Pt (IV) complex containing nitrogen donor, preparation method of Pt (IV) complex and application of Pt (IV) complex in preparation of antitumor drugs

Abstract

The invention discloses a Pt (IV) complex containing a nitrogen donor, a preparation method of the Pt (IV) complex and application of the Pt (IV) complex in preparation of antitumor drugs, and belongs to the field of medicines. A Pt (IV) metal anticancer agent based on an N-containing donor (leflunomide) as an axial ligand for regulating and controlling the drug resistance of the liver cancer by constructing a LOXL3-DHODH axis signal channel is a complex 1 with a chemical structural formula shown as a formula I or a complex 2 with a chemical structural formula shown as a formula II; the complex 1 and the complex 2 have good cytotoxic activity on a cisplatin-resistant hepatoma cell line at a cellular level, and have lower toxicity on normal hepatoma cells than cisplatin. Therefore, the problems of toxicity and drug resistance of traditional platinum drugs can be effectively solved, and the anti-cancer effect of platinum-based drugs is improved. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly to a Pt(IV) complex containing a nitrogen donor, a preparation method thereof, and its use in the preparation of anti-tumor drugs. Background Art

[0002] Liver cancer is one of the main causes of cancer-related deaths globally. Platinum-based chemotherapy resistance is a key factor in the poor prognosis and recurrence of liver cancer, and alleviating or overcoming platinum drug resistance is an urgent problem to be solved clinically. Alterations in drug metabolism, DNA damage repair, and apoptotic protein dysregulation are important pathways for cisplatin resistance. Therefore, using safe and effective platinum-based chemotherapy reagents or identifying targets that make liver cancer cells sensitive to chemotherapy to provide new treatment approaches is an effective strategy to address liver cancer drug resistance and reduce side effects. Lysyl oxidase-like 3 (LOXL3) plays an important role in cancer and can confer chemotherapy resistance through a series of mechanisms. Currently, in multi-target and Pt(IV) prodrugs based on Pt(II) drugs, the selection of bioactive axial ligands is limited to O donors, and their preparation is mainly achieved by adding hydroxyl groups at their axial positions after oxidation with hydrogen peroxide, which limits the application of some bioactive non-O donor-based small biomolecules in the preparation of Pt(IV) prodrugs for cancer treatment. By constructing a Pt(IV) metal anti-cancer agent based on the LOXL3-DHODH axis signaling pathway and regulating liver cancer drug resistance, with a nitrogen donor (leflunomide) as the axial ligand, it is expected to enhance the DNA damage effect through the synergistic mechanism of dihydroorotate dehydrogenase inhibition and DNA damage, and promote ferroptosis and other ways to increase chemotherapy sensitivity and reverse tumor drug resistance, thereby effectively inhibiting the progression of liver cancer. Summary of the Invention

[0003] The object of the present invention is to provide a Pt(IV) complex containing a nitrogen donor, a preparation method thereof, and its use in the preparation of anti-tumor drugs to solve the problems existing in the above-mentioned prior art. The preparation method of this Pt(IV) complex has a simple process, convenient operation, and good product stability; it has good cytotoxic activity against cisplatin-resistant liver cancer cell lines at the cellular level and lower toxicity to normal liver cancer cells than cisplatin. Therefore, the present invention can effectively solve the toxicity and drug resistance problems of traditional platinum drugs and improve the anti-cancer effect of platinum-based drugs.

[0004] To achieve the above object, the present invention provides the following solution:

[0005] The present invention provides a Pt(IV) complex containing a nitrogen donor, which comprises Complex 1 shown in Formula I or Complex 2 shown in Formula II of the following chemical structural formula:

[0006]

[0007] Wherein, Any of the following structures:

[0008]

[0009] The present invention also provides a method for preparing the complex 1 as described above, comprising the following steps:

[0010] S1. Dissolve the divalent platinum complex and N-chlorosuccinimide separately in water, then mix them, and stir at room temperature in the dark to obtain the intermediate product I;

[0011] Among them, the divalent platinum complex is any of the following structures:

[0012]

[0013] S2. Dissolve 5-methyl-N-[4-(trifluoromethyl)phenyl]-4-isoxazolecarboxamide (leflunomide) and anhydrous potassium carbonate in N,N-dimethylformamide (DMF), stir at room temperature, remove impurities, then add the intermediate product I, and continue stirring for reaction;

[0014] S3. After the reaction is completed, add dichloromethane, mix and let stand, then rotary evaporate until viscous, extract with dichloromethane, collect the precipitate, wash it, and dry it under vacuum to obtain the Pt(IV) complex.

[0015] The chemical formula of the above leflunomide is as follows:

[0016]

[0017] Preferably, the mass-volume ratio of the divalent platinum complex to water is 30 mg:(1 - 10) mL, and the mass-volume ratio of N-chlorosuccinimide to water is (0.01 - 0.05) g:(10 - 20) mL;

[0018] and / or the stirring time at room temperature in the dark is 2 - 6 h.

[0019] Preferably, after the reaction of the divalent platinum complex and N-chlorosuccinimide at room temperature is completed, it also includes the steps of removing impurities and drying under vacuum.

[0020] Preferably, the molar ratio of 5-methyl-N-[4-(trifluoromethyl)phenyl]-4-isoxazolecarboxamide (leflunomide) to anhydrous potassium carbonate is 1:1;

[0021] and / or the stirring time at room temperature is 2 - 4 h, and the intermediate product I is added to the product after removing impurities according to a molar ratio of 3:1, and continue stirring for reaction for 10 - 20 h.

[0022] The present invention also provides a method for preparing the complex 2 as described above, comprising the following steps:

[0023] Dissolve and mix the divalent platinum complex and N-chlorosuccinimide in absolute ethanol, heat under reflux, centrifuge the reaction mixture after cooling, dissolve the precipitate in N,N-dimethylformamide, drop it into dichloromethane, centrifuge after standing, wash with dichloromethane, and then take the precipitate and dry it to obtain the intermediate product II.

[0024] Among them, the divalent platinum complex is any one of the following structures:

[0025]

[0026] Dissolve 5-methyl-N-[4-(trifluoromethyl)phenyl]-4-isoxazolecarboxamide (leflunomide) and anhydrous potassium carbonate in N,N-dimethylformamide, stir at room temperature, remove impurities, add the intermediate product II, continue stirring and reacting. After the reaction is completed, add dichloromethane, mix and stand, rotary evaporate until viscous, extract with dichloromethane, collect the precipitate, wash and dry under vacuum to obtain the Pt(IV) complex.

[0027] Preferably, the ratio of the divalent platinum complex, N-chlorosuccinimide and absolute ethanol is (35 - 40) mg : (30 - 35) mg : (1 - 2) mL; and / or the conditions for heating under reflux are: heating in an oil bath for 4 - 8 h in a dark environment and condensing and refluxing at 80 °C.

[0028] Preferably, the molar ratio of 5-methyl-N-[4-(trifluoromethyl)phenyl]-4-isoxazolecarboxamide to anhydrous potassium carbonate is 1:1; and / or the stirring time at room temperature is 2 - 4 h. After removing impurities, add the intermediate product II according to a molar ratio of 3:1 and continue stirring and reacting for 10 - 20 h.

[0029] The Pt(IV) complex with nitrogen donors prepared by the present invention can target and inhibit the activity of dihydro-lactate dehydrogenase, induce DNA damage, reverse or improve the toxicity and drug resistance of traditional divalent platinum complexes in clinical use. The Pt(IV) complex of the present invention is not limited to Complex 1 and Complex 2 with the above chemical structures, and also includes the stereoisomers of Complex 1 and Complex 2, or the pharmaceutically acceptable salts of Complex 1 and Complex 2. These substances can be used as active ingredients or main active ingredients, supplemented with pharmaceutically acceptable excipients for the inhibition of dihydro-lactate dehydrogenase activity and anti-tumor preparations.

[0030] The present invention also provides the application of the Pt(IV) complex with nitrogen donors in any one of the following:

[0031] (1) Application in the preparation of anti-tumor drugs;

[0032] (2) Application in the preparation of drugs for alleviating the drug resistance of platinum-based anti-tumor drugs.

[0033] Preferably, the tumor includes liver cancer.

[0034] The present invention discloses the following technical effects:

[0035] (1) The Pt(IV) complex containing a nitrogen donor provided by the present invention, which is a tetravalent platinum complex, exhibits good proliferation inhibitory activity against tumor cells resistant to cisplatin at the cellular level, can induce obvious damage to the DNA of tumor cells, can promote the death of tumor cells by inducing apoptosis, and has strong potential for treating liver cancer.

[0036] (2) The preparation method of the present invention has simple steps, convenient operation, good operation safety, the prepared target tetravalent platinum complex product has good stability, high yield, and has significant functions of inhibiting the proliferation of tumor cells and reducing the toxicity to normal cells, and has a wide application prospect. Description of the Drawings

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0038] Figure 1 Chemical structural formulas of Complex 1 (A) prepared in Example 1 and Complex 2 (B) prepared in Example 2;

[0039] Figure 2 For the preparation of Complex 1 in Example 1 1 1H-NMR spectrum (DMSO-d6, 400 MHz);

[0040] Figure 3 For the preparation of Complex 1 in Example 1 13 13C{H}-NMR spectrum (DMSO-d6, 400 MHz);

[0041] Figure 4 For the preparation of Complex 2 in Example 2 1 1H-NMR spectrum (DMSO-d6, 400 MHz);

[0042] Figure 5 For the preparation of Complex 2 in Example 2 13 13C{H}-NMR spectrum (DMSO-d6, 400 MHz);

[0043] Figure 6 High-resolution mass spectrum of Complex 1 prepared in Example 1

[0044] Figure 7 High-resolution mass spectrum of complex 2 prepared for Example 2;

[0045] Figure 8 Effect of control group cells and complex 1 prepared in Example 1 on the cell cycle of cisplatin-resistant hepatocellular carcinoma cell line SMMC7721. Detailed implementation manners

[0046] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be construed as a limitation on the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0047] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0048] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0049] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the specification of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are also obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0050] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0051] Preparation of anti-tumor tetravalent platinum complex 1 (hereinafter referred to as complex 1) in Example 1:

[0052] S1. Weigh 300 mg of cisplatin and place it in a single-neck flask, then dissolve it with 10 mL of water. Subsequently, add 0.0136 g of NCS (N-chlorosuccinimide) dissolved in ultrapure water (16 mL) to the single-neck flask, and stir the mixture in the dark at room temperature for 4 h. Remove the solid residue by high-speed centrifugation, add anhydrous ethanol to the solid obtained by rotary evaporation of the supernatant, ultrasonically form a suspension, centrifuge the suspension, and retain the yellow solid precipitate. Finally, wash the yellow solid precipitate three times with anhydrous ethanol and obtain intermediate I after vacuum drying.

[0053] S2. Dissolve leflunomide (5-methyl-N-[4-(trifluoromethyl)phenyl]-4-isoxazolecarboxamide) (151.30 mg, 0.56 mM) in 1.5 mL of DMF (N,N-dimethylformamide), add 78 mg (0.56 mM) of anhydrous potassium carbonate thereto, stir the mixture at room temperature for 3 h, and centrifuge to filter out solid impurities. Then, add OCl2Pt (70.49 mg, 0.19 mM) in a ratio of 3:1, and stir the mixture at room temperature for 16 h.

[0054] S3. After the reaction is completed, add dichloromethane (20 mL) to the reaction supernatant, place it in the refrigerator and let it stand overnight, then rotary evaporate until it becomes viscous, add dichloromethane (30 mL) for extraction, and collect the precipitate. Wash it three times with dichloromethane and obtain complex 1 after vacuum drying.

[0055] 1 The 1H-NMR spectrum (DMSO-d6, 400 MHz) is as Figure 2 shown, and the data analysis is as follows: 1 1H NMR (400 MHz, DMSO-d6) δ 12.42 (s, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.54 (d, J = 8.5 Hz, 2H), 2.03 (s, 3H).

[0056] 13 The 13C{H}-NMR spectrum (DMSO-d6, 400 MHz) is as Figure 3 shown, and the data analysis is as follows: 13 13C NMR (126 MHz, DMSO) δ 191.34 (s), 188.97 (s), 167.08 (s), 165.95 (s), 144.68 (s), 126.66 - 126.17 (m), 125.05 (s), 118.89 (s), 118.48 (s), 27.59 (s).

[0057] The high-resolution mass spectrum is as Figure 6 shown.

[0058] The chemical structural formula of complex 1 can be determined by the above identification asFigure 1 as shown in A.

[0059] Preparation of antitumor tetravalent platinum complex 2 in Example 2 (hereinafter referred to as complex 2):

[0060] S1. Mix the weighed oxaliplatin (396 mg, 1 mM) and NCS (320 mg, 2.4 mM) in a single-necked flask, add anhydrous ethanol (15 mL) for dissolution, heat in an oil bath at 80 °C for 6 h under dark conditions with reflux condensation. After cooling the mixture, centrifuge it, dissolve the lower precipitate in DMF (1 mL), drop it into dichloromethane (30 mL), let it stand and then centrifuge, wash twice with dichloromethane, and take the lower precipitate, and dry it in vacuo to obtain intermediate product II.

[0061] S2. Dissolve leflunomide (75.65 mg, 0.28 mM) in DMF (1 mL), and add anhydrous potassium carbonate (39 mg, 0.28 mM) thereto. Stir the mixture at room temperature for 3 h. Then, centrifuge and filter the solid impurities. Add OXACl2Pt (42.14 mg, 0.09 mM) in a molar ratio of 3:1 and stir at room temperature for 16 h.

[0062] S3. After the reaction is completed, add dichloromethane (20 mL) to the reaction supernatant, place it in the refrigerator and let it stand overnight, then rotary evaporate until it becomes viscous, add dichloromethane (30 mL) for extraction, and collect the precipitate. Wash it three times with dichloromethane and dry it in vacuo to obtain complex 2.

[0063] 1 The 1H-NMR spectrum (DMSO-d6, 400 MHz) is as Figure 4 shown, and the data analysis is as follows: 1 1H NMR (400 MHz, DMSO-d6) δ 12.42 (s, 2H), 7.69 (d, J = 8.5 Hz, 4H), 7.54 (d, J = 8.5 Hz, 4H), 2.58 (s, 2H), 2.03 (s, 6H), 2.01 (s, 2H), 1.50 (t, J = 14.4 Hz, 4H), 1.14 - 1.03 (m, 2H).

[0064] 13 The 13C{H}-NMR spectrum (DMSO-d6, 400 MHz) is as Figure 5 shown, and the data analysis is as follows: 1313C NMR (126 MHz, DMSO) δ 188.98 (s), 167.08 (s), 163.56 (s), 144.69 (s), 126.32 (dd, J = 11.0, 7.2 Hz), 125.05 (s), 118.48 (s), 77.57 (s), 62.12 (d, J = 76.2 Hz), 27.77 (d, J = 46.3 Hz).

[0065] The high-resolution mass spectrum is as Figure 7 shown.

[0066] The chemical structural formula of the identified complex 2 can be determined as shown in Figure 1 B therein.

[0067] Example 3

[0068] In this example, the complexes 1 and 2 prepared in Example 1 and Example 2 of the present invention will be subjected to performance tests through specific experiments.

[0069] 1. Toxic activity tests of complexes 1 and 2 at the cellular level

[0070] Complex 1, Complex 2, OCl2Pt, OXACl2Pt and leflunomide were respectively dissolved in DMSO solution to prepare concentrated stock solutions, and cisplatin and oxaliplatin concentrated stock solutions were prepared in PBS.

[0071] The IC 50 values of each complex were determined using the MTT assay. SMMC7721, HepG2, HuH-7 and L-02 cells were respectively inoculated on 96-well plates and incubated overnight in a constant temperature and humidity incubator with 5% CO2 at 37°C. The old culture medium was removed, and Complex 1, Complex 2, cisplatin, oxaliplatin, OCl2Pt, OXACl2Pt, cisplatin + leflunomide, oxaliplatin + leflunomide, leflunomide, etc. diluted with cell culture medium according to a geometric gradient were respectively added to the wells of the 96-well plates. Three parallel wells were set for each concentration, and incubated for 72 h; the cell viability was tested using the thiazolyl blue (MTT) method; 20 μL of MTT solution (5 mg / mL) was added to each well and incubated at 37°C for 4 hours. The culture medium and MTT mixed solution were aspirated, and DMSO (150 μL) was added to each well to dissolve the purple formazan, and shaken horizontally for 10 min to make the solution evenly mixed. The OD value of each well at 570 nm was measured with an enzyme-linked immunosorbent assay reader, and the IC 50 value was calculated. The test results are shown in Table 1 below.

[0072] Table 1 IC 50 values (72 h) of Complex 1 and Complex 2 and the control drugs cisplatin and oxaliplatin against different cells

[0073]

[0074] Compared with Complex 2 prepared by the present invention, Complex 1 shows effective anti-proliferative activity against most of the tested cancer cells, especially strong selective inhibitory activity against hepatocellular carcinoma cell line SMMC7721; the inhibitory activity of Complex 2 against different tumor cells is lower than that of the cisplatin treatment group. The axial ligand leflunomide has little toxicity to the tested cells. The activity of the mixed solution of cisplatin and leflunomide is weaker than that of cisplatin, and the activity of the physical mixture of oxaliplatin and leflunomide is weaker than that of oxaliplatin. Although the activity of Complex 1 is slightly weaker than that of the control group cisplatin, its toxicity to normal hepatocellular carcinoma is weaker than that of the cisplatin treatment group, indicating its potential to reduce drug toxicity. According to the toxicity and activity screening data, the SMMC7721 cell line was selected for subsequent experiments.

[0075] 2. Effect of Complex 1 on cell cycle arrest of cisplatin-resistant hepatocellular carcinoma cell line SMMC7721

[0076] Operation steps: The cell cycle test was detected by flow cytometry using PI single staining method. SMMC7721 cells were seeded in 6-well culture plates and allowed to adhere and grow for 24 hours. After removing the original culture medium, the tumor cells were treated with the culture medium containing Complex 1, cisplatin, and leflunomide for 72 hours; the control treatment group was incubated with an equal volume of fresh culture medium. Then, 2 mL of complete culture medium containing Complex 1 (3 μM), leflunomide (50 μM), and cisplatin (1.5 μM) was added to each well and the cells were cultured continuously. After 24 hours, the cells were digested with trypsin and collected, washed with PBS (4 °C) buffer, and then fixed in 70% ethanol (4 °C) for 24 hours. After the fixation operation was completed, centrifugation was performed to collect the cell pellet. Rnase (100 μL) was added to the cell pellet to resuspend the cells, and the cells were incubated in a 37 °C water bath for 30 minutes. Subsequently, PI (500 μL) was added for staining, and the cells were incubated at 4 °C in the dark for another 30 minutes. The supernatant was removed, 500 μL of PBS was added, and the cell cycle was analyzed by flow cytometry. The specific test results are as Figure 5 shown.

[0077] As Figure 8It can be seen that after the SMMMC7721 cells were incubated in the culture medium containing the complex for 24 h, the percentages of cell cycle arrest in the S phase were 32.73% (Control), 82.85% (Complex 1, 3 μM), 49.51% (Leflunomide, 50 μM), and 59.07% (Cisplatin, 1.5 μM), respectively. Compared with the control group, cisplatin arrested the cell cycle in the S phase, which was consistent with the literature; the ligand leflunomide had no obvious inhibitory effect on the cell cycle, which was consistent with the data of the toxicity and activity experiments; Complex 1 had an obvious inhibitory effect in the S phase, changing from 32.73% to 82.85%, further indicating that Complex 1 could block DNA replication and thus inhibit cell proliferation. It shows that Complex 1 maintained the DNA damage effect of the platinum-based complex parent body.

[0078] The embodiments described above are only used to describe the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A Pt(IV) complex containing a nitrogen donor, characterized in that, It includes complex 1 with a chemical structural formula as shown in Formula I or complex 2 as shown in Formula II: Among them, is any one of the following structures:

2. A method for preparing the Pt(IV) complex as described in claim 1, characterized in that, It includes the following steps: Dissolve the divalent platinum complex and N-chlorosuccinimide in water respectively and then mix them, stir at room temperature in the dark to obtain Intermediate Product I; wherein, the divalent platinum complex is any one of the following structures: Dissolve 5-methyl-N-[4-(trifluoromethyl)phenyl]-4-isoxazolecarboxamide and anhydrous potassium carbonate in N,N-dimethylformamide, stir at room temperature, after impurity removal, add the Intermediate Product I, continue stirring and reacting, after the reaction ends, add dichloromethane, mix and let stand, then rotary evaporate until viscous, and extract with dichloromethane, collect the precipitate, wash and dry in vacuum to obtain the Pt(IV) complex.

3. The preparation method according to claim 2, characterized in that, The mass-volume ratio of cisplatin to water is 30 mg:(1-10) mL, and the mass-volume ratio of N-chlorosuccinimide to water is (0.01-0.05) g:(10-20) mL; and / or the stirring time at room temperature in the dark is 2-6 h.

4. The preparation method according to claim 2, characterized in that, After the reaction of the divalent platinum compound and N-chlorosuccinimide at room temperature ends, it also includes the steps of impurity removal and vacuum drying.

5. The preparation method according to claim 2, characterized in that, The molar ratio of 5-methyl-N-[4-(trifluoromethyl)phenyl]-4-isoxazolecarboxamide to anhydrous potassium carbonate is 1:1; and / or the stirring time at room temperature is 2-4 h, and the Intermediate Product I is added in a molar ratio of 3:1 to the product after impurity removal, and continue stirring and reacting for 10-20 h.

6. A method for preparing the Pt(IV) complex as described in claim 1, characterized in that, It includes the following steps: Dissolve the divalent platinum compound and N-chlorosuccinimide in absolute ethanol and mix them, heat under reflux, after the reaction mixture cools, centrifuge, dissolve the precipitate in N,N-dimethylformamide, drop it into dichloromethane, let stand and then centrifuge, wash with dichloromethane, and take the precipitate to dry to obtain Intermediate Product II; Dissolve 5-methyl-N-[4-(trifluoromethyl)phenyl]-4-isoxazolecarboxamide and anhydrous potassium carbonate in N,N-dimethylformamide, stir at room temperature, after impurity removal, add the Intermediate Product II, continue stirring and reacting, after the reaction ends, add dichloromethane, mix and let stand, then rotary evaporate until viscous, and extract with dichloromethane, collect the precipitate, wash and dry in vacuum to obtain the Pt(IV) complex.

7. The preparation method according to claim 6, characterized in that, The ratio of oxaliplatin, N-chlorosuccinimide and absolute ethanol is (35-40) mg:(30-35) mg:(1-2) mL; and / or the conditions for heating under reflux are: oil bath heating in the dark for 4-8 h, and condensation reflux at 80 °C.

8. The preparation method according to claim 6, characterized in that, The molar ratio of 5-methyl-N-[4-(trifluoromethyl)phenyl]-4-isoxazolecarboxamide to anhydrous potassium carbonate is 1:1; and / or the stirring time at room temperature is 2-4 h, after impurity removal, add the Intermediate Product II in a molar ratio of 3:1, and continue stirring and reacting for 10-20 h.

9. Application of the Pt(IV) complex with a nitrogen donor as described in claim 1 in any one of the following: (1) Application in the preparation of anti-tumor drugs; (2) Application in the preparation of drugs for alleviating the drug resistance of platinum-based anti-tumor drugs.

10. The application according to claim 9, wherein The tumor includes liver cancer.

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