Anti-cancer agent TYJ2517 targeting MCF-7 human breast cancer cells as well as preparation and application of anti-cancer agent TYJ2517

Abstract

The invention provides an isoalantolactone derivative TYJ2517, namely 3, 4, 5-trimethoxybenzyl aminomethyl 3-demethyleneisoalantolactone as well as preparation and application thereof, and relates to the field of medicinal compounds, the structure of the isoalantolactone derivative is shown as follows: although many anti-cancer drugs have anti-cancer ability, the drugs generate toxicity to normal cells, and the drugs cannot generate toxicity to the normal cells. Therefore, the development of selective anti-cancer active drugs safe to normal cells is particularly important. According to the invention, TYJ2517 is synthesized, the IC50 value of the TYJ2517 to MCF-7 cells is 3.6 + / -0.8 [mu] M, and the IC50 value of the TYJ2517 to L-02 normal cells exceeds 100 [mu] M. A cell drug uptake capability test result shows that the TYJ2517 uptake capability of L-02 normal cells is low, and the TYJ2517 uptake capability of MCF-7 cancer cells is high. When the administration time is 1 hour, the selective anti-cancer index (SI) of the TYJ2517 to MCF-7 breast cancer cells and L-02 normal cells exceeds 40. Mechanism research shows that the activity of the TYJ2517 for resisting MCF-7 cancer cells is a result of inhibiting the expression quantity of PARP1 protein kinase and ROS, inducing early and late apoptosis of the MCF-7 cells and hindering migration and colony of the MCF-7 cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical compounds, in particular to an anti-cancer agent TYJ2517 targeting MCF-7 human breast cancer cells and its preparation and use. BACKGROUND

[0002] Isoalantolactone (ISA or IAL) (CAS No. 470-17-7) is a sesquiterpene lactone compound with significant biological activities such as analgesic, anti-tumor, anti-inflammatory, antibacterial and anthelmintic activities [1-2] , mainly existing in plants of the Asteraceae family, such as ragweed plants of the genus Ambrosia [3] . Its chemical name is (3aR,4aS,8aR,9aR)-8a-methyl-3,5-dimethylenedecahydronaphtho[2,3-b]furan-2(3H)-one, and its chemical structure is shown in structural formula 1. From the chemical structure, isoalantolactone (molecular formula C 15 H 20 O2, molecular weight 232.32 g / mol) has a characteristic α-methylene-γ-lactone ring structure, and chemical modification of this structure is an attractive field of pharmaceutical chemistry research.

[0003] Isoalantolactone does not contain polar groups, so it is almost insoluble in water, which limits its application [4] . Therefore, there are many studies on improving water solubility by modifying its structure. Kumar et al. modified it into an amino adduct to improve water solubility and pharmacokinetics, while retaining its anti-tumor activity [5] . The α-methylene group is the main site for modification of isoalantolactone [5-12] , so by forming various hybrid molecules through the exocyclic α-methylene group of isoalantolactone and quinoline, isoquinoline, caffeine, theobromine and theophylline, the shortcomings of isoalantolactone are changed [8，12] .

[0004] Studies have found that the anti-allergic, antioxidant and anticancer activities of isoalantolactone are related to reactive oxygen species (ROS)-mediated pathways [13-16] . Isoalantolactone has selective toxicity to cancer cells in vitro and in vivo, and its anticancer effect is related to the overexpression of ROS [17-18] . A large number of studies have shown that phytochemicals targeting ROS metabolism can selectively kill cancer cells by increasing ROS levels above the toxicity threshold. Since the ROS level of cancer cells is higher than that of normal cells, the toxicity threshold of cancer cells is more easily reached than that of normal cells

[19] .

[0005] The poly ADP-ribose polymerase (PARP) family contains 17 members, which are involved in a variety of cellular processes such as DNA replication and repair. Recent studies have shown that PARP1 inhibition and its DNA capture ability are key to anti-tumor activity. PARP-1 is a key nuclease that repairs DNA damage caused by oxidative stress. PARP-1 promotes NOX complex-mediated ROS production by up-regulating the transcriptional activity of Ets-1, leading to oxidative DNA damage and cell death

[20] .

[0006] No studies on the toxicity of isoalantolactone have been published so far, but the sesquiterpene lactone mixture containing isoalantolactone was positive in human skin patch tests

[21] . Isoalantolactone has an α, β-unsaturated moiety, which is an α-methyl-γ-lactone as a Michael acceptor, and interacts with nucleophilic substances such as sulfhydryl groups (R-SH) in enzymes and other proteins, leading to the destruction of macromolecules and redox functions [21-23] , and its therapeutic activity can also cause serious toxicity.

[0007] Based on the fact that isoalantolactone is a promising natural anti-tumor drug precursor that has been proven, it is expected that more safe and efficient anti-cancer drugs can be found by chemically modifying its parent molecule.

[0008] References:

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[0010] [2] He G, Zhang X, Chen Y, et al. Isoalantolactone inhibits LPS-induced inflammation via NF-κB inactivation in peritoneal macrophages and improves survival in sepsis [J]. Biomedicine & Pharmacotherapy, 2017, 90(90): 598-607.

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[0018]

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[0019]

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[0020]

[12] Stepanova V A, Patrushev S S, Rybalova T V, et al. Crosscopling reaction to access a library of eudesmane-type methylene lactones with quinoline or isoquinoline substituent [J]. Journal Of Molecular Structure, 2022, 1247: 131373.

[0021]

[13] Chen W, Li P, Liu Y, et al. Isoalantolactone induces apoptosis through ROS-mediated ER stress and inhibition of STAT3 in prostate cancer cells [J]. Journal Of Experimental & Clinical Cancer Research, 2018, 37(1): 309.

[0022]

[14] Ding Y H, Song Y D, Wu Y X, et al. Isoalantolactone suppresses LPS induced inflammation by inhibiting TRAF6 ubiquitination and alleviates acute lung injury [J]. Acta Pharmacologica Sinica, 2018, 40(1): 64-74.

[0023]

[15] Hu F, Yahg P. Isoalantolactone exerts anticancer effects on human HEC-1-B endometrial cancer cells via induction of ROS mediated apoptosis and inhibition of MEK / ERK signalling pathway [J]. Acta Biochimica Polonica, 2022, 69(2): 453-458.

[0024]

[16] Wu Z C, Hui X G, Hno L, et al. Antiproliferative effects of isoalantolactone in human liver cancer cells are mediated through caspase-dependent apoptosis, ROS generation, suppression of cell migration and invasion and targeting Ras / Raf / MEK signalling pathway [J]. Acta Biochimica Polonica, 2022, 69(2): 299-304.

[0025]

[17] Rasul A, Di J, Millimouno F M, et al. Reactive oxygen species mediate isoalantolactone-induced apoptosis in human prostate cancer cells [J]. Molecules, 2013, 18(8): 9382-9396.

[0026]

[18] Cai H, Meng X, Li Y, et al. Growth inhibition effects of isoalantolactone on K562 / A02 cells: Caspase-dependent apoptotic pathways, S phase arrest, and downregulation of Bcr / Ab1 [J]. Phytotherapy Research, 2014, 28(11): 1679-1686.

[0027]

[19] Ho B Y, Wu Y M, Chang K J, et al. Dimerumic acid inhibits SW620 cell invasion by attenuating H2O2-mediated MMP-7 expression via JNK / C-Jun and ERK / C-Fos activation in an AP-1-dependent manner [J]. International Journal of Biological Sciences, 2011, 7(6): 869-880.

[0028]

[20] Hervieu M, Breusa S, Allio M, et al. PARP-1 inhibition increases oxidative stress in Ets-1-expressing MDA-MB-231 breast cancer cells [J]. Cancer Reports (Hoboken), 2025, 8(1): e70119.

[0029]

[21] Amorim M H, Gil da Costa R M, Lopes C, et al. Sesquiterpene lactones: Adverse health effects and toxicity mechanisms [J]. Critical Reviews In Toxicology, 2013, 43(7): 559-579.

[0030]

[22] Zhang S, Won Y K, Ong C N, et al. Anti-cancer potential of sesquiterpene lactones: Bioactivity and molecular mechanisms [J]. Current Medicinal Chemistry Anticancer Agents, 2005, 5(3): 239-249.

[0031]

[23] Ghantous A, Gali-Muhtasib H, Vuorela H, et al. What makes sesquiterpene lactones reach cancer clinical trials?[J]. Drug Discovery Today, 2010, 15(15-16): 668-678.

[0032]

[0033] Structure 1: Structure of isoalantolactone SUMMARY

[0034] In view of the deficiencies of the prior art, in the process of modifying the parent structure of isoalantolactone, it is found that the outer ring double bond of the α-methylene-γ-lactone group is modified with 3,4,5-trimethoxybenzylamine as a Michael electron donor to synthesize 3,4,5-trimethoxybenzylaminomethyl 3-desmethylisoalantolactone (TYJ2517). The present application provides TYJ2517, i.e. 3,4,5-trimethoxybenzylaminomethyl 3-desmethylisoalantolactone, and a preparation method and use thereof. The compound is obtained by a chemical synthesis method, and its activity is determined by an MTT method, and it is determined that TYJ2517 has very strong inhibitory proliferation activity on MCF-7, HepG2, SW620, AGS, SU-DHL-10 and other five kinds of human cancer or human lymphoma cells, and has very weak toxicity on L-02 normal human liver cells, and has very excellent selective anticancer activity. Anti-cancer drugs generally have a short retention time in the body, and if the cells do not take the drugs in time, the drugs cannot fully exert their anticancer effect. Therefore, the ability of the cells to take the drugs is determined by a simulation experiment, i.e. MCF-7 cells and L-02 normal cells are respectively treated with a culture medium containing TYJ2517 or isoalantolactone for 0.5, 1, 2, 4, 6 and 8 hours, and then fresh culture medium is replaced, and the cells are respectively cultured for another 48 hours, and then the half inhibitory concentration IC50 of the cells is determined by an MTT method. The results show that the IC50 of MCF-7 cells is 0.5 μg / mL, and the IC50 of L-02 normal cells is 10 μg / mL, which indicates that the TYJ2517 has very strong anticancer activity and very weak toxicity on normal cells. 50The results show that L-02 normal cells hardly uptake TYJ2517 at 0.5, 1, 2, 4, 6 and 8 hours of treatment time, while the ability of MCF-7 cancer cells to uptake TYJ2517 is very significant, which shows that TYJ2517 has strong targeting MCF-7 cancer cell killing ability, and it is speculated that TYJ2517 can target and kill MCF-7 cancer cells without affecting L-02 normal liver cells when used in clinical treatment. The ability of L-02 normal cells to uptake isoalantolactone is stronger than that of MCF-7 cancer cells, and isoalantolactone has strong toxicity to L-02 normal cells. The selective anti-MCF-7 cancer cell level of the compound TYJ2517 is more than 200 times that of isoalantolactone, and TYJ2517 has targeting effect on MCF-7 breast cancer cells when it is distributed in vivo. In addition, the present application provides the results that the water solubility of TYJ2517 at pH 2.0, 7.4 and 9.0 is higher than that of isoalantolactone, and the results that the proliferation inhibitory activity of TYJ2517 on MCF-7 human breast cancer cells is achieved by inhibiting the expression amount of PARP1 and ROS, inducing early and late MCF-7 cell apoptosis, hindering the migration, invasion and colony of MCF-7 cells.

[0035] To achieve the above object, the present application is realized by the following technical solutions:

[0036] The structure of TYJ2517 is shown in structural formula 2:

[0037]

[0038] Structural formula 2: 3,4,5-trimethoxybenzylaminomethyl 3-demethylenisoalantolactone (TYJ2517)

[0039] Example 1: Synthesis method

[0040] The technical solutions of the present application are clearly and completely described. Based on the examples in the present application, all other examples obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0041] Preparation of 3,4,5-trimethoxybenzylaminomethyl 3-demethylenisoalantolactone (TYJ2517)

[0042]

[0043] In room temperature condition, first, isoalantolactone (40 mg, 0.172 mmol) was added in a single mouth round bottom flask fixed on a magnetic stirrer, 5 mL of absolute ethanol (EtOH) was added as reaction solvent, after 10 min stirring, catalyst triethylamine (Et3N) 400 μL (2.87 mmol) was added, after 30 min stirring, 3,4,5-trimethoxybenzylamine (50.89 mg, 0.258 mmol) was added, continue stirring for 48 h for complete reaction, reaction was followed by thin layer chromatography. After completion of reaction, reaction was quenched with 30 mL distilled water, then extracted with 20 mL ethyl acetate, repeated 3 times, combined ethyl acetate layer was extracted with 30 mL saturated NaCl solution once, organic layer was collected and dried over anhydrous magnesium sulfate, concentrated under reduced pressure, purified by silica gel column chromatography (petroleum ether: ethyl acetate = 15: 1), to get the target compound (TYJ2517) 38.17 mg.

[0044] White powder, yield 88.91%, melting point: 104-108 °C; 1 H NMR (300 MHz, Chloroform-d) δ 6.57 (s, 2H), 4.76 (q, J = 1.6 Hz, 1H), 4.49 (td, J = 4.2, 1.8 Hz, 1H), 4.39 (q, J = 1.6 Hz, 1H), 3.84 (d, J = 7.9 Hz, 9H), 3.81 - 3.70 (m, 2H), 3.05 (dd, J = 11.4, 7.0 Hz, 1H), 2.93 (q, J = 6.8 Hz, 1H), 2.77 (dd, J = 11.4, 7.1 Hz, 1H), 2.49 (dtd, J = 12.1, 6.0, 4.1 Hz, 1H), 2.36 - 2.29 (m, 1H), 2.16 (dd, J = 15.5, 1.9 Hz, 1H), 2.04 - 1.95 (m, 1H), 1.94 - 1.62 (m, 3H), 1.56 (t, J = 3.3 Hz, 2H), 1.50 (td, J = 8.2, 7.0, 3.6 Hz, 2H), 1.25 (d, J = 2.8 Hz, 1H), 1.20 (d, J = 12.9 Hz, 1H), 0.79 (s, 3H). 13 C NMR (75 MHz, Chloroform-d) δ 178.19, 153.28 (2C), 149.24, 136.94, 135.76, 106.50, 104.88 (2C), 78.33, 60.85, 56.11 (2C), 54.49, 47.60, 46.54, 44.90, 42.24, 41.49, 39.23, 36.74, 34.83, 22.68, 21.14, 17.82. HRMS (ESI) m / z calcd for C25 H 35 NO5 + (M+H) + 430.2593, found 430.2598.

[0045] Example 2: Bioactivity experiment

[0046] The experimental procedure is as follows:

[0047] ①TYJ2517 and the control substance isoalantolactone were prepared into stock solutions with a final concentration of 1000 times with dimethyl sulfoxide (DMSO) and stored in a sterile environment in the dark and refrigerated, and before use, the stock solution was diluted to 1000 times of the test solution with the corresponding culture medium, and was ready for use.

[0048] ②Before the experiment, human colorectal adenocarcinoma cells SW620, human gastric cancer early differentiation cells SGC-7901, human gastric cancer cells AGS, human B cell lymphoma cells SU-DHL-10 and human normal liver cells L-02 were cultured with RPMI-1640 culture medium (containing 10% FBS and 1% penicillin-streptomycin double antibody); human hepatocellular carcinoma cells HepG-2, human cervical squamous carcinoma cells SiHa, human uterine cancer cells HeLa and human lung cancer cells A549 were cultured with DMEM culture medium containing high glucose (containing 10% FBS and 1% penicillin-streptomycin double antibody); human breast cancer cells MCF-7 were cultured with DMEM basic (1X) culture medium (containing 10% FBS and 1% penicillin-streptomycin double antibody).

[0049] ③Take the logarithmic growth period of SW620, SGC-7901, AGS, MCF-7, HepG-2, SiHa, HeLa, A549, SU-DHL-10 and L-02, and inoculate 8000-10000 cells per hole in a 96-well plate.

[0050] ④After culturing overnight, replace the above test solution with different concentrations, continue to culture for 48 hours, discard the culture medium, add 150 μL of 0.01 g / L MTT solution to each well, continue to culture for 4 h, discard the culture medium, dissolve the generated formazan with DMSO, and measure the absorbance value at 492 nm wavelength. The inhibition rate is calculated according to the following formula.

[0051] Inhibition rate % = [(1-experimental group absorbance value) / control group absorbance value] X 100%

[0052] According to the inhibition rate value, the half inhibitory concentration (IC 50 ) of the test compound on cells is calculated again. All experiments are repeated more than 3 times, and the final result is expressed as mean ± SD.

[0053] For the present application, the IC50 The values ​​(μM) are shown in Table 1.

[0054] Anticancer drugs generally have a short retention time in the body. If cells do not take up the drug in time, the drug cannot exert its full anticancer effect. In this study, MCF-7 cells and L-02 cells seeded in 96-well plates were treated with medium containing TYJ2517 or the control drug isotretinoin for 0.5, 1, 2, 4, 6, and 8 hours, respectively. After removing the old medium and replacing it with fresh medium, the cells were cultured for another 48 hours. The IC50 was determined by the MTT assay. 50 Values. Based on the IC50 values ​​of each compound against normal L-02 cells. 50 Value and IC50 value against MCF-7 cancer cells 50 The selectivity index SI was calculated, and then the multiples of the selectivity index SI were calculated based on the selectivity index SI of TYJ2517 and the selectivity index SI of isotrolactone.

[0055] For the present invention, its IC 50 The values ​​(μM) are shown in Table 2; their SI values ​​and times are shown in Table 3.

[0056] Table 1: IC50 of compound TYJ2517 or control isotretinoin on tumor cells and normal cells after treatment for 48 h. 50 Value (μM)

[0057]

[0058] Table 2: IC50 values ​​of MCF-7 cells and L-02 cells after treatment with TYJ2517 or isotretinoin for different time periods. 50 Value (μM)

[0059]

[0060] Table 3: Selectivity index SI [IC] of TYJ2517 or the reference substance isotretinoin 50(L-02) / (IC 50(MCF-7) ] and the multiples of the selectivity index (times, [SI (TYJ2517) / SI (ISA) ])

[0061]

[0062] As shown in Table 1, TYJ2517 exhibits almost no toxicity to L-02 normal cells (>100 μM). Among the nine cancer cell types, TYJ2517 showed the strongest inhibitory effect on MCF-7 cell proliferation, with an IC50 concentration of [missing value]. 50The IC50 value of TYJ2517 against SW620, SU-DHL-10, HepG2 and AGS cells was 3.6 ± 0.8 μM. 50 The IC50 values of all the compounds were below 10 μM, and the anti-proliferation effect was strong. The control, alantolactone, had a strong toxicity against L-02 normal cells (IC50 = 14.6 ± 0.7 μM), and the IC50 values of the compound against most cancer cells were below 10 μM. 50 The IC50 values of all the compounds were below 10 μM, and the anti-proliferation effect was strong. The control, alantolactone, had a strong toxicity against L-02 normal cells (IC50 = 14.6 ± 0.7 μM), and the IC50 values of the compound against most cancer cells were below 10 μM. 50 The IC50 values of all the compounds were below 10 μM, and the anti-proliferation effect was strong. The control, alantolactone, had a strong toxicity against L-02 normal cells (IC50 = 14.6 ± 0.7 μM), and the IC50 values of the compound against most cancer cells were below 10 μM.

[0063] As can be seen from Table 2, the uptake ability of the compounds by the cells, the anti-proliferation activity of TYJ2517 against MCF-7 cells was still strong at any time period of 0.5, 1, 2, 4, 6 and 8 h, and the IC50 value was in the range of 2.4-5.4 μM, which was not different from the effect of 48 h of continuous treatment, while the toxicity of TYJ2517 against normal cells L-02 was very low (IC50 > 100 μM), meaning that L-02 normal cells hardly took up TYJ2517, and the MCF-7 cancer cells significantly inhibited their proliferation ability after taking up TYJ2517. However, the uptake ability of alantolactone by MCF-7 cancer cells was weak, and the IC50 value was 18.7-27.7 μM, especially when the treatment time was ≤ 6 h, the effect was more obvious, while the uptake of alantolactone by normal cells L-02 was strong (IC50 = 5.5-8.5 μM), meaning that the uptake ability of alantolactone by L-02 normal cells was stronger than that by MCF-7 cancer cells, and if used in the clinic, the anti-MCF-7 cancer effect would not be obvious but would have side effects on normal liver cells. 50 50 The IC50 values of all the compounds were below 10 μM, and the anti-proliferation effect was strong. The control, alantolactone, had a strong toxicity against L-02 normal cells (IC50 = 14.6 ± 0.7 μM), and the IC50 values of the compound against most cancer cells were below 10 μM. 50 The IC50 values of all the compounds were below 10 μM, and the anti-proliferation effect was strong. The control, alantolactone, had a strong toxicity against L-02 normal cells (IC50 = 14.6 ± 0.7 μM), and the IC50 values of the compound against most cancer cells were below 10 μM. 50 The IC50 values of all the compounds were below 10 μM, and the anti-proliferation effect was strong. The control, alantolactone, had a strong toxicity against L-02 normal cells (IC50 = 14.6 ± 0.7 μM), and the IC50 values of the compound against most cancer cells were below 10 μM.

[0064] Example 3: PARP1 kit assay

[0065] ​1. Take MCF-7 and L-02 cells in the logarithmic growth phase from a 100-mm culture dish, discard the culture medium, add 1 mL of phosphate-buffered saline (PBS) for washing, discard the PBS buffer, add 1 mL of trypsin for digestion, and after complete digestion, centrifuge at 1000 rpm for 5 min, discard the supernatant, add culture medium to prepare a cell suspension, count the cells, and divide at 1×10⁻⁶. 6 Cells were seeded at a density of 1 cell / well in a 6-well culture plate.

[0066] 2. After the cells have adhered to the wall for 24 hours after seeding, add different concentrations of TYJ2517 or the control isotretinoin to treat the cells. After 48 hours, remove the culture medium, wash with PBS, remove the PBS, scrape off the cells from each well and put them into 1.5 ml centrifuge tubes.

[0067] 3. After centrifuging at 1000 rpm for 5 minutes, discard the PBS buffer.

[0068] 4. Add 0.2 mL / tube of cell lysis buffer (RIPA buffer, Solabio, catalog number: R0010), vortex, centrifuge at 1000 rpm for 5 minutes, and take the supernatant for PARP1 protein assay.

[0069] 5. Follow the instructions for the PARP1 kit.

[0070] 6. Repeat the experiment three or more times and process the data.

[0071] For this invention, the PARP1 protein assay results for TYJ2517 and isothomoleactone are as shown in the appendix to the specification. Figure 1 As shown. Figure 1 This is a graph showing the expression of PARP1 protein kinase in MCF-7 and L-02 cells after treatment with TYJ2517, isothomolelactone, and the positive control drug olaparib.

[0072] according to Figure 1 The results showed that TYJ2517 at different concentrations exhibited concentration-dependent inhibition of PARP1 protein kinase in MCF-7 breast cancer cells. At a concentration of 10 μM, TYJ2517 showed the most significant inhibitory effect on PARP1 protein kinase, far superior to 10 μM isotretinoin, and its inhibitory effect was close to that of the clinically used PARP1 inhibitor olaparib. Figure 1 As shown in Figure C, the expression level of PARP1 protein kinase in L-02 cells was lower than that in MCF-7 cancer cells. According to... Figure 1 B shows that the decreasing trend of PARP1 protein kinase in L-02 cells is not as obvious as that in MCF-7 cancer cells, but it still inhibits the expression of PARP1 protein kinase in a concentration-dependent manner.

[0073] Example 4: ROS kit assay

[0074] 1. Take the MCF-7 cells and L-02 cells in logarithmic growth phase on a 100-mm culture dish, discard the culture medium, add 1 mL of PBS buffer for rinsing, discard the PBS buffer, add 1 mL of trypsin for digestion, after complete digestion, centrifuge at 1000 rpm for 5 min to discard the supernatant, add the culture medium to prepare a cell suspension, count, and seed in a 6-well culture plate at a cell density of 1 x 10 6 cells / well.

[0075] 2. After 24 hours of cell seeding and cell adhesion, add TYJ2517 or control artesunate for treatment, and after 48 hours, remove the culture medium, wash with PBS buffer, and remove the PBS buffer.

[0076] 3. Add 1.5 mL / well of DCFH-DA (1:1000) diluted with serum-free medium to 10 μM, and then incubate in the incubator for 30 minutes.

[0077] 4. Remove the culture medium containing the DCFH-DA probe, wash with 1 mL of PBS buffer for 3 times, and fix with methanol.

[0078] 5. Take photos under a microscope.

[0079] 6. Repeat the experiment more than 3 times, and process the data.

[0080] For the present application, the ROS kit assay results of TYJ2517 and artesunate are shown in the attached drawings. Figure 2 Figure 2 is a graph of the ROS expression results of the compound TYJ2517 and artesunate on MCF-7 cells and L-02 cells.

[0081] According to Figure 2 The results show that the MCF-7 cells treated with TYJ2517 have a significantly increased intracellular ROS level in the treatment concentration range of 1-10 μM, showing a very significant difference (P<0.0001) compared with the control group; while 10 μM artesunate slightly increases the ROS compared with the control group, but there is no statistically significant difference. The analysis of the results of L-02 normal cells shows that the compound TYJ2517 has a weak ROS induction effect in normal cells, while artesunate shows a significant ROS induction effect in normal cells, showing a very significant difference (P<0.0001).

[0082] Example 5: Apoptosis experiment

[0083] ​1. Take MCF-7 and L-02 cells in logarithmic growth phase on 100-mm culture dishes, discard the culture medium, add 1 mL of PBS buffer for rinsing, discard the PBS buffer, add 1 mL of trypsin for digestion, collect the cells in a 1.5 mL centrifuge tube after complete digestion, centrifuge at 1000 rpm for 5 min to discard the supernatant, prepare a cell suspension, count, take 5x10 5 cells and plant in each 30-mm culture dish.

[0084] 2. After the cells adhere, add TYJ2517 or isoalantolactone for treatment, discard the culture medium after 48 h, digest the cells, combine, centrifuge at 1000 rpm for 5 min.

[0085] 3. Collect the cells and wash twice with PBS buffer.

[0086] 4. Resuspend the cells with 200 μL of 1x binding buffer (staining buffer), and centrifuge at 1000 rpm for 5 min.

[0087] 5. Discard the supernatant, add 2.5 μL of Annexin V-FITC and stain in the dark for 15-20 min, add 5 μL of 50 μg / mL PI solution for staining 5 min before machine, and store in the dark.

[0088] 6. Add 400 μL of 1x binding buffer to each sample, and detect the apoptosis of the cells by flow cytometry.

[0089] For the present application, the apoptosis results of MCF-7 cells or L-02 cells treated with different concentrations of TYJ2517 and isoalantolactone for 48 h are shown in the drawings as described in the specification. Figure 3 Figure 3 is a result graph of the apoptosis of MCF-7 cells and L-02 cells caused by the compound TYJ2517 and isoalantolactone.

[0090] According to Figure 3 ​The results of apoptosis can be seen that after treatment of MCF-7 cells with TYJ2517, the number of cells in early apoptosis increased from 0% in the blank group to 27.1% (3 μM TYJ2517 treatment), but as the concentration of compound TYJ2517 increased, the number of early apoptotic cells decreased, respectively 2.82% and 2.68% (5 μM and 10 μM TYJ2517 treatment), while the number of late apoptotic cells increased from 27.7% (3 μM TYJ2517 treatment) to 57.7% and 62.8% (5 μM and 10 μM TYJ2517 treatment). When treated with 10 μM isoalantolactone, it mainly affected the number of late apoptotic cells (28.0%), and almost did not affect the number of early apoptotic cells (1.71%), but at the same concentration of 10 μM, TYJ2517 had a more significant effect on late apoptosis of MCF-7 cells (28.0%→62.8%). For L-02 normal cells, the effect of 10 μM isoalantolactone on the number of late apoptotic cells was the most significant, 25.6%, while the same concentration of TYJ2517 had a weak effect on early apoptosis, and TYJ2517 did not affect the late apoptosis of L-02 normal cells at any concentration, which was comparable to the blank group. From the results, it can be concluded that TYJ2517 induces early apoptosis of MCF-7 cells at a low concentration of 3 μM, and induces late apoptosis of MCF-7 cells at a higher concentration of 5 μM and 10 μM, affecting the proliferation of cells. Compound TYJ2517 hardly induces apoptosis of L-02 normal cells. In contrast, isoalantolactone has a strong late-apoptosis-promoting effect on L-02 normal cells.

[0091] Example 6: Cell scratch test and cell invasion test

[0092] Cell scratch test: MCF-7 and L-02 cells in the logarithmic growth phase were taken, the culture medium was discarded, and the PBS buffer was washed and discarded. 1 mL of trypsin was added for digestion. After complete digestion, the cells were collected, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the culture medium was added to prepare a cell suspension, which was counted, and 5×10 5 cells / well were inoculated in a 6-well culture plate. After 24 hours of culture in the cell culture box, the cells were slightly tilted and forced to scratch the hole with 2 μL of sterile gun head, and the time at this moment was recorded as 0 h. Then, PBS was used to wash 3 times to remove cell debris, and the cells were treated with different concentrations of TYJ2517 or isoalantolactone and incubated for 48 h. The migration of the cells was observed and photographed under an inverted microscope at 0, 24, and 48 h, respectively.

[0093] Cell invasion experiment: MCF-7 and L-02 cells in logarithmic growth phase were taken, the culture medium was discarded, and the cells were washed with PBS buffer solution, the PBS buffer solution was discarded, 1 mL trypsin was added for digestion, the cells were collected after complete digestion, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and a cell suspension (1 × 10 5 The upper chamber of the Transwell (transfer disc) was added with 100 μL of the cell suspension, 200 μL of cell culture solution containing 20% FBS was added to the lower chamber, after the cells adhered, different concentrations of drugs were added to the lower chamber, and incubation was continued for 24 h, the cells in the upper chamber were wiped off with a cotton swab, 200 μL of methanol was added for fixation for 10 min, distilled water was used for washing, and staining was performed with a Saum dye, five different fields were selected under a microscope for counting.

[0094] The migration of MCF-7 and L-02 cells (cell scratch experiment and cell invasion experiment) by the compounds TYJ2517 and isoalantolactone is shown in the specification Figure 4 . Figure 4 is a graph showing the migration and invasion of MCF-7 and L-02 cells by the compounds TYJ2517 and isoalantolactone.

[0095] Figure 4 The migration experiment results show that the compound TYJ2517 can inhibit the migration and invasion ability of MCF-7 cells in a concentration-dependent manner. Under the condition of the same dose of 10 μM, the anti-migration and anti-invasion effects of TYJ2517 are significantly better than those of isoalantolactone (P < 0.0001). The compound TYJ2517 does not have a significant effect on the migration and invasion of L-02 normal cells, while isoalantolactone shows a significant anti-migration and anti-invasion effect on L-02 normal cells (P < 0.0001).

[0096] Example 7: Cell colony experiment

[0097] MCF-7 and L-02 cells in logarithmic growth phase were taken, the culture medium was discarded, and the cells were washed with PBS buffer solution, the PBS buffer solution was discarded, 1 mL trypsin was added for digestion, the cells were collected after complete digestion, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and a cell suspension was prepared, counted, and 1 × 10 4 cells / well of cells were inoculated in a 6-well plate, and new culture medium was replaced every 2-3 days for continuous culture, when the colonies grew to a size suitable for counting (> 50 cells), the culture plate was removed from the incubator. Then the cells were fixed with ethanol and stained with 1 × Giemsa dye, washed, dried, and counted. All experiments were independently repeated at least three times.

[0098] The effects of compound TYJ2517 and isoalantolactone on MCF-7 and L-02 cell colony are shown in the following figures. Figure 5 Figure 5 Figure 1 is a graph showing the effects of compound TYJ2517 and isoalantolactone on MCF-7 and L-02 cell colony.

[0099] The results of the colony experiment show that TYJ2517 inhibits the formation of MCF-7 cell colony in a concentration-dependent manner, and almost no MCF-7 cell colony is formed when the concentration of TYJ2517 is 10 μM. The MCF-7 cell colony state produced by 10 μM isoalantolactone has no difference from the blank control group. For L-02 normal cells, TYJ2517 has no effect on the formation of the colony, while 10 μM isoalantolactone significantly inhibits the formation of the colony.

[0100] Example 8: Water solubility determination

[0101] Compound TYJ2517 or isoalantolactone was added to methanol to prepare a clear solution of 10 mM, and then diluted with methanol to different concentrations of 400, 200, 100, 50, 25, 12.5, 6.25 and 3.125 μM. The peak area of the sample at different concentrations was recorded by high performance liquid chromatography (HPLC). The concentration-peak area standard curve was obtained with the concentration as the abscissa and the peak area as the ordinate. Three phosphate buffers with pH values of 2.0, 7.4 and 9.0 were prepared. Compound TYJ2517 or isoalantolactone (2.0 mg) and 500 μL of phosphate buffer (pH = 2.0, 7.4, 9.0) were added to a 1.5 mL centrifuge tube, respectively, to prepare a supersaturated suspension. The suspension was vortex mixed for 2 min and shaken on a shaker for 30 min. Then the sample was centrifuged at 12000 rpm for 30 min, filtered with a 0.22 μm microporous filter, and the supernatant was collected and analyzed by HPLC. The water solubility of compound TYJ2517 and isoalantolactone was calculated according to the standard curve.

[0102] The water solubility results of compound TYJ2517 and isoalantolactone are shown in Table 4.

[0103] Table 4: Water solubility of compound TYJ2517 and control isoalantolactone (μmol / L)

[0104]

[0105] As can be seen from Table 4, under different pH (2.0, 7.4 and 9.0) conditions, the water solubility of TYJ2517 is significantly improved compared with isoalantolactone.

[0106] ​In summary, the synthesized compound 3,4,5-trimethoxybenzylaminomethyl 3- demethylisoalantolactone (TYJ2517) showed very strong anti-proliferation activity in five human-derived tumor cells including MCF-7, and showed different degrees of proliferation inhibition in different cells, and its IC 50 value in MCF-7 cells was 3.6±0.8 μM, which was much better than that of the control isoalantolactone (IC 50 value = 21.3±3.8 μM); and the IC 50 value of TYJ2517 to L-02 normal cells was more than 100 μM, while the control isoalantolactone had strong toxicity to L-02 normal cells (IC 50 value = 14.6±0.7 μM). Anti-cancer drugs generally have a short retention time in vivo, and if the cells do not take up the drug in time, the drug cannot fully exert its anti-cancer effect, so the drug uptake capacity experiment was also carried out. After MCF-7 cells and L-02 cells were treated with TYJ2517 or control isoalantolactone-containing medium for 0.5, 1, 2, 4, 6 and 8 hours, they were replaced with fresh medium, and then cultured for 48 hours, and the IC 50 values were determined by MTT method. The results showed that L-02 normal cells hardly took up TYJ2517, while MCF-7 cancer cells took up TYJ2517 and had very significant inhibition of MCF-7 cell proliferation. However, MCF-7 cancer cells had weak uptake of isoalantolactone, especially in short time (≤4h) treatment, while normal cells L-02 had strong uptake of isoalantolactone, meaning that L-02 normal cells had stronger uptake of isoalantolactone than MCF-7 cancer cells, and if used in the clinic, the anti-cancer effect would not be obvious but would have side effects on normal cells. In particular, when the uptake time was 1h, the selective anti-cancer index SI of TYJ2517 to breast cancer cells MCF-7 and normal cells L-02 was more than 40, while the selective index of isoalantolactone was only 0.2 (SI < 1 indicates strong toxicity to normal cells), and TYJ2517 increased the selective anti-cancer level by more than 200 times, and it was speculated that TYJ2517 circulating in the blood had targeting to MCF-7 breast cancer cells when distributed in vivo, while isoalantolactone circulating in the blood did not have MCF-7 cancer cell targeting when distributed in vivo, and it had strong toxicity to normal liver cells.

[0107] Further mechanism study found that the inhibitory activity of TYJ2517 on the proliferation of MCF-7 human breast cancer cells was through inhibiting the expression of PARP1 protein kinase and ROS, inducing early and late apoptosis of MCF-7 cancer cells, hindering the migration and colony of MCF-7 cancer cells, and the ability was better than that of the control alantolactone, and TYJ2517 had little effect on the expression of PARP1 and ROS of L-02 normal cells, cell migration, invasion and colony formation. The control alantolactone had greater toxicity to L-02 normal cells.

[0108] In summary, TYJ2517 has strong anti-proliferation activity on MCF-7 cancer cells and very low toxicity on L-02 normal cells, that is, it has strong selective anti-cancer activity, and the uptake ability of MCF-7 cancer cells is much stronger than that of L-02 normal cells, and various indicators are significantly better than its lead alantolactone, which is a high-efficiency and low-toxicity anti-MCF-7 human breast cancer candidate compound with great development prospects.

[0109] The above examples are only used to illustrate the technical solutions of the present application, but not to limit it. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should still be able to modify the technical solutions recorded in the foregoing embodiments, or make equivalent substitutions for some technical features, without departing from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. TYJ2517, i.e. 3, 4, 5-trimethoxybenzylaminomethyl 3-demethylisoalantolactone, has the following structure:

2. Use of the isoalantolactone derivative TYJ2517 according to claim 1.

3. The isoalantolactone derivative TYJ2517 according to claim 1, wherein, Applied to anticancer drugs.

4. The isoalantolactone derivative TYJ2517 according to claim 1, wherein, It has strong inhibitory proliferation activity on MCF-7, SW620, SU-DHL-10, HepG2 and AGS and other five kinds of human cancer cells, but has weak toxicity on L-02 human normal liver cells, and has strong selective anticancer activity.

5. The isoalantolactone derivative TYJ2517 according to claim 1, wherein, showed different abilities of inhibiting proliferation in different cancer cells, and had little toxicity to L-02 normal cells (IC 50 > 100 μM), and the strongest ability of inhibiting proliferation to MCF-7, with an IC 50 value of 3.6 ± 0.8 μM, which was better than that of the control artupl 21.3 ± 3.8 μM, and the control artupl had strong toxicity to L-02 normal cells (IC s0 value = 14.6 ± 0.7 μM), and the compound TYJ2517 had low toxicity and high efficiency of anticancer activity.

6. The isoalantolactone derivative TYJ2517 according to claim 1, wherein, TYJ2517 still has strong anti-cancer activity to MCF-7 cells in any time of 0.5-8h, and its IC 50 values are in the range of 2.4-5.4μM, which has no difference with the effect of 48h, while its anti-proliferation ability to normal cells L-02 is very low (IC 50 >100μM), L-02 normal cells hardly uptake TYJ2517, while MCF-7 cancer cells have very significant inhibition of proliferation ability after uptake of TYJ2517. MCF-7 cancer cells have weak ability to uptake arturn in (IC 50 values are in the range of 18.7-27.7μM), especially in short time (≤6h), while normal cells L-02 have strong ability to uptake arturn in (IC 50 values are in the range of 5.5-8.5μM), and L-02 normal cells have stronger ability to uptake arturn in than MCF-7 cancer cells. When the uptake time is 1h, the selective index SI of TYJ2517 to breast cancer cells MCF-7 and normal cells L-02 is more than 40, while the selective index of arturn in is only 0.2 (SI<1 means toxicity to normal cells), and the selective index of TYJ2517 is increased by more than 200 times. TYJ2517 has targeting property to MCF-7 breast cancer cells in vivo distribution, while arturn in has no targeting property to MCF-7 cancer cells, and arturn in has toxicity to L-02 normal liver cells.

7. The isoalantolactone derivative TYJ2517 according to claim 1, wherein, The inhibitory proliferation activity of TYJ2517 on MCF-7 cancer cells is caused by inhibiting poly(ADP-ribose) polymerase 1 (PARP1) and inhibiting the expression of reactive oxygen species (ROS).

8. The isoalantolactone derivative TYJ2517 according to claim 1, wherein, The inhibitory proliferation activity of TYJ2517 on MCF-7 cancer cells is caused by inducing early and late apoptosis of MCF-7 cells.

9. The isoalantolactone derivative TYJ2517 according to claim 1, wherein, The inhibitory proliferation activity of TYJ2517 on MCF-7 cancer cells is caused by hindering cell migration, cell invasion and cell colony.

10. The isoalantolactone derivative TYJ2517 according to claim 1, wherein, Under different pH (2.0, 7.4 and 9.0) conditions, the solubility of TYJ2517 in water is significantly improved compared with isoalantolactone.

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