1-bromo-3-methoxypropane.

[0084] 1 H NMR (500MHz, CDCl3) δ8.3(s,1H),7.7(dd,J=8.5,2.7Hz,1H),7.3(s,1H),7.2(q,J=7.7Hz,1H) ,7.0-6.9(m,1H),6.9(d,J=9.2Hz,2H),6.8-6.8(m,1H),6.7(d,J=8.2Hz,1H),6.7(s,1H),6.3 (s,1H),4.8(dd,J=13.1,5.6Hz,1H),4.1(t,J=6.3Hz,2H),3.6(t,J=6.2Hz,2H),3.4(s,3H),3 .2(dd,J=13.1,3.7Hz,1H),2.9-2.8(m,1H),2.7(dd,J=15.8,4.6Hz,1H),2.1(p,J=6.3Hz,2H).

[0085] Example 11

[0086] Preparation of 1,3-difluoro-14-(3-fluorophenyl)-10-(3-(pyrrolidone-1-yl)propoxy)-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one

[0087]

[0088] The method was similar to that in Example 1, except that 2-iodoethanol was replaced with 1-(3-bromopropyl)pyrrolidine.

[0089] 1H NMR(500MHz, CDCl3)δ8.6(s,1H),7.7(dd,J=8.4,2.8Hz,1H),7.3(d,J=6.4Hz,1H),7.12(q,J=7.7Hz,1H),7 .0(ddd,J=10.3,8.1,2.9Hz,1H),6.9(d,J=8.3Hz,2H),6.8(td,J=8.2,2.4Hz,1H),6.7(d,J=8.2Hz,1H),6.7 (d,J=10.2Hz,1H),6.3(s,1H),4.8(dd,J=13.1,5.4Hz,1H),4.0(t,J=6.3Hz,2H),3.2(td,J=12.3,4.8Hz,1 H), 2.8 (d, J = 15.5Hz, 1H), 2.8 (t, J = 7.7Hz, 2H), 2.7-2.6 (m, 5H), 2.1 (p, J = 6.6Hz, 2H), 1.9 (q, J = 3.4Hz, 4H).

[0090] Example 12

[0091] Preparation of 1,3-difluoro-14-(3-fluorophenyl)-10-(3-(piperidin-1-yl)propoxy)-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one

[0092]

[0093] The method was similar to that in Example 1, except that 2-iodoethanol was replaced with 1-(3-bromopropyl)piperidine hydrobromic acid.

[0094] 1H NMR (500MHz, CDCl3) δ8.5(s,1H),7.6(dd,J=8.4,2.9Hz,1H),7.3(s,1H),7.1(q,J=7.6Hz,1H),7.0(ddd,J=10.4,8.0,2.9 Hz,1H),6.9(d,J=8.3Hz,2H),6.8(td,J=8.3,2.4Hz,1H),6.7(d,J=8.1Hz,1H),6.7(d,J=10.2Hz,1H),6.3(s,1H),4.8(dd ,J=13.1,5.4Hz,1H),4.0(t,J=6.2Hz,2H),3.2(td,J=12.3,4.8Hz,1H),2.8(td,J=13.6,11.4,5.5Hz,1H),2.7(d,J=4.7H z, 1H), 2.6 (d, J = 8.1Hz, 2H), 2.6 (d, J = 32.1Hz, 4H), 2.1 (q, J = 7.4, 6.9Hz, 2H), 1.7 (p, J = 5.7Hz, 4H), 1.5 (t, J = 6.2Hz, 2H).

[0095] Example 13

[0096] Preparation of 1,3-difluoro-14-(3-fluorophenyl)-10-(3-morpholinopropoxy)-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one

[0097]

[0098] The method was similar to that in Example 1, except that 2-iodoethanol was replaced with 4-(3-bromopropyl)morpholine hydrobromic acid.

[0099] 1H NMR (500MHz, CDCl3) δ8.5(s,1H),7.7(dd,J=8.3,2.9Hz,1H),7.3(d,J=5.2Hz,1H),7.1(q,J=7.7Hz,1H),7.0(ddd,J=10.5,8.0, 3.0Hz,1H),6.9(d,J=6.7Hz,2H),6.8(td,J=8.2,2.4Hz,1H),6.7(dd,J=8.1,2.1Hz,1H),6.7(dd,J=10.2,2.4Hz,1H),6.3(s,1H ),4.8(dd,J=13.2,5.4Hz,1H),4.0(t,J=6.3Hz,2H),3.7(t,J=4.7Hz,4H),3.3(t,J=5.1Hz,1H),3.2(td,J=12.3,4.8Hz,1H),2. 8(ddd,J=16.6,11.1,5.5Hz,1H), 2.7(dd,J=15.6,4.7Hz,1H), 2.5(t,J=7.4Hz,2H), 2.5(t,J=4.6Hz,3H), 2.0(p,J=6.7Hz,2H).

[0100] Example 14

[0101] Preparation of 1,3-difluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindolo[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl-2-methoxyacetic acid ester

[0102]

[0103] 1,3-Difluoro-14-(3-fluorophenyl)-10-hydroxy-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one (1 mmol), EDCI (2 mmol), and 4-dimethylaminopyridine (2 mmol) were mixed and dissolved in DMF. The mixture was stirred in an ice bath for 30 min. Methoxyacetic acid (1.5 mmol) was weighed, dissolved in DMF, and added to the mixed solution. The mixture was stirred in an ice bath for another 3 h. After the reaction was complete, water was added to quench the reaction. The mixture was extracted with ethyl acetate, evaporated to dryness, and purified by column chromatography to give a white solid with a yield of 77%.

[0104] 1H NMR (500MHz, CDCl3) δ8.4(s,1H),7.7(d,J=8.3Hz,1H),7.4(d,J=8.6Hz,1H),7.2( d,J=7.5Hz,2H),7.0(dd,J=14.5,8.6Hz,2H),6.8(t,J=8.3Hz,1H),6.8(d,J=8.2H z,1H),6.7(d,J=10.2Hz,1H),6.4(s,1H),4.8(dd,J=13.3,5.6Hz,1H),4.3(s,2H) ,3.6(s,3H),3.3(td,J=12.5,4.7Hz,1H),2.9(s,1H),2.7(dd,J=15.8,4.6Hz,1H).

[0105] Example 15

[0106] Preparation of 1,3-difluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindolo[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl 2-hydroxyacetic acid ester

[0107]

[0108] The method was similar to that in Example 14, except that methoxyacetic acid was replaced with glycolic acid.

[0109] 1 H NMR (500MHz, CDCl3) δ8.4(s,1H),7.8(d,J=8.3Hz,1H),7.3(d,J=8.6Hz,1H),7.1( d,J=7.5Hz,2H),7.0(dd,J=14.5,8.6Hz,2H),6.9(t,J=8.3Hz,1H),6.8(d,J=8.2H z,1H),6.7(d,J=10.2Hz,1H),6.4(s,1H),5.3(s,1H),4.8(dd,J=13.3,5.6Hz,1H) ,4.3(s,2H),3.3(td,J=12.5,4.7Hz,1H),2.9(s,1H),2.7(dd,J=15.8,4.6Hz,1H).

[0110] Example 16

[0111] Preparation of 1,3-difluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yldimethylglycine ester

[0112]

[0113] The method was similar to that in Example 14, except that methoxyacetic acid was replaced with N,N-dimethylglycine.

[0114] 1 H NMR (500MHz, CDCl3) δ8.4 (s, 1H), 7.7-7.6 (m, 1H), 7.4 (d, J = 8.7Hz, 1H), 7.2-7.1 (m, 2H), 7. 0(td,J=7.8,3.9Hz,1H),7.0-6.9(m,1H),6.8(td,J=8.2,2.4Hz,1H),6.8(dd,J=8.2,2.2Hz, 1H),6.7(dt,J=10.3,2.5Hz,1H),6.3(s,1H),4.8(dd,J=13.2,5.6Hz,1H),3.5(s,2H),3.25 (td,J=12.3,4.7Hz,1H),2.9(q,J=9.8,5.5Hz,1H),2.7(dd,J=15.6,4.6Hz,1H),2.5(s,6H).

[0115] Example 17

[0116] Preparation of 1,3-difluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl-3-(dimethylamino)propionate

[0117]

[0118] The method was similar to that in Example 14, except that methoxyacetic acid was replaced with N,N-dimethyl-β-alanine.

[0119] 1H NMR(500MHz, CDCl3)δ8.5(s,1H),7.7(d,J=8.5Hz,1H),7.3(d,J=8.7Hz,1H),7.2-7.1 (m,2H),7.0-6.9(m,1H),6.9(d,J=8.3Hz,1H),6.9-6.8(m,1H),6.8(d,J=8.2Hz,1H),6 .7(d,J=10.2Hz,1H),6.3(s,1H),4.8(dd,J=13.3,5.5Hz,1H),3.2(td,J=12.9,12.5,4 .7Hz,1H),2.8(t,J=13.5Hz,1H),2.8(s,4H),2.7(dd,J=15.5,4.5Hz,1H),2.3(s,6H).

[0120] Example 18

[0121] Preparation of 1,3-difluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl-4-(dimethylamino)butyrate

[0122]

[0123] The method was similar to that in Example 14, except that methoxyacetic acid was replaced with 4-(dimethylamino)butyric acid. 1 H NMR (500MHz, CDCl3) δ8.4(s,1H),7.6(d,J=8.5Hz,1H),7.4(d,J=8.6Hz,1H),7. 2-7.0(m,2H),7.0-6.9(m,1H),6.9-6.8(m,3H),6.7(d,J=10.2Hz,1H),6.3(s,1 H),4.8(dd,J=13.3,5.5Hz,1H),3.2(td,J=12.9,12.5,4.7Hz,1H),2.8(t,J=13 .5Hz,1H),2.8(s,4H),2.7(dd,J=15.5,4.5Hz,1H),2.3(s,6H),1.4-1.3(m,2H).

[0124] Example 19

[0125] Preparation of 3-((1,3-difluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl)oxy)-N,N,N-trimethyl-3-oxopropyl-1-amine iodide

[0126]

[0127] 1,3-Difluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl-3-(dimethylamino)propionate (1 mmol), methyl iodoforme (1.2 mmol), and DCM (5 mL) were added sequentially to a round-bottom flask and stirred overnight at room temperature. The next day, TLC monitoring showed complete conversion of the starting material. An equal volume of diethyl ether was added to the reaction system, and a white solid precipitated. The precipitate was filtered, washed with diethyl ether, and dried to obtain a white solid in 86% yield.

[0128] 1 H NMR(500MHz,CD3OD)δ7.6(dd,J=8.5,2.9Hz,1H),7.4(d,J=8.8Hz,1H),7.3-7.2(m,1H),7.2 (d,J=2.3Hz,1H),7.1(q,J=7.9Hz,1H),7.0(dd,J=8.8,2.3Hz,1H),6.8(td,J=8.3,2.5Hz,1H ),6.7(d,J=8.2Hz,1H),6.7(d,J=10.5Hz,1H),6.5(s,1H),4.7(dd,J=12.8,5.2Hz,1H),3.8 (t,J=7.5Hz,2H),3.3(t,J=7.5Hz,3H),3.2(s,9H),2.7(dd,J=15.5,4.2Hz,1H),2.5(s,1H).

[0129] Example 20

[0130] Preparation of 4-((1,3-difluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl)oxy)-4-oxobutyric acid

[0131]

[0132] The method was similar to that in Example 14, except that methoxyacetic acid was replaced with succinic acid.

[0133] 1H NMR (500MHz, CDCl3) δ8.8(s,1H),7.6(d,J=8.2Hz,1H),7.2(d,J=8.8Hz,1H),7.2- 7.1(m,2H),7.0(t,J=9.1Hz,1H),6.9-6.8(m,2H),6.7(d,J=8.2Hz,1H),6.7(d,J=1 0.1Hz,1H),6.3(s,1H),4.7(dd,J=13.4,5.4Hz,1H),3.1(dt,J=12.4,6.4Hz,1H),2 .9-2.8(m,2H),2.8(d,J=6.6Hz,2H),2.7(d,J=16.3Hz,1H),2.5(d,J=15.6Hz,1H).

[0134] Example 21

[0135] Preparation of 1,3-difluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl-2-(2-methoxyethoxy)acetic acid ester

[0136]

[0137] The method was similar to that in Example 14, except that methoxyacetic acid was replaced with 2-(2-methoxyethoxy)acetic acid.

[0138] 1 H NMR (500MHz, CDCl3) δ8.4 (s, 1H), 7.7 (d, J = 8.3Hz, 1H), 7.4-7.3 (m, 1H), 7.2-7.1 (m,2H),7.0-6.9(m,2H),6.8(t,J=8.4Hz,1H),6.8(d,J=8.2Hz,1H),6.7(d,J=10. 1Hz,1H),6.4(s,1H),4.8(dd,J=13.0,5.6Hz,1H),4.5-4.4(m,2H),3.9-3.8(m,2H ),3.7-3.6(m,2H),3.5-3.4(m,3H),3.3-3.2(m,1H),2.9(s,1H),2.8-2.6(m,1H).

[0139] Example 22

[0140] Preparation of 1,3-difluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl-2-(2-(2-methoxyethoxy)acetic acid ester

[0141]

[0142] The method was similar to that in Example 14, except that methoxyacetic acid was replaced with 2-[2-(2-methoxyethoxy)ethoxy]acetic acid.

[0143] 1 H NMR (500MHz, CDCl3) δ8.4 (s, 1H), 7.7-7.6 (m, 1H), 7.4 (d, J = 8.7Hz, 1H), 7.2 (d, J = 5.7Hz, 2H), 7 .0(q,J=9.5,8.9Hz,2H),6.8(t,J=8.1Hz,1H),6.8(d,J=8.3Hz,1H),6.7(d,J=10.2Hz,1H),6.4( s,1H),4.81(dd,J=13.3,5.6Hz,1H),4.4(s,2H),3.9-3.8(m,2H),3.8-3.7(m,2H),3.7-3.6(m,2 H),3.6-3.5(m,2H),3.4(s,3H),3.3(td,J=12.6,12.0,4.6Hz,1H),2.9(s,1H),2.8-2.6(m,1H).

[0144] Example 23

[0145] Preparation of 1,3-difluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl 2,5,8,11-tetraoxetane-13-ester

[0146]

[0147] The method was similar to that in Example 14, except that methoxyacetic acid was replaced with 2,5,8,11-tetraoxetane-13-acid.

[0148] 1H NMR (500MHz, CDCl3) δ8.4(s,1H),7.7(d,J=8.0Hz,1H),7.4(d,J=8.8Hz,1H),7.2(s,2H),7.0(q, J=9.4Hz,2H),6.8(t,J=8.2Hz,1H),6.8(d,J=8.1Hz,1H),6.7(d,J=10.1Hz,1H),6.4(s,1H),4.8( dd,J=13.8,5.7Hz,1H),4.4(s,2H),3.9-3.8(m,2H),3.7(t,J=4.9Hz,2H),3.7-3.7(m,4H),3.7- 3.6(m,2H),3.6-3.5(m,2H),3.4(s,3H),3.3-3.2(m,1H),2.9(d,J=18.0Hz,1H),2.8-2.6(m,1H).

[0149] Example 24

[0150] Preparation of 1,3-difluoro-14-(3-fluorophenyl)-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl-3-(piperidin-1-yl)propionate

[0151]

[0152] The method was similar to that in Example 14, except that methoxyacetic acid was replaced with 1-piperidinpropionic acid.

[0153] 1 H NMR (500MHz, CDCl3) δ8.8(s,1H),7.7(dd,J=8.3,2.9Hz,1H),7.3(d,J=8.7Hz,1H),7.2-7.1(m,2H),7.0(dd d,J=10.5,8.0,2.9Hz,1H),6.9(dd,J=8.8,2.3Hz,1H),6.8(td,J=8.2,2.5Hz,1H),6.7(dd,J=8.1,2.1Hz,1 H),6.7(dt,J=10.1,2.4Hz,1H),6.3(s,1H),4.8(dd,J=13.1,5.5Hz,1H),3.3-3.2(m,1H),2.8(dp,J=12.2, 6.6, 6.0Hz, 5H), 2.6 (dd, J = 15.6, 4.6Hz, 1H), 2.5 (s, 3H), 2.4 (s, 1H), 1.6 (d, J = 5.7Hz, 2H), 1.5-1.4 (m, 2H).

[0154] Example 25

[0155] Preparation of 1,3-difluoro-14-(3-fluorophenyl)-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl-3-(pyrrolidine-1-yl)propionate

[0156]

[0157] The method was similar to that in Example 14, except that methoxyacetic acid was replaced with 3-pyrrolidin-1-propionic acid.

[0158] 1 H NMR (500MHz, CDCl3) δ8.4(s,1H),7.7(dd,J=8.3,2.9Hz,1H),7.3(d,J=8.7Hz,1H),7.2-7.1(m,1H),7.2(d, J=2.2Hz,1H),7.0(ddd,J=10.5,8.0,2.9Hz,1H),6.9(dd,J=8.7,2.3Hz,1H),6.8(td,J=8.1,2.5Hz,1H),6.8 (dd,J=8.2,2.2Hz,1H),6.7(dt,J=10.2,2.4Hz,1H),6.3(s,1H),4.8(dd,J=13.2,5.5Hz,1H),3.3-3.2(m,1H ),2.9(t,J=7.4Hz,2H),2.8(s,1H),2.8(t,J=7.4Hz,2H),2.7-2.7(m,1H),2.7-2.5(m,4H),1.9-1.8(m,4H).

[0159] Example 26

[0160]

[0161] Under ice bath conditions, 1,3-difluoro-14-(3-fluorophenyl)-10-hydroxy-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one (1 mmol), dimethylcarbamoyl chloride (1.2 mmol), and DCM (30 mL) were sequentially added to a round-bottom reaction flask. After stirring for 10 min, N,N-diisopropylethylamine (3 mmol) was added to the reaction system, and the mixture was allowed to warm naturally to room temperature and stirred overnight. The reaction was quenched with water after complete reaction, extracted with ethyl acetate, evaporated to dryness, and purified by column chromatography to give a pale yellow solid in 82% yield.

[0162] 1H NMR (500MHz, CDCl3) δ8.6(s,1H),7.7(d,J=8.3Hz,1H),7.2(d,J=8.7Hz,1H),7.2-7.1(m,2 H),7.0(t,J=8.9Hz,1H),6.9(d,J=8.7Hz,1H),6.8(t,J=8.3Hz,1H),6.7(d,J=8.2Hz,1H), 6.7(d,J=10.2Hz,1H),6.3(s,1H),4.8(dd,J=13.2,5.5Hz,1H),3.2(dt,J=12.3,6.2Hz,1H ), 3.1 (s, 3H), 3.0 (s, 3H), 2.8 (td, J = 13.7, 11.3, 5.7Hz, 1H), 2.6 (dd, J = 15.7, 4.6Hz, 1H).

[0163] Example 27

[0164] Preparation of 1,3-difluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yldiethylcarbamate

[0165]

[0166] The method was similar to that in Example 26, except that dimethylcarbamoyl chloride was replaced with N,N-diethylchloroformamide.

[0167] 1 H NMR (500MHz, CDCl3) δ8.9 (s, 1H), 7.7-7.6 (m, 1H), 7.1 (q, J = 8.4Hz, 3H), 7.0-6.9 (m, 1H), 6.9 (d ,J=8.8Hz,1H),6.8(t,J=8.4Hz,1H),6.7(d,J=8.2Hz,1H),6.7(d,J=10.2Hz,1H),6.2(s,1H),4. 7(dd,J=13.1,5.3Hz,1H),3.4(dq,J=46.3,7.2Hz,4H),3.2(td,J=12.4,4.8Hz,1H),2.7(td,J= 13.6, 11.2, 5.7Hz, 1H), 2.6 (dd, J = 15.6, 4.6Hz, 1H), 1.3 (d, J = 7.1Hz, 3H), 1.2 (t, J = 7.3Hz, 3H).

[0168] Example 28

[0169] Preparation of 1,3-difluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-ylpiperazine-1-carboxylic acid ester

[0170]

[0171] Step 1: The method is similar to that in Example 26, except that dimethylcarbamoyl chloride is replaced with 4-chlorocarbonyl-piperazine-1-carboxylic acid tert-butyl ester.

[0172] Step 2: The product from Step 1 of Example 28 (1 mmol), trifluoroacetic acid (10 mmol), and DCM (20 mL) were added sequentially to a round-bottom flask and stirred at room temperature for 3 hours. The reaction was monitored by TLC and found to be complete. The reaction mixture was then transferred to a separatory funnel containing 200 mL of ethyl acetate, washed sequentially with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain a pale yellow solid in 69% yield.

[0173] 1 H NMR (500MHz, CDCl3) δ8.4(s,1H),7.7(d,J=8.2Hz,1H),7.3(d,J=9.0Hz,1H),7. 2(d,J=7.1Hz,2H),7.0(q,J=9.4Hz,2H),6.8(t,J=8.3Hz,1H),6.8(d,J=8.3Hz,1 H),6.7(d,J=10.2Hz,1H),6.3(s,1H),4.8(dd,J=13.8,5.5Hz,1H),3.8(s,2H),3 .6(s,2H),3.2(q,J=12.6Hz,1H),3.0(s,4H),2.8(s,1H),2.7(d,J=15.8Hz,1H).

[0174] Example 29

[0175] Preparation of 1,3-difluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl[1,4'-dipiperidine]-1'-carboxylic acid ester

[0176]

[0177] The method was similar to that in Example 26, except that dimethylcarbamoyl chloride was replaced with [1,4']bipiperidine-1'-formyl chloride.

[0178] 1H NMR (500MHz, CDCl3) δ8.8(s,1H),7.7(d,J=8.1Hz,1H),7.1(dd,J=13.8,7.9Hz,2H),7.1(d,J=10.3 Hz,1H),7.0(t,J=9.2Hz,1H),6.9(d,J=8.8Hz,1H),6.8(t,J=8.3Hz,1H),6.7(d,J=8.2Hz,1H),6.7( d,J=10.1Hz,1H),6.3(s,1H),4.74(dd,J=13.1,5.5Hz,1H),4.3(dd,J=54.0,13.5Hz,2H),3.2(td,J =12.5,4.6Hz,1H),3.0(t,J=12.8Hz,1H),2.9-2.5(m,8H),2.0-1.9(m,2H),1.6(d,J=104.2Hz,8H).

[0179] Example 30

[0180] Preparation of 1,3-difluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl dihydrophosphate

[0181]

[0182] Step 1: 1,3-Difluoro-14-(3-fluorophenyl)-10-hydroxy-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one (0.5 mmol), tetrazolium (1.8 mmol), dibenzyl N,N'-diisopropylphosphonamide (1.0 mmol), tert-butyl hydroperoxide (2.5 mmol), and DMF (20 mL) were added sequentially to a round-bottom flask. The mixture was stirred at room temperature for 1 hour, followed by stirring for another 0.5 hours with 80 mL of 10% sodium metabisulfite aqueous solution. The reaction was monitored by TLC until complete. The reaction was quenched with ice water and then extracted three times with ethyl acetate. The combined organic layers were washed twice with brine, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain a white solid in 58% yield.

[0183] Step 2: Dissolve 0.3 mmol of the product from Step 1 in Example 30 in 10 mL of dry DCM, add 0.2 mL of trimethylbromosilane under argon atmosphere, and react overnight. After the reaction is complete, evaporate the solvent to dryness, recrystallize with ethyl acetate to give a pale green solid in 49% yield.

[0184] 1H NMR (500MHz, CD3OD) δ7.7-7.6(m,1H),7.4(d,J=8.8Hz,1H),7.3(d,J=10.5Hz,2H),7.1(t,J=7.6Hz,1H),7.0(d,J=8.8Hz,1H),6.8(t,J=7.7Hz, 1H), 6.7 (dd, J = 26.5, 9.3Hz, 2H), 6.5 (s, 1H), 4.7 (dd, J = 13.0, 5.1Hz, 1H), 3.3 (dd, J = 12.3, 4.1Hz, 1H), 2.8 (dd, J = 15.0, 3.8Hz, 1H), 2.5 (s, 1H).

[0185] Example 31

[0186] Preparation of 1,3-difluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl phosphate dimethyl ester

[0187]

[0188] The method was similar to that in Example 26, except that dimethylcarbamoyl chloride was replaced with dimethyl chlorophosphate.

[0189] 1 H NMR (500MHz, CDCl3) δ9.0 (s, 1H), 7.7-7.6 (m, 1H), 7.2 (s, 1H), 7.1 (q, J = 7.8Hz, 1H), 7.0 (dd, J = 8 .9,2.4Hz,1H),7.0(ddd,J=10.5,8.0,2.9Hz,1H),6.8(td,J=8.2,2.5Hz,1H),6.8(dd,J=8.1,2.2 Hz,1H),6.7(dt,J=10.3,2.4Hz,1H),6.3(s,1H),4.8(dd,J=13.1,5.5Hz,1H),3.9(d,J=11.2Hz,6 H), 3.2 (td, J = 12.3, 4.7Hz, 1H), 2.8 (ddd, J = 15.8, 10.9, 5.2Hz, 1H), 2.6 (dd, J = 15.7, 4.6Hz, 1H).

[0190] Test Example 1: MTT assay to determine the inhibitory rate of a compound on tumor cell growth

[0191] Test method: Hepatocellular carcinoma cell lines (Huh7, HepG2, SK-Hep-1) were cultured in RPMI 1640 or MEM medium containing 10% fetal bovine serum at 37°C and 5% CO2 in a cell culture incubator. When the cells reached the logarithmic growth phase, 5000–8000 cells per well were seeded into 96-well plates. After 24 hours of culture, the old medium was removed, and medium containing the test sample (100 mmol·L⁻¹) was added. 1 The target compound DMSO stock solution was prepared to experimental concentrations of 18, 6, 2, 0.67, 0.22, 0.07, 0.024, and 0.008 μmol·L⁻¹. 1 Each experimental concentration was tested in triplicate, with a blank control group included. After culturing the cells for 72 hours, 10 μL of MTT solution was added, and after incubation for 4 hours, the supernatant in the 96-well plate was aspirated, and 150 μL of DMSO was added to each well. The plates were then shaken for 20 minutes. The absorbance (OD value) of each well in the 96-well plate was measured at 570 nm using a microplate reader, and the cell proliferation inhibition rate (IR) was calculated. The cell proliferation inhibition rate % was calculated as: (average OD value of control wells / average OD value of experimental wells) / (average OD value of control wells / OD value of blank wells). The half-maximal inhibitory concentration (IC50) was calculated using GraphPad Prism 7.00. 50 Values ​​(means ± SD, n = 3), detailed data are shown in Tables 1 and 2. Data are calculated as the mean ± standard deviation of three independent experiments.

[0192] The test results are shown in Table 1.

[0193] Table 1. Antiproliferative activity of compounds against hepatocellular carcinoma cell lines

[0194]

[0195] As can be seen from Table 1, the compounds of the present invention have excellent anti-proliferative activity against liver cancer cell lines Huh7, HepG2, and SK-Hep-1, and are comparable to or even better than comparative compounds A and B.

[0196] Test Example 2: Determination of water solubility of compounds by HPLC

[0197] Test method: A standard curve was established using methanol to prepare a known concentration of the compound. The compound powder and ultrapure water were mixed in an Eppendorf tube, then vortexed vigorously for 1 minute, followed by sonication at room temperature for 10 minutes. The mixture was then incubated with shaking for 72 hours to ensure saturation at room temperature. The compound suspension was centrifuged at 10,000 RPM for 15 minutes, and the supernatant was filtered through a 0.22 mm syringe filter. The concentration of the compound in the supernatant was determined by HPLC.

[0198] The test results are shown in Table 2.

[0199] Table 2. Determination of water solubility of compounds

[0200]

[0201] As can be seen from Table 2, the compounds of the present invention have good to excellent water solubility, which is superior to that of comparative compound A and comparative compound B.

[0202] Test Example 3: Inhibition experiment of compound 19 on topoisomerase I (Topo1)

[0203] Test method: Add 2 μL of 10× DNA Top1 buffer (720 mM KCl, 350 mM Tris-HCl (pH 8.0), 50 mM spermidine, 50 mM DTT, and 50 mM MgCl2), 2 μL of 0.1% BSA, 0.2 μL of each concentration of the test compound, 0.5 μL of Top1 (1 μ / mL, Takara Biotechnology, Dalian), and 0.5 μL of deionized water to a 1.5 mL Eppendorf tube and incubate at 37 °C for 20 min. Then add 0.4 μL of pBR322 plasmid DNA (Takar Biotechnology, Dalian, China), and add deionized water to make a final volume of 20 μL, and continue incubating at 37 °C for 15 min. Finally, add 4 μL of loading buffer (0.25% bromophenol blue and 50% glycerol) to stop the reaction. Then, 10 μL of the test solution was added to 0.8% agarose gel in 1×TAE, and electrophoresis was performed at 110V for 1 hour. The DNA gel was stained with ethidium bromide (0.5 μg / mL water) for 30 min, and the DNA bands were photographed using a Shenhua Technology imaging system.

[0204] Test results are as follows Figure 1 As shown. (Through) Figure 1 It can be seen that compound 19 of the present invention exhibits good to excellent inhibitory effects on topoisomerase I at specific concentrations, and its inhibitory effect on Topo1 at the same concentration is superior to that of camptothecin, evodiamine, comparative compound A and comparative compound B.

[0205] Test Example 4: Compound 19's Effect on Huh7 Cell Cycle Arrest

[0206] Assay method: Cell cycle was detected using flow cytometry (BD LSRFortessa) according to the instructions. Simply put, Huh cells were seeded in 6-well plates (1×10⁶ cells / wells). 6Cells were cultured in wells ( / well) for 12 hours. Then, cells were treated with different concentrations of chemicals for 24 hours. Cells were collected and fixed with pre-cooled 75% ethanol at 4°C for 2 hours. Cells were washed twice with PBS and stained with 10 μL propidium iodide (PI) in the dark for 0.5 hours. For each sample, at least 2 × 10⁶ cells were analyzed by flow cytometry. 4 Cells were analyzed using ModfitLT32 software (Verity Software House).

[0207] Test results are as follows Figure 2 As shown. (Through) Figure 2 It can be seen that compound 19 of the present invention, at a nanomolar concentration, arrests the cell cycle of the hepatocellular carcinoma cell line Huh7 in the G2 / M phase, and its effect is better than that of comparative compound B and comparable to that of comparative compound A.

[0208] Test Example 5: Experiment on apoptosis induction of Huh7 cells by compound 19

[0209] Test method: Huh7 cells (1×10⁻⁶) 5 Cells were seeded overnight in six-well plates (number cells / well) and then treated with different concentrations of the specified compound for 24 hours. Cells were then collected and washed three times with PBS. Following the instructions of the apoptosis detection kit (Yeasen, Cat.) #:40302ES60, China), cells were centrifuged, resuspended in 100 μL of 1× binding buffer, transferred to flow cytometry tubes, and incubated in the dark for 5 min with 5 μL of Annexin-V-FITC. Cells were then resuspended in 5 μL of PI staining solution and 400 μL of PBS for flow cytometry analysis.

[0210] Test results are as follows Figure 3 As shown. (Through) Figure 3 It can be seen that compound 19 of the present invention can significantly induce apoptosis of the hepatocellular carcinoma cell line Huh7 at nanomolar concentrations, and the effect is better than that of comparative compound A and comparative compound B.

[0211] Test Example 6: Drug Metabolism Study in Rats

[0212] Test Methods: Three male SD rats weighing 220–300 g were provided by the Guangdong Provincial Medical Laboratory Animal Center. After being housed in a suitable environment for one week, 10 mg / kg of compound 19 was dissolved in 2.5% DMSO, 2.5% HS-15, and physiological saline, and injected intraperitoneally at a concentration of 5 mL / kg. Subsequently, blood samples were collected from the rats' hearts at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h after administration. The samples were placed in EDTA-K2 tubes, and plasma was separated by centrifugation and stored at -80℃. 20 μL of methanol was added to 100 μL of the plasma sample, vortexed, and then 300 μL of a methanol solution containing 40 ng / mL propranolol as an internal standard was added, vortexed, and centrifuged at 14,000 g for half an hour at 4℃. The supernatant was then used for analysis. The analysis was performed using a liquid chromatography-mass spectrometry (LC-MS / MS) system. Chromatographic conditions: Mobile phase A was aqueous (containing 0.1% formic acid); mobile phase B was organic (containing 0.1% formic acid). Flow rate was 0.3 mL / min, and column temperature was 25℃. Pharmacokinetic parameters were analyzed using DAS 2.0 software.

[0213] The test results are shown in Table 3.

[0214] Table 3. Pharmacokinetic parameters of rats after intraperitoneal injection of 19 (10 mg / kg)

[0215]

[0216] Table 3 lists the pharmacokinetic parameters of compound 19 of the present invention in rats after intraperitoneal injection. The results show that compound 19 of the present invention has good pharmacokinetic properties and can be rapidly absorbed into the bloodstream (Tmax = 1.67 h).

[0217] Test Example 7: Antitumor activity of compound 19 of the present invention in a nude mouse xenograft tumor model

[0218] Test method: Male BALB / c nude mice (certificate USC2023xs059, 4-5 weeks old) were housed in an SPF animal laboratory with a relative humidity of ~70%, a temperature of 20-26℃, and a 12-hour light-dark cycle. One week after immunization, Huh7 cells (1.75 × 10⁻⁶) were subcutaneously injected into the right axilla. 7 / mL) 0.1mL / animal. When all tumors exceed 100mm 3Mice were randomly divided into a control group, a sorafenib group, a compound B group, a compound 19-1 group, and a compound 19-2 group (n=5). All groups received intraperitoneal injections of the compounds dissolved in physiological saline containing 2.5% HS-15 and 2.5% DMSO, once daily for 14 consecutive days. Tumor volume and body weight were recorded every 2 days. Tumor volume was calculated using the formula L×W. 2 / 2, where W is the tumor width and L is the tumor length. The tumor growth inhibition (TGI) formula is [1-(T-T0) / (C-C0)]×100%, where C and C0 are the tumor volumes of the control group at the first and last measurements, and T and T0 are the tumor volumes of the treatment group. Mice were sacrificed on day 15, tumors were removed, and body weight was recorded. Liver, kidney, heart, lung, and spleen were stained with H&E. The TGI of the sorafenib group was 62.47%, the TGI of the compound B group was 45.46%, the TGI of the compound 19 10mpk group was 46.41%, and the TGI of the compound 19 40mpk group was 83.27%.

[0219] Test results are as follows Figure 4 As shown. By Figure 4 It can be seen that compound 19 exhibits excellent antitumor effects in the human hepatocellular carcinoma Huh7 xenograft model. At the same dose, the anti-hepatocellular carcinoma activity of compound 19 is comparable to that of control compound B. Moreover, the tumor-suppressing effect of compound 19 at a dose of 40 mg / kg is significantly better than that of control compound B at a dose of 10 mg / kg.

[0220] The above results indicate that the evodiamine derivatives with the structure shown in formula (I) provided by the present invention, or their stereoisomers, geometric isomers, tautomers, nitrides, hydrates, solvates, or metabolites, exhibit excellent inhibitory activity against Huh7, HepG2, and SK-Hep-1 tumor cell lines, and at the same time, they can exhibit good antitumor activity at the animal level.

[0221] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of protection of this application is limited to these examples; within the framework of this application, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of one or more embodiments of this application as described above, which are not provided in detail for the sake of brevity.

[0222] One or more embodiments in this application are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of this application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of one or more embodiments in this application should be included within the protection scope of this application.

Claims

1. A water-soluble erythroside derivative, characterized in that, Its structural formula is as follows: 。 2. The application of a water-soluble evodiamine derivative as described in claim 1, characterized in that, The water-soluble evodiamine derivative is used to prepare drugs for treating liver cancer.

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