An ester group-containing evodiamine derivative, a preparation method and application thereof

Abstract

The application belongs to the technical field of medicines, and particularly relates to an ester group substituted evodiamine derivative, a preparation method and application, and a structural formula is as follows: The application shows excellent inhibitory activity on HT-29, HTC-116, LOVO, RKO, HGC-27, MGC-803, SGC-7901, Huh7, SK-EP1 and MCF-7 tumor cell lines, and can show good antitumor activity at an animal level.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to an ester-substituted evodiamine derivative, its preparation method, and its application. Background Technology

[0002] Topoisomerases (Topo 1 and Topo 2) are key enzymes regulating the dynamic changes in DNA topology during DNA synthesis and transcription. They are highly expressed in tumor cells and are a popular target for anti-tumor chemotherapy drug development. In current cancer clinical treatment, although precision medicine, represented by targeted therapy and immunotherapy, has developed rapidly, antibiotic chemotherapy drugs, represented by selective Topo 1 inhibitors such as camptothecin derivatives (topotecan, irinotecan, and belotecone) and selective Topo 2 inhibitors such as doxorubicin derivatives (doxorubicin, daunorubicin, idarubicin, epirubicin, and pentorubicin), remain one of the main treatment methods. However, the long-term use of these drugs over the past 30 years has led to very serious drug resistance, and coupled with the inherent toxicity of the drugs themselves, this has greatly limited their clinical efficacy and application scope.

[0003] Evodiamine, a traditional Chinese herbal medicine, has been widely used for thousands of years to treat gastrointestinal diseases, amenorrhea, headaches, and postpartum hemorrhage. Evodiamine is an alkaloid isolated from the fruit of Evodiamine, possessing a novel quinazoline-carboline skeleton. Numerous studies have reported its dual inhibitory effects on topoisomerases 1 and 2 (Topo 1 and Topo 2), as well as its broad-spectrum antitumor activity, making it a promising candidate to succeed camptothecin and doxorubicin as a next-generation blockbuster chemotherapy drug among natural alkaloids, and it is highly sought after in both academic and pharmaceutical circles. During the current strategic opportunity period for the revitalization and development of traditional Chinese medicine, the creation of new drugs based on the traditional Chinese medicine erythrodiamine is also a significant measure in building a technological innovation system for traditional Chinese medicine. However, research has found that erythrodiamine's poor water solubility, weak target inhibitory activity, low antitumor activity, and poor metabolic stability significantly limit its further clinical development.

[0004] Existing technologies have reported on structural modifications and structure-activity relationship studies of the evodiamine skeleton, and a series of evodiamine derivatives have been developed. However, due to limitations in water solubility, antitumor activity, and drug-likeness, no evodiamine-based drugs have yet been marketed. Furthermore, ester-substituted evodiamine derivatives generally exhibit better drug-likeness. For example, patent CN105418610A and literature (Euro.J.Med.Chem.2022,239,114530) disclose typical ester-substituted evodiamine derivative compounds A and B, respectively, and their applications in disease treatment. To overcome the existing technical challenges in the development of evodiamine-based drugs, there is an urgent need to invent new evodiamine-based drug molecules.

[0005] Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an ester-substituted evodiamine derivative, its preparation method and application, so as to improve its tumor-inhibiting effect.

[0007] This invention provides an ester-substituted erythromycin derivative with the following structural formula:

[0008]

[0009] R is

[0010] Compounds with the following structure are preferred:

[0011]

[0012] More preferably, R is

[0013] The structure of Boc is

[0014] This invention provides a method for preparing the ester-substituted evodiamine derivative, comprising the following steps: mixing compound I and ROH, reacting to obtain the ester-substituted evodiamine derivative, wherein the structural formula of compound I is [insert structural formula here]. The condensing agent for the reaction is EDCI, and the catalyst for the reaction is 4-dimethylaminopyridine.

[0015] Preferably, the solvent for the reaction is dichloromethane.

[0016] Preferably, compound I is dissolved in the solvent DMF.

[0017] Preferably, the molar amount of compound I is less than the molar amount of ROH.

[0018] This invention provides an application of the ester-containing substituted evodiamine derivative, wherein the ester-containing substituted evodiamine derivative is used to prepare a drug for treating and inhibiting tumors.

[0019] Preferably, the ester-containing substituted evodiamine derivative is used to prepare a drug that can inhibit topoisomerase 1 and / or 2, and more preferably, the tumor is colon cancer.

[0020] The beneficial effects of the present invention are that the evodiamine derivatives having the structures shown in formula (I) and formula (I) provided by the present invention, or their stereoisomers, geometric isomers, tautomers, nitrides, hydrates, solvates, or their metabolites, exhibit excellent inhibitory activity against HT-29, HTC-116, LOVO, RKO, HGC-27, MGC-803, SGC-7901, Huh7, SK-EP1 and MCF-7 tumor cell lines, and at the same time, they can exhibit good antitumor activity at the animal level. Attached Figure Description

[0021] Figure 1 The compound is shown to inhibit the activity of topoisomerase I.

[0022] in, Figure 1 A represents the inhibitory activity of compounds 14-23 against topoisomerase I. Figure 1 B represents the inhibitory activity of compound 3-11 against topoisomerase I. Figure 1 C represents the inhibitory activity of compound 10 at different concentrations against topoisomerase I.

[0023] Figure 2 The effect of compound 10 on the cell cycle of LOVO and RKO cells.

[0024] Figure 3 The effect of compound 10 on apoptosis in LOVO and RKO cells.

[0025] Figure 4 This study describes the antitumor effect of compound 10 in a human colon cancer cell xenograft model of HT-29.

[0026] in, Figure 4 A represents the effect of compound 10 on the tumor volume of human colon cancer cells HT-29. Figure 4 B represents the effect of compound 10 on the body weight of experimental animals inoculated with human colon cancer cells HT-29. Figure 4 C represents the effect of compound 10 on the tumor weight of human colon cancer cells HT-29. Figure 4 D is a photograph showing the effect of compound 10 on the tumor volume of human colon cancer cells HT-29. Detailed Implementation

[0027] Example 1: Preparation of 3-fluoro-14-(3-fluorophenyl)-10-methoxy-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one

[0028]

[0029] Step 1: 2-Bromo-5-fluorobenzoic acid (20 g, 91.32 mmol), copper powder (9.132 mmol), and potassium carbonate (182.64 mmol) were placed in a dry 500 mL round-bottom flask. 100 mL of water was added and the mixture was stirred magnetically. 3-fluoroaniline (109.58 mmol) was added, and the mixture was heated at 105 °C for 5 h. The reaction solution was cooled to approximately 60 °C, activated carbon was added, and the mixture was stirred for 2 h. The solution was filtered while hot to obtain the filtrate. The pH was adjusted to 3, and the solid was obtained by filtration. After drying, a white solid was obtained, with a yield of 82.8%.

[0030] Step 2: 5-Fluoro-2-((3-fluorophenyl)amino)benzoic acid (8.83 g, 74.02 mmol), 5-methoxytryptamine (74.02 mmol), EDCI (88.82 mmol), and HOBt (88.82 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 80.2%.

[0031] Step 3: Weigh 29.8 g (70.757 mmol) of 5-fluoro-2-((3-fluorophenyl)amino)-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)benzamide into a 500 mL round-bottom flask, add 100 mL of DMF, 212.27 mmol of triethyl orthoformate, and 70.757 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 beat with acetonitrile. After drying, a pale yellow solid is obtained with a yield of 82.3%.

[0032] Example 2: Preparation of (S)-3-fluoro-14-(3-fluorophenyl)-10-hydroxy-8,13,13b,14-tetrahydroindole[[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one and (R)-3-fluoro-14-(3-fluorophenyl)-10-hydroxy-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one.

[0033]

[0034] Step 1: Dissolve 2 g (4.64 mmol) of 3-fluoro-14-(3-fluorophenyl)-10-methoxy-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one in DMF. Vacuum was removed, and the mixture was stirred in an ice bath for 10 min. The mixture was then cooled to 0 °C, and 11.6 mmol of bis(trimethylsilyl)amino potassium (11.6 mmol) was added. The ice bath was removed, and after half an hour, 11.6 mmol of (1S)-(+)-10-camphorsulfonyl chloride (11.6 mmol) dissolved in DMF was added. The reaction was carried out at 65 °C for 15 h. After the reaction was complete, the mixture was extracted with ethyl acetate and recrystallized from dichloromethane and methanol to obtain (13bR)-13-(((4S)-7,7-dimethyl-2-oxobis) Cyclo[2.2.1]heptane-1-yl)methyl)sulfonyl)-3-fluoro-14-(3-fluorophenyl)-10-methoxy-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one, a pale yellow solid, was obtained in 45.5% yield. The recrystallization mother liquor was separated by column chromatography to obtain (13bS)-13-(((4S)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1-yl)methyl)sulfonyl)-3-fluoro-14-(3-fluorophenyl)-10-methoxy-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one, a pale yellow solid, in 40.2% yield.

[0035] Step 2: Dissolve (13bS)-13-(((4S)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1-yl)methyl)sulfonyl)-3-fluoro-14-(3-fluorophenyl)-10-methoxy-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one (0.698 g, 1.619 mmol) in anhydrous DCM, under argon protection, and stir at -40°C for 30 min. Then add boron tribromide. (9.713 mmol), slowly heated to room temperature, continued the reaction for 2 h, quenched with water in an ice bath, extracted and column chromatography to give the product (13bS)-13-(((4S)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1-yl)methyl)sulfonyl)-3-fluoro-14-(3-fluorophenyl)-10-hydroxy-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one yellow solid, yield 41.5%.

[0036] Step 3: Dissolve (13bS)-13-(((4S)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1-yl)methyl)sulfonyl)-3-fluoro-14-(3-fluorophenyl)-10-hydroxy-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one (1 g, 1.556 mmol) in anhydrous tetrahydrofuran, then... Tetrabutylammonium fluoride (3.11 mmol) was added, and the mixture was heated under reflux at 70 °C for 12 h under argon protection. The reaction was quenched with water, extracted with ethyl acetate, washed three times with water, and evaporated to dryness under vacuum to give (S)-3-fluoro-14-(3-fluorophenyl)-10-hydroxy-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one, a pale yellow solid, in 92.5% yield.

[0037] Step 4: The technical method of this step is similar to that of Step 2 in Example 1, except that the (13bS)-13-(((4S)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1-yl)methyl)sulfonyl)-3-fluoro-14-(3-fluorophenyl)-10-methoxy-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinoline from Step 2 is removed. The quinazolin-5(7H)-one was replaced with an equimolar amount of (13bR)-13-(((4S)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1-yl)methyl)sulfonyl)-3-fluoro-14-(3-fluorophenyl)-10-methoxy-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one, with a yield of 43.6%.

[0038] Step 5: The technical method of this step is similar to that of Step 2 in Example 1, except that the (13bS)-13-(((4S)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1-yl)methyl)sulfonyl)-3-fluoro-14-(3-fluorophenyl)-10-hydroxy-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinoline from Step 3 is removed. The quinazolin-5(7H)-one was replaced with an equimolar amount of (13bR)-13-(((4S)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1-yl)methyl)sulfonyl)-3-fluoro-14-(3-fluorophenyl)-10-hydroxy-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one, with a yield of 91.8%.

[0039] Example 3: Preparation of (S)-3-fluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyridin[2,1-b]quinazolin-10-yl(tert-butyloxycarbonyl)glycine

[0040]

[0041] N-(tert-butoxycarbonyl)glycine (0.05 g, 0.287 mmol), EDCI (0.478 mmol), and 4-dimethylaminopyridine (0.478 mmol) were mixed and dissolved in DCM. The mixture was stirred in an ice bath for 30 min. (S)-3-fluoro-14-(3-fluorophenyl)-10-hydroxy-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one (0.239 mmol) was weighed, dissolved in DMF, and added to the mixed solution. The mixture was stirred in an ice bath for 3 h. After the reaction was complete, water was added to quench the reaction, and the mixture was extracted with ethyl acetate, evaporated to dryness, and purified by column chromatography to give a white solid with a yield of 76.6%. 1 H NMR (400MHz, CDCl3) δ8.79 (s, 1H), 7.72 (dd, J = 8.6, 2.9Hz, 1H), 7.27-7.22 (m ,1H),7.22-7.16(m,1H),7.13(d,J=2.1Hz,1H),7.08-6.99(m,2H),6.90-6.7 8(m,4H),6.33(s,1H),5.27-5.11(m,1H),4.85-4.76(m,1H),4.16(d,J=5.8H z,2H),3.27-3.15(m,1H),2.99-2.86(m,1H),2.67-2.57(m,1H),1.46(s,9H).

[0042] Example 4: Preparation of (S)-3-fluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl(tert-butyloxycarbonyl)-L-alanine ester

[0043]

[0044] The preparation method is similar to that in Example 3. 1H NMR (400MHz, CDCl3) δ8.70 (s, 1H), 7.73 (dd, J = 8.6, 2.9Hz, 1H), 7.29-7.24 (m, 1H), 7. 24-7.16(m,1H),7.14-7.11(m,1H),7.09-7.04(m,1H),7.04-6.99(m,1H),6.90-6.79( m,4H),6.33(s,1H),5.28-5.05(m,1H),4.86-4.77(m,1H),4.66-4.43(m,1H),3.28-3 .16(m,1H),2.98-2.87(m,1H),2.69-2.59(m,1H),1.56(d,J=7.2Hz,3H),1.46(s,9H).

[0045] Example 5: Preparation of (S)-3-fluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl(tert-butyloxycarbonyl)-L-valine ester

[0046]

[0047] The preparation method is similar to that in Example 3. 1 H NMR(600MHz, CDCl3)δ8.63(s,1H),7.72(dd,J=8.5,2.9Hz,1H),7.26(s,1H),7.22-7.18(m,1H),7.10(d,J=1.8Hz,1 H),7.06(dd,J=8.7,2.9Hz,1H),7.04-7.00(m,1H),6.86(d,J=7.3Hz,2H),6.80(t,J=7.9Hz,2H),6.32(s,1H),5.12( d,J=8.9Hz,1H),4.85-4.79(m,1H),4.45(dd,J=8.8,4.6Hz,1H),3.27-3.19(m,1H),2.92(dd,J=16.0,9.4Hz,1H),2. 64(dd,J=15.5,4.0Hz,1H),2.33(dd,J=12.1,6.5Hz,1H),1.45(s,9H),1.07(d,J=6.8Hz,3H),1.02(d,J=6.9Hz,3H).

[0048] Example 6: Preparation of (S)-3-fluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl(tert-butyloxycarbonyl)-L-leucine ester

[0049]

[0050] The preparation method is similar to that in Example 3. 1 H NMR (600MHz, CDCl3) δ8.71-8.60(m,1H),7.73(dd,J=8.5,2.9Hz,1H),7.27(d,J=7.9Hz,1H),7.22-7.18(m,1H),7.11( d,J=1.8Hz,1H),7.09-7.05(m,1H),7.02(dd,J=8.9,4.4Hz,1H),6.86(ddd,J=9.4,5.8,2.1Hz,2H),6.84-6.79(m,2H), 6.33(s,1H),5.13(d,J=8.9Hz,1H),4.82(q,J=5.6Hz,1H),4.47(dd,J=8.8,4.6Hz,1H),3.27-3.19(m,1H),2.97-2.89( m, 1H), 2.66 (q, J = 4.6Hz, 1H), 2.34 (td, J = 13.2, 6.6Hz, 1H), 1.46 (s, 9H), 1.08 (d, J = 6.8Hz, 3H), 1.03 (d, J = 6.9Hz, 3H).

[0051] Example 7: Preparation of (S)-3-fluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl(2S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanoate

[0052]

[0053] The preparation method is similar to that in Example 3. 1H NMR(500MHz, CDCl3)δ8.78(s,1H),7.72(dd,J=8.4,1.9Hz,1H),7.26(s,1H),7.19(dd,J=15.1,7.8Hz ,1H),7.11(s,1H),7.08-6.97(m,2H),6.91-6.74(m,4H),6.33(s,1H),5.12(dd,J=32.3,8.9Hz,1H), 4.81(dd,J=13.0,5.3Hz,1H),4.67-4.45(m,1H),3.22(dd,J=16.2,7.5Hz,1H),2.93(d,J=10.8Hz,1H ), 2.64 (d, J = 15.2Hz, 1H), 2.09 (d, J = 46.9Hz, 1H), 1.46 (s, 9H), 1.39-1.23 (m, 2H), 1.12-0.94 (m, 6H).

[0054] Example 8: Preparation of (S)-3-fluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyridine[2,1-b]quinazolin-10-yl(tert-butoxycarbonyl)-L-methionine ester

[0055]

[0056] The preparation method is similar to that in Example 3. 1 H NMR (500MHz, CDCl3) δ8.41(s,1H),7.75(dd,J=8.5,2.6Hz,1H),7.29(d,J=8.7Hz,1H),7.26(s,1H),7.22(dd,J=1 4.9,7.6Hz,1H),7.15(s,1H),7.12-7.07(m,1H),7.04(dd,J=8.8,4.5Hz,1H),6.93-6.87(m,2H),6.83(t,J=9.9Hz ,2H),6.34(s,1H),5.24(d,J=7.2Hz,1H),4.84(dd,J=13.2,5.6Hz,1H),4.66(d,J=4.2Hz,1H),3.24(td,J=12.6,4 .5Hz,1H),2.98(d,J=10.6Hz,1H),2.67(dd,J=15.2,7.5Hz,3H),2.32(d,J=6.5Hz,1H),2.14(s,3H),1.47(s,9H).

[0057] Example 9: Preparation of (S)-3-fluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyridine[2,1-b]quinazolin-10-yl(tert-butoxycarbonyl)-L-phenylalanine ester

[0058]

[0059] The preparation method is similar to that in Example 3. 1 H NMR(400MHz, CDCl3)δ8.63(s,1H),7.74(dd,J=8.6,2.9Hz,1H),7.36-7.31(m,1H),7.31-7.26(m,2H) ,7.26-7.16(m,4H),7.10-7.04(m,1H),7.02(dd,J=9.0,4.7Hz,1H),7.00-6.97(m,1H),6.88(d,J=8.5 Hz,1H),6.85-6.80(m,2H),6.78(dd,J=8.8,2.3Hz,1H),6.33(s,1H),5.13(d,J=7.1Hz,1H),4.83(dd, J=12.8,5.2Hz,2H),3.29-3.17(m,3H),3.00-2.89(m,1H),2.64(dd,J=15.6,4.1Hz,1H),1.44(s,1H).

[0060] Example 10: Preparation of (S)-3-fluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyridin[2,1-b]quinazolin-10-yl(tert-butyloxycarbonyl)-L-glutamate ester

[0061]

[0062] The preparation method is similar to that in Example 3. 1H NMR (500MHz, CDCl3) δ9.10 (s, 1H), 7.70 (d, J = 7.8Hz, 1H), 7.26-7.13 (m, 2H), 7.12-6.97 (m, 3H) ,6.88(d,J=8.0Hz,1H),6.80(dd,J=16.8,10.3Hz,3H),6.31(d,J=3.9Hz,1H),6.26(s,1H),5.73 (s,1H),5.61(d,J=7.4Hz,1H),4.78(d,J=12.4Hz,1H),4.51(s,1H),3.18(d,J=5.0Hz,1H),2.8 8(d,J=11.1Hz,1H),2.59(d,J=15.0Hz,1H),2.49-2.29(m,3H),2.16-2.06(m,1H),1.45(s,9H).

[0063] Example 11: Preparation of (S)-3-fluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl O-benzyl-N-(tert-butoxycarbonyl)-L-serine ester

[0064]

[0065] The preparation method is similar to that in Example 3. 1 H NMR (500MHz, CDCl3) δ8.71 (s, 1H), 7.73 (d, J = 7.6Hz, 1H), 7.32 (m, 5H), 7.25-7.15 (m, 2H), 7.10-6.9 9(m,3H),6.86(d,J=7.8Hz,1H),6.80(d,J=8.7Hz,3H),6.30(s,1H),5.54(d,J=8.6Hz,1H),4.81(d, J=10.7Hz,1H),4.69(d,J=8.3Hz,1H),4.59(dd,J=30.7,12.0Hz,2H),4.09(d,J=6.9Hz,1H),3.81(d ,J=7.3Hz,1H),3.20(t,J=12.0Hz,1H),2.91(d,J=11.3Hz,1H),2.60(d,J=9.7Hz,1H),1.45(s,9H).

[0066] Example 12: Preparation of (S)-3-fluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl(tert-butoxycarbonyl)-L-tryptophan ester

[0067]

[0068] The preparation method is similar to that in Example 3. 1 H NMR (500MHz, CDCl3) δ8.67(s,1H),8.40(s,1H),7.72(d,J=8.4Hz,1H),7.63(d,J=7.7Hz,1H),7.34(d,J=7 .8Hz,1H),7.22-7.13(m,3H),7.10(t,J=7.3Hz,1H),7.07-7.02(m,2H),7.02-6.96(m,1H),6.86(d,J=7.9H z,1H),6.83-6.73(m,3H),6.69(d,J=7.4Hz,1H),6.26(s,1H),5.23(d,J=7.8Hz,1H),4.87(d,J=5.7Hz,1H) ,4.79(d,J=8.2Hz,1H),3.50-3.33(m,2H),3.14(d,J=12.1Hz,1H),2.84(d,1H),2.52(s,1H),1.44(s,9H).

[0069] Example 13: Preparation of (S)-3-fluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl(tert-butoxycarbonyl)-L-histidine ester

[0070]

[0071] The preparation method is similar to that in Example 3. 1 H NMR (400MHz, CDCl3) δ8.79 (s, 1H), 7.72 (dd, J = 8.6, 2.9Hz, 1H), 7.27-7.22 (m ,1H),7.22-7.16(m,1H),7.13(d,J=2.1Hz,1H),7.08-6.99(m,2H),6.90-6.7 8(m,4H),6.33(s,1H),5.27-5.11(m,1H),4.85-4.76(m,1H),4.16(d,J=5.8H z,2H),3.27-3.15(m,1H),2.99-2.86(m,1H),2.67-2.57(m,1H),1.46(s,9H).

[0072] Example 14: Preparation of (R)-3-fluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyridin[2,1-b]quinazolin-10-yl(tert-butyloxycarbonyl)glycine

[0073]

[0074] N-(tert-butoxycarbonyl)glycine (0.05 g, 0.287 mmol), EDCI (0.478 mmol), and 4-dimethylaminopyridine (0.478 mmol) were mixed and dissolved in DCM. The mixture was stirred in an ice bath for 30 min. (R)-3-fluoro-14-(3-fluorophenyl)-10-hydroxy-8,13,13b,14-tetrahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-5(7H)-one (0.239 mmol) was weighed, dissolved in DMF, and added to the mixed solution. The mixture was stirred in an ice bath for 3 h. After the reaction was complete, water was added to quench the reaction, and the mixture was extracted with ethyl acetate, evaporated to dryness, and purified by column chromatography to give a white solid with a yield of 73.9%. 1 H NMR (600MHz, CDCl3) δ8.75(s,1H),7.73(dd,J=8.5,2.8Hz,1H),7.25(d,J=10.3Hz,1H),7.20(dd,J=15. 1,8.2Hz,1H),7.13(d,J=1.9Hz,2H),7.08-7.03(m,1H),7.02(dd,J=8.8,4.5Hz,1H),6.88(d,J=8.4Hz,1 H),6.86-6.79(m,3H),6.33(s,1H),5.17(s,1H),4.81(q,J=5.6Hz,1H),4.16(d,J=5.4Hz,2H),3.22(dt ,J=12.8,4.8Hz,1H),2.93(dd,J=17.8,7.7Hz,1H),2.63(dd,J=15.4,4.0Hz,1H),1.45(d,J=6.1Hz,9H).

[0075] Example 15: Preparation of (R)-3-fluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl(tert-butyloxycarbonyl)-L-alanine ester

[0076]

[0077] The preparation method is similar to that in Example 14. 1H NMR(600MHz, CDCl3)δ8.71(s,1H),7.71(dd,J=8.5,2.8Hz,1H),7.25(s,1H),7.23-7.17(m,1H),7 .11(s,1H),7.05(dd,J=8.8,2.9Hz,1H),7.02(dd,J=12.1,3.7Hz,1H),6.90-6.84(m,2H),6.84-6 .79(m,2H),6.32(s,1H),5.16(s,1H),4.85-4.79(m,1H),4.62-4.48(m,1H),3.21(td,J=12.5,4. 7Hz, 1H), 2.92 (d, J = 9.4Hz, 1H), 2.62 (dd, J = 15.5, 4.3Hz, 1H), 1.54 (d, J = 7.0Hz, 3H), 1.44 (s, 9H).

[0078] Example 16: Preparation of (R)-3-fluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl(tert-butyloxycarbonyl)-L-valine ester

[0079]

[0080] The preparation method is similar to that in Example 14. 1 H NMR (600MHz, CDCl3) δ8.78(s,1H),7.73(dd,J=8.5,2.9Hz,1H),7.30-7.23(m,1H),7.20(q,J=8.0Hz,1H),7.11(s,1H ),7.08-7.03(m,1H),7.02(dd,J=8.9,4.5Hz,1H),6.92-6.83(m,2H),6.81(t,J=7.5Hz,2H),6.34(s,1H),5.15(d,J= 9.0Hz,1H),4.82(dd,J=13.2,5.7Hz,1H),4.46(dd,J=9.2,4.8Hz,1H),3.23(td,J=12.4,4.7Hz,1H),3.01-2.85(m,1 H), 2.65 (d, J = 15.3Hz, 1H), 2.34 (dd, J = 12.2, 6.2Hz, 1H), 1.46 (s, 9H), 1.08 (d, J = 6.8Hz, 2H), 1.03 (d, J = 6.9Hz, 3H).

[0081] Example 17: Preparation of (R)-3-fluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl(tert-butyloxycarbonyl)-L-leucine ester

[0082]

[0083] The preparation method is similar to that in Example 14. 1 H NMR (600MHz, CDCl3) δ8.58(s,1H),7.74(dd,J=8.6,2.9Hz,1H),7.26(d,J=6.1Hz,1H),7.23-7.17(m,1H),7.13 (d,J=2.1Hz,1H),7.10-7.05(m,1H),7.05-7.01(m,1H),6.90-6.85(m,2H),6.85-6.79(m,2H),6.34(s,1H),5. 02(d,J=8.7Hz,1H),4.83(dd,J=13.2,5.7Hz,1H),4.54(td,J=9.0,4.3Hz,1H),3.30-3.16(m,1H),3.00-2.88( m,1H),2.70-2.61(m,1H),1.88-1.77(m,2H),1.66(t,J=9.0Hz,1H),1.46(s,9H),1.01(dd,J=5.8,2.0Hz,6H).

[0084] Example 18: Preparation of (R)-3-fluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl(2S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanoate

[0085]

[0086] The preparation method is similar to that in Example 14. 1H NMR (600MHz, CDCl3) δ8.56(s,1H),7.74(dd,J=8.5,2.6Hz,1H),7.27(d,J=8.7Hz,1H),7.20(dd,J=14.8,7.6Hz,1 H),7.12(s,1H),7.09-7.05(m,1H),7.03(dd,J=8.8,4.4Hz,1H),6.87(d,J=8.5Hz,2H),6.85-6.77(m,2H),6.34(s ,1H),5.11(dd,J=41.0,9.2Hz,1H),4.83(q,J=5.6Hz,1H),4.66-4.47(m,1H),3.29-3.17(m,1H),2.94(s,1H),2.6 6(q,J=4.1Hz,1H),2.09(d,J=54.9Hz,1H),1.64-1.52(m,1H),1.45(s,9H),1.35-1.27(m,1H),1.10-0.95(m,6H).

[0087] Example 19: Preparation of (R)-3-fluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyridine[2,1-b]quinazolin-10-yl(tert-butoxycarbonyl)-L-methionine ester

[0088]

[0089] The preparation method is similar to that in Example 14. 1 H NMR(400MHz, CDCl3)δ8.42(s,1H),7.73(dd,J=8.6,2.8Hz,1H),7.28(s,1H),7.25-7.19(m,1H),7.15(t,J =8.5Hz,1H),7.06(ddd,J=11.3,8.6,3.8Hz,2H),6.92-6.86(m,1H),6.86-6.80(m,2H),6.33(s,1H),5.24( t,J=12.9Hz,1H),4.82(dd,J=13.3,5.1Hz,1H),4.64(d,J=4.7Hz,1H),3.22(td,J=12.9,4.8Hz,1H),3.03- 2.90(m,1H),2.72-2.60(m,3H),2.38-2.27(m,1H),2.13(d,J=5.1Hz,1H),2.12-2.02(m,4H),1.44(s,9H).

[0090] Example 20: Preparation of (R)-3-fluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyridine[2,1-b]quinazolin-10-yl(tert-butoxycarbonyl)-L-phenylalanine ester

[0091]

[0092] The preparation method is similar to that in Example 14. 1 H NMR (600MHz, CDCl3) δ8.58(s,1H),7.73(dd,J=8.5,2.9Hz,1H),7.33(d,J=7.1Hz,1H),7.27(t,J=7.3 Hz,1H),7.26-7.19(m,4H),7.06(dd,J=8.7,2.8Hz,1H),7.04-7.01(m,1H),6.99(dd,J=14.0,3.2Hz, 1H),6.87(s,1H),6.82(d,J=7.1Hz,2H),6.80-6.75(m,1H),6.31(s,1H),5.11(d,J=4.1Hz,1H),4.82 (dd,J=13.0,5.7Hz,2H),3.21(dd,J=19.9,5.3Hz,3H),2.93(s,1H),2.68-2.60(m,1H),1.43(s,9H).

[0093] Example 21: Preparation of (R)-3-fluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyridine[2,1-b]quinazolin-10-yl(tert-butyloxycarbonyl)-L-glutamate ester

[0094]

[0095] The preparation method is similar to that in Example 14. 1 H NMR(500MHz, CDCl3)δ8.97(s,1H),7.78-7.65(m,1H),7.26-7.15(m,2H),7.13-6.99(m,3H),6.94-6.73(m,4H),6.43-6.17(m,2H),5.81-5.49 (m,2H),4.79(d,J=12.1Hz,1H),4.52(s,1H),3.32-3.08(m,1H),2.91(s,1H),2.61(d,J=15.0Hz,1H),2.40(s,3H),2.10(s,1H),1.45(s,9H).

[0096] Example 22: Preparation of (R)-3-fluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl O-benzyl-N-(tert-butoxycarbonyl)-L-serine ester

[0097]

[0098] The preparation method is similar to that in Example 14. 1 H NMR (500MHz, CDCl3) δ8.58 (s, 1H), 7.74 (d, J = 8.1Hz, 1H), 7.36-7.27 (m, 5H), 7.25-7.16 (m, 2H), 7.08 (s, 1H) ,7.03(ddd,J=13.4,8.3,3.4Hz,2H),6.87(d,J=8.1Hz,1H),6.81(t,J=7.8Hz,3H),6.31(s,1H),5.53(d,J=8 .8Hz,1H),4.86-4.76(m,1H),4.69(d,J=8.6Hz,1H),4.59(dd,J=29.8,12.1Hz,2H),4.09(d,J=7.5Hz,1H),3 .81(d,J=7.4Hz,1H),3.21(t,J=12.3Hz,1H),2.91(dd,J=14.9,11.3Hz,1H),2.68-2.53(m,1H),1.46(s,9H).

[0099] Example 23: Preparation of (R)-3-fluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl(tert-butoxycarbonyl)-L-tryptophan ester

[0100]

[0101] The preparation method is similar to that in Example 14. 1H NMR (500MHz, CDCl3) δ8.55(s,1H),8.33(s,1H),7.72(d,J=8.4Hz,1H),7.63(d,J=7.7Hz,1H),7.34(d,J=7.9 Hz,1H),7.18(dd,J=16.4,8.4Hz,3H),7.10(t,J=7.3Hz,1H),7.04(d,J=11.6Hz,2H),7.00(dd,J=8.6,4.4Hz, 1H),6.90-6.73(m,4H),6.68(t,J=7.9Hz,1H),6.25(s,1H),5.24(d,J=7.9Hz,1H),4.87(d,J=5.7Hz,1H),4. 82-4.72(m,1H),3.56-3.31(m,2H),3.14(t,J=12.0Hz,1H),2.86(d,J=6.3Hz,1H),2.51(s,1H),1.44(s,9H).

[0102] Example 24: Preparation of (R)-3-fluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl(tert-butoxycarbonyl)-L-histidine ester

[0103]

[0104] The preparation method is similar to that in Example 14. 1 H NMR (400MHz, CDCl3) δ8.79 (s, 1H), 7.72 (dd, J = 8.6, 2.9Hz, 1H), 7.27-7.22 (m ,1H),7.22-7.16(m,1H),7.13(d,J=2.1Hz,1H),7.08-6.99(m,2H),6.90-6.7 8(m,4H),6.33(s,1H),5.27-5.11(m,1H),4.85-4.76(m,1H),4.16(d,J=5.8H z,2H),3.27-3.15(m,1H),2.99-2.86(m,1H),2.67-2.57(m,1H),1.46(s,9H).

[0105] Comparative Example 1: Preparation of (S)-3-fluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl N-(tert-butoxycarbonyl)-O-(tert-butyl)-L-serine ester

[0106]

[0107] The preparation method is similar to that in Example 3. 1 H NMR (500MHz, CDCl3) δ8.4(s,1H),7.8-7.7(m,1H),7.3(d,J=8.8Hz,1H),7.2-7.2(m,1H),7.1(d,J =2.2Hz,1H),7.1-7.1(m,1H),7.1-7.0(m,1H),6.9-6.9(m,2H),6.8-6.8(m,2H),6.3(s,1H),5.5(d ,J=9.0Hz,1H),4.8(dd,J=13.3,5.7Hz,1H),4.6(d,J=9.1Hz,1H),4.0(dd,J=9.0,3.0Hz,1H),3.7( dd,J=8.9,3.0Hz,1H),3.3-3.2(m,1H),3.0(s,1H),2.7(d,J=19.7Hz,1H),1.5(s,9H),1.2(s,9H).

[0108] Comparative Example 2: Preparation of (R)-3-fluoro-14-(3-fluorophenyl)-5-oxo-5,7,8,13,13b,14-hexahydroindole[2',3':3,4]pyrido[2,1-b]quinazolin-10-yl N-(tert-butoxycarbonyl)-O-(tert-butyl)-L-serine ester

[0109]

[0110] The preparation method is similar to that in Example 14. 1 H NMR (400MHz, CDCl3) δ8.4 (s, 1H), 7.76 (dd, J = 8.6, 2.9Hz, 1H), 7.28-7.23 (m, 1H), 7.25-7 .16(m,1H),7.14-7.11(m,1H),7.09-7.05(m,1H),7.04-6.99(m,1H),6.90-6.79(m,4H), 6.33(s,1H),5.25-5.04(m,1H),4.85-4.76(m,1H),4.66-4.43(m,1H),3.97(d,J=5.8Hz, 2H),3.28-3.16(m,1H),2.99-2.85(m,1H),2.70-2.60(m,1H),1.45(s,9H),1.23(s,9H).

[0111] Test Example 1: MTT assay to determine the inhibitory rate of a compound on tumor cell growth

[0112] Test Method: Colon cancer cell lines (HT-29, HCT-116, LOVO, RKO), gastric cancer cell lines (HGC-27, MGC-803, SGC-7901), and liver cancer cell lines (HUH7, SK-EP1) 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) 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.

[0113] Test results:

[0114] Table 1. Antiproliferative activity of compounds against colon cancer cell lines.

[0115]

[0116] Table 2. Antiproliferative activities of compounds against different cancer cell lines.

[0117]

[0118] As can be seen from Tables 1 and 2, the compounds of the present invention have good to excellent inhibitory effects on colon cancer cell lines HT-29, HCT-116, LOVO, RKO, HGC-27, MGC-803, SGC-7901, Huh7, SK-EP1 and MCE7, and some compounds have better activity than typical compound A and typical compound B.

[0119] Test Example 2: Inhibition Experiment of Compounds on Topoisomerase I (Top1)

[0120] Test Method: Groups: DNA, DNA+Top1, CPT, EVO, target compound 10 (200, 100, 50, 25 μM), and partially synthesized compound (100 μM). For each group, add 2 μL of 10× DNA Topoisomerase I Buffer (350 mM Tris-HCl, (pH 8.0), 720 mM KCl, 50 mM DTT, 50 mM spermidine, 50 mM MgCl2), 2 μL of 0.1% BSA, and 10 μL of ultrapure water. Add 0.2 μL of different concentrations of positive control and test compound (DNA and DNA+Top1 groups added with the corresponding volume of DMSO), 0.5 μL of Top1 (0.5 units, not added to the DNA group), mix thoroughly, and incubate at 37°C for 20 minutes. Then add 0.5 μL of pBR322 plasmid DNA, supplemented with ultrapure water, to a final volume of 20 μL. After incubation at 37°C for 15 min, the reaction was terminated with 4 μL of loading buffer. Then, 10 μL of sample was added to a 0.8% agarose gel TAE (Tris-acetate-EDTA) and electrophoresed at a constant voltage of 110V for 1 hour. After electrophoresis, the DNA gel was stained with green fluorescent dye (10 μL / 100 mL ultrapure water) for 30 min, and the DNA bands were photographed using a ChampGel 6000 (Beijing Sage Creation, China).

[0121] Experimental results are as follows Figure 1 As shown, through Figure 1 It can be seen that the compounds of the present invention exhibit good to excellent inhibitory effects on topoisomerase I at specific concentrations.

[0122] Test Example 3: Compound Effects on LOVO and RKO Cell Cycle Arrest Experiments

[0123] Test method: LOVO and RKO cells were seeded in 6-well plates (5 × 10⁻⁶ cells per well). 5Cells / well). After culturing for 24 hours and allowing cells to adhere, add TPT and different concentrations (LOVO and RKO concentration gradients of 10, 30, and 90 nmol / L) of compound 10 to each well. After 24 hours of culture, collect all liquid from the wells, digest with 0.5 ml of trypsin for 3 minutes, add 1 ml of complete culture medium to stop the digestion, collect the liquid into a 15 ml centrifuge tube, centrifuge at 1500 rpm for 3 minutes in a pre-chilled 4°C centrifuge, remove the supernatant, add 1 mL of PBS, mix well by pipetting, transfer to a 1.5 mL centrifuge tube, and centrifuge at 1000 rpm for 3 minutes in a pre-chilled 4°C centrifuge. Wash three times with plain PBS, centrifuge, and repeat three times. Fix the cells with 500 μL of 70% pre-chilled ethanol (diluted with PBS) and store at 4°C for two hours to overnight. After fixation, centrifugation was continued to remove the fixative, and the fixative was washed away with PBS. For staining, 100 μL of RNase A solution was added to the cell pellet, the cells were mixed and resuspended, and incubated at 37°C for 30 min. Then, 400 μL of PI staining solution was added to each sample, mixed, and incubated at 4°C in the dark for 30 min. The cells were then analyzed using a microarray, and the red fluorescence at an excitation wavelength of 488 nm was recorded. Data analysis was performed using Modfit software.

[0124] Experimental results are as follows Figure 2 As shown, through Figure 2 It can be seen that compound 10 of the present invention, at a nanomolar concentration, arrests cell cycle in the G2 / M phase of colon cancer cell lines LOVO and RKO.

[0125] Test Example 4: Compound-induced apoptosis experiment in LOVO and RKO cells

[0126] Experimental method: LOVO and RKO cells were seeded in 6-well plates (5 × 10⁻⁶ cells per well). 5Cells per well. On the second day, after cell attachment, cells were treated with prepared target compound 10 (concentration gradients of 10, 30, and 90 nmol / L) and drug-free medium, respectively. After culturing at 37°C for 24 h, all liquid in the six-well plate was collected, washed once with 1 mL PBS, and then digested with 500 μL of EDTA-free trypsin for 3 min. The digestion was stopped with 1 mL of medium, and the plate was centrifuged for 3 min at 1500 rpm in a pre-chilled 4°C centrifuge. After centrifugation, the plate was placed at 4°C, and the supernatant was aspirated, leaving some liquid to avoid removing cells. Approximately 1 mL of PBS (pre-chilled at 4°C) was added, and the cells were resuspended by pipetting and transferred to 1.5 mL centrifuge tubes. The plates were centrifuged again, and the supernatant was carefully removed. Resuspend cells by pipetting in 100 μL of Binding Buffer (diluted 1:9 with deionized water); transfer to flow cytometry tubes. Do not add Annexin V / FITC or propidium iodide solution (PI) to the negative control tubes (for voltage adjustment); add only 5 μL of Annexin V / FITC to the Annexin V / FITC single staining tubes (for compensation adjustment); add 5 μL of Annexin V / FITC to the blank control and drug-containing flow cytometry tubes, mix well, and incubate at room temperature in the dark for 5 min; add 5 μL of propidium iodide solution (PI) to the PI single staining tubes and other tubes. Immediately after adding PI, analyze the cells using a flow cytometer.

[0127] Experimental results are as follows Figure 3 As shown, through Figure 3 It can be seen that compound 10 of the present invention can significantly induce apoptosis in colon cancer cell lines LOVO and RKO at nanomolar concentrations.

[0128] Test Example 5: Drug Metabolism Study in Rats

[0129] The compound provided in the aforementioned examples, at a concentration of 50 mg / kg, was dissolved in physiological saline containing 2.5% polyoxyethylene castor oil and 2.5% DMSO, and then administered via intraperitoneal injection. Blank blood samples were collected before administration, and rat cardiac blood samples were collected at predetermined time points after administration: 10 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, and 24 h post-administration. 20 μL of the working solution was added to 100 μL of blank plasma sample, vortexed, and then 300 μL of a methanol solution containing 40 ng / mL propranolol internal standard was added, vortexed, and centrifuged at 14000 rpm for half an hour at 4°C. The supernatant was then analyzed by LC-MS / MS. Chromatographic conditions: Mobile phase A was aqueous phase, 5% methanol (containing 0.1% formic acid); mobile phase B was organic phase, 95% methanol (containing 0.1% formic acid). The flow rate was 0.4 mL / min. -1 Column temperature: room temperature. Pharmacokinetic parameters were calculated using DAS 2.0 software, with 3 rats per group. Data are expressed as the mean for each group, and AUC is obtained.0-t AUC 0-∞ MRT 0-∞ C max T max 、 and t 1 / 2 Parameters such as these.

[0130] Table 3. Pharmacokinetic parameters of rats after intraperitoneal injection of 10 (50 mg / kg)

[0131]

[0132] Table 3 lists the pharmacokinetic parameters of the compounds of the present invention in rats after intraperitoneal injection. The results show that the compounds of the present invention possess favorable pharmacokinetic properties, including ideal clearance (CL) and half-life (T). 1 / 2 ), peak concentration (C) max ) and exposure (AUC) 0-t ).

[0133] Test Example 6: Antitumor activity of the compounds of the present invention in a nude mouse xenograft tumor model

[0134] Nude mice were placed in an SPF-grade animal facility after inoculation, and their growth was closely monitored, with tumor diameter measured daily. When the tumor size reached 100 mm... 3 Around 10:00 AM, 24 nude mice with good growth and similar tumor volume were selected and randomly divided into three groups of 8 mice each. These three groups were labeled as a saline control group, a topotecan (0.5 mg / kg) administration group, and a compound 10 (40 mg / kg) administration group. The mice were administered the compound intraperitoneally once daily. From the start of administration, the weight and tumor size of each group were measured daily, and the tumor volume was calculated. Administration was continued for 14 consecutive days, and tumor growth was monitored for approximately 14 days. After 14 days, all mice were sacrificed, tumor tissue was obtained, the tumor weight was measured, and the tumors were photographed and preserved. The experimental indicator was to investigate the effect of the compound in the example on tumor growth, specifically the tumor inhibition rate (TGI) (%). The tumor diameter was measured three times a week using calipers. The tumor volume (V) was calculated using the formula: V = 1 / 2 × a × b 2Where a and b represent the length and width respectively. T / C(%) = (T - T0) / (C - C0) × 100%, where T and C are the tumor volumes at the end of the experiment; T0 and C0 are the tumor volumes at the start of the experiment. When the tumor shows regression, the tumor growth inhibition rate (TGI)(%) = 100 - (T - T0) / T0 × 100. If the tumor shrinks compared to the initial volume, i.e., T < T0 or C < C0, it is defined as partial regression (PR) of the tumor; if the tumor completely disappears, it is defined as complete regression (CR) of the tumor. The comparison between the tumor volumes of the two groups is performed using a two-tailed Student's t-test, and P < 0.05 is defined as having a statistically significant difference.

[0135] The results of this test example are listed in Figure 4 where BLK represents the blank control, TPT represents topotecan, i.p. represents intraperitoneal injection, and qd represents once-a-day administration.

[0136] It can be seen from Figure 4 that the compound of the present invention exhibits excellent anti-tumor activity in the human colon cancer cell HT-29 xenograft model. Compared with the model group, the compound 10 and TPT groups significantly reduced the tumor weight (p < 0.0001) and volume. It shows that the target compound can significantly inhibit the growth of tumors and has a statistical difference compared with the model group. In addition, the body weights of the mice in the dosing group did not show a significant decrease, while the body weights of the mice in the TPT group decreased significantly compared with the model group (p < 0.0001), which indicates that the compound 10 has less toxicity when administered at a dose of 40 mg / kg and has better safety than TPT.

[0137] The above results show that the evodiamine derivatives with the structures shown in formula (I) and formula (I) provided by the present invention, or their stereoisomers, geometric isomers, tautomers, N-oxides, hydrates, solvates, or their metabolites exhibit excellent inhibitory activities against HT-29, HTC-116, LOVO, RKO, HGC-27, MGC-803, SGC-7901, Huh7, SK-EP1 and MCF-7 tumor cell lines. At the same time, they can exhibit good anti-tumor activities at the animal level.

[0138] Those of ordinary skill in the art should understand that: the discussion of any above embodiment is only exemplary and is not intended to imply that the protection scope of the present application is limited to these examples; under the idea of the present application, the technical features between the above embodiments or different embodiments can also be combined, and the steps can be implemented in any order, and there are many other variations in different aspects of one or more embodiments of the present application as described above, and they are not provided in detail for the sake of brevity.

[0139] 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. An ester-substituted erythromycin derivative, characterized by the following structural formula: as follows: ， R is , , , , , , , , , , or .

2. The ester-substituted evodiamine derivative as described in claim 1, characterized in that, R is .

3. A method for preparing an ester-substituted evodiamine derivative as described in claim 1 or 2, characterized in that, The process includes the following steps: Compound I is mixed with ROH and reacted to obtain an ester-substituted erythromycin derivative. The structural formula of Compound I is [insert structural formula here]. The condensing agent for the reaction is EDCI, and the catalyst for the reaction is 4-dimethylaminopyridine.

4. The preparation method according to claim 3, characterized in that, The solvent for the reaction is dichloromethane.

5. The preparation method according to claim 3, characterized in that, Compound I is dissolved in the solvent DMF.

6. The preparation method according to claim 3, characterized in that, The molar amount of compound I is less than the molar amount of ROH.

7. The application of an ester-containing substituted evodiamine derivative as described in claim 1 or 2, characterized in that, The ester-substituted evodiamine derivative is used to prepare drugs for inhibiting colon cancer.

8. The application of an ester-substituted evodiamine derivative as described in claim 1 or 2, characterized in that, The ester-substituted evodiamine derivative is used to prepare a drug that inhibits the following cell lines: HT-29, HTC-116, LOVO, RKO, HGC-27, MGC-803, SGC-7901, Huh7, SK-HEP-1, and MCF-7 tumor cell lines.

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