Platinum (IV) prodrug taking small molecule TrxR inhibitor as ligand as well as preparation method and application of platinum (IV) prodrug

Abstract

The invention belongs to the technical field of medicinal chemistry, and particularly discloses a platinum (IV) prodrug with a small molecule TrxR inhibitor as a ligand and a preparation method and application of the platinum (IV) prodrug, the synthesized platinum (IV) prodrug shows good anti-proliferative activity on cancer cells such as MDA-MB-231, MCF-7, 4T1 and MDA-MB-231 / CDDP, and the anticancer activity of a compound 6b is optimal. In an in-vivo anti-tumor activity test, the compound 6b can effectively inhibit proliferation of cis-platinum sensitive and cis-platinum drug-resistant triple negative breast cancer xenotransplantation tumors, the tumor inhibition rates of the compound 6b are 73.8% and 66.3%, and the curative effect of the compound 6b is superior to that of a positive drug cis-platinum. In addition, the compound 6b can effectively inhibit proliferation of homotransplantation tumors of mouse breast cancer cells 4T1, and the tumor inhibition rate of the compound 6b is 76.8% and is obviously higher than that of a cis-platinum treatment group. And the compound 6b shows a powerful immunoregulation effect in vivo, which indicates that the design successfully realizes effective combination of chemotherapy and immunotherapy, and the compound disclosed by the invention has potential application prospects in treatment of triple negative breast cancer.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology and relates to the design and synthesis of a platinum (IV) prodrug, particularly to a method for synthesizing a platinum (IV) prodrug using a small molecule TrxR inhibitor as a ligand and its application in the field of antitumor therapy. Background Technology

[0002] Breast cancer is the most common type of cancer among women and has become one of the leading causes of cancer death in women. Triple-negative breast cancer (TNBC) accounts for approximately 15% to 20% of all breast cancer cases. It is characterized by high malignancy, a high rate of metastasis, and the lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Furthermore, poor prognosis, tumor metastasis, and limited treatment options are also major causes of death in TNBC patients. Currently, platinum-based anticancer drugs, such as cisplatin (CDDP), are widely used in clinical chemotherapy for various solid tumors, including lung cancer, ovarian cancer, and triple-negative breast cancer, due to their broad-spectrum anticancer activity and high efficacy. However, these anticancer drugs have many drawbacks in clinical application, such as poor selectivity, severe side effects, and drug resistance, which limit their clinical application. Therefore, developing a novel platinum-based anticancer drug with strong targeting, high efficacy, and low toxicity for the chemotherapy of TNBC has become an urgent priority.

[0003] Studies have confirmed that platinum (IV) complexes are kinetically more chemically inert than platinum (II) drugs, effectively reducing off-target reactions with biological substances and thus lowering toxicity. They can also serve as prodrugs for platinum (II) drugs. Research has found that thioredoxin reductase (TrxR), a key reductase, plays a crucial role in maintaining intracellular redox balance. Given that TrxR expression is higher in malignant cancer cells than in non-cancer tissues, TrxR is considered a promising target for cancer therapy. Therefore, developing a novel platinum (IV) prodrug targeting TrxR holds promise for providing a new strategy for the treatment of TNBC. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a platinum (IV) prodrug with a small molecule TrxR inhibitor as a ligand. This type of compound can overcome the disadvantages of existing anti-tumor drugs, such as large toxic side effects and poor oral bioavailability, while treating TNBC. Another objective of the present invention is to provide a method for synthesizing this type of platinum (IV) complex with a small molecule TrxR inhibitor as a ligand.

[0005] This invention is achieved through the following technical solution: A platinum (IV) prodrug using a small molecule TrxR inhibitor as a ligand has the following chemical structure:

[0006] In the formula, n = 1 or 2; R1 = -CF3, -OCF3, or -OCH3.

[0007] Furthermore, the chemical structures are shown in formulas (6a)-(6f): .

[0008] A further improvement to the present invention is as follows: A method for preparing a platinum (IV) prodrug using a small molecule TrxR inhibitor as a ligand includes the following steps: (1) Compound 1 and compound 2 were reacted under the conditions of concentrated hydrochloric acid and glacial acetic acid to synthesize compound 3; (2) React compound 3 with succinic anhydride or glutaric anhydride under organic base conditions to synthesize compound 4; (3) Compound 4 was reacted with platinum (IV) complex 5 under condensing agent and organic base conditions to synthesize compound 6; The synthesis route is shown below:

[0009] In the formula, n = 1 or 2; R1 = -CF3, -OCF3, or -OCH3.

[0010] Furthermore, in step (1), the molar ratio of compound 1 to compound 2 is 2~3:1, and the volume ratio of concentrated hydrochloric acid to glacial acetic acid is 1:8~12; Furthermore, the reaction is carried out at room temperature for 40-50 hours.

[0011] Furthermore, in step (2), the organic base is triethylamine, DMF is used as solvent, and the molar ratio of compound 3 to succinic anhydride or glutaric anhydride and the organic base is 1:2~4:1~2; Furthermore, the reaction temperature is 40~60℃ and the time is 8~12h.

[0012] Furthermore, in step (3), the condensing agent is TBTU, the organic base is triethylamine, DMF is used as the solvent, and the molar ratio of compound 4 to platinum (IV) complex 5, condensing agent and organic base is 1:0.8~1.2:1~2:1~2; Furthermore, the reaction temperature is 20~40℃ and the time is 8~12h.

[0013] Furthermore, steps (1) to (3) also include a separation and purification process.

[0014] A further improvement of the present invention is as follows: The above-mentioned application of platinum (IV) prodrugs with small molecule TrxR inhibitors as ligands in the preparation of anti-breast cancer drugs.

[0015] This invention introduces a small-molecule TrxR inhibitor into the axial ligand of a platinum (IV) prodrug, resulting in a platinum (IV) prodrug with a small-molecule TrxR inhibitor as the ligand, serving as a compound with targeting function. Studies have shown that this type of platinum (IV) complex exhibits good anti-breast cancer activity. Compound 6b, for example, demonstrates superior anti-proliferative activity against MDA-MB-231, MCF-7, and 4T1 cancer cells compared to cisplatin, and exhibits low toxicity against normal human breast cells MCF-10A. Furthermore, compound 6b also shows good inhibitory activity against cisplatin-resistant MDA-MB-231 / CDDP cells. Compound 6b demonstrates superior antitumor efficacy compared to cisplatin in cisplatin-sensitive or resistant TNBC xenograft models and mouse breast cancer allograft models, with no significant toxic side effects observed. In vitro and in vivo antitumor studies indicate that compound 6b has potential for targeted therapy of breast cancer. Attached Figure Description

[0016] Figure 1 This is a diagram showing the in vivo anti-4T1 breast cancer cell results of compound 6b in this embodiment of the invention; Among them, (A) tumor images of each treatment group; (B) changes in tumor volume of each treatment group; (C) tumor mass of each treatment group; (D) curves of changes in mouse body weight of each treatment group; **Compared with the control group, P < 0.01.

[0017] Figure 2 This is a schematic diagram illustrating the in vivo anti-4T1 breast cancer cell activity of compound 6b in an embodiment of the present invention. Among them, (A) the serum cytokine levels of different groups of BALB / c mice were detected by ELISA, and compared with the control group, **P < 0.01; (B) CD4 levels in tumor tissue + T cells, CD8 + Immunofluorescence of T cells, CRT and CHOP proteins, scale bar: 50 μm.

[0018] Figure 3 This is a schematic diagram illustrating the in vivo anti-MDA-MB-231 cancer cell activity of compound 6b in an embodiment of the present invention. Among them, (AE) images of tumors removed in each group; (BF) changes in tumor volume in each treatment group; (CG) tumor mass in each treatment group; (DH) changes in body weight of mice in each group recorded over 21 days; **p<0.01; (IJ) immunohistochemical staining of Ki67, GPX4 and TrxR1 proteins in tumor tissue, scale bar: 50 μm.

[0019] Figure 4 The images show H&E staining of major organs of mice, including the heart, liver, spleen, kidneys, and lungs, after 21 days of treatment with compound 6b in this embodiment of the invention. Detailed Implementation

[0020] The present invention will now be described in detail with reference to specific embodiments.

[0021] This invention relates to and synthesizes a platinum (IV) prodrug using a small molecule thioredoxin reductase (TrxR) inhibitor as a ligand, the chemical structure of which is shown in the following formula:

[0022] In the formula, n = 1 or 2; R1 = -CF3, -OCF3, or -OCH3.

[0023] The following specific examples use compounds 6a-6f as an example to illustrate the specific preparation process and activity verification.

[0024]

[0025] Example 1: Preparation of compounds 3a-3c Compound 1 (17.6 mmol) (3-trifluoromethylbenzaldehyde, 3-trifluoromethoxybenzaldehyde, or 3-methoxybenzaldehyde) and compound 2 (8 mmol) were dissolved in glacial acetic acid (20 mL) and concentrated hydrochloric acid (2 mL), and stirred at room temperature for 48 h. The reaction was monitored by thin-layer chromatography (TLC). After the reaction was complete, 10N NaOH solution was added to adjust the pH to 10, and then DCM (200 mL) was added. The mixture was washed with saturated sodium chloride aqueous solution. The organic phase was dried over anhydrous Na2SO4, concentrated under vacuum, and the crude product was purified by silica gel column chromatography using petroleum ether and ethyl acetate as eluents to give compounds 3a, 3b, or 3c.

[0026]

[0027] 3a, yellow solid, 1.65 g, yield 50.2%. 11H NMR (500 MHz, CDCl3) δ 7.80 (s, 2H), 7.62 (d, J J = 5.8 Hz, 4H), 7.56 (t, J J = 6.6 Hz, 4H), 4.15 (d, J J = 1.3 Hz, 4H), 1.66 (s, 1H). 13 13C NMR (125 MHz, CDCl3) δ 187.33, 136.10, 135.78, 134.48, 133.44, 131.13 (q, J CF J = 32.5 Hz), 129.18, 126.83 (q, J CF J = 3.7 Hz), 125.67 (q, J CF J = 3.5 Hz), 124.92, 122.75, 120.58, 47.97. C 21 1H 15 F6NO [M + H] + : 412.1136; found: 412.1145. Purity: 98.79% (by HPLC). 3b, yellow solid, 1.72 g, yield 48.6%. 1 1H NMR (500 MHz, CDCl3) δ 7.75 (s, 2H), 7.46 (t, J J = 7.8 Hz, 2H), 7.31 (d, J J = 7.7 Hz, 2H), 7.24 – 7.21 (m, 4H), 4.14 (d, J J = 1.6 Hz, 4H), 1.69 (s, 1H). 13 13C NMR (125 MHz, CDCl3) δ 187.43, 149.30, 137.02, 135.99, 134.46, 130.04, 128.75, 122.54, 121.49, 121.45, 119.45, 47.95. C 21 1H 15 F6NO3 [M + H] +: 444.1034; found:444.1045. Purity: 98.86% (by HPLC). 3c, yellow solid, 1.4 g, yield 52.2%. 1 H NMR (600 MHz, DMSO-d6) δ 7.56 (s, 2H),7.38 (t, J = 7.9 Hz, 2H), 7.05 – 7.02 (m, 4H), 6.99 (dd, J = 8.2, 2.1 Hz,2H), 3.99 (s, 4H), 3.80 (s, 6H), 2.83 (s, 1H). 13 C NMR (150 MHz, DMSO-d6) δ187.05, 158.66, 135.66, 135.61, 133.14, 129.09, 122.07, 115.01, 114.32,54.57, 47.00. C 21 H 21 NO3 [M + H] + : 336.1600; found: 336.1606. Purity: 99.27% ​​(byHPLC). Example 2: Preparation of compounds 4a-4b, 4c-4d and 4e-4f Compounds 3a (2 mmol), 3b (2 mmol), or 3c (2 mmol) were dissolved in dry DMF (8 mL), and succinic anhydride (6 mmol) or glutaric anhydride (6 mmol) and triethylamine (3 mmol) were added. The mixture was stirred overnight at 50 °C. The reaction was monitored by TLC. After the reaction was complete, the solvent was removed by rotary evaporation, and DCM (100 mL) was added. The mixture was washed with a saturated aqueous sodium chloride (NaCl) solution. The organic phase was dried over anhydrous Na₂SO₄, concentrated under vacuum, and the crude product was purified by silica gel column chromatography using dichloromethane and methanol as eluents to give compounds 4a-4b, 4c-4d, and 4e-4f.

[0028]

[0029] 4a, yellow solid, 865 mg, yield 84.6%. 1 H NMR (600 MHz, DMSO- d6 ) δ 11.96 (s, 1H), 7.93 (d, J= 18.5 Hz, 2H), 7.89 – 7.86 (m, 2H), 7.83 – 7.79 (m, 4H),7.75 (t, J = 7.6 Hz, 2H), 4.86 (d, J = 29.2 Hz, 4H), 2.41 (t, J = 5.3 Hz, 2H), 2.31 (t, J = 6.1 Hz, 2H). C 25 H 19 F6NO4[M + H] + : 512.1297; found: 512.1293.Purity: 98.42% (by HPLC). 4b, yellow solid, 905 mg, yield 86.2%. 1 H NMR (600 MHz, DMSO- d6 ) δ 11.96 (s,1H), 7.92 (s, 2H), 7.86 – 7.81 (m, 4H), 7.79 – 7.73 (m, 4H), 4.82 (s, 4H), 2.20 (t, J = 7.3 Hz, 2H), 2.06 (t, J = 7.4 Hz, 2H), 1.58 – 1.53 (m, 2H).C 26 H 21 F6NO4[M + H] + : 526.1453; found: 526.1452. Purity: 98.94% (by HPLC). 4c, yellow solid, 943 mg, yield 86.8%. 1 H NMR (600 MHz, DMSO- d6 ) δ 11.89 (s,1H), 7.72 (s, 2H), 7.65 (t, J = 7.7 Hz, 2H), 7.63 – 7.57 (m, 4H), 7.48 (d, J = 7.3 Hz, 2H), 4.84 (d, J = 24.1 Hz, 4H), 2.42 (t, J = 6.2 Hz, 2H), 2.31 (t, J= 6.1 Hz, 2H). C 25 H 19 F6NO6[M + H] + : 544.1195; found: 544.1192. Purity: 99.03%(by HPLC). 4 days, yellow solid, 930 mg, yield 83.5%. 1 H NMR (600 MHz, DMSO- d6 ) δ 11.94 (s, 1H), 7.71 (d, J = 13.0 Hz, 2H), 7.65 (t, J = 7.6 Hz, 2H), 7.60 – 7.58 (m,4H), 7.47 (d, J = 6.5 Hz, 2H), 4.81 (s, 4H), 2.20 (t, J = 6.4 Hz, 2H), 2.06(t, J = 6.7 Hz, 2H), 1.57 – 1.54 (m, 2H). C 26 H 21 F6NO6[M + H] + : 558.1351; found:558.1350. Purity: 98.97% (by HPLC). 4e, yellow solid, 785 mg, yield 90.2%. 1 H NMR (500 MHz, DMSO- d6 ) δ 11.97 (s,1H), 7.68 (s, 2H), 7.44 – 7.40 (m, 2H), 7.15 – 7.11 (m, 4H), 7.04 (d, J = 8.3Hz, 2H), 4.84 (d, J = 11.3 Hz, 4H), 3.81 (d, J = 5.3 Hz, 6H), 2.41 (t, J =6.1 Hz, 2H), 2.32 (t, J = 6.1 Hz, 2H). C 25 H 25 NO6[M + H] +: 436.1760; found:436.1761. Purity: 97.87% (by HPLC). 4f, yellow solid, 805 mg, yield 89.6%. 1 H NMR (600 MHz, DMSO- d6 ) δ 11.97 (s, 1H), 7.67 (d, J = 9.7 Hz, 2H), 7.42 (t, J = 7.9 Hz, 2H), 7.12 – 7.10 (m, 4H), 7.04 (d, J = 9.6 Hz, 2H), 4.82 (d, J = 10.1 Hz, 4H), 3.81 (s, 6H), 2.22 (t, J = 7.2 Hz, 2H), 2.07 (t, J = 7.4 Hz, 2H), 1.59 – 1.54 (m, 2H). C 26 H 27 NO6[M + H] + : 450.1917; found: 450.1919. Purity: 99.03% (by HPLC).

[0030] Example 3: Preparation of compounds 6a-6f Compounds 4a-4b (0.294 mmol), 4c-4d (0.294 mmol), or 4e-4f (0.294 mmol) were dissolved in dry DMF (3 mL), and TBTU (0.441 mmol), triethylamine (0.441 mmol), and complex 5 (0.294 mmol) were added. The mixture was stirred overnight at 30 °C. The reaction was monitored by TLC. After the reaction was complete, the reaction mixture was diluted with 100 mL of DCM and then washed three times with an aqueous sodium chloride solution. The organic phase was dried over anhydrous Na₂SO₄, concentrated under vacuum, and the crude product was purified by silica gel column chromatography using dichloromethane and methanol as eluents to give the title compounds 6a-6f. Compound 6a, yellow solid, 115 mg, yield 46.4%. 1 H NMR (600 MHz, DMSO- d6 ) δ 7.93(d, J = 14.7 Hz, 2H), 7.87 (t,J = 8.2 Hz, 2H), 7.82 (d, J = 7.7 Hz, 2H),7.78 – 7.73 (m, 4H), 6.25 – 5.92 (m, 6H), 4.86 (d, J = 26.6 Hz, 4H), 2.41 –2.37 (m, 4H). 13 C NMR (150 MHz, DMSO- d6 ) δ 185.44, 179.00, 170.01, 134.78,134.55, 134.23, 134.03, 133.49, 133.28 (d, J CF = 5.7 Hz), 133.12, 129.46,129.35, 129.14 (d, J CF = 8.0 Hz), 128.93 (d, J CF = 8.4 Hz), 126.84, 126.35,125.34 (d, J CF = 15.4 Hz), 124.29, 122.48, 120.67, 45.40, 41.67, 30.41, 27.30. 195 Pt NMR (129 MHz, DMSO- d6 ) δ 547.36. C 25 H 24 Cl3F6N3O4PtNa [M + Na] + : 867.0282;found: 867.0281. Purity: 96.95% (by HPLC). Compound 6b, yellow solid, 126 mg, yield 51.2%. 1 H NMR (600 MHz, DMSO- d6 ) δ 7.93(d, J = 7.8 Hz, 2H), 7.89 – 7.82 (m, 4H), 7.79 – 7.75 (m, 4H), 6.21 – 6.00(m, 6H), 4.83 (d, J = 19.8 Hz, 4H), 2.27 (t, J= 7.2 Hz, 2H), 2.11 (t, J =7.2 Hz, 2H), 1.59 – 1.54 (m, 2H). 13 C NMR (150 MHz, DMSO- d6 ) δ 186.78, 180.51,171.58, 135.85, 135.63, 135.30, 134.96, 134.62, 134.27 (d, J CF = 30.6 Hz),130.59, 130.39, 130.08 (d, J CF = 31.5 Hz), 127.94, 127.39, 126.38 (d, J CF =3.7 Hz), 125.35, 123.54, 46.51, 42.39, 35.81, 31.47, 21.32. 195 Pt NMR (129MHz, DMSO- d6 ) δ 550.48. C 26 H 26 Cl3F6N3O4PtNa [M + Na] + : 881.0439; found:881.0433. Purity: 98.15% (by HPLC). Compound 6c, yellow solid, 115 mg, yield 44.9%. 1 H NMR (600 MHz, DMSO- d6 ) δ 7.71(s, 2H), 7.67 – 7.64 (m, 2H), 7.63 – 7.58 (m, 4H), 7.47 (d, J = 7.8 Hz, 2H), 6.24 – 6.01 (m, 6H), 4.85 (d, J = 21.6 Hz, 4H), 2.42 – 2.38 (m, 4H). 13 C NMR (150 MHz, DMSO-) d6) δ 185.45, 179.02, 169.99, 147.93, 136.00, 135.78, 134.09,133.94, 133.39, 133.16, 130.36, 130.24, 128.67, 122.56, 122.24, 121.37,121.28, 120.36, 118.66, 45.40, 41.69, 30.41, 27.32. 195 Pt NMR (129 MHz, DMSO- d6 ) δ 547.27. C 25 H 24 Cl3F6N3O6PtNa [M + Na] + : 899.0180; found: 899.0182. Purity:98.72% (by HPLC). Compound 6d, yellow solid, 113 mg, yield 43.1%. 1 H NMR (600 MHz, DMSO- d6 ) δ 7.70(d, J = 21.8 Hz, 2H), 7.67 – 7.60 (m, 6H), 7.46 (d, J = 25.1 Hz, 2H), 6.11(dd, J = 82.0, 44.3 Hz, 6H), 4.82 (d, J = 11.4 Hz, 4H), 2.27 (t, J = 7.3 Hz, 2H), 2.11 (t, J = 7.4 Hz, 2H), 1.59 – 1.54 (m, 2H). 13 C NMR (150 MHz, DMSO- d6 )δ 186.77, 180.48, 171.55, 148.96, 137.05, 136.83, 135.15, 134.88, 134.51,131.47, 131.28, 129.74, 123.63, 123.24, 122.44, 122.32, 121.41, 119.71,46.48, 42.42, 35.81, 31.49, 21.32. 195 Pt NMR (129 MHz, DMSO-d6 ) δ 549.94.C 26 H 26 Cl3F6N3O6PtNa [M + Na] + : 913.0337; found: 913.0327. Purity: 97.60% (byHPLC). Compound 6e, yellow solid, 130 mg, yield 57.5%. 1 H NMR (500 MHz, DMSO- d6 ) δ 7.68(s, 2H), 7.43 (dd, J = 13.8, 7.6 Hz, 2H), 7.16 – 7.10 (m, 4H), 7.04 (d, J =8.7 Hz, 2H), 6.31 – 5.92 (m, 6H), 4.85 (d, J = 8.6 Hz, 4H), 3.81 (d, J = 4.6Hz, 6H), 2.44 – 2.36 (m, 4H). 13 C NMR (125 MHz, DMSO- d6 ) δ 186.55, 180.05,170.92, 159.84, 136.72, 136.17, 135.95, 133.29, 132.99, 130.53, 130.46,123.11, 116.27, 116.11, 115.86, 55.76, 46.59, 43.03, 31.47, 28.45. 195 Pt NMR (129 MHz, DMSO-) d6 ) δ 546.58. C 25 H 30 Cl3N3O6PtNa [M + Na] + : 791.0746; found:791.0735. Purity: 98.97% (by HPLC). Compound 6f, yellow solid, 117 mg, yield 50.9%. 1 H NMR (500 MHz, DMSO- d6 ) δ 7.66(d, J= 12.7 Hz, 2H), 7.46 – 7.41 (m, 2H), 7.16 – 7.09 (m, 4H), 7.04 (d, J =7.6 Hz, 2H), 6.35 – 5.94 (m, 6H), 4.82 (s, 4H), 3.81 (d, J = 6.0 Hz, 6H), 2.26 (t, J = 7.2 Hz, 2H), 2.11 (t, J = 7.3 Hz, 2H), 1.59 – 1.54 (m, 2H). 13 CNMR (125 MHz, DMSO- d6 ) δ 186.83, 180.46, 171.49, 159.82, 136.72, 136.58,136.18, 135.96, 133.40, 133.32, 130.55, 130.38, 123.11, 116.23, 116.13,115.85, 55.79, 46.65, 42.75, 35.77, 31.56, 21.36. 195 Pt NMR (129 MHz, DMSO- d6 )δ 549.53. C 26 H 32 Cl3N3O6PtNa [M + Na] + : 805.0902; found: 805.0894. Purity: 98.93%(by HPLC). Example 4: In vitro antiproliferative activity assay

[0031] 1. Experimental Methods The cytotoxicity of compounds 6a-6f (conjugated from small molecule TrxR inhibitors coupled with platinum (IV) complexes) and their corresponding ligands 4a, 4b, 4c, 4d, 4e, and 4f against three types of TNBC cells (MDA-MB-231, MDA-MB-231 / CDDP, MCF-7) and mouse breast cancer cells 4T1 was detected using the CCK-8 assay kit. First, the antiproliferative activity of the small molecule TrxR inhibitors coupled with platinum (IV) complexes 6a-6f is shown in Table 1, with cisplatin, the small molecule TrxR inhibitors (3a), 3b, and (3c) serving as positive controls. All cells were seeded in 96-well plates (2000 cells per well, 100 μL) and incubated overnight at 37 °C under humid conditions containing 5% CO2. The cells were then treated with different concentrations of the test compounds under the same conditions for 72 hours. After 72 hours of incubation, add 10 μL of fresh CCK-8 solution and incubate at 37 °C for 2 hours. Record the sample readings at 450 nm using a microplate reader and calculate the IC50 using SPSS software. 50 value.

[0032] Table 1. Antiproliferative activity of compounds 6a-6f against different cancer cell lines.

[0033] IC 50 mean ± SD (μM) a Half-inhibitory concentration, expressed as mean ± standard deviation for each compound in three parallel experiments. Mixture b Cisplatin and 4b in equimolar combination therapy. CDDP c Cisplatin. FI d The ratio of the half-maximal inhibitory concentration (WMC) of cisplatin to the WMC of compound 6b was increased. (FI) e The ratio of the half-maximal inhibitory concentration (WMC) of the mixture to the WMC of compound 6b is increased. RF f Drug resistance factors.

[0034] 2. Experimental Results In vitro experimental results showed that the antiproliferative activity of target compounds 6a-6f was superior to that of their respective positive control drugs. In MDA-MB-231 cell experiments, compound 6b (IC50) showed the best antiproliferative activity. 50 = 0.34 μM) has a greater in vitro antiproliferative effect than complex 6a (IC50). 50Compound 6a (0.59 μM) was 1.74 times stronger than 4b alone, CDDP, and CDDP / 4b in combination (molar ratio 1:1), and was 9.24 times, 13.79 times, and 3.44 times stronger, respectively. Interestingly, compounds 6c, 6d, 6e, and 6f also showed similar effects. Interestingly, similar consistent trends were observed with compounds 6a-6f in MCF-7 and 4T1 cell experiments. Furthermore, compound 6b also exhibited good anti-proliferative activity against cisplatin-resistant MDA-MB-231 / CDDP cells, with an IC50 concentration of [missing value]. 50 The resistance factor was 0.40 μM, with a resistance factor of only 1.18, compared to 8.02 for cisplatin, indicating significant resistance. Notably, the IC50 values ​​for Pt(IV) complexes 6a-6b, 6c-6d, and 6e-6f were significantly different. 50 The difference in values ​​can be attributed to the presence of axial ligands in the Pt(IV) complex. This suggests that incorporating longer carbon chains into the linker may contribute to enhanced anticancer activity of the Pt(IV) complex. The above experimental results indicate that the platinum(IV) prodrug 6b, constructed by introducing a small molecule TrxR inhibitor into the axial position of the platinum(IV) complex, effectively overcomes cisplatin resistance, improves efficacy, and reduces toxicity. Therefore, compound 6b is a potential candidate drug for the treatment of TNBC.

[0035] Example 5: In vivo antitumor activity experiment 1. Model Establishment All animal experiments in this invention were approved by the Institutional Animal Care and Use Committee of Guangxi Normal University and conducted in accordance with its guidelines; the approved animal care and ethics review certificate number is 202506-004. Twenty-five 4-week-old female mice were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd., and housed under pathogen-free conditions. Logarithmically growing 4T1 breast cancer cells were subcutaneously inoculated into the right side of BALB / c mice to establish a 4T1 cell allogeneic xenograft model. When the tumor volume reached approximately 150 mm³, all mice were randomly divided into five experimental groups (n=5 per group). Group 1 was the control group; Group 2 received cisplatin (2.5 mg / kg); Group 3 received 4b (30 mg / kg); Group 4 received a combination of CDDP and 4b (2.5 + 30 mg / kg); and Group 5 received 6b (7.2 mg / kg). Administered intravenously every two days for three weeks. The control group received saline under the same conditions, and tumor volume and body weight were monitored every two days.

[0036] 2. Experimental Results like Figure 1As shown in AC, the 4T1 tumor model treated with 6b exhibited superior anti-tumor effects, with a tumor growth inhibition rate (TGI) of 76.8%, higher than the cisplatin treatment group (45.6%), the 4b treatment group (49.7%), and the cisplatin and 4b combined treatment group (60.7%). In the later treatment phase, compared with the control group, the mice in the 6b and 4b combined treatment groups did not show a significant decrease in body weight; conversely, the mice in the cisplatin and combined treatment groups showed a significant decrease in body weight. Figure 1 D). In addition, such as Figure 2 The ELISA results in A showed that in the 6b treatment group, the levels of pro-inflammatory cytokines (such as TNF-α, IFN-γ, and IL-6) were effectively increased, and this had a greater impact on the tested cytokines compared to other treatments. Figure 2 As shown in B, CD4 + and CD8 + T cell levels were significantly higher in the 6b treatment group than in other treatment groups. Notably, compared to other groups, the 6b treatment group showed a significant increase in the immunogenic cell death (ICD)-related marker CRT and the ERS-related marker CHOP. In summary, these results suggest that the antitumor activity of complex 6b may be related to the activation of ER stress-mediated immune responses. Example 6: In vivo antitumor activity experiment

[0037] 1. Model Establishment Twenty-five 4-5 week old female nude mice were purchased from Hangzhou Qizhen Experimental Animal Technology Co., Ltd. and housed under pathogen-free conditions. Logarithmically growing MDA-MB-231 cells were subcutaneously inoculated into the right side of BALB / c nude mice to establish an MDA-MB-231 xenograft model. When the tumor volume reached approximately 120 mm... 3 Mice were randomly divided into five experimental groups based on body weight and tumor size: Group 1 was the control group; Group 2 received cisplatin (2.5 mg / kg); Group 3 received 4b (30 mg / kg); Group 4 received a combination of CDDP and 4b (2.5 + 30 mg / kg); and Group 5 received 6b (7.2 mg / kg). Administered intravenously every two days for three weeks. Tumor volume and body weight changes were monitored every two days, and tumor inhibition rates were calculated based on the treatment results of each compound. The MDA-MB-231 / CDDP xenograft model was constructed using the same method as the MDA-MB-231 model.

[0038] 2. Experimental Results like Figure 3As shown in AD, in vivo antitumor activity experiments showed that the tumor growth inhibition (TGI) rate in the 6b treatment group reached 73.8%, which was superior to 55.4% in the cisplatin treatment group, 51.2% in the 4b treatment group, and 62.9% in the cisplatin and 4b combination treatment group, indicating that the complex 6b can more effectively inhibit the growth of MDA-MB-231 xenograft tumors.

[0039] like Figure 3 As shown in EF, in the MDA-MB-231 / CDDP xenograft model, the cisplatin treatment group exhibited significantly lower activity due to severe drug resistance, with an inhibition rate of 28.7%. In contrast, the tumor growth inhibition rates in the 4b treatment group and the cisplatin + 4b combination therapy group were 42.6% and 52.1%, respectively. The experiment observed that complex 6b effectively inhibited the growth of MDA-MB-231 / CDDP xenografts, with a tumor growth inhibition rate of 66.3%, significantly higher than other treatment groups. Figure 3 CG). Furthermore, no significant weight loss was observed in the 6b and 4b treatment groups, while significant weight loss was observed in both the cisplatin treatment group and the combination therapy group in the later stages of the MDA-MB-231 and MDA-MB-231 / CDDP models. Figure 3 D and H). Crucially, immunohistochemical analysis of tumor tissue showed that complex 6b treatment significantly downregulated the expression of Ki-67, TrxR1, and GPX4. Figure 3 I and J). Further hematoxylin-eosin (H&E) staining of major organs such as the heart, liver, spleen, kidneys, and lungs of nude mice showed that no morphological changes or adverse reactions occurred after treatment with complex 6b. Figure 4 ).

[0040] The above description of the embodiments is only for illustrating the technical concept and features of the present invention. Its purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. Those skilled in the art can obviously easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative effort. Therefore, the above embodiments should not be used to limit the scope of protection of the present invention. All improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A platinum (IV) prodrug using a small molecule TrxR inhibitor as a ligand, characterized in that, The chemical structure is shown in the following formula: ； In the formula, n = 1 or 2; R1 = -CF3, -OCF3, or -OCH3.

2. The platinum (IV) prodrug with a small molecule TrxR inhibitor as a ligand according to claim 1, characterized in that: The chemical structures are shown in formulas (6a)-(6f): 。 3. The method for preparing a platinum (IV) prodrug using a small molecule TrxR inhibitor as a ligand as described in claim 1, characterized in that, Includes the following steps: (1) Compound 1 and compound 2 were reacted under the conditions of concentrated hydrochloric acid and glacial acetic acid to synthesize compound 3; (2) React compound 3 with succinic anhydride or glutaric anhydride under organic base conditions to synthesize compound 4; (3) Compound 4 was reacted with platinum (IV) complex 5 under condensing agent and organic base conditions to synthesize compound 6; The synthesis route is shown below: ； In the formula, n = 1 or 2; R1 = -CF3, -OCF3, or -OCH3.

4. The method for preparing a platinum (IV) prodrug using a small molecule TrxR inhibitor as a ligand according to claim 3, characterized in that: In step (1), the molar ratio of compound 1 to compound 2 is 2~3:1, and the volume ratio of concentrated hydrochloric acid to glacial acetic acid is 1:8~12; And / or, the reaction is carried out at room temperature for 40-50 hours.

5. The method for preparing a platinum (IV) prodrug using a small molecule TrxR inhibitor as a ligand according to claim 3, characterized in that: In step (2), the organic base is triethylamine, DMF is used as solvent, and the molar ratio of compound 3 to succinic anhydride or glutaric anhydride and the organic base is 1:2~4:1~2; And / or, the reaction temperature is 40~60℃ and the time is 8~12h.

6. The method for preparing a platinum (IV) prodrug using a small molecule TrxR inhibitor as a ligand according to claim 3, characterized in that: In step (3), the condensing agent is TBTU, the organic base is triethylamine, DMF is used as solvent, and the molar ratio of compound 4 to platinum (IV) complex 5, condensing agent and organic base is 1:0.8~1.2:1~2:1~2; And / or, the reaction temperature is 20~40℃ and the time is 8~12h.

7. The method for preparing a platinum (IV) prodrug using a small molecule TrxR inhibitor as a ligand according to claim 3, characterized in that: Steps (1) to (3) also include the separation and purification process.

8. The use of a platinum (IV) prodrug with a small molecule TrxR inhibitor as a ligand as described in claim 1 in the preparation of an anti-breast cancer drug.

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