A lung cancer cell inhibitor, its preparation method and application

Abstract

The present invention discloses a lung cancer cell inhibitor, the chemical structural formula of which is shown as follows: #imgabs0# The present invention also discloses a preparation method and application of the lung cancer cell inhibitor. The lung cancer cell inhibitor exhibits excellent anti-lung cancer activity and targeting in vitro and in vivo, has potential medicinal value, and can be used for the preparation of various anti-lung cancer drugs.

Description

Technical Field

[0001] The present invention relates to complexes, and specifically to an inhibitor of lung cancer cells. Meanwhile, the present invention also relates to a preparation method and application of the inhibitor of lung cancer cells. Background Art

[0002] Since 1965, when Rosenberg accidentally discovered that cisplatin has anti-tumor activity, it still has important significance for cancer treatment and the development of platinum-based anti-tumor drugs, and cisplatin is still considered to be one of the important drugs used in clinical cancer treatment. However, the application of cisplatin and its derivatives in clinical practice is accompanied by serious adverse reactions, including neurotoxicity, nephrotoxicity, hematological toxicity and drug resistance, etc. (Guo, Z.; et al. Chem. Soc. Rev., 2013, 42: 202–224.). Therefore, there is an urgent need to develop a zinc metal anti-cancer complex with high efficiency and low toxicity.

[0003] Literature reports that metal complexes of 8-hydroxyquinoline and its derivatives have good anti-tumor and antibacterial activities (Chen, Z. F., et al. Science China: Chemistry, 2017, 47(02): 172-182.), however, the research on this type of complex against cisplatin-resistant human lung adenocarcinoma cell lines (A549 / DDP or A549R) is lacking. Summary of the Invention

[0004] One of the technical problems to be solved by the present invention is to provide an inhibitor of lung cancer cells with high activity in vivo and in vitro and targeting cisplatin-resistant human lung cancer cells, which is a 5-substituted-8-hydroxyquinoline platinum complex synthesized with 5-(ethoxymethyl)-8-hydroxyquinoline as the active ligand.

[0005] Specifically, the chemical structural formula of the inhibitor of lung cancer cells is shown as the following formula:

[0006]

[0007] Another technical problem to be solved by the present invention is to provide a preparation method of the above-mentioned inhibitor of lung cancer cells.

[0008] Specifically, the preparation method of the inhibitor of lung cancer cells,

[0009] Weigh 0.1 mmol of the ligand 5-(ethoxymethyl)-8-hydroxyquinoline and 0.1 mmol of the metal salt cis-Pt(DMSO)2Cl2, dissolve them in 1.5 - 10.0 mL of methanol or a mixed solution of methanol and acetone, water or DMSO (the volume ratio is any ratio), carry out a coordination reaction at 45 - 85 °C for 1 - 7 days, filter, and dry in a vacuum drying oven at 45 °C to obtain yellow blocky crystals PtL1 respectively.

[0010] The third technical problem to be solved by the present invention is to provide the application of the above-mentioned lung cancer cell inhibitor.

[0011] Specifically, it relates to the application of the above-mentioned lung cancer cell inhibitor in the preparation of drugs for targeted treatment of lung cancer. Further, it relates to the application of the above-mentioned lung cancer cell inhibitor in the preparation of drugs for targeted treatment of cisplatin-resistant lung cancer strains.

[0012] In the present invention, 5-(ethoxymethyl)-8-hydroxyquinoline (H-L1), 5-bromo-8-hydroxyquinoline (H-L2), and 8-hydroxyquinoline (H-L3) are used as active ligands to synthesize 5-substituted-8-hydroxyquinoline platinum complexes [Pt(L1)(DMSO)Cl] (PtL1), [Pt(L2)(DMSO)Cl] (PtL2), and [Pt(L3)(DMSO)Cl] (PtL3) targeting human cisplatin-resistant lung cancer strains, and their activities and toxicity experiments on human lung cancer A549, cisplatin-resistant strain A549R, and normal HL-7702 cells are investigated. The experimental results show that PtL1 targets and inhibits the growth of human lung adenocarcinoma cisplatin-resistant strain A549R, and its IC 50 value is 1.3 ± 0.7 μM, and its activity is much greater than that of H-L1, cis-Pt(DMSO)2Cl2, H-L2, H-L3, cisplatin, PtL2, and PtL3. Moreover, its inhibitory effect on normal HL-7702 cells is very small (>50 μM), indicating that the 5-(ethoxymethyl)-8-hydroxyquinoline platinum complex PtL1 can target and inhibit the proliferation of human lung adenocarcinoma cisplatin-resistant strain A549R and also has better anti-cancer activity. More importantly, the in vivo tumor inhibition experiment shows that PtL1 has a good tumor inhibition effect on the nude mouse model bearing human lung cancer A549, and the inhibition rate is as high as 68.2%, far higher than the inhibitory effect of the clinical drug cisplatin (50.0%). In summary, the 5-(ethoxymethyl)-8-hydroxyquinoline platinum complex PtL1 exhibits excellent in vitro and in vivo anti-tumor activities, has potential medicinal value, and is expected to be used in the preparation of various anti-tumor drugs. Brief Description of the Drawings

[0013] Figure 1 It is the X-ray single crystal structure diagram of the complex PtL1 prepared in Example 1 of the present invention;

[0014] Figure 2 It is the X-ray single crystal structure diagram of the complex PtL2 prepared in Example 1 of the present invention;

[0015] Figure 3 It is the X-ray single crystal structure diagram of the complex PtL3 prepared in Example 1 of the present invention;

[0016] Figure 4 It is the infrared spectrum diagram of the complex PtL1 prepared in Example 1 of the present invention;

[0017] Figure 5 Infrared spectrum of the complex PtL2 prepared in Example 1 of the present invention;

[0018] Figure 6 Infrared spectrum of the complex PtL3 prepared in Example 1 of the present invention. Detailed implementation manners

[0019] The present invention will be further described below with specific examples, but the present invention is not limited to these examples.

[0020] The metal salt cis-Pt(DMSO)2Cl2 involved in the synthesis method of the present invention can be prepared with reference to existing literature (Al-Allaf, T.A.K.; et al. Transit. Met. Chem., 1998, 23: 403-406.).

[0021] Example 1

[0022] In a 15-cm long thick-walled pressure-resistant tube, add 0.1 mmol of ligand H-L1, H-L2 or H-L3, 0.1 mmol of metal salt cis-Pt(DMSO)2Cl2, and 4.5 mL of methanol. Carry out a coordination reaction at 55 °C for 5 days, filter, and dry in a vacuum drying oven at 45 °C to obtain yellow bulk crystals PtL1–PtL3 respectively. The yields of PtL1, PtL2, and PtL3 are 78.3%, 83.6%, and 62.4% respectively.

[0023] Synthesis route:

[0024]

[0025] Identification of the obtained product:

[0026] (1) X-ray single crystal structure diagram of the complex PtL1, and its spectrum is as shown in Figure 1 shown.

[0027] (2) X-ray single crystal structure diagram of the complex PtL2, and its spectrum is as shown in Figure 2 shown.

[0028] (3) X-ray single crystal structure diagram of the complex PtL2, and its spectrum is as shown in Figure 3 shown.

[0029] (4) Infrared spectrum of the complex PtL1, as shown in Figure 4 shown.

[0030] IR (KBr): 3922, 3131, 3013, 2917, 1627, 1599, 1576, 1506, 1460, 1400, 1322, 1168, 1131, 1022, 980, 936, 920, 816, 762, 734, 683, 648, 569, 448 cm -1 .

[0031] (5) The infrared spectrum of the complex PtL2 is as follows Figure 5 shown.

[0032] IR (KBr): 3130, 3009, 2916, 1619, 1583, 1573, 1499, 1459, 1400, 1366, 1320, 1266, 1233, 1200, 1137, 1123, 1088, 1047, 1037, 981, 957, 934, 922, 815, 767, 755, 694, 664, 544, 450 cm -1 .

[0033] (6) The infrared spectrum of the complex PtL3 is as follows Figure 6 shown.

[0034] IR (KBr): 3130, 3004, 2915, 1592, 1578, 1507, 1472, 1399, 1322, 1283, 1243, 1175, 1125, 1035, 978, 928, 856, 820, 804, 777, 748, 699, 650, 632, 585, 530, 452, 418 cm -1 .

[0035] (7) The elemental analysis results are shown in Table 1.

[0036] Table 1 Elemental analysis results of the complexes PtL1–PtL3 in the examples

[0037]

[0038] Therefore, it can be determined that the obtained yellow complexes PtL1–PtL3 have the following structural formulas:

[0039]

[0040] Example 2

[0041] In a 15-cm long thick-walled pressure-resistant tube, 0.1 mmol of ligand H-L1, 0.1 mmol of metal salt cis-Pt(DMSO)2Cl2, and a mixed solution of 10 mL of methanol and water (volume ratio: 95:1) were added. A coordination reaction was carried out at 55 °C for 5 days, followed by filtration and drying in a vacuum drying oven at 45 °C, to obtain yellow block crystals of PtL1 respectively, with a yield of 70.1%.

[0042] Example 3

[0043] In a 15-cm long thick-walled pressure-resistant tube, 0.1 mmol of ligand H-L1, 0.1 mmol of metal salt cis-Pt(DMSO)2Cl2, and a mixed solution of 1.5 mL of methanol and DMSO (volume ratio 1:1) were added. A coordination reaction was carried out at 55 °C for 5 days, followed by filtration and drying in a vacuum drying oven at 45 °C, to obtain yellow block crystals of PtL1 respectively, with a yield of 66.9%.

[0044] Example 4

[0045] In a 15-cm long thick-walled pressure-resistant tube, 0.1 mmol of ligand H-L1, 0.1 mmol of metal salt cis-Pt(DMSO)2Cl2, and a mixed solution of 8.0 mL of methanol and DMF (volume ratio 50:3) were added. A coordination reaction was carried out at 55 °C for 5 days, followed by filtration and drying in a vacuum drying oven at 45 °C, to obtain yellow block crystals of PtL1 respectively, with a yield of 76.1%.

[0046] To fully illustrate the uses of the three 8-hydroxyquinoline derivative platinum(II) complexes PtL1–PtL3 targeting human lung cancer cisplatin-resistant cells in pharmaceuticals, the applicant conducted in vitro and in vivo anti-tumor activity experiments on them.

[0047] I. Proliferation inhibition activity experiments of the three 8-hydroxyquinoline derivative platinum(II) complexes PtL1–PtL3 targeting human lung cancer cisplatin-resistant cells on three human cell lines

[0048] 1. Cell lines and cell culture

[0049] Three human cell lines, namely human lung cancer A549 and cisplatin-resistant strain A549R, and normal HL-7702 cells, were selected.

[0050] All human cell lines were cultured in RPMI-1640 culture medium containing 100 U / mL penicillin, 10 wt% calf blood, and 100 U / mL streptomycin, and incubated in an incubator at 37 °C with a volume concentration of 5% CO2.

[0051] 2. Preparation of the test compounds

[0052] The purity of all compounds used should be ≥95.0%. Dilute their DMSO stock solutions with physiological buffer to a final concentration of 20 μmol / L (the final concentration of DMSO ≤ 1.0%), and test the inhibitory effect of each compound on the growth of normal cells or selected tumor cells at this concentration.

[0053] 3. Cell growth inhibition assay (MTT method)

[0054] (1) Take normal cells or tumor cells in the logarithmic growth phase. After digestion with trypsin, prepare a cell suspension with a concentration of 5000 cells / mL using a culture medium containing 10% calf serum. Inoculate 190 μL per well into a 96-well culture plate to make the density of the cells to be tested reach 1000 - 10000 per well (fill the edge wells with sterile PBS).

[0055] (2) Incubate at 37°C with 5% CO2 for 24 h until the cell monolayer covers the bottom of the well. Add 10 μL of the drug at a certain concentration gradient to each well, and set 4 replicates for each concentration gradient.

[0056] (3) Incubate at 37°C with 5% CO2 for 48 h, and observe under an inverted microscope.

[0057] (4) Add 10 μL of MTT solution (5 mg / mL PBS, i.e., 0.5% MTT) to each well and continue to culture for 4 h.

[0058] (5) Terminate the culture, carefully aspirate the culture medium in the wells, add 150 μL of DMSO to each well to fully dissolve the formazan precipitate. After mixing with an oscillator, measure the optical density value of each well at a wavelength of 570 nm and a reference wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0059] (6) At the same time, set a zero-adjustment well (culture medium, MTT, DMSO), and a control well (cells, culture medium, MTT, the same concentration of drug dissolution medium, DMSO).

[0060] (7) Based on the measured optical density value (OD value), judge the number of viable cells. The larger the OD value, the stronger the cell activity. Use the formula:

[0061]

[0062] Calculate the inhibition rate of each compound on the growth of the selected cells, and then calculate the IC 50 value of each test compound for each selected cell line using the Bliss method. The results are shown in Table 2 below.

[0063] Table 2. IC 50 values (μM) of compounds for various cell lines

[0064]

[0065]

[0066] From the IC in Table 2 50 activity screening results, it can be seen that PtL1 targets and inhibits the growth of cisplatin-resistant human lung adenocarcinoma cell line A549R. Its IC 50 value is 1.3 ± 0.7 μM, and its activity is much greater than that of H-L1, cis-Pt(DMSO)2Cl2, H-L2, H-L3, cisplatin, PtL2, PtL3 and all 8-hydroxyquinoline platinum complexes reported in the literature (Y.-C. Liu, et al. Med. Chem. Commun., 2016, 7, 1802–1811.). Moreover, its inhibitory effect on normal HL-7702 cells is very small (>50 μM), indicating that the 5-(ethoxymethyl)-8-hydroxyquinoline platinum complex PtL1 can target and inhibit the proliferation of cisplatin-resistant human lung adenocarcinoma cell line A549R and also has better anti-cancer activity. In summary, the 5-(ethoxymethyl)-8-hydroxyquinoline platinum complex PtL1 exhibits superior in vitro anti-tumor activity and targeting, has potential medicinal value, and is expected to be used in the preparation of various anti-tumor drugs.

[0067] II. In vivo tumor suppression experiment on nude mice bearing tumors

[0068] Collect the human lung cancer cell line A549 in the logarithmic growth phase and adjust it to a suspension of viable cell concentration of 5×10 6 cells / mL with serum-free medium. Use a 1.0 mL syringe to draw 0.2 mL of the suspension, which contains approximately 1×10 7 viable cells, and then inoculate it subcutaneously into the right axilla of nude mice. Wait until the subcutaneous tumor grows to about 1 cm 3 in size, and use it as the tumor source for making the subcutaneous transplantation tumor model and passage it on nude mice. After the human lung cancer A549 has been passaged 4 times on nude mice and its growth is stable, select tumor-bearing mice with vigorous tumor growth and no ulceration, sacrifice them by cervical dislocation, disinfect the animal skin with 75.0% medical alcohol, dissect the tissue mass, remove the necrotic part, cut the tumor tissue into small pieces about 1.5 mm 3 in size, and inoculate them subcutaneously into the right axilla of nude mice with a trocar. Measure the tumor diameter of the transplanted tumor with an electronic vernier caliper. When the tumor volume grows to 100 - 300 mm 3 in size, randomly divide the animals into groups.

[0069] Nude mice bearing human lung cancer A549 tumors are randomly divided into a vehicle group, a PtL1 drug addition group, and a blank control group, with 6 animals in each group. Start intraperitoneal injection of drugs on the day of grouping and administer the drugs once every other day. Measure the tumor diameter and body weight with an electronic vernier caliper every three days. Sacrifice the animals by cervical dislocation on the 21st day, dissect the tumors, weigh them, take pictures, and calculate the tumor suppression rate.

[0070] Tumor volume calculation formula: V = a × b 2 / 2, where a is the major axis and b is the minor axis;

[0071] Relative tumor volume RTV = V t / V0, where V t is the volume at each measurement, and V0 is the volume at grouping;

[0072] Relative tumor growth rate T / C% = (T RTV / C RTV ) × 100%;

[0073] Tumor growth inhibition rate (%) = (average tumor weight of the vehicle group - average tumor weight of the treatment group) / average tumor weight of the vehicle group × 100%.

[0074] Table 3. Inhibitory rate of complex PtL1 on A549 - bearing nude mice in vivo

[0075]

[0076] The in - vivo antitumor experiment in Table 3 shows that PtL1 has a good antitumor effect on the nude mouse model bearing human lung cancer A549, with an inhibition rate as high as 68.2%, far higher than the inhibitory effect of the clinical drug cisplatin (50.0%). The in - vivo anticancer data of cisplatin are from relevant reports in the literature ((a) J. Qi, Y. Zheng, B. Li, L. Wei, J. Li, X. Xu, S. Zhao, X. Zheng and Y. Wang, Eur. J. Med. Chem., 2022, 237, 114415. (b) J. Zhou, L. Rao, G. Yu, T. R. Cook, X. Chen and F. Huang, Chem. Soc. Rev., 2021, 50, 2839 - 2891. (c) Y. Li, S. Shi, S. Zhang, Z. Gan, X. Wang, X. Zhao, Y. Zhu, M. Cao, X. Wang and W. Li, Dalton Trans., 2021, 50, 11180–11188. (d) F. Seidi, Y. Zhong, H. Xiao, Y. Jin and D. Crespy, Chem. Soc. Rev., 2022, 51, 6652 - 6703.). In conclusion, the 5 - (ethoxymethyl) - 8 - hydroxyquinoline platinum complex PtL1 exhibits excellent in - vivo antitumor activity, has potential medicinal value, and is expected to be used in the preparation of various antitumor drugs.

[0077] In summary, the 5-(ethoxymethyl)-8-hydroxyquinoline platinum complex PtL1 of the present invention exhibits excellent anti-tumor activity and selectivity in vitro and in vivo, as well as good cytotoxic selectivity, indicating that the design concept and synthesis method of the novel anti-tumor 5-(ethoxymethyl)-8-hydroxyquinoline platinum complex PtL1 synthesized in the present invention are feasible. The anti-tumor activity exhibited by the 5-(ethoxymethyl)-8-hydroxyquinoline platinum complex PtL1 endows it with good potential medicinal value and is expected to be used in the preparation of various anti-tumor drugs.

Claims

1. Lung cancer cell inhibitor, characterized in that, The chemical structural formula is shown as follows: 。 2. The preparation method of the lung cancer cell inhibitor according to claim 1, characterized in that, Weigh 0.1 mmol of the ligand 5-(ethoxymethyl)-8-hydroxyquinoline and 0.1 mmol of the metal salt cis-Pt(DMSO)2Cl2, dissolve them in 1.5 - 10.0 mL of methanol or a mixed solution of methanol and acetone, water or DMSO, conduct a coordination reaction at 45 - 85 °C for 1 - 7 days, filter, and dry in a vacuum drying oven at 45 °C to obtain the yellow blocky crystal PtL1 respectively.

3. Use of the lung cancer cell inhibitor according to claim 1 in the preparation of a lung cancer drug for targeted treatment of cisplatin-resistant strains.

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