Multi-target carboplatin complex as well as synthesis method and anticancer activity application thereof

Abstract

The invention discloses a multi-target carboplatin-platinum (IV) complex as well as a preparation method and application thereof, and belongs to the field of medicinal chemistry. The complex has two different bioactive ligands in the axial direction, carboplatin is used as a raw material, carboplatin is oxidized into oxyplatin in hydrogen peroxide, and then the oxyplatin, axial ligand NHS ester and anhydride are subjected to continuous two-time esterification to obtain a'multi-target 'platinum (IV) anticancer prodrug candidate. 1 / 1 NHS ester and platinum oxide are exchanged in DMSO in the first esterification, and 10 / 1 anhydride and a monosubstituted complex are exchanged in DMF in the second esterification; the reaction raw materials are easy to obtain, the synthesis steps are simple, and the reaction conditions are mild. The multi-target carboplatin complex shows good physiological activity on HeLa, C33A, SiHa and other cervical cancer cells, and is expected to be applied to potential anti-cancer drugs.

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and particularly relates to a synthesis method of multi-target Pt(IV) complexes and its anti-tumor application. Background Art

[0002] Cancer remains one of the main factors leading to human death. Radiotherapy and chemotherapy play a crucial role in cancer treatment, and nearly half of cancer patients use platinum drugs in chemotherapy. Platinum drugs mainly achieve anti-cancer effects by interfering with DNA replication, transcription, etc. to induce apoptosis of tumor cells. However, platinum drugs are prone to off-target, resulting in dose-limiting toxic side effects, and the problem of drug resistance also limits their widespread use. To improve the anti-cancer efficacy, platinum drugs are often combined with other drugs for chemotherapy clinically to overcome the drug resistance of single drugs. However, the respective pharmacokinetic properties of different drugs will complicate the treatment results. Existing studies have confirmed that the discovery of Pt(IV) prodrugs is expected to solve the problems of side effects and drug resistance of Pt(II) drugs. Pt(IV) prodrugs can be reduced and activated in cancer cells, releasing two axial ligands and a square planar Pt(II) drug, enabling the combination of the therapeutically active Pt(II) drug and its bioactive ligands to form an ideal combination for multi-action therapy. Although cancer cells are more sensitive to platinum drugs compared to healthy tissue cells, during the application of platinum drugs, only a small part of the drugs enter cancer cells, and a considerable part of the drugs will interact with DNA and other nucleophiles in normal cells, resulting in off-target. This off-target platinumation causes dose-limiting toxic side effects in the human body, and drug resistance severely restricts the widespread application of platinum drugs.

[0003] In the past few decades, it has been proven that inflammation is prone to cause cancer and promote all stages of tumorigenesis. Cyclooxygenase (COX) is a key enzyme in the biosynthesis of prostaglandins derived from arachidonic acid. COX-1 is expressed in almost all tissues and is responsible for regulating the basal level of prostaglandins in normal physiological processes. While COX-2 can be induced in inflamed and tumorigenic tissues and can mediate the overexpression of prostaglandin E2 (PGE2). PGE2 plays an important role in tumorigenesis, invasion, metastasis, and drug resistance. Widely used non-steroidal anti-inflammatory drugs such as diclofenac and naproxen can inhibit COX activity, thereby preventing the formation of prostaglandins and inflammatory effects.

[0004] Histones are proteins that control chromatin and nucleosome structure. Histone deacetylases (HDACs) cause DNA to wrap more tightly around histones through histone deacetylation, making this DNA less accessible to gene transcription factors. This inhibits the expression of proteins related to cell cycle arrest, tumor immunity, apoptosis of damaged cells, etc., all of which contribute to the development of cancer. Histone deacetylase inhibitors (HDACis) can selectively restore the expression of these cancer suppressors and other anti-cancer genes by controlling the tightness of DNA wrapping around histones. HDACis can also indirectly inhibit the expression of angiogenesis factors and help block the blood supply to tumors. 4-Phenylbutyric acid (PhB) is also a known HDACi that binds to coenzyme A (CoA) through a thiol adduct and inhibits histone deacetylase (HDAC), thereby affecting cell metabolism. Its synergistic effect with DNA-binding drugs has attracted attention as an anti-cancer compound.

[0005] To overcome these limitations and improve anti-cancer efficacy, clinicians use platinum-based drugs in combination with other drugs for chemotherapy. The main advantage of combination chemotherapy is that several anti-proliferative drugs with different mechanisms of action and different cellular targets attack the tumor, thus increasing the chance of killing cancer cells and overcoming resistance to single drugs. Currently, researchers are conducting (pre)clinical studies on the combination of several non-steroidal anti-inflammatory drugs such as aspirin and ibuprofen with chemotherapy drugs to understand their anti-tumor potential. However, each drug has different pharmacokinetic properties, and combined use can complicate the overall treatment outcome. Summary of the Invention

[0006] To overcome the above technical deficiencies, the object of the present invention is to provide the synthesis, characterization, chemical and biological properties of multi-target Pt(IV) complexes. The Pt(IV) complexes are prepared by coupling a non-steroidal drug such as ibuprofen, diclofenac or naproxen at an axial position and using a hydroxyl group to couple PhB at a second axial position to form multi-target prodrugs. A series of biological studies were then carried out to investigate their anti-proliferative effects on various different cancer cells (such as HeLa, SiHa and C33A) and the Pt accumulation in C33A cells. The research results show that the tri-target active group Pt(IV) complex based on carboplatin has greatly enhanced anti-tumor activity compared to carboplatin and exhibits better anti-tumor potential.

[0007] The multi-target Pt(IV) complex of the present invention has the following structural formula:

[0008]

[0009] Among them, the axial ligands of carboplatin, ibuprofen, diclofenac, and naproxen are all cyclooxygenase (COX) inhibitors; the axial ligand phenylbutyric acid is a histone deacetylase (HDAC) inhibitor.

[0010] The present invention also provides a method for synthesizing the above-mentioned multi-target Pt(IV) complex. The technical solution is to first convert carboplatin into a mono-coordinated dual-target tetravalent platinum prodrug, and then convert it into a multi-target tetravalent platinum prodrug compound.

[0011] The method for synthesizing the multi-target Pt(IV) complex includes the following steps: using carboplatin, naproxen / diclofenac / ibuprofen, and phenylbutyric acid as raw materials, and obtaining the multi-target carboplatin complex through Pt(II) oxidation, NHS protection, anhydride formation, and ester exchange.

[0012] The synthetic route is shown as follows:

[0013] (1) Pt(II) oxidation

[0014]

[0015] (2) NHS protection

[0016]

[0017] (3) Anhydride formation

[0018]

[0019]

[0020] (4) Ester exchange

[0021]

[0022] Further, in step 1) of the above technical solution, the concentration of hydrogen peroxide is 20-30%, and the reaction temperature is 60-80°C.

[0023] Further, in step 2) of the above technical solution, the molar ratio of diclofenac / phenylbutyric acid, NHS, and dicyclohexylcarbodiimide is 1:1:1.

[0024] Further, in step 3) of the above technical solution, the molar ratio of phenylbutyric acid / cinnamic acid / naproxen to dicyclohexylcarbodiimide is 1:0.5.

[0025] Further, in step 4) of the above technical solution, the molar ratio of intermediate D / F to oxoplatinum B is 1:1, and the reaction temperature is selected from 60-70°C; the molar ratio of intermediate N / O to anhydride G / M / K is 10:1, and the reaction temperature is selected from 75-90°C.

[0026] The present invention also provides the use of the foregoing multi-target carboplatin complex in the preparation of anti-tumor drugs.

[0027] Further, in the above technical solution, the anti-tumor effect is against HeLa, MCF-7, HepG2, HCT116, C33A, Siha, and A549 tumor cells; the complex has the best activity against cervical cancer cells HeLa, C33A, and Siha.

[0028] Advantages of the invention

[0029] 1. The present invention uses carboplatin, phenylbutyric acid, ibuprofen, diclofenac, and naproxen as raw materials, and combines different compounds targeting tumor targets to innovatively construct three new multi-target carboplatin complexes.

[0030] 2. The multi-target carboplatin complex of the present invention was tested for its activity against HeLa, MCF-7, HepG2, HCT116, C33A, Siha, and A549. Among them, the carboplatin complex showed significant cell activity against cervical cancer cells HeLa, C33A, and Siha.

[0031] 3. The multi-target carboplatin complex of the present invention was tested for the total cellular platinum accumulation in C33A. Among them, the multi-target carboplatin complex had a five-fold higher accumulation in C33A cancer cells compared to carboplatin, showing extremely strong cancer cell killing performance. Brief description of the drawings

[0032] Figure 1 It is the graph of the platinum accumulation in C33A cells for the multi-target carboplatin complex in the example; that is, the Pt content in whole cells after treating C33A cells (1×10 6 ) with three prodrugs under the same concentration (compound concentration: 5 μM). Specific examples

[0033] The present invention will be further described below through specific examples. These examples should be understood as only for illustrating the present invention and not for limiting the protection scope of the present invention. After reading the content recorded in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent changes and modifications also fall within the scope defined by the claims of the present invention.

[0034] In this reaction, carboplatin was used as the template substrate for modification. Different tumor target inhibitors were respectively spliced with carboplatin to find the best reaction conditions, and three prodrugs of multi-target carboplatin complexes with excellent activity were obtained.

[0035] Example 1

[0036] Experimental operation steps of Compound 1:

[0037] Slowly add 30% H2O2 (70 mL) solution dropwise to a round-bottom flask containing carboplatin a (1.00 g, 2.69 mmol) using a separatory funnel in the dark. Heat the mixture to 50 °C and stir the reaction for 1 h in the dark. Filter off the formed white precipitate, and vacuum dry to obtain a white solid. Wash it successively with cold water, absolute ethanol, and anhydrous ether to obtain 0.92 g of white solid oxoplatinum b.

[0038] Weigh diclofenac c (2.96 g, 10 mmol) and NHS (1.15 g, 10 mmol) and dissolve them in 50 mL of CHCl3. Add DCC (2.06 g, 10 mmol) while stirring, and then stir the reaction at room temperature for 7 h. After the reaction is completed, filter the solution to remove the reaction by-product dicyclohexylurea. Concentrate the filtrate under vacuum to obtain a white solid, and then purify the solid by column chromatography to obtain 1.86 g of NHS-diclofenac ester d.

[0039] Weigh phenylbutyric acid e (3.28 g, 20 mmol) and dissolve it in 50 mL of CH2Cl2. Add DCC (1.15 g, 10 mmol) while stirring, and then stir the reaction at room temperature for 7 h. After the reaction is completed, filter the solution to remove the reaction by-product dicyclohexylurea. Then concentrate the filtrate, dissolve the remaining solid in ethyl acetate, and place it in a -20 °C refrigerator overnight. Filter it the next day to remove the insoluble matter. Concentrate the filtrate under vacuum to obtain a white solid, and then purify the solid by column chromatography to obtain 2.14 g of phenylbutyric anhydride g.

[0040] Weigh NHS-diclofenac ester d (196.6 mg, 0.5 mmol) and oxoplatinum b (202.5 mg, 0.5 mmol) and dissolve them in 10 mL of DMSO. Place the mixture in an oil bath at 65 °C and stir the reaction in the dark for 48 h. After the reaction is completed, filter it, and then rotary evaporate under reduced pressure using an oil pump to remove most of the solvent DMSO to obtain a yellow viscous solid. Then freeze-dry the remaining solid to remove the residual DMSO. Next, dissolve the treated residual solid and phenylbutyric anhydride g (1.55 g, 5 mmol) in 10 mL of DMF. Place the mixture in an oil bath at 80 °C and stir the reaction in the dark for 48 h. After the reaction is completed, rotary evaporate under reduced pressure using an oil pump to remove most of the DMF, and then freeze-dry the remaining solid to remove the residual DMF. Finally, purify it by column chromatography to obtain 49 mg of product 1.

[0041] Experimental procedure for compound 2:

[0042] Slowly add 30% H2O2 (70 mL) solution dropwise to a round-bottom flask containing carboplatin a (1.00 g, 2.69 mmol) using a separatory funnel in the dark. Heat the mixture to 50 °C and stir the reaction for 1 h in the dark. Filter off the formed white precipitate, vacuum dry to obtain a white solid, and wash successively with cold water, absolute ethanol, and anhydrous diethyl ether to obtain 0.92 g of white solid platinum oxide b.

[0043] Weigh phenylbutyric acid e (1.64 g, 10 mmol) and NHS (1.15 g, 10 mmol) and dissolve them in 50 mL of CHCl3. While stirring, add DCC (2.06 g, 10 mmol), and then stir the reaction at room temperature for 7 h. After the reaction is completed, filter the solution to remove the reaction by-product dicyclohexylurea, vacuum concentrate the filtrate to obtain a white solid, and then purify the solid by column chromatography to obtain 1.25 g of NHS-phenylbutyrate f.

[0044] Weigh ibuprofen l (3.31 g, 20 mmol) and dissolve it in 50 mL of CH2Cl2. While stirring, add DCC (1.15 g, 10 mmol), and then stir the reaction at room temperature for 7 h. After the reaction is completed, filter the solution to remove the reaction by-product dicyclohexylurea, then concentrate the filtrate, dissolve the remaining solid in ethyl acetate, and place it in a -20 °C refrigerator overnight. Filter it the next day to remove the insoluble matter, vacuum concentrate the filtrate to obtain a white solid, and then purify the solid by column chromatography to obtain 2.42 g of ibuprofen anhydride m.

[0045] Weigh NHS-phenylbutyrate f (130.6 mg, 0.5 mmol) and platinum oxide b (202.5 mg, 0.5 mmol) and dissolve them in 10 mL of DMSO. Place the mixture in an oil bath at 65 °C and stir the reaction in the dark for 48 h. After the reaction is completed, filter, and then rotary evaporate under reduced pressure using an oil pump to remove most of the solvent DMSO to obtain a yellow viscous solid. Then freeze-dry the remaining solid to remove the residual DMSO. Next, dissolve the treated residual solid and ibuprofen anhydride m (1.56 g, 5 mmol) in 10 mL of DMF. Place the mixture in an oil bath at 80 °C and stir the reaction in the dark for 48 h. At this time, the reaction solution is a clear and transparent light yellow solution. After the reaction is completed, rotary evaporate under reduced pressure using an oil pump to remove most of the DMF, then freeze-dry the remaining solid to remove the residual DMF, and finally purify by column chromatography to obtain 30 mg of product 2.

[0046] Experimental procedure for compound 3:

[0047] Slowly add 30% H2O2 (70 mL) solution dropwise to a round-bottom flask containing carboplatin a (1.00 g, 2.69 mmol) using a separatory funnel under light protection. Heat the mixture to 50 °C and stir the reaction for 1 h under light protection. Filter off the formed white precipitate, vacuum dry to obtain a white solid, and wash it successively with cold water, absolute ethanol, and anhydrous diethyl ether to obtain 0.92 g of white solid oxoplatinum b.

[0048] Weigh phenylbutyric acid e (1.64 g, 10 mmol) and NHS (1.15 g, 10 mmol), dissolve them in 50 mL of CHCl3, add DCC (2.06 g, 10 mmol) while stirring, and then stir the reaction at room temperature for 7 h. After the reaction is completed, filter the solution to remove the reaction by-product dicyclohexylurea, vacuum concentrate the filtrate to obtain a white solid, and then purify the solid by column chromatography to obtain 1.25 g of NHS-phenylbutyrate f.

[0049] Weigh naproxen j (4.61 g, 20 mmol), dissolve it in 50 mL of CH2Cl2, add DCC (1.15 g, 10 mmol) while stirring, and then stir the reaction at room temperature for 7 h. After the reaction is completed, filter the solution to remove the reaction by-product dicyclohexylurea, then concentrate the filtrate, dissolve the remaining solid in ethyl acetate, and place it in a -20 °C refrigerator overnight. Filter it the next day to remove the insoluble matter, vacuum concentrate the filtrate to obtain a white solid, and then purify the solid by column chromatography to obtain 2.96 g of naproxen anhydride k.

[0050] Weigh NHS-phenylbutyrate f (130.6 mg, 0.5 mmol) and oxoplatinum b (202.5 mg, 0.5 mmol), dissolve them in 10 mL of DMSO, place the mixture in an oil bath at 65 °C and stir the reaction under light protection for 48 h. After the reaction is completed, filter it, then use a rotary evaporator under reduced pressure with an oil pump to remove a large amount of DMSO to obtain a yellow viscous solid. Then freeze-dry the remaining solid to remove the residual DMSO. Next, dissolve the treated residual solid and naproxen anhydride k (2.21 g, 5 mmol) in 10 mL of DMF, place the mixture in an oil bath at 80 °C and stir the reaction under light protection for 48 h. After the reaction is completed, use a rotary evaporator under reduced pressure with an oil pump to remove a large amount of DMF, then freeze-dry the remaining solid to remove the residual DMF. Add the residual solid to diethyl ether, mix well, centrifuge at 8000 r for 5 min, then separate the yellow precipitate, wash it several times with a small amount of dichloromethane and anhydrous diethyl ether, air dry and store to obtain 80 mg of product 3.

[0051] Multi-target carboplatin complex 1 (49 mg); yield 12%; 11H NMR (400 MHz, DMSO-d6) δ 7.50 (d, J = 8.0 Hz, 2H), 7.34 - 7.10 (m, 8H), 7.04 - 7.00 (m, 1H), 6.36 (br, 6H), 5.76 (s, 1H), 3.75 (s, 2H), 2.51 - 2.48 (m, 6H), 2.26 - 2.21 (m, 2H), 1.82 - 1.72 (m, 4H); 13 13C NMR (100 MHz, DMSO-d6) δ 179.5, 177.9, 176.30, 143.1, 141.8, 137.3, 131.0, 130.2, 129.0, 128.3, 128.2, 127.1, 125.6, 120.3, 115.6, 67.0, 59.8, 55.6, 54.9, 34.5, 31.1, 27.1, 25.1, 15.8, 14.1; 195 195Pt NMR (86 MHz, DMSO-d6) δ 1946.5; HRMS (ESI) m / z: [M + Na] + Calcd for C 30 H 33 Cl2N3O8Pt 852.1175; Found 852.1176

[0052] The product was characterized as follows:

[0053] Multitarget carboplatin complex 2 (30 mg); Yield 8%; 1 1H NMR (400 MHz, DMSO-d6) δ 7.24 - 7.28 (m, 2H), 7.14 - 7.19 (m, 5H), 7.01 - 7.04 (m, 2H), 6.44 (br, 6H), 2.51 - 2.54 (m, 4H), 2.38 (d, J = 7.2 Hz, 2H), 2.21 (t, J = 7.6 Hz, 3H), 1.65 - 1.83 (m, 4H), 1.24 - 1.30 (m, 6H), 0.85 (d, J = 6.8 Hz, 6H); 13 13C NMR (100 MHz, DMSO-d6) δ 181.3, 179.7, 176.4, 176.3, 141.9, 139.4, 139.1, 129.7, 128.7, 128.4, 127.3, 125.7, 55.5, 46.0, 44.3, 34.7, 33.3, 31.8, 30.6, 27.2, 22.2, 20.0, 15.7; 195 195Pt NMR (86 MHz, DMSO-d6) δ 1961.7; HRMS (ESI) m / z: [M + H] +Calcd for C 29 H 40 N2O8Pt 740.2508;Found 740.2508

[0054] Multitarget carboplatin complex 3 (80 mg); Yield 21%; 1 H NMR (400 MHz, DMSO-d6) δ 7.72 (d, J = 9.2 Hz, 1H), 7.63 - 7.68 (m, 2H), 7.37 (dd, J = 1.6, 8.4 Hz, 1H), 7.23 (t, J = 7.6 Hz, 3H), 7.08 - 7.15 (m, 4H), 6.38 (br, 6H), 3.83 (t, J = 4.4 Hz, 3H), 2.18 - 2.54 (m, 9H), 1.61 - 1.74 (m, 4H), 1.47 (d, J = 6.8 Hz, 3H); 13 C NMR (100 MHz, DMSO-d6) δ 181.2, 179.7, 176.3, 157.0, 141.9, 137.3, 133.1, 129.1, 128.4, 128.3, 128.2, 126.9, 126.5, 125.7, 125.6, 118.4, 105.7, 55.6, 55.2, 46.3, 34.7, 34.5, 31.6, 31.0, 27.2, 19.8, 15.6; 195 Pt NMR (86 MHz, DMSO-d6) δ 1958.1; HRMS (ESI) m / z: [M + H] + Calcd for C 30 H 36 N2O9Pt 764.2144; Found 764.2145

[0055] Example 2

[0056] This example shows the results of the in vitro anti - proliferation experiment (half - inhibitory concentration) of the compound described in Example 1 against various cancer cells and the detection of platinum accumulation in cells. The materials and consumables listed in this example can be obtained from commercial sources unless otherwise specified. The cells are from the cell and microorganism resource library of CTCC or other relevant institutions. The experimental methods in this example are standard operating procedures in molecular biology, cell biology, virology, etc., which can be easily understood and operated by researchers in the field. The specific steps are as follows:

[0057] 1. Cells:

[0058] Cell lines used: human liver cancer cell line HepG-2, human breast cancer cell line MCF-7, human colon cancer cell line HCT116, human lung cancer cell line A549, human cervical cancer cell lines HeLa, C33A and Siha. Among them: the cell lines of HeLa, C33A, Siha, HepG-2, MCF-7, A549, and HCT116 were all cultured in DMEM complete medium. The above cell lines were all cultured in a cell incubator at 37 °C and 5% CO2 saturated humidity.

[0059] 2. Main reagents:

[0060] DMEM medium; fetal bovine serum FBS

[0061] 3. Steps for in vitro anti-proliferation experiment of tumor cells:

[0062] Step 1 Seeding: Observe the cell state after passage. When it is in the logarithmic growth phase, digest, centrifuge, and pipette evenly, count with a cell counter, dilute the cell suspension to 3 - 8×10 3 cells / 100 μL, and inoculate into a 96-well plate, 100 μL per well.

[0063] Step 2 Culturing cells: Place the 96-well plate with inoculated cells in a cell incubator at 37 °C and 5% CO2 saturated humidity for 12 - 24 h. After the cells are completely adhered, perform drug treatment.

[0064] Step 3 Drug administration: Under light-proof conditions in a laminar flow hood, dilute the stock solution of the compound to be measured above with blank culture medium to a 10-fold drug solution with corresponding concentration gradients; carefully aspirate the original culture medium, add 180 μL of fresh complete medium; add 20 μL of the drug solution to the corresponding wells of the 96-well plate, that is, dilute 10-fold to reach the corresponding drug concentration gradients, and the DMSO concentration is less than 0.1%. Set 5 replicates for each concentration, and set a blank control group at the same time. Gently shake to mix well, mark it, and place it in a cell incubator at 37 °C and 5% CO2 saturated humidity for 48 h.

[0065] Step 4 Color development: After 48 h, aspirate the culture medium from the 96-well plate, add 100 μL of fresh complete medium and 10 μL of CCK8 (5 mg / mL) solution, gently shake in parallel to mix well, and place it in a cell incubator at 37 °C and 5% CO2 saturated humidity for continued culture for 1 - 5 h.

[0066] Step 5 Measurement: Finally, use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance value (OD value) at λ = 450 nm; use GraphPad Prism 8.01 software to estimate the IC 50 value from the concentration-response curve, and use curve fitting for non-linear regression analysis.

[0067] 4. Experimental Procedures for Determining Pt Content in Whole Cells

[0068] Step 1: Seeding the cells: Digest and centrifuge the MCF-7 cells in the logarithmic growth phase, collect them in a centrifuge tube and count. Adjust the cell suspension concentration to 1×10 6 cells / 2 mL. Inoculate the cells into a 6-well plate, 2 mL per well, that is, 1×10 6 cells / well. Place them in a cell incubator at 37°C and 5% CO2 with saturated humidity, and culture the cells overnight.

[0069] Step 2: Administering the drug: After the cells are completely adherent, administer the drug. Dilute the stock solution of the compound to be measured to 5 μM with the culture medium, and administer the drug to the cells. Set up a carboplatin control group and a drug group, with 3 replicate wells in each group. Place them in a cell incubator at 37°C and 5% CO2 with saturated humidity, and culture the cells overnight.

[0070] Step 3: Collection: Aspirate the culture medium, wash the 6-well plate 3 times with cold PBS, add 500 μL of 0.25% trypsin to digest until the cells become round and the edges become clear. Aspirate the trypsin, add 2 mL of complete culture medium, and gently pipette until the cells detach. Transfer the cells to a centrifuge tube, centrifuge at 2000 r for 5 min, discard the supernatant, wash 2 more times with PBS, add 1 mL of PBS, gently pipette to mix evenly and count, centrifuge at 12000 r for 1 min, collect the cells and transfer them to an ep tube for lyophilization. Then seal and store at -80°C in the refrigerator for later use.

[0071] Step 4: Determination: After taking out the lyophilized cells, accurately measure 200 μL of concentrated nitric acid with a pipette gun for overnight digestion, add 1 mL of ultrapure water for dilution, and use ICP-MS to determine the Pt content.

[0072] 5. Detection Results:

[0073] The results of the in vitro anti-proliferative activities of the three prodrugs against tumor cells at gradient concentrations are as follows;

[0074]

[0075] The anti-proliferative activities of the three prodrugs against three cervical cancer cells are as follows:

[0076]

[0077] After treating C33A cells (1×10 6 ) with the three prodrugs at the same concentration (compound concentration: 5 μM), the Pt content in whole cells ( Figure 1 ).

[0078] 6. Conclusion:

[0079] 1. The most obvious conclusion from the cytotoxicity data is that complexes 1 - 3 all exhibited certain anti - cancer activities, and most of them showed a certain improvement compared to carboplatin itself.

[0080] 2. Regarding drug selectivity, we found that the synthesized Carboplatin multi - target Pt(IV) compounds had higher anti - tumor activities against cervical cancer cell lines compared to carboplatin. Especially, the improvement of compound 3 was the most significant. The IC 50 values of carboplatin against C33A, HeLa, and SiHa were 33.4 μM, 22.7 μM, and 79.6 μM respectively. While compound 3 showed high effectiveness against all three cervical cancer cell lines. Its IC 50 values against C33A, HeLa, and SiHa were 10.0 μM, 18.3 μM, and 20.6 μM respectively. Among the three cervical cancer cell lines, the inhibitory ability against C33A cells was the highest, more than three times that of carboplatin, and it also showed certain anti - tumor effects against HeLa and SiHa.

[0081] 3. Use ICP - MS technology to detect the intracellular Pt content in C33A cells treated with 5 μM of the compound for 24 h. The results are as Figure 1 shown. The Pt contents in whole cells treated with compounds 1 - 3 were 28.6 ng Pt / 10 6 cells, 23.0 ng Pt / 10 6 cells, and 32.1 ng Pt / 10 6 cells respectively, which were about 5 times that of the carboplatin treatment group at the same dose. Since cell uptake or efflux is the first step in its anti - tumor mechanism, the accumulation of small - molecule platinum - based chemotherapeutic drugs in tumor cells plays a key role in their anti - proliferative activities.

[0082] As mentioned above, the above is only the preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, should be covered within the protection scope of the present invention.

Claims

1. Three multi-target Pt(IV) complexes, characterized in that: The structural formula is as follows:

2. The multi-target Pt(IV) complex according to claim 1, characterized in that: The axial ligands of carboplatin, diclofenac, ibuprofen and naproxen, are all cyclooxygenase (COX) inhibitors; the axial ligand phenylbutyric acid is a histone deacetylase (HDAC) inhibitor.

3. The method for synthesizing a multi-target Pt(IV) complex according to claim 1 or 2, characterized in that: The method comprises the following steps: using carboplatin, naproxen / diclofenac / ibuprofen and phenylbutyric acid as raw materials, and obtaining a multi-target carboplatin complex through Pt (II) oxidation, NHS protection, anhydride formation and esterification exchange.

4. The method for synthesizing the multi-target carboplatin complex according to claim 3, characterized in that: The specific steps include: (1) Pt(II) oxidation (2) NHS protection (3) Anhydride (4) Esterification 5. The method for synthesizing the multi-target carboplatin complex according to claim 3, characterized in that: In step 1), the concentration of hydrogen peroxide is 20-30%, and the reaction temperature is 60-80°C.

6. The method for synthesizing the multi-target carboplatin complex according to claim 3, characterized in that: In step 2), the molar ratio of diclofenac / phenylbutyric acid, NHS and dicyclohexylcarbodiimide is 1:1:

1.

7. The method for synthesizing the multi-target carboplatin complex according to claim 3, characterized in that: In step 3), the molar ratio of phenylbutyric acid / cinnamic acid / naproxen to dicyclohexylcarbodiimide is 1:0.

5.

8. The method for synthesizing the multi-target carboplatin complex according to claim 3, characterized in that: In step 4), the molar ratio of the intermediate D / F to oxygen platinum B is 1:1, and the reaction temperature is selected from 60-70°C; the molar ratio of the intermediate N / O to the anhydride G / M / K is 10:1, and the reaction temperature is selected from 75-90°C.

9. Use of the multi-target carboplatin complex according to claim 1 or 2 in the preparation of anti-tumor drugs.

10. Use of the multi-target carboplatin complex according to claim 9 in the preparation of anti-carboplatin resistant tumor drugs, characterized in that: Anti-tumor agents include HeLa, MCF-7, HepG2, A549, HCT116, C33A, and SiHa tumor cells.

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