A compound having a hydroxychloroquine tetravalent platinum structure and its preparation method and application

Abstract

The present invention discloses a compound having a hydroxychloroquine tetravalent platinum structure, a preparation method thereof, and an application thereof. The compound having a hydroxychloroquine tetravalent platinum structure provided by the present invention has a structure as shown in formula (1). The present invention introduces hydroxychloroquine, which has the ability to inhibit autophagy, into a tetravalent platinum structure, and designs and prepares a hydroxychloroquine tetravalent platinum complex. This type of compound exerts an anti-tumor effect through a synergistic anti-cancer mechanism of DNA damage, autophagy inhibition, tumor microenvironment regulation, and immune activation, overcoming the problem of drug resistance of current divalent platinum (II) drugs, significantly improving anti-tumor activity, and reducing toxicity. It is a new type of platinum (IV) drug with the potential to inhibit tumor proliferation and tumor metastasis.

Description

Technical Field

[0001] The invention belongs to the fields of medicinal chemistry and organic chemistry, and particularly relates to a compound with a hydroxychloroquine tetravalent platinum structure, a preparation method and an application thereof. Background Art

[0002] In recent years, the incidence and mortality of cancer have risen dramatically, making it a major cause of premature death. Platinum(II) drugs, primarily cisplatin, oxaliplatin, and carboplatin, are commonly used in cancer treatment. However, these drugs suffer from limitations such as poor targeting, strong side effects, and the development of drug resistance.

[0003] Hydroxychloroquine (HCQ) is not only widely used as an antimalarial drug, but also for the treatment of autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. Recently, the anti-tumor effects of HCQ have garnered widespread attention. Studies have shown that HCQ has a clear autophagy inhibitory effect, readily aggregating in the acidic lysosomes, inhibiting lysosomal enzyme function and hindering the formation of autophagosomes, thereby inhibiting autophagy and inducing tumor cell apoptosis. HCQ also has the ability to activate the immune system and suppress the hypoxic, inflammatory tumor microenvironment, offering significant potential for development in the field of tumor treatment.

[0004] Platinum(IV) has d 2 sp 3 The hexacoordinate structure stabilizes platinum (IV) compounds, resulting in higher blood stability and lower toxicity than platinum (II) drugs. Platinum (IV) compounds contain two ligands in the axial position, facilitating structural modification and enabling the introduction of diverse functional structural fragments to prepare multifunctional platinum compounds, providing an effective strategy for the development of new platinum drugs. Currently, the development of novel multifunctional platinum (IV) compounds has become a hot topic in anti-tumor drug research and development. Summary of the Invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art of the above-mentioned platinum (II) drugs, the primary purpose of the present invention is to provide a compound having a tetravalent platinum structure of hydroxychloroquine.

[0006] Another object of the present invention is to provide a method for preparing the compound having the tetravalent platinum structure of hydroxychloroquine.

[0007] The present invention introduces hydroxychloroquine HCQ, which has the ability to inhibit autophagy, into a quadrivalent platinum system to construct a hydroxychloroquine quadrivalent platinum compound, thereby obtaining a novel quadrivalent platinum compound with the effects of DNA damage, autophagy inhibition, tumor microenvironment regulation, and immune activation. This compound is expected to address the drawbacks and defects of clinical divalent platinum drugs and develop into a novel platinum drug for clinical application.

[0008] Another object of the present invention is to provide the use of the above-mentioned compound having a hydroxychloroquine tetravalent platinum structure in the preparation of anti-tumor drugs.

[0009] The purpose of the present invention is achieved through the following solutions:

[0010] A compound having a hydroxychloroquine tetravalent platinum structure, the structure of which is shown in formula (1):

[0011]

[0012] in, Compound A includes cisplatin, oxaliplatin, carboplatin, heptaplatin, nedaplatin, lobaplatin, miplatin, picoplatin, One of the following;

[0013] R 3 It is one of —(CH2)—, —(CH2)2—, —(CH2)3—, and —(CH2)4—.

[0014] The preparation method of the compound having the tetravalent platinum structure of hydroxychloroquine is as follows:

[0015]

[0016] The reaction is carried out in a mixed solution containing a condensing agent and an organic base, wherein the molar ratio of compound (2), compound (3), condensing agent and organic base is 1:(2.0-4.0):(2.0-4.0):(2.0-4.0); and the amount ratio of compound (2) to solvent is 1 g:(30-80 mL).

[0017] The preparation method comprises the following steps: mixing a condensing agent, a compound (3) and a solvent under an inert atmosphere, reacting for 5-30 minutes; adding an organic base, reacting for 5-30 minutes; then adding the compound (2), reacting in the dark, and obtaining the compound (1) after purification.

[0018] The inert atmosphere includes at least one of nitrogen, helium, and argon.

[0019] The condensing agent is at least one of O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and dicyclohexylcarbodiimide (DCC).

[0020] The organic base is at least one of triethylamine (TEA), N,N-diisopropylethylamine (DIEA), and 4-dimethylaminopyridine (DMAP).

[0021] The solvent is at least one of N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and acetone.

[0022] The temperature of the light-proof reaction is 25-120° C., and the time is 12-72 hours.

[0023] The compound (2) is prepared by the following steps: dispersing compound A in water, adding hydrogen peroxide, reacting, crystallizing, and separating to obtain compound (2);

[0024] The dosage ratio of the compound A to hydrogen peroxide is 1 g: 30-80 mL, wherein the mass concentration of hydrogen peroxide is 30%;

[0025] The reaction temperature is 30-80°C and the reaction time is 2-10h;

[0026] The crystallization is specifically carried out by placing the mixed solution after the reaction at 4°C for crystallization for 8-20 hours, filtering and separating the solid, adding water, dissolving at 50-100°C, and then placing it at 4°C for crystallization for 8-20 hours.

[0027] The compound (3) is prepared by the following steps: mixing hydroxychloroquine (HCQ), compound (4) and a solvent, and reacting them to obtain compound (3);

[0028] Among them, the structure of compound (4) is as follows:

[0029] Among them, R 3 The meaning of is the same as compound (1);

[0030] The solvent is at least one of tetrahydrofuran (THF), acetonitrile, dichloromethane, and acetone;

[0031] The dosage ratio of hydroxychloroquine to the solvent is 1 g: 30-80 mL; the molar ratio of hydroxychloroquine to compound (4) is 1:1-5;

[0032] The reaction temperature is 30-100° C. and the reaction time is 4-24 hours.

[0033] Application of the compound having the tetravalent platinum structure of hydroxychloroquine in the preparation of anti-tumor drugs.

[0034] The tumor includes at least one of breast cancer, liver cancer, lung adenocarcinoma, bladder cancer, kidney cancer, pancreatic cancer, ovarian cancer, cervical cancer, and digestive tract cancer.

[0035] The anti-tumor drugs include anti-tumor metastasis drugs.

[0036] The anti-tumor drug is a pharmaceutical composition, which comprises an effective therapeutic amount of a compound having a hydroxychloroquine tetravalent platinum structure and pharmaceutically acceptable excipients.

[0037] The pharmaceutical composition further includes but is not limited to at least one of platinum drugs, 5-fluorouracil drugs, paclitaxel drugs, and doxorubicin drugs.

[0038] The dosage form of the pharmaceutical composition is one of tablets, capsules, aerosols, dispersible tablets, oral liquids, suppositories, pills, large infusions, small injections, freeze-dried powder injections, ointments, and liniments.

[0039] The mechanism of the present invention is:

[0040] The present invention introduces hydroxychloroquine (HCQ) as a functional group into a tetravalent platinum system to obtain a hydroxychloroquine tetravalent platinum compound with autophagy regulation function, thereby obtaining a drug with multiple action mechanisms of DNA damage, autophagy inhibition, tumor microenvironment regulation and immune activation, and exerting effective anti-tumor proliferation and tumor metastasis effects.

[0041] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0042] The present invention introduces hydroxychloroquine, which has the ability to inhibit autophagy, into a tetravalent platinum structure to design and prepare a hydroxychloroquine tetravalent platinum complex. This compound exerts its anti-tumor effects through a synergistic anti-cancer mechanism involving DNA damage, autophagy inhibition, tumor microenvironment regulation, and immune activation. This is of great significance for overcoming drug resistance to divalent platinum drugs, enhancing anti-tumor activity, and reducing toxicity. The tetravalent platinum complex of the present invention has the potential to be developed into a drug for preventing tumor proliferation and metastasis, providing not only a new option for anticancer drug research and development, but also a new approach for the development of novel platinum drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The antiproliferative activity of the compound (HCQPt(IV)1-1) obtained in Example 1 against 4T1 tumors in BALB / c mice. (a) Schematic diagram of the dosing schedule for the mouse experiment; (b) Changes in tumor volume over time; (c) Tumor weights in each group at the end of the experiment; (d) Tumor images; (e) H&E staining of tumor tissue; (f) Body weight changes during treatment; (gk) Organ indexes of the spleen, heart, liver, lung, and kidney, respectively. (n=6) *P<0.05, **P<0.01, ***P<0.001, ns: not significant.

[0044] Figure 2 It is the amount of drug accumulated in the liver and spleen.

[0045] Figure 3 H&E staining results of liver, spleen and kidney.

[0046] Figure 4Results of in vivo anti-tumor metastasis experiments; (a) representative photos of lung metastases in each group; (b) statistical analysis of the number of lung nodules in each group, **P < 0.01, ***P < 0.001; (c) H&E staining results of lung tissue.

[0047] Figure 5 Transwell assay results; (a) representative images; (b) relative mobility analysis, ***P < 0.001.

[0048] Figure 6 Results of the scratch experiment; (a) representative images; (b) analysis of the scratch healing rate results.

[0049] Figure 7 is the uptake of platinum drugs in tumor cells and tissues; (a) drug uptake in tumor cells in vitro; (b) drug uptake in tumor tissues in vivo; (c) drug distribution in tumor cells in vitro, ***P < 0.001.

[0050] Figure 8 Results of drug-induced apoptosis in 4T1 cells tested by Annexin V-FITC / PI double staining; among them, (a) Blank, (b) CDDP (10 μM), (c) OXP (10 μM), and (d) HCQPt(IV)1-1 (10 μM).

[0051] Figure 9 Results of drug-induced mitochondrial damage in 4T1 cells tested by JC-1 staining; (a) Blank, (b) CDDP (10 μM), (c) OXP (10 μM), (d) HCQPt(IV)1-1 (10 μM).

[0052] Figure 10 Western Blot analysis of Bcl-2, Bax, Caspase3 and c-Caspase3 expression; (a) protein blot; (b) relative grayscale analysis.

[0053] Figure 11 Western blot analysis of autophagy-related proteins and TEM observation images of autophagosomes in 4T1 cells treated with platinum drugs (10 μM) for 24 hours; (a) Protein blot of P62 and LC3II / I; (b) Relative grayscale analysis; (c) TEM image of autophagosomes in cells, autophagosomes are marked with red arrows, **P < 0.01, ***P < 0.001.

[0054] Figure 12Results of immunohistochemical staining of P62 in 4T1 tumor tissues; (a) Representative images; (b) Quantitative analysis of P62 expression, ***P < 0.001, **P < 0.01.

[0055] Figure 13 Western blot analysis of HIF-1α, ERK1 / 2, COX-2, iNOS and Caspase1 proteins in 4T1 cells after treatment with platinum drugs; (a) Western blot image; (b) relative gray value analysis, *P<0.05, ***P<0.001.

[0056] Figure 14 Results of immunohistochemical staining of HIF-1α in 4T1 tumor tissues; (a) representative micrographs; (b) quantitative analysis of HIF-1α expression, **P<0.01.

[0057] Figure 15 Western blot analysis of VEGFA and MMP-9 proteins in 4T1 cells after treatment with platinum drugs; (a) Western blot image; (b) relative gray value analysis, ***P<0.001.

[0058] Figure 16 The immunohistochemical staining results of CD34 and MMP-9 in 4T1 tumor tissues; (a) Representative micrographs, ***P<0.001.

[0059] Figure 17 PD-L1 and CD4 expression in 4T1 tumor tissues of BALB / c mice + ,CD8 + Immunohistochemical staining of T cells; (a) representative images; (b) PD-L1 expression and CD4 + 、CD8 + Quantification data of T cells; tumor tissues were obtained from in vivo anti-tumor experiments; **P<0.01, ***P<0.001.

[0060] Figure 18 Schematic diagram of the anti-tumor mechanism of the hydroxychloroquine tetravalent platinum compound of the present invention. DETAILED DESCRIPTION

[0061] The present invention will be described in further detail below with reference to the Examples and accompanying drawings, but the embodiments of the present invention are not limited thereto. Where specific conditions are not specified in the Examples, conventional conditions or conditions recommended by the manufacturer were followed. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0062] Unless otherwise specified, all reagents used in the examples can be purchased from the market.

[0063] Example 1: Preparation of Hydroxychloroquine Tetravalent Platinum Compound

[0064] (1) Synthesis of Hydroxychloroquine Tetravalent Platinum HCQPt(IV)1-1

[0065]

[0066] To a 250mL round-bottom flask, add 1.0g of oxaliplatin and 30mL of distilled water and stir to disperse. Slowly add 50mL of 30% hydrogen peroxide dropwise to the reaction system, raise the temperature to 60°C, and stir for 4 hours. Stop the reaction, let it stand at 4°C to crystallize for 12 hours, and filter to isolate the resulting white solid. Add 70mL of distilled water, heat to 80°C to dissolve, let it stand at 4°C to crystallize for 12 hours, and filter to obtain tetravalent platinum oxide compound 2-1 (white crystals, 0.79g, 70%).

[0067]

[0068] To a 250mL round-bottom flask, add 1.0g of cisplatin CDDP and 30mL of distilled water, stir to disperse, then slowly add 50mL of 30% hydrogen peroxide dropwise to the reaction system. Raise the temperature to 60°C and stir for 4 hours. Stop the reaction, allow to crystallize at 4°C for 12 hours, and filter to isolate the resulting yellow solid. Add 70mL of distilled water, heat to 80°C to dissolve, allow to crystallize at 4°C for 12 hours, and filter to obtain tetravalent platinum oxide compound 2-2 (yellow crystals, 0.65g, 58%).

[0069]

[0070] Dissolve 203 mg of hydroxychloroquine (HCQ) in 50 mL of tetrahydrofuran (THF) and introduce 67 mg of succinic anhydride. Stir the solution at 80°C for at least 12 hours, monitoring the reaction by TLC. After completion of the reaction, remove the solvent from the mixture under reduced pressure to yield compound 3-1. This compound was added directly to the next reaction without purification.

[0071]

[0072] Compound 3-1 (287 mg) and TBTU (212 mg) were dissolved in 5 mL of dry DMF and stirred for 15 minutes. TEA (92 μL) was added and stirred for a further 15 minutes, followed by compound 2-1 (129 mg). The mixture was reacted at 50°C in the dark under nitrogen for 48 hours. After completion of the reaction, the solvent was removed under reduced pressure, and the product was purified by silica gel column chromatography to yield the product HCQPt(IV)1-1 (pale yellow solid, 117 mg, 29.3%).

[0073] HCQPt(IV)1-1 1 H. 13 C NMR (δ in ppm) and MS data: 1 H NMR(500MHz,DMSO-d6)δ8.64-8.52(m,2H),8.46-8.40(m,2H),8.17-8.11(m,1H),7.91(s,1H),7.68-7.62(m, 2H),7.10-7.04(m,1H),6.93-6.85(m,1H),6.18-6.12(m,1H),5.78-5.72(m,1H),4.31-3.78(m,5H),3.75-3.5 2(m,7H),3.51-3.32(m,4H),3.28-2.71(m,4H),2.69-2.52(m,4H),2.49-2.31(m,5H),2.17-1.99(m,2H),1.94 -1.72(m,4H),1.71-1.58(m,2H),1.56-1.47(m,2H),1.46-1.36(m,2H),1.35-1.21(m,8H),1.20-0.91(m,7H). 13 C NMR (126MHz, DMSO-d6) δ180.1,174.2,163.9,154.0,145.7,142.1,137.4,126.6,126.0,121.6,116.4,99.3,65 .4,64.0,61.9,61.3,60.7,55.0,49.2,46.2,32.1,31.4,31.0,30.0,24.0,20.0,19.4,15.6,8.3.MS-ESI:calcd for[M+H3O] + :1286(M=C 52 H 72 Cl2N8O 12 Pt),found:1286.

[0074] (2) Synthesis of Hydroxychloroquine Tetravalent Platinum HCQPt(IV)1-2

[0075]

[0076] Compound 3-1 (287 mg) and TBTU (212 mg) were dissolved in 5 mL of dry DMF and stirred for 15 minutes. TEA (92 μL) was added and stirred for a further 15 minutes, followed by the addition of compound 2-2 (100 mg). The reaction was incubated at 50°C in the dark under nitrogen for 48 hours. After completion of the reaction, the solvent was removed under reduced pressure, and the product was purified by silica gel column chromatography to yield the product HCQPt(IV)1-2 (pale yellow solid, 125 mg, 35%).

[0077] HCQPt(IV)1-2 1 H. 13 C NMR (δ in ppm) and MS data: 1 H NMR(500MHz,DMSO-d6)δ8.92-8.66(m,2H),8.55(d,J=7.0Hz,1H),8.52-7.93(m,4H),7.78(dd,J=9.1,2.1Hz,2H),7.39-6.98(m,6H,NH3),6.97-6.8 9(m,1H),4.14-3.79(m,4H),3.67(d,J=7.0Hz,2H),3.51-3.24(m,10H),3. 09(d,J=7.2Hz,2H),2.54(s,10H),2.09-1.56(m,6H),1.42-1.06(m,12H). 13 CNMR(126MHz,DMSO-d6)δ184.5,179.2,156.7,144.8,140.2,134.3,130.0,127.7,122.0,11 8.4,104.0,70.1,68.8,65.2,59.8,53.4,39.2,36.9,35.5,24.8,20.4,13.1.MS-ESI:calcd for[M+H3O] + :1189(M=C 44 H 64 Cl4N8O8Pt),found:1189.

[0078] (3) Synthesis of Compounds HCQPt(IV)1-3

[0079]

[0080] Compound 3-1 (143 mg) and TBTU (106 mg) were dissolved in 5 mL of dry DMF and stirred for 15 minutes. TEA (92 μL) was added and stirred for a further 15 minutes, followed by compound 2-2 (100 mg). The mixture was reacted at 50°C in the dark under nitrogen for 48 hours. After completion of the reaction, the solvent was removed under reduced pressure, and the product was purified by silica gel column chromatography to obtain product 1-3 (pale yellow solid, 70 mg, 31%).

[0081] HCQPt(IV)1-3 1 H. 13 C NMR (δ in ppm) and MS data: 1 H NMR(500MHz,DMSO)δ9.07(dd,J=86.9,47.0Hz,1H),8.86-8.66(m,1H),8.66-8.47(m,1H),8.49-8.22(m,1H),8.21- 7.78(m,1H),7.53(dd,J=71.6,24.7Hz,1H),6.90-6.49(m,1H),4.60-4.02(m,3H),4.04-3.77(m,2H),3.68(d,J=31 .2Hz,2H),3.58(s,1H),3.44(d,J=7.0Hz,2H),3.11(d,J=56.3Hz,3H),2.83(dd,J=53.2,27.2Hz,2H),2.39(d,J=17 .9Hz,1H),1.84(d,J=75.2Hz,2H),1.58(s,3H),1.27(d,J=4.0Hz,4H),1.17(s,1H),1.11-1.04(m,2H),1.00(s,1H). 13 CNMR(126MHz,DMSO)δ171.2,154.5,150.9,145.6,134.8,134.5,124.7,117.1,99.8, 64.3,55.1,52.9,52.7,47.9,46.7,31.9,28.5,28.1,19.0,14.5,8.3.MS-ESI:calcd for[M] + :752(M=C 22 H 36 Cl3N5O5Pt),found:752.

[0082] (4) Synthesis of Compounds HCQPt(IV)1-4

[0083]

[0084] 3-1 (140 mg) and TBTU (100 mg) were dissolved in 5 mL of dry DMF and stirred for 15 minutes. TEA (92 μL) was added and stirred for a further 15 minutes, followed by the addition of compound 2-1 (129 mg). The mixture was reacted at 50°C in the dark under nitrogen for 48 hours. After completion of the reaction, the solvent was removed under reduced pressure, and the product was purified by silica gel column chromatography to obtain product 1-4 (pale yellow solid, 65 mg, 26%).

[0085] HCQPt(IV)1-4 1 H NMR (δ in ppm) and MS data: 1 H NMR (500MHz, DMSO-d6) δ8.50-8.04(m,1H),7.96-7.76(m,2H),7.41(dt,J=23.1,7. 7Hz,2H),6.56-6.07(m,1H),4.03(q,J=6.1Hz,1H),3.50(s,2H),3.16(s,2H),3.00( dd,J=7.4,5.5Hz,1H),2.57(d,J=22.1Hz,6H),2.45(d,J=14.9Hz,4H),2.01(d,J=1 2.4Hz,3H),1.69-1.43(m,6H),1.37-1.20(m,4H),1.15-0.80(m,4H).MS-ESI:calcd for[M+K] + :888(M=C 30 H 44 ClN5O9Pt),found:888.

[0086] In order to better understand the essence of the present invention, the following is an example of hydroxychloroquine tetravalent platinum compound HCQPt (IV) 1-1, with its pharmacological experimental results of the inhibitory effect on tumors in vivo and in vitro experiments, illustrating that these compounds have potential uses in the pharmaceutical field. Pharmacological Examples provide partial activity data of some compounds. It must be noted that the pharmacological examples of the present invention are for illustrating the present invention rather than for limiting the present invention. Simple improvements to the present invention carried out according to the essence of the present invention all belong to protection scope of the present invention.

[0087] (1) Antitumor activity experiment

[0088] In this experiment, the MTT method was used to determine the cell viability. The half-inhibitory concentration (IC 50 ) value, which measures the in vitro anticancer activity of the complex.

[0089] 100 μL of tumor cells in the logarithmic growth phase were inoculated into a 96-well plate with a cell density of 3000-5000 / well, and the last column was reserved as a zero well. Place in a 37°C cell culture incubator for 12 hours, then add 100 μL of compound culture medium solution with gradient concentrations to the 96-well plate and continue to culture for 72 hours. Add 20 μL of 5 mg / mL MTT solution to each well of the 96-well plate, culture for 4 hours, remove the plate, remove the culture medium, add 150 μL of DMSO, and shake in a 37°C shaker in the dark for 20 minutes. Measure the absorbance OD value of each well at 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader to calculate its IC 50 Each group of experiments was set up with three parallel samples.

[0090] The cell lines used in this experiment include: human lung adenocarcinoma cell line A549, cisplatin-resistant lung adenocarcinoma cell line A549R, human liver cancer cell line HepG2, mouse breast cancer cell line 4T1, human breast cancer cell line MCF7, and human normal liver cell line LO2.

[0091] The test results are shown in Table 1 below:

[0092] Table 1 In vitro antitumor activity data of compounds

[0093]

[0094] a RF: Resistance factor RF=IC 50 (A549R) / IC 50 (A549). b SI: Selection index SI = IC 50 (LO2) / IC 50 (HepG2). c ND: Not detected. c OXP-HCQ: a mixture of OXP and HCQ (1:2, molar ratio).

[0095] It can be seen from Table 1 that compounds 1-1 to 1-4 obtained in Example 1 all have good antitumor activity, especially HCQPt(IV)1-1 shows the strongest antitumor efficacy, with IC 50 The values ​​were all lower than 7.88 μM, which was relatively better than its precursor OXP. At the same time, HCQ had a weak effect on tumor cells (IC 50 >50μM), and the antitumor activity of the mixture OXP-HCQ (molar ratio of 1:2) was not significantly enhanced compared with OXP. These facts indicate that the structure of HCQ in the platinum(IV) system has a significant impact on its antitumor activity.

[0096] Drug resistance is a key constraint to the clinical anti-tumor application of platinum-based drugs. To determine the potential of the anti-tumor compound HCQ platinum (IV) to overcome drug resistance, the drug's resistance factor (RF) was calculated. Compound HCQPt(IV)1-1, obtained in Example 1, showed significant potential in overcoming CDDP resistance. Its RF = 0.65 was 8.6-fold lower than CDDP (RF = 5.63) and significantly lower than OXP, OXP-HCQ (RF = 1.12, 1.38), and the platinum (IV) reference drug STP (RF = 2.68). Subsequently, to evaluate the drug's in vitro toxicity, the selectivity index (SI) for normal cells was calculated. Compound HCQPt(IV)1-1 showed low toxicity against normal cells, with an SI of 2.97, significantly higher than CDDP, OXP, OXP-HCQ, and STP (SI = 0.42-1.24).

[0097] In summary, the anti-tumor effect of the compound HCQPt(IV)1-1 obtained in Example 1 is superior to that of its prodrug OXP, and it can overcome the drug resistance of CDDP and reduce its toxicity to normal cells in vitro.

[0098] (II) Antitumor activity experiment in mice

[0099] In order to further determine the potential of hydroxychloroquine tetravalent platinum complex as an anti-tumor drug, its in vivo anti-tumor ability was evaluated. This section selected the hydroxychloroquine oxaliplatin tetravalent platinum compound HCQPt(IV)1-1 obtained in Example 1 with significant in vitro anti-tumor ability as the research object, and used cisplatin and oxaliplatin as positive references to evaluate the anti-tumor activity of hydroxychloroquine tetravalent platinum drugs. The animal model selected was BALB / c female mice bearing 4T1 tumors. BALB / c mice (18-20g) were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. All animals were fed in accordance with the National Institutes of Health's Guide to the Care and Use of Laboratory Animals.

[0100] After in vitro expansion of 4T1 cells, the cells were digested and collected, washed three times with saline, and resuspended in saline. Tumor cells were inoculated into the right axilla of female BALB / c mice at a density of 1 × 10 6 . On the 4th day after inoculation, the tumor was palpable. The mice were randomly divided into 4 groups, with 6 mice in each group: blank control group, compound HCQPt(IV)1-1 group (2mgPt / kg), oxaliplatin OXP group (2mgPt / kg), cisplatin CDDP group (2mgPt / kg). The drug was administered on the 4th, 7th, 10th and 13th days, for a total of 4 times, by tail vein injection (as Figure 1(a)). During treatment, changes in mouse tumor volume were recorded to assess tumor growth rate; changes in mouse body weight were recorded to evaluate drug toxicity. Tumor growth inhibition rate (TGI) of the test drug = (1-tumor weight of the drug-treated group / tumor weight of the saline group) × 100%. Mice were sacrificed on day 15, and serum, tumor tissue, and organ tissues (heart, lung, liver, spleen, and kidney) were collected. Tumor tissue was weighed. Tissue samples were fixed in formalin and embedded in paraffin, and evaluated by hematoxylin and eosin (H&E) staining and immunohistochemical analysis.

[0101] Compound HCQPt(IV)1-1 showed strong antitumor activity and low toxicity in vivo. 3 , P < 0.001), it effectively suppressed the tumor volume to 543 mm 3 ( Figure 1 In (b), the tumor growth inhibition rate (TGI) was 44.2%, which was stronger than that of its prodrug OXP (TGI = 25.1%, P < 0.05). Subsequently, H&E staining of tumor tissue further demonstrated that HCQPt(IV)1-1 caused significant degeneration / necrosis of tumor cells and nuclear dispersion, with similar effects to CDDP and OXP.

[0102] Systemic toxicity was detected by monitoring body weight during the experiment. Figure 1 The results in (f) showed that compared with the blank group, platinum (II) drugs CDDP (P < 0.001) and OXP (P < 0.01) caused a significant decrease in mouse body weight, while HCQPt (IV) 1-1 had a smaller effect on mouse body weight (P = ns). Organ index (ratio of organ weight to body weight) ( Figure 1 Figures (g)-(k) further show that the platinum (II) drugs CDDP and OXP caused a sharp decrease in spleen index, which may be the key reason for the decline in anti-tumor immunity in mice (P<0.001), while HCQPt(IV)1-1 did not cause significant changes (P=ns). In addition, liver damage was also observed in the CDDP-treated group (P<0.001). Then, atomic absorption spectroscopy (AAS) was used to measure the accumulation of platinum in the spleen and liver. HCQPt(IV)1-1 had lower accumulation levels in the liver and spleen than CDDP and OXP ( Figure 2 ), which may be the main reason for the reduced toxicity of HCQPt(IV)1-1 on the liver and spleen. The low toxicity of compound HCQPt(IV)1-1 was also verified by H&E staining of major organs. Compared with the blank group, compound HCQPt(IV)1-1 did not cause obvious histological differences in liver, spleen and kidney tissues ( Figure 3 ).

[0103] In summary, the compound HCQPt(IV)1-1 obtained in Example 1 showed stronger antitumor activity in vivo than the precursor OXP and had lower toxicity, and has the potential to be further researched and developed as a new antitumor drug.

[0104] (III) In vivo and in vitro anti-tumor metastasis experiments

[0105] Transwell assay: The in vitro anti-metastatic activity was determined using the Transwell assay. 4T1 cells were starved for 24 hours, harvested, resuspended in serum-free medium, and seeded in the upper chamber of a Transwell chamber (0.2 ml medium, 5 × 10 4 0.6 ml of culture medium (10% serum concentration, 10 μM drug concentration; the drugs were HCQPt(IV)1-1, CDDP, and OXP obtained in Example 1) was added to the lower chamber and incubated for 24 hours. Subsequently, the migrated cells in the upper chamber were fixed with 4% paraformaldehyde for 20 minutes, stained with 0.1% crystal violet for 20 minutes, and photographed using an inverted microscope.

[0106] Scratch healing assay: The anti-metastatic activity was further evaluated by in vitro scratch healing assay. 4T1 cells were starved and incubated for 24 hours, and the cells were collected and plated at 8×10 5 Cells were seeded at a density of 10 μM / well in a 6-well culture plate and incubated for 12 hours. Once the cell density reached 90%, a wound was applied to each well. Next, HCQPt(IV)1-1 (10 μM), CDDP (10 μM), and OXP (10 μM), obtained in Example 1, were added, respectively, and incubated for another 24 hours. The 10 μM concentration in the parentheses indicates the final concentration of the drug in the mixed solution after addition. Images were taken at 0, 12, and 24 hours to analyze the wound healing rate.

[0107] In vivo anti-lung metastasis experiment: The lung is one of the main sites of tumor metastasis. Female BALB / c mice (18-20 g) were used as experimental subjects. 4T1 cells (6×10 5 A lung metastasis model was established using a 2-mg Pt / kg Pt-containing oxaliplatin (HCQPt(IV)1-1) solution (n=5). Mice were then randomly divided into four groups (a blank group, the compound HCQPt(IV)1-1 obtained in Example 1, a CDDP group, and an OXP group, n=5). Pt was administered at a dose of 2 mg Pt / kg on days 3, 6, and 9. On day 10, the mice were sacrificed, and lung tissues were collected and fixed with Bouin's fixative for 24 hours. Nodules in each group were counted and analyzed. Lung tissue was also stained with H&E.

[0108] In vivo lung metastasis inhibition test results ( Figure 4) showed that compared with the blank group, compound HCQPt(IV)1-1 had a significant inhibitory effect on lung nodules, with an inhibition rate of 49.6%, significantly superior to CDDP (21.6%) and OXP (18.1%) (P<0.01). Furthermore, H&E staining results showed that the number of lung nodules in the HCQPt(IV)1-1-treated group was lower and smaller. These findings reveal the strong anti-tumor metastasis activity of compound HCQPt(IV)1-1 in vivo.

[0109] Subsequent Transwell and wound wound assays further confirmed its anti-tumor metastasis ability. Figure 5 The Transwell results showed that the migration rate of the compound HCQPt(IV)1-1 treated group was reduced to 33.9% of the blank group, which was better than the reference drug CDDP group (46.5%, P<0.01) and OXP group (71.2%, P<0.001). Figure 6 The scratch test results showed that the wound healing inhibition rate of compound HCQPt(IV)1-1 was also higher than that of CDDP and OXP. This effectively confirmed the in vitro anti-migration ability of hydroxychloroquine tetravalent platinum compound HCQPt(IV)1-1.

[0110] In summary, the compound HCQPt(IV)1-1 obtained in Example 1 showed excellent anti-metastatic activity both in vivo and in vitro, and has great potential for research as an anti-metastatic drug.

[0111] (IV) Drug uptake experiments in tumor cells and tissues

[0112] The uptake of chemotherapeutic drugs in tumor cells and tissues, as well as their distribution in subcellular structures, significantly influences their anti-tumor activity. Therefore, it is of great significance to investigate their enrichment levels within cells and tissues, their distribution in subcellular structures, and their relationship with biological activity.

[0113] Take 4T1 cells in good condition and in logarithmic growth phase and place them in six-well plates (10 6 / well), cultured in a 37°C, 5% carbon dioxide incubator for 3 hours until adhered, added the compound to a concentration of 10 μM, continued to culture for 24 hours, collected the cells, washed three times with PBS (1 mL×3), and collected the cells by centrifugation. 70% concentrated nitric acid (LC) was added to the cells for nitration to prepare samples, and the content of platinum was quantitatively determined by atomic absorption spectroscopy (AAS) to calculate the drug uptake in tumor cells. The cells were treated in the same way, and the cell membrane, cytoplasm and DNA were separated using an organelle separation kit, and nitrated with 70% concentrated nitric acid (LC) to prepare samples, and the distribution of the drug in the subcellular organelles of tumor cells was detected by AAS. The tumor tissue used in the in vivo activity test experiment was taken, nitrated with 70% concentrated nitric acid (LC), prepared samples, and the drug uptake in the tumor tissue was detected by AAS.

[0114] like Figure 7 As shown, compared with the platinum (II) drugs CDDP and OXP, the hydroxychloroquine platinum (IV) compound HCQPt (IV) 1-1 obtained in Example 1 has a higher uptake level in both tumor cells in vitro and tumor tissues in vivo. This may be due to the introduction of the hydroxychloroquine ligand in its structure, which effectively increases the lipophilicity of the drug and thus increases its uptake. It is well known that DNA damage induced by platinum drugs plays a crucial role in promoting tumor cell apoptosis, so we further tested the distribution of the drug in tumor cells. Figure 7 The results in (c) showed that the platinum content in the DNA of the HCQPt(IV)1-1 treatment group was 1.8 and 3.5 times that of the CDDP and OXP groups (P<0.001), which would be conducive to the DNA damage mechanism of the HCQPt(IV)1-1.

[0115] (V) Activating the mitochondrial-mediated Bcl-2 / Bax / caspase3 pathway to induce tumor cell apoptosis

[0116] In order to study the properties of hydroxychloroquine platinum (IV) compounds inducing apoptosis in tumor cells, we used the Annexin V-FITC / PI double staining method to detect the ability of the compounds HCQPt (IV) 1-1, CDDP, and OXP obtained in Example 1 to induce apoptosis in 4T1 cells. Figure 8 The results confirmed that the compound HCQPt(IV)1-1 obtained in Example 1 effectively induced apoptosis in 4T1 cells in vitro, and its apoptosis-inducing ability was comparable to that of CDDP and OXP. Next, we used JC-1 staining to examine the ability of the drug to induce mitochondrial damage in tumor cells. Figure 9 The results showed that the compound HCQPt(IV)1-1 obtained in Example 1 caused a significant loss of membrane potential (ΔΨm) in tumor cells, and its ability to damage mitochondria was comparable to that of CDDP and OXP.

[0117] To further explore its mechanism of inducing apoptosis, we performed Western Blot detection of proteins related to the mitochondrial apoptosis pathway. Figure 10 4T1 cells were treated with compound CDDP, OXP and the compound HCQPt(IV)1-1 obtained in Example 1 for 24 hours. Figure 10 The results confirmed that treatment with the compound HCQPt(IV)1-1 obtained in Example 1 significantly downregulated the anti-apoptotic protein Bcl-2 and upregulated the pro-apoptotic protein Bax in 4T1 cells. Subsequently, the expression of the apoptosis execution proteins Caspase3 and c-Caspase3 increased. Therefore, the hydroxychloroquine platinum (IV) compound HCQPt(IV)1-1 obtained in Example 1 can induce tumor cell apoptosis by activating the mitochondrial-mediated Bcl-2 / Bax / Caspase3 pathway.

[0118] (6) Inhibiting the autophagy process

[0119] LC3 is an important indicator for the formation of autophagosomes, that is, when autophagy is activated, LC3-I is gradually converted into LC3-II, and the autophagosomes fuse with lysosomes and form autophagolysosomes, start degradation process, and complete autophagy. And P62 is the indicator protein of autophagy degradation process. Hydroxychloroquine, as a kind of effective autophagy inhibitor, can be accumulated in lysosomes, interfere with lysosomal acidic environment, cause lysosomal dysfunction, hinder autophagosome-lysosome fusion degradation process, inhibit autophagy, cause autophagosome accumulation and promote tumor cell apoptosis. In order to study whether the anti-tumor activity of the hydroxychloroquine platinum (IV) complex obtained in embodiment 1 is relevant with autophagy inhibition, we tested the expression changes of autophagy-related proteins LC3II / LC3I and P62, and observed the quantity of autophagosomes by TEM.

[0120] like Figure 11 As shown in Figures (a) and (b), the LC3II / LC3I ratio in the group treated with the compound HCQPt(IV)1-1 obtained in Example 1 was significantly increased compared to the blank group (P<0.01), indicating the formation of autophagosomes in tumor cells. Simultaneously, the expression of protein P62 was upregulated, indicating that the lysosomal degradation process was blocked, confirming that the autophagy process in cells was inhibited by the hydroxychloroquine tetravalent platinum drug. Figure 11 The TEM results in (c) show that the number of autophagosomes in tumor cells increased significantly after treatment with the compound HCQPt(IV)1-1 obtained in Example 1. Figure 12 The results of immunofluorescence staining of tumor tissues further confirmed that the expression of P62 in tumor tissues of the group treated with the compound HCQPt(IV)1-1 obtained in Example 1 was significantly increased, which is consistent with the blocking mechanism of hydroxychloroquine on autophagy.

[0121] Based on this, it can be inferred that the hydroxychloroquine platinum (IV) compound obtained in Example 1 can induce lysosomal dysfunction, destroy the formation and degradation of autophagic lysosomes, and thus inhibit the autophagy of tumor cells and promote the apoptosis of tumor cells.

[0122] (VII) Inhibiting hypoxia and inflammatory tumor microenvironment

[0123] Hypoxia and chronic inflammation are key characteristics of tumors, playing a crucial role in promoting immunosuppression and angiogenesis, both of which are crucial factors in tumor progression and metastasis. The formation of hypoxia and inflammation in the tumor microenvironment is closely related to autophagy.

[0124] like Figure 13 and 14 As shown, the hydroxychloroquine platinum (IV) compound HCQPt (IV) 1-1 obtained in Example 1 can effectively downregulate the expression of HIF-1α in both in vitro tumor cells (P < 0.05) and in vivo tumor tissues (P < 0.01), which demonstrates its ability to regulate the hypoxic microenvironment by inhibiting HIF-1α. At the same time, the expression of ERK1 / 2 was also downregulated, and the levels of key inflammation-related proteins iNOS, caspase1, and COX-2 were also significantly reduced, indicating that the hydroxychloroquine platinum (IV) compound HCQPt (IV) 1-1 obtained in Example 1 can effectively inhibit the inflammatory tumor microenvironment, which will further affect the immune activation and metastasis of the tumor.

[0125] (8) Inhibit tumor angiogenesis and metastasis

[0126] Angiogenesis is a key factor influencing tumor proliferation and metastasis, providing nutrients for tumor growth and a crucial pathway for tumor cell metastasis. Vascular endothelial growth factor (VEGF) is a key enzyme in angiogenesis. Matrix metalloproteinases (MMPs) control the degradation of extracellular matrix proteins and synergize with VEGF in promoting tumor angiogenesis and metastasis.

[0127] Immunohistochemistry and Western Blot were used to detect the expression of VEGFA, MMP-9 and CD34 in tumor tissues. + Expression. Figure 15 and Figure 16 As shown in the figure, the VEGFA in the tumor cells of the group treated with the compound HCQPt(IV)1-1 obtained in Example 1 was inhibited (P<0.01), and the expression of CD34 in the tumor tissues was inhibited. +Microvessel density was also significantly lower than that of the blank group (P < 0.001). Furthermore, MMP-9 was downregulated in both in vitro tumor cells (P < 0.05) and in in vivo tumor tissues (P < 0.01). This suggests that the compound HCQPt(IV)1-1 obtained in Example 1 inhibits angiogenesis and, consequently, tumor metastasis by inhibiting VEGFA and MMP-9 in tumor tissues.

[0128] (9) Activate immunity

[0129] Immunosuppression is a major characteristic of cancer, which can promote the survival of tumor cells and cancer metastasis, thereby further leading to chemotherapy failure. Currently, more and more studies have confirmed that inhibiting protective autophagy, inhibiting hypoxia and inflammatory TME can inhibit the immune checkpoint PD-L1 in tumor cells, increase the number of tumor infiltrating lymphocytes (TILs), and activate immune responses. Therefore, we detected the expression of PD-L1 in tumor tissues and CD4 + 、CD8 + The number of T cells.

[0130] Figure 17 The results showed that HCQPt(IV)1-1 could significantly inhibit the expression of PD-L1 in tumors, reducing it to 63.3% of the blank group (P < 0.001), while platinum (II) drugs CDDP and OXP failed to inhibit the expression of PD-L1 (154.9% and 143.2%). + and CD8 + The density of T cells was significantly increased compared to the blank group, reaching 2.8 and 2.4 times that of the blank group, respectively (P<0.001), while the effects of CDDP and OXP on T cells were almost negligible. Therefore, the compound HCQPt(IV)1-1 can activate T cell immune responses in tumor tissues by inhibiting PD-L1 expression.

[0131] In summary, the hydroxychloroquine platinum (IV) compound obtained by the present invention has significant anti-tumor proliferation and anti-metastasis activities. Its mechanism of action is as follows Figure 18 This class of compounds has been shown to accumulate at high levels in tumor cells, causing DNA damage and upregulating γ-H2AX and p53. The introduction of the autophagy inhibitor hydroxychloroquine (HCQ) disrupts lysosomal function in tumor cells, inhibiting autophagy. This mechanism synergizes with DNA damage to promote apoptosis. Furthermore, it downregulates ERK1 / 2, HIF-1α, iNOS, caspase-1, and COX-2, further suppressing the hypoxic and inflammatory tumor microenvironment. It also inhibits angiogenesis by suppressing VEGFA, MMP-9, and CD34, effectively inhibiting tumor metastasis.

[0132] Example 2

[0133] A pharmaceutical composition comprising the following ingredients: 0.07 g of the hydroxychloroquine tetravalent platinum compound HCQPt(IV)1-1 obtained in Example 1, 0.02 g of microcrystalline cellulose, 0.05 g of starch, 0.03 g of lactose, 0.01 g of povidone, 0.02 g of sodium carboxymethyl starch, and 0.02 g of micropowdered silica gel.

[0134] The hydroxychloroquine tetravalent platinum compound of the present invention can be used alone or in combination with marketed platinum-based anti-tumor drugs such as 5-fluorouracil and paclitaxel to prepare a pharmaceutical composition with anti-tumor activity. The pharmaceutical composition can be in the form of tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, sustained-release tablets, capsules, hard capsules, soft capsules, sustained-release capsules, oral liquids, mixtures, lozenges, granules, electuary, pills, powders, ointments, suspensions, solutions, injections, powder injections, freeze-dried powder injections, suppositories, liniments, ointments, plasters, creams, sprays, aerosols, drops, patches, and the like.

[0135] As an alternative implementation, the pharmaceutically acceptable excipients described in the present invention include, but are not limited to, liquid or solid fillers, diluents, excipients (such as cocoa butter and suppositories), solvents, or packaging materials. Pharmaceutically acceptable excipients can be aqueous or non-aqueous. Conventional excipients include colloids, such as gelatin; starches, such as corn starch and potato starch; sugars, such as lactose, glucose, and sucrose; cellulosic materials and mixtures thereof, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate. Pharmaceutically acceptable excipients include, but are not limited to, tragacanth powder, malt, talc, oils (such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil, etc.), alcohols (such as propylene glycol, ethanol, glycerol, sorbitol, mannitol, polyethylene glycol, etc.), esters (such as ethyl oleate, ethyl laurate, agar), buffers (such as magnesium hydroxide, aluminum hydroxide, boric acid and sodium borate and phosphate buffer), alginic acid, pyrogen-free water, isotonic saline, and Ringer's solution.

[0136] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A compound having a hydroxychloroquine tetravalent platinum structure, characterized in that, The structure is shown in formula (1): in, is compound A, which is one of cisplatin and oxaliplatin; R 3 It is one of —(CH2)—, —(CH2)2—, —(CH2)3—, and —(CH2)4—.

2. the preparation method of the compound with hydroxychloroquine tetravalent platinum structure according to claim 1, is characterized in that, The synthetic route is as follows: The reaction is carried out in a mixed solution containing a condensing agent and an organic base, wherein the molar ratio of compound (2), compound (3), condensing agent and organic base is 1:(2.0-4.0):(2.0-4.0):(2.0-4.0); and the amount ratio of compound (2) to solvent is 1 g:30-80 mL.

3. according to the preparation method of the compound with hydroxychloroquine tetravalent platinum structure of claim 2, it is characterized in that, The following steps are involved: Under an inert atmosphere, a condensing agent, compound (3) and a solvent are mixed and reacted for 5-30 minutes; an organic base is added and reacted for 5-30 minutes; then compound (2) is added and reacted in the dark, and compound (1) is obtained after purification.

4. according to the preparation method of the compound with hydroxychloroquine tetravalent platinum structure of claim 3, it is characterized in that: The inert atmosphere is at least one of nitrogen, helium, and argon; The condensing agent is at least one of TBTU, HATU and DCC; The organic base is at least one of triethylamine, N,N-diisopropylethylamine, and 4-dimethylaminopyridine; The solvent is at least one of N,N-dimethylformamide, dimethyl sulfoxide, and acetone; The temperature of the light-proof reaction is 25-120° C., and the time is 12-72 hours.

5. according to the preparation method of the compound with hydroxychloroquine tetravalent platinum structure of claim 3, it is characterized in that, The compound (2) is prepared by the following steps: Dispersing compound A in water, adding hydrogen peroxide, reacting, crystallizing, and separating to obtain compound (2); The dosage ratio of the compound A to hydrogen peroxide is 1 g: 30-80 mL, wherein the mass concentration of hydrogen peroxide is 30%; The reaction temperature is 30-80°C and the reaction time is 2-10h; The crystallization is specifically carried out by placing the mixed solution after the reaction at 4°C for crystallization for 8-20 hours, filtering and separating the solid, adding water, dissolving at 50-100°C, and then placing it at 4°C for crystallization for 8-20 hours.

6. according to the preparation method of the compound with hydroxychloroquine tetravalent platinum structure of claim 3, it is characterized in that, The compound (3) is prepared by the following steps: Mixing hydroxychloroquine, compound (4) and a solvent, and reacting to obtain compound (3); Among them, the structure of compound (4) is as follows: Among them, R 3 The meaning of is the same as compound (1); The solvent is at least one of tetrahydrofuran, acetonitrile, dichloromethane, and acetone; The dosage ratio of hydroxychloroquine to the solvent is 1 g: 30-80 mL; the molar ratio of hydroxychloroquine to compound (4) is 1:1-5; The reaction temperature is 30-100° C. and the reaction time is 4-24 hours.

7. Use of the compound having the tetravalent platinum structure of hydroxychloroquine according to claim 1 in the preparation of antitumor drugs.

8. The application according to claim 7, characterized in that: The tumor includes at least one of breast cancer, liver cancer, lung adenocarcinoma, bladder cancer, kidney cancer, pancreatic cancer, ovarian cancer, cervical cancer, and digestive tract cancer; The anti-tumor drugs include anti-tumor metastasis drugs.

9. The use according to claim 7, characterized in that: The anti-tumor drug is a pharmaceutical composition, which comprises an effective therapeutic amount of a compound having a hydroxychloroquine tetravalent platinum structure and pharmaceutically acceptable excipients.

10. The application according to claim 9, characterized in that: The pharmaceutical composition further comprises at least one of platinum drugs, 5-fluorouracil drugs, paclitaxel drugs, and doxorubicin drugs.

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