Tetravalent platinum complex for inhibiting cholesterol metabolism, preparation method and application thereof

Abstract

The present invention discloses a tetravalent platinum complex for inhibiting cholesterol metabolism, a preparation method and an application thereof, and belongs to the field of medical technology. The tetravalent platinum complex of the present invention is obtained by connecting and coordinating a cholesterol metabolism inhibitor with a platinum anticancer drug; the platinum anticancer drug includes a cisplatin derivative or an oxaliplatin derivative. The present invention achieves the anti-tumor purpose by introducing a cholesterol regulator into the structure of a platinum drug, and destroying the material and energy basis required for the growth and reproduction of tumor cells by regulating cholesterol metabolism. In the process of regulating cholesterol metabolism, the immune cells in the tumor microenvironment are affected, and the anti-tumor immune response is activated, thereby achieving the purpose of synergistic anti-tumor, while overcoming the drug resistance and systemic toxicity of traditional platinum drugs.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a tetravalent platinum complex for inhibiting cholesterol metabolism, a preparation method and an application thereof. Background Art

[0002] Cancer is the second leading cause of death worldwide, and drug resistance is a major obstacle to cancer treatment (Fojo A. Cancer 1987). Anticancer drugs, represented by cisplatin (CDDP), are the most commonly used chemotherapeutic agents for various malignancies (Huang, X.; Huang, R.; Gou, S. Bioconjugate Chem. 2016). However, acquired and intrinsic resistance pose a significant challenge to platinum-based anticancer therapy. CDDP resistance is due to multiple factors, including inefficient cellular uptake, DNA damage repair, and inactivation through interactions with sulfur-containing molecules such as glutathione (GSH) and thioredoxin (TrxR) (Wang, X.Y. et al. J. Med. Chem. 2019). To overcome platinum drug resistance, researchers have developed various tetravalent platinum complexes as prodrugs of CDDP or its analogs. These complexes are inert in blood and normal tissues but become active in tumor cells after reduction to the divalent platinum species and axial ligand. Due to its chemical inertness and fewer off-target reactions, it produces fewer side effects than CDDP (Raveendran, R. et al. Chem. Sci. 2016). However, the anti-tumor activity of most tetravalent platinum complexes originates from DNA damage. Therefore, it is necessary to develop new tetravalent platinum complexes with unique mechanisms of action to eliminate resistance to traditional platinum drugs.

[0003] Cholesterol, a key component of cell membranes, plays a crucial role in promoting tumorigenesis and suppressing tumor immune responses (Huang, CF; Freter, C. Int. J. Mol. Sci. 2015). Cholesterol metabolism maintains cholesterol homeostasis primarily through endogenous cholesterol synthesis and exogenous cholesterol uptake. Upregulated cholesterol synthesis and uptake in tumor cells, along with the abnormal accumulation of numerous metabolites, lead to enhanced cell proliferation, survival, invasion, metastasis, and adaptation to the tumor microenvironment, thereby conferring drug resistance, enabling tumor cells to evade apoptosis and continue to divide and proliferate (Gu, L. FEBS Journal 2019). Abnormal cholesterol metabolism in tumor cells not only contributes to drug resistance, but its derivatives can also modulate tumor immune recognition or immune escape, playing a key role in immune surveillance (Huang, CF; Freter, C. Int. J. Mol. Sci. 2015). Cholesterol may play a role in immune regulation by modulating T cell immune responses and anti-tumor immune responses, or by inducing immunosuppression in tumor cells themselves. Studies have found that apolipoprotein E (ApoE) can bind to LDLR on the surface of tumor cells, recruit MDSCs to inhibit CD8 + T cell infiltration of tumors. Cholesterol metabolism can affect the recruitment and activation of tumor-associated macrophages (TAMs), and plays an important regulatory role in the malignant progression of tumors. In addition, cholesterol and its metabolites have an important regulatory effect on the differentiation and function of dendritic cells (DCs). However, simple cholesterol inhibitors are generally lipid-lowering drugs, which have weak cytotoxicity or direct killing effects on tumor cells and cannot be used as effective anti-tumor preparations. At present, no one has tried to couple cholesterol inhibitors with platinum drugs as single-molecule chemical immunomodulators for the treatment of tumors. Therefore, introducing cholesterol inhibitors into platinum drug molecules, inducing cell death by inhibiting cholesterol metabolism and damaging DNA in tumor cells, and regulating the tumor microenvironment and anti-tumor immune response, and treating tumors through the synergistic effect of chemotherapy and immunotherapy is a new way to overcome the resistance of traditional platinum drugs. Summary of the Invention

[0004] To address the shortcomings of existing platinum-based anti-tumor drugs, the present invention provides a class of tetravalent platinum complexes that inhibit cholesterol metabolism, as well as their preparation methods and applications. By incorporating a cholesterol regulator into the platinum drug structure, the present invention modulates cholesterol metabolism to disrupt the material and energy foundation required for tumor cell growth and reproduction, achieving anti-tumor effects. Furthermore, in the process of regulating cholesterol metabolism, the drug modulates immune cells in the tumor microenvironment, activating anti-tumor immune responses and achieving synergistic anti-tumor effects. This approach also overcomes the drug resistance and systemic toxicity of traditional platinum-based drugs.

[0005] The technical solutions of the present invention are as follows:

[0006] The present invention first protects a tetravalent platinum complex for inhibiting cholesterol metabolism, wherein the tetravalent platinum complex is obtained by connecting and coordinating a cholesterol metabolism inhibitor with a platinum derivative; the platinum derivative includes a cisplatin derivative or an oxaliplatin derivative;

[0007] The tetravalent platinum complex includes a single R platinum complex containing one cholesterol metabolism inhibitory group or a double R platinum complex containing two cholesterol metabolism inhibitory groups;

[0008] The general structural formula of the single R platinum complex is:

[0009]

[0010] The general structural formula of the double R platinum complex is:

[0011]

[0012] Wherein, R is a cholesterol metabolism inhibitory group.

[0013] Furthermore, the cholesterol metabolism inhibitory group is selected from one of the groups having the following structural formula:

[0014]

[0015] Furthermore, the tetravalent platinum complex comprises one of the following structural formulas:

[0016]

[0017]

[0018]

[0019] The present invention also protects a method for synthesizing the single R platinum complex, which comprises the following steps:

[0020] S1-1, mixing a platinum compound with hydrogen peroxide to react to obtain a platinum derivative (i.e., a hydroxylation product of the platinum compound);

[0021] S1-2. Add a cholesterol metabolism inhibitor, N-hydroxysuccinimide, dicyclohexylcarbodiimide, and triethylamine to acetonitrile, stir at room temperature for reaction, and remove the acetonitrile by rotary evaporation. Dissolve the rotary evaporation product in dichloromethane, rinse, collect the organic layer, and add anhydrous Na2SO4 to remove water to obtain an intermediate compound; dissolve the intermediate compound in anhydrous dimethyl sulfoxide to obtain an intermediate compound solution;

[0022] S1-3. Add the platinum derivative obtained in step S1-1 dropwise to the intermediate compound solution obtained in step S1-2, stir the reaction, filter, collect the filtrate, add excess ether to remove dimethyl sulfoxide, extract with methanol, wash, and dry to obtain a mono-R platinum complex that inhibits cholesterol metabolism.

[0023] Furthermore, in step S1-1, the platinum compound is cisplatin or oxaliplatin; the mass volume ratio of the platinum compound to hydrogen peroxide is 400 g:10-30 mL; the temperature of the mixing reaction is 30-70° C., and the time is 2-8 hours;

[0024] In step S1-2, the molar volume ratio of the cholesterol metabolism inhibitor, N-hydroxysuccinimide, dicyclohexylcarbodiimide, triethylamine, and acetonitrile is 0.3 mmol:0.3 mmol:0.3 mmol:0.3 mmol:15-45 mL; the stirring speed is 160-450 r / min for 12-48 h; and the mass volume ratio of the intermediate compound to anhydrous dimethyl sulfoxide is 100 mg:3-5 mL;

[0025] In step S1-3, the stirring reaction temperature is 30-70° C., the time is 12-72 h, and the stirring speed is 180-450 r / min; the mass volume ratio of the platinum derivative to the intermediate compound solution is 100 mg:3-8 mL; and the drying is vacuum drying.

[0026] The present invention also protects a method for synthesizing the bis-R platinum complex, which comprises the following steps:

[0027] S2-1, mixing a platinum compound with hydrogen peroxide to react to obtain a platinum derivative;

[0028] S2-2. Mix the cholesterol metabolism inhibitor with the platinum derivative obtained in S2-1, O-benzotriazole-N,N,N′,N′-tetramethyluronium tetrafluoroboric acid, and triethylamine in an N,N-dimethylformamide solution, stir the mixture for reaction, filter, collect the filtrate, remove N,N-dimethylformamide by vacuum rotary evaporation, add ethanol and water, centrifuge, collect the precipitate to obtain the desired product, and vacuum dry to obtain a bis-R platinum complex that inhibits cholesterol metabolism.

[0029] Furthermore, in step S2-1, the platinum compound is cisplatin or oxaliplatin; the mass volume ratio of the platinum compound to hydrogen peroxide is 400 g:10-30 mL; the temperature of the mixing reaction is 30-70° C., and the time is 2-8 hours;

[0030] In step S2-2, the molar volume ratio of the cholesterol metabolism inhibitor, platinum derivative, O-benzotriazole-N,N,N′,N′-tetramethyluronium tetrafluoroboric acid, triethylamine, and N,N-dimethylformamide solution is 0.3 mmol: 0.66 mmol: 0.66 mmol: 0.66 mmol: 6-25 mL; the stirring reaction temperature is room temperature, the time is 24-96 h, and the stirring speed is 200-600 r / min.

[0031] The present invention also protects the use of the tetravalent platinum complex in the preparation of anti-tumor drugs.

[0032] Furthermore, the tumors include ovarian cancer, colorectal cancer, melanoma, pancreatic cancer, breast cancer, lung cancer, liver cancer, head and neck squamous cell carcinoma, bladder cancer, cervical cancer, gallbladder cancer, bone tumor, osteosarcoma, prostate cancer, lung cancer resistant cells, and ovarian cancer resistant cell lines.

[0033] The present invention also protects the use of a tetravalent platinum complex for inhibiting cholesterol metabolism, and the use of the tetravalent platinum complex for inhibiting cholesterol metabolism in the preparation of a metal small molecule chemoimmunotherapy agent for tumor chemoimmunotherapy.

[0034] The present invention also protects a metal small molecule chemoimmunotherapy agent for tumor chemoimmunotherapy, wherein the metal small molecule chemoimmunotherapy agent comprises the tetravalent platinum complex, and the platinum complex comprises a single R platinum complex or a double R platinum complex.

[0035] The tetravalent platinum complex of the present invention is obtained by connecting and coordinating a cholesterol metabolism inhibitor R with a platinum derivative cisplatin or an oxaliplatin derivative.

[0036] The single R platinum complex or double R platinum complex of the present invention can effectively regulate cholesterol metabolism in tumor cells and overcome the drug resistance of tumor cells.

[0037] The active ingredient of the metal small molecule chemoimmunotherapy agent for tumor chemoimmunotherapy of the present invention is a single R platinum complex or a double R platinum complex, and the complex has the following effects on immune cells: (1) it can inhibit the expression of M2 phenotype and promote the expression of M1 phenotype in macrophages; (2) it can promote the maturation of DC cells; (3) it can promote the infiltration of T lymphocytes in tumors; (4) it can inhibit the activation of MDSCs and Treg cells in the tumor microenvironment; and (5) it can reshape the tumor immune microenvironment.

[0038] The beneficial technical effects of the present invention are:

[0039] The present invention synthesizes a platinum complex by introducing a cholesterol regulator into the structure of a platinum drug. The synthesized tetravalent platinum complex has excellent anti-tumor activity both in vivo and in vitro. It treats cancer from two aspects: inhibiting cholesterol metabolism and activating anti-tumor immune responses. It participates in the anti-tumor process through multiple pathways, which is different from the mechanism of action of traditional platinum anti-tumor drugs.

[0040] This invention, for the first time, destroys the material and energy basis required for tumor cell growth and reproduction by regulating cholesterol metabolism, thereby achieving the purpose of anti-tumor. In the process of cholesterol metabolism regulation, it affects the immune cells in the tumor microenvironment, inhibits the polarization of macrophages to M2 type, activates DC, inhibits the expression of MDSCs and Treg T cells in the tumor microenvironment, and promotes CD8 + T lymphocyte infiltration reshapes the tumor microenvironment. As a chemoimmunotherapeutic agent, the tetravalent platinum complex of the present invention not only has the above-mentioned immunomodulatory function, but also retains the chemotherapeutic properties of platinum drugs. Therefore, its therapeutic effect is better than that of simple chemotherapy drugs and immunomodulators. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is the chemical structural formula of the compounds FP and DFP involved in the present invention.

[0042] Figure 2 is the FP in Example 1 of the present invention 1 H NMR (600 MHz, DMSO-d6) spectrum.

[0043] Figure 3 is the FP in Example 1 of the present invention 13 C NMR (101 MHz, DMSO-d6) spectrum.

[0044] Figure 4 is the DFP in Example 1 1 H NMR (600 MHz, DMSO-d6) spectrum.

[0045] Figure 5 is the DFP in Example 1 13 C NMR (101 MHz, DMSO-d6) spectrum.

[0046] Figure 6 This is an immunoblot analysis of the cholesterol regulation of A549 / CDDP cells by FP and DFP.

[0047] Figure 7 Immunofluorescence in A549 / CDDP tumor cells.

[0048] Figure 8Immunofluorescence analysis of the effect of DFP (1.5 mg Pt per kg) on ​​cholesterol-regulating proteins in tumor tissues of the LLC lung cancer mouse model.

[0049] Figure 9 Verification of the death mode of A549 / CDDP by FP and DFP.

[0050] Figure 10 The graph shows the changes in cell morphology and the IL-1β content in the cell supernatant after FP and DFP were cultured with A549 / CDDP lung cancer cells for 24 hours.

[0051] Figure 11 The graph shows the inhibitory effect of DFP (1.5 mg Pt per kg) on ​​the growth of LLC lung cancer transplanted tumors in C57BL / 6 mice (16 days).

[0052] Figure 12 Figure 2 is a flow cytometric graph showing the effect of DFP (1.5 mg Pt per kg) on ​​the levels of antigen-presenting cells in LLC lung cancer transplanted tumor tissues of C57BL / 6 mice (day 16).

[0053] Figure 13 This is a flow cytometric graph of the immune activation of CD8+ T cells in lung cancer transplanted tumor tissues of C57BL / 6LLC mice induced by DFP.

[0054] Figure 14 This figure shows the inhibitory effect of DFP (1.5 mg Pt per kg) on ​​immunosuppressive Terg T cells in LLC lung cancer transplanted tumor tissues of C57BL / 6 mice.

[0055] Figure 15 Graph showing the inhibitory effect of DFP (1.5 mg Pt per kg) on ​​MDSCs (GRE-1) cells (16 days).

[0056] Figure 16 Diagram of the action mechanism of FP and DFP. DETAILED DESCRIPTION

[0057] The present invention will be described in detail below with reference to the accompanying drawings and embodiments.

[0058] The platinum complex involved in the present invention is a platinum (IV) complex as a skeleton, and an active small molecule that can affect the platinum drug resistance pathway is modified at its axial position to synthesize a platinum (IV) complex that can overcome the resistance of platinum anti-tumor drugs (the specific mechanism is as follows Figure 16In this invention, a cholesterol regulator is introduced into a platinum complex to achieve anti-tumor effects by regulating lipid metabolism, disrupting the material and energy base required for tumor cell growth and reproduction. Studies have shown that the complex can not only directly kill tumor cells through chemical reactions, but also indirectly kill tumor cells by inhibiting cholesterol metabolism, reducing immunosuppressive myeloid cell subsets, promoting the maturation and expansion of immunostimulatory cells, reshaping the tumor microenvironment, and activating the immune response. In other words, FP and DFP possess both immunomodulatory and chemotherapeutic functions, enabling chemoimmunotherapy of tumors, making them chemoimmunotherapeutic agents distinct from traditional chemotherapy agents.

[0059] In one embodiment of the present invention, fenofibric acid (FA) is linked to a cisplatin (CDDP) derivative to obtain tetravalent platinum complexes FP and DFP, which achieve the purpose of tumor chemoimmunotherapy. Small molecule chemoimmunotherapy agents that inhibit cholesterol metabolism by platinum complexes and reshape the tumor immunosuppressive microenvironment to overcome tumor resistance have not been reported or used to date. The structural formulas of the prepared tetravalent platinum complexes FP and DFP are as follows: Figure 1 shown.

[0060] The synthesis method of the single R platinum complex of the present invention comprises the following steps:

[0061] S1-1, mixing a platinum compound with hydrogen peroxide to react to obtain a platinum derivative (i.e., a hydroxylation product of the platinum compound);

[0062] S1-2. Add a cholesterol metabolism inhibitor, N-hydroxysuccinimide, dicyclohexylcarbodiimide, and triethylamine to acetonitrile, stir at room temperature for reaction, and remove the acetonitrile by rotary evaporation. Dissolve the rotary evaporation product in dichloromethane, rinse, collect the organic layer, and add anhydrous Na2SO4 to remove water to obtain an intermediate compound; dissolve the intermediate compound in anhydrous dimethyl sulfoxide to obtain an intermediate compound solution;

[0063] S1-3. Add the platinum derivative obtained in step S1-1 dropwise to the intermediate compound solution obtained in step S1-2, stir the reaction, filter, collect the filtrate, add excess ether to remove dimethyl sulfoxide, extract with methanol, wash, and dry to obtain a mono-R platinum complex that inhibits cholesterol metabolism.

[0064] In one embodiment of the present invention, in step S1-1, the platinum compound is cisplatin or oxaliplatin; the mass volume ratio of the platinum compound to hydrogen peroxide is 400g:10mL, 400g:15mL, 400g:20mL or 400g:30mL; the temperature of the mixing reaction is 30°C, 40°C, 50°C or 70°C, and the time is 2h, 4h, 6h or 8h;

[0065] In one embodiment of the present invention, in step S1-2, the molar volume ratio of the cholesterol metabolism inhibitor, N-hydroxysuccinimide, dicyclohexylcarbodiimide, triethylamine, and acetonitrile is 0.3mmol:0.3mmol:0.3mmol:0.3mmol:15-45mL, 0.3mmol:0.3mmol:0.3mmol:0.3mmol:20mL, 0.3mmol:0.3mmol:0.3mmol:0.3mmol:25mL, 0.3mmol:0.3mmol:0.3mmol l:0.3mmol:0.3mmol:35mL or 0.3mmol:0.3mmol:0.3mmol:0.3mmol:45mL; the stirring speed is 160r / min, 200r / min, 300r / min or 450r / min, and the time is 12h, 20h, 30h, 40h or 48h; the mass volume ratio of the intermediate compound to anhydrous dimethyl sulfoxide is 100mg:3mL, 100mg:3.5mL, 100mg:4mL or 100mg:5mL;

[0066] In one embodiment of the present invention, in step S1-3, the stirring reaction temperature is 30°C, 40°C, 60°C or 70°C, the time is 12h, 24h, 48h or 72h, and the stirring speed is 180r / min, 200r / min, 300r / min or 450r / min; the mass volume ratio of the platinum derivative to the intermediate compound solution is 100mg:3mL, 100mg:4mL, 100mg:5mL or 100mg:8mL; and the drying is vacuum drying.

[0067] The present invention also protects a method for synthesizing the bis-R platinum complex, which comprises the following steps:

[0068] S2-1, mixing a platinum compound with hydrogen peroxide to react to obtain a platinum derivative;

[0069] S2-2. Mix the cholesterol metabolism inhibitor with the platinum derivative obtained in S2-1, O-benzotriazole-N,N,N′,N′-tetramethyluronium tetrafluoroboric acid, and triethylamine in an N,N-dimethylformamide solution, stir the mixture for reaction, filter, collect the filtrate, remove N,N-dimethylformamide by vacuum rotary evaporation, add ethanol and water, centrifuge, collect the precipitate to obtain the desired product, and vacuum dry to obtain a bis-R platinum complex that inhibits cholesterol metabolism.

[0070] In one embodiment of the present invention, in step S2-1, the platinum compound is cisplatin or oxaliplatin; the mass volume ratio of the platinum compound to hydrogen peroxide is 400g:10mL, 400g:15mL, 400g:20mL or 400g:30mL; the temperature of the mixing reaction is 30°C, 45°C, 65°C or 70°C, and the time is 2h, 4h, 6h or 8h;

[0071] In one embodiment of the present invention, the molar volume ratio of the cholesterol metabolism inhibitor, platinum derivative, O-benzotriazole-N,N,N′,N′-tetramethyluronium tetrafluoroboric acid, triethylamine, and N,N-dimethylformamide solution is 0.3mmol:0.66mmol:0.66mmol:0.66mmol:6mL, 0.3mmol:0.66mmol:0.66mmol:0.66mmol:25mL, 0.3mmol:0.66mmol:0.66mmol:0.66mmol:18mL or 0.3mmol:0.66mmol:0.66mmol:0.66mmol:20mL; the stirring reaction temperature is room temperature, the time is 24h, 48h, 72h or 96h, and the stirring speed is 200r / min, 300r / min, 450r / min or 600r / min.

[0072] Example 1

[0073] The synthesis route of tetravalent platinum complex FP is as follows:

[0074]

[0075] The specific synthesis method is:

[0076] S1. React 400 mg of cisplatin (CDDP) with 18 mL of hydrogen peroxide H2O2 at 60°C for 4 h to obtain a platinum derivative;

[0077] S2, will (0.3mmol), N-hydroxysuccinimide (NHS) (0.3mmol), dicyclohexylcarbodiimide (DCC) (0.3mmol), triethylamine (0.3mmol)) were added to 30mL of acetonitrile and stirred at room temperature for 24h at 450r / min. After the reaction, the acetonitrile was removed by rotary evaporation. The product was dissolved in 20mL of DCM and rinsed three times with water. The organic layer was collected and anhydrous Na2SO4 was added to remove water to obtain an intermediate compound. According to the mass volume ratio of the intermediate compound to anhydrous dimethyl sulfoxide of 100mg:5mL, the intermediate compound was dissolved in anhydrous dimethyl sulfoxide to obtain an intermediate compound solution;

[0078] S3. The platinum derivative obtained in S1 was added dropwise to the intermediate compound solution obtained in S2 (the mass volume ratio of the platinum derivative to the intermediate compound solution was 100 mg:5 mL). The mixture was stirred at 30°C for 72 hours. After the reaction was complete, the filtrate was collected and excess ether was added to remove DMSO. The product was extracted with methanol, washed twice with methanol and ether, and dried under vacuum to obtain the target product.

[0079] Example 1 FP 1 H NMR (600 MHz, DMSO-d6) and 13 C NMR (101 MHz, DMSO-d6) spectrum is shown in Figure 2-3 shown.

[0080] The yield of FP is 31%. The H NMR spectrum of FP is shown in Figure 2. 1 δ7.74(d,2H),7.64(d,2H),7.13(d,2H),2.84(s,4H),1.76(s,4H). FP characterization by C NMR 13 C NMR (600MHz, Chloroform-D, ppm): 193.54,176.03,168.33.80,167.80,157.49,137.29, 135.07,131.06,131.04,130.34,130.23,127.56,117.49,116.92,74.00,24.38,24.14.

[0081] Example 2

[0082] The tetravalent platinum complex DFP has the following synthesis route:

[0083]

[0084] The specific synthesis method is:

[0085] S1. React 400 mg of cisplatin (CDDP) with 18 mL of hydrogen peroxide H2O2 at 60°C for 4 h to obtain a platinum derivative;

[0086] S2, the platinum derivative obtained in S1 (0.3 mmol) and A mixture of 1,2-dimethylformamide (DMF, 15 mL) and 1,2-dimethylformamide (DMF, 15 mL) was stirred at 550 rpm for 72 h. After the reaction was complete, the filtrate was collected and the DMF was removed under high vacuum. Ethanol and water were added to the residue to obtain the desired product, which was then dried under vacuum to obtain the target product.

[0087] DFP in Example 2 1 H NMR (600 MHz, DMSO-d6) and 13 C NMR (101 MHz, DMSO-d6) spectrum is shown in Figure 4-5 shown.

[0088] The yield of DFP is 70%. 1 H NMR of DFP (400 MHz, DMSO-d6): δ (ppm) 7.72 (d, J = 12 Hz, 4H), 7.65 (d, J = 6 Hz, 4H), 7.62 (d, J = 12 Hz, 4H), 7.00 (d, J = 12 Hz, 4H), 6.58 (s, 6H), 1.56 (s, 12H). 13 C NMR (100MHz, DMSO-d6): δ (ppm) 193.77, 180.62, 160.20, 137.37, 136.97, 132.09, 131.59, 129.27, 129.06, 118.22, 80.13, 26.30.195Pt NMR (86MHz, DMSO-d6, δppm): 1174.16.

[0089] Example 3

[0090] Cytotoxicity test of compounds

[0091] The compounds prepared in Examples 1-2 of the present invention were tested for cytotoxic activity. Breast cancer cells (MFC-7, MDA-MB-231, MDA-MB-435), pancreatic cancer cells (PNAC-1), and cervical cancer cells (HeLa) were used as models, respectively. The compounds synthesized in Examples 1-2 and cisplatin were used as test substances. After the test substances were applied to the cells, the survival of the cells was observed, and the cell survival rate was tested using the MTT method.

[0092] The specific operation steps are as follows: cells are plated at 2×10 3 Cells were seeded at a density of 100 μL in a 96-well plate containing 100 μL of culture medium and cultured for 24 h. FP and DFP stock solutions were prepared in DMSO. The stock solutions were diluted to different concentrations and aliquoted at 200 μL per well (DMSO < 0.1%). After incubation for 72 h, MTT (20 μL, 5 mg mL -1) and incubate at 37°C for 4 hours. Remove the culture medium and add DMSO (150 μL) to dissolve the purple crystals. Shake the plate for 15 minutes and measure the absorbance of the solution at 570 nm using a microplate reader. Similar methods can be used to test the toxicity of FP and DFP to other cell types. The results are shown in Table 1.

[0093] Table 1. IC of FA, CDDP, FA and CDDP (1:1, 1:2), FP and DFP on different cells 50 (μM) value

[0094] Complex A549 A549 / CDDP A2780 / CDDP PNAC-1 MDA-MB-231 LO-2 FP 0.15±0.0.2 0.82±0.01 0.44±0.05 0.31±0.24 0.21±0.01 0.75±0.13 DFP 3.59±0.22 5.98±0.31 4.46±0.99 3.59±0.22 1.71±0.59 5.34±1.13 FA >64 >64 >64 >64 >64 >64 CDDP 11.92±0.25 >64 >64 32.24±0.42 30.09±1.20 12.51±3.54 CDDP+FA 26.97±1.1 >64 >64 >64 >64 18.33±1.49 CDDDP+2FA 37.57±5.04 >64 >64 >64 >64 43.88±3.02 Oxoplatin 57.3±4.16 >64 >64 >64 >64 32.19±2.65

[0095] As can be seen from Table 1, FP and DFP have good anti-tumor activity against cisplatin-resistant strains and can overcome cisplatin resistance.

[0096] Example 4

[0097] Effects of compounds on proteins related to cholesterol metabolism

[0098] (1) The present invention tested the expression levels of LDLR, ABAC1, and ACAT1 proteins in A549 / CDDP cells by Western blotting technology and immunofluorescence method.

[0099] The specific implementation method is as follows: A549 / CDDP cells were cultured at 2×10 6 The cells were seeded at a density of 100 cells / mL in a 6-cm cell culture dish and cultured in DMEM medium containing 10% fetal bovine serum for 24 h. The medium was then replaced with fresh medium containing FA, CDDP, FP, and DFP (1 μM), respectively, and incubated for another 24 h. The cells were washed twice with cold PBS, collected, lysed on ice, centrifuged at 4°C for 15 min (12,000 g), the supernatant was collected, added to the loading buffer and normalized, boiled at 96°C for 10 min, and the samples were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% BSA at room temperature for 1 h and then incubated with LDLR, ABAC1, and ACAT1 protein antibodies at 4°C overnight. The membrane was washed and incubated with secondary antibodies at room temperature for 1 h to observe protein bands.

[0100] See the results Figure 6 The results showed that FP and DFP had significant inhibitory effects on LDLR and ACAT1, and could upregulate ABAC1, indicating that FP and DFP could effectively inhibit the uptake and transport of cholesterol.

[0101] (2) The cholesterol content in the cells and the supernatant was detected by fluorescence quantitative method, and the cholesterol ester content in the cells was tested by immunofluorescence method.

[0102] The specific implementation method is as follows: BODIPY-cholesterol (GC42964, Glpbio, Montclair, CA, USA) was used to detect cholesterol efflux. A549 / CDDP cells were cultured at 2×10 5 Cells were seeded in 6-well plates at a density of 10 cells / mL and incubated overnight. Cells were rinsed with cold 1× PBS. Cells were stained in a serum-free culture medium containing 0.1 mM BODIPY-cholesterol for 1 hour. The culture medium was then removed and the cells were cultured with culture medium containing 1 μM FA, CDDP, FP, and DFP for 24 hours. Cells and supernatants were collected, and intracellular cholesterol efflux and total cholesterol were measured by fluorescence, recorded using a fluorometer (Em 480 nm, Em 508 nm). A549 / cDDP cells were seeded in 20 mm confocal microplates (Nest) and cultured at 37°C for 24 hours. Cells were treated with FA, CDDP, FP, and DFP (1 μM) for 24 hours. After Nile Red treatment at 37°C for 20 minutes, the cells were washed twice with cold PBS and fixed with 4% paraformaldehyde for 10 minutes at room temperature. Nuclei were stained with Hoechst 3342. Finally, A549 / cDDP cells were scanned under a fluorescence microscope.

[0103] Figure 7 Confocal microscopy revealed that FP significantly inhibited lipid droplet accumulation, suggesting that FP and DFP can act as regulators of cholesterol metabolism. Fluorescence quantitative experiments revealed that FP and DFP effectively inhibited intracellular cholesterol synthesis and promoted cholesterol efflux, consistent with the results of Western blot experiments.

[0104] (3) Immunofluorescence technology was used to test the effect of DFP on the levels of LXRα, ApoE and LDLR in tumor tissues of LLC lung cancer mouse model.

[0105] The specific method is as follows: After DFP treatment of LLC lung cancer mice, tumor tissue was collected and fixed with 4% paraformaldehyde. The fixed tumor tissue samples were then cryoprotected in 30% sucrose and cut into 5 μm-thick sections using a Cryosta (MicromHM 525, Thermo Scientific, Waltham, MA, USA). Paraffin-embedded tumor tissue sections were used for histological staining. Sections were deparaffinized and hydrated by soaking in xylene twice for 10 minutes, ethanol for 3 minutes, 95% ethanol / H2O for 3 minutes, 90% ethanol / H2O for 3 minutes, 80% ethanol for 3 minutes, 70% ethanol / H2O for 3 minutes, 60% ethanol / H2O for 3 minutes, 50% ethanol / H2O for 3 minutes, and ddH2O for 3 minutes. Tissue sections were then permeabilized with 0.5% Triton X-100 in PBS for 15 minutes. The sections were washed with cold PBS for 5 minutes and blocked with 0.5% BSA in PBS at room temperature for 30 minutes. Antigen retrieval buffer was heated at 95°C for 15 minutes. Subsequently, the sections were incubated with primary antibody (1:100 dilution) at 4°C overnight, then washed 2×5 minutes with PBS buffer and incubated with secondary antibody (1:100) at room temperature for 1 hour. The sections were observed using a confocal fluorescence microscope. The results are shown in Figure 8 .

[0106] Depend on Figure 8 The results showed that DFP increased ApoE expression, indicating that DFP activated LXRα, leading to ApoE activation. In contrast, DFP significantly inhibited LDLR, while CDDP had little effect on LDLR. DFP's downregulation of LDLR in tumor tissues suggests that DFP's antitumor activity may be partially due to its inhibition of cholesterol uptake in tumor cells.

[0107] Example 5

[0108] Effects of compounds on pyroptotic proteins, cell morphology, and IL-1β

[0109] (1) Western blotting was used to test the expression levels of NLRP3, Cleaved-CASP1, GSDMD, and GSDMD-N proteins in A549 / CDDP cells. The specific method is as follows: A549 / CDDP cells were plated at 2×10 6The cells were seeded at a density of 100 cells / mL in a 6-cm cell culture dish and cultured in 1640 medium containing 10% fetal bovine serum for 24 h. The medium was then replaced with fresh medium containing FA, CDDP, FP, and DFP (1 μM), respectively, and incubated for another 24 h. The cells were washed twice with cold PBS, collected, lysed on ice, centrifuged at 4°C for 15 min (12,000 g), the supernatant was collected, added to the loading buffer and normalized, boiled at 96°C for 10 min, and the samples were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% BSA at room temperature for 1 h and then incubated with protein antibodies at 4°C overnight. The membrane was washed and incubated with secondary antibodies at room temperature for 1 h to observe protein bands.

[0110] See the results Figure 9 The results showed that after A549 / CDDP tumor cells were treated with FP and DFP respectively, the expression of GSDMD was significantly downregulated, while the expression of GSDMD-N, caspase-1 and NLRP3 was significantly upregulated, indicating that FP and DFP caused pyroptosis in A549 / CDDP cells.

[0111] (2) Observe the changes in cell morphology under an optical microscope.

[0112] In a specific embodiment, A549 / CDDP cells were seeded in a 20 mm confocal microplate (Nest) and cultured at 37°C for 24 hours. The cells were treated with FA, CDDP, FP, and DFP (1 μM) for 24 hours. The supernatant was removed, the cells were washed three times with cold PBS, and fresh culture medium was added. Cell morphology was observed under a confocal microscope with bright field microscope.

[0113] (3) The release effect of IL-1β in cells was tested by ELISA method.

[0114] The specific implementation method is as follows: A549 / CDDP cells were cultured at 2×10 5 Cells were seeded in 6-well plates at a density of 100 cells / mL and cultured at 37°C for 24 hours. Cells were treated with FA, CDDP, FP, and DFP (1 μM) for 24 hours, and the supernatant was collected. The IL-1β secreted in the supernatant of the cell culture was detected using a kit. The results are shown in Figure 10 .

[0115] The above results showed that after FP and DFP treatment, the expression of GSDMD was significantly downregulated, while the expression of GSDMD-N, caspase-1 and NLRP3 was significantly upregulated, indicating that these complexes caused pyroptosis in A549 / CDDP cells; under an optical microscope, it was observed that pores were formed on the membrane during cell pyroptosis, causing water influx, cell swelling, and cell membrane rupture; IL-1β was significantly increased after FP and DFP treatment, further proving that FP and DFP can induce cell pyroptosis.

[0116] Example 6

[0117] Antitumor activity of DFP in mice

[0118] LLC cells were suspended in PBS at a rate of 2 × 10 cells per mouse. 5 9 days after tumor cell implantation, the tumor grew to 80-150 mm. 3 Tumor-bearing mice were randomly divided into three groups after inoculation: (a) control group, (b) CDDP group, and (c) DFP group. Drugs were administered via tail vein injection every 2 days for a total of 16 days. The weight and tumor volume of mice were recorded every 2 days. The diameter of the tumor was measured with a vernier caliper and calculated according to the formula to monitor tumor growth: tumor volume (mm 3 )=0.5×length×width 2 On the 16th day, the mice were killed for further testing of other indicators. Figure 11 .

[0119] The results showed that DFP had stronger anti-tumor activity than CDDP, with significant reductions in tumor volume and weight. During DFP treatment, the body weight of mice remained almost unchanged.

[0120] Example 7

[0121] Effects of DFP on antigen presenting cells (APC) in vivo

[0122] The effect of DFP on CD86, CD206 and CD80 in tumor tissues of LLC lung cancer mouse model was tested by flow cytometry.

[0123] The specific implementation method is as follows: After the LLC lung cancer mouse model was administered for 16 days, the mice were killed, the tumor tissues were collected, and single cell suspensions were prepared by mechanical grinding. The cell suspension was then collected by low-temperature centrifugation at 1500 rpm for 5 minutes, RBC lysis buffer was added for 5 minutes at room temperature to remove red blood cells, and the cells were washed twice with cold PBS. According to the instructions, macrophages in the tumor tissue were co-stained with CD11b-FITC, CD206-APC, and PE-CD80 antibodies, and the lymph nodes around the tumor tissue were stained with CD11c-FITC, CD86-APC, and then immediately analyzed by flow cytometry. The results are shown in Figure 2. Figure 12 shown.

[0124] Depend on Figure 12 The results show that DFP will CD206 +The cell ratio decreased from 15.25% to 1.01%, which was significantly lower than that of the control (Ctrl), and CD86 increased from 3.82% to 29.91%, indicating that DFP reduced M2 macrophages and increased M1 macrophages in tumor tissues, which would reduce the effect of CD8 + The immunosuppressive activity of cytotoxic T cells; the content of DCs was detected in the lymph nodes, and the content of DCs in DFP-injected mice was significantly increased. The above data show that DFP can effectively activate antigen-presenting cells in the body and then activate T cells.

[0125] Example 8

[0126] The effect of DFP on T cells in tumor tissue of LLC lung cancer mouse model was tested by flow cytometry. The specific implementation method is the same as above. The cell suspension was co-stained with CD3-FITC, CD80-PE and CD4-APC antibodies according to the instructions, and then immediately analyzed by flow cytometry. The results are shown in Figure 2. Figure 13 shown.

[0127] Depend on Figure 13 It can be seen that compared with the mice treated with PBS (2.78%) and CDDP (3.45%), the number of CD8 + T cells (blue squares) were effectively activated (6.17%); CD4 + T cells (red squares) were also slightly activated. These results suggest that DFP's inhibition of cholesterol metabolism can stimulate a strong immune response in vivo.

[0128] Example 9

[0129] The effect of DFP on immunosuppressive cells in tumor tissue of LLC lung cancer mouse model was tested by immunofluorescence technique. Figure 14-15 .

[0130] Depend on Figure 14-15 It can be seen that compared with the Ctrl and CDDP groups, DFP effectively inhibited GR-1 red, indicating that DFP can inhibit the infiltration of MDSCs in tumor tissues. At the same time, DFP also significantly inhibited the green fluorescence intensity of FOXP3, indicating that DFP can also inhibit the infiltration of Treg cells in tumor tissues.

[0131] In summary, the cholesterol inhibitor for tumor chemoimmunotherapy provided by the present invention is a tetravalent platinum complex obtained by connecting fenofibric acid (FA) to a cisplatin (CDDP) derivative. They can not only directly kill tumor cells through chemical reactions, but also indirectly kill tumor cells by inhibiting cholesterol metabolism, reducing immunosuppressive myeloid cell subpopulations, promoting the maturation and expansion of immunostimulatory cells, reshaping the tumor microenvironment and activating the immune response. That is, the cholesterol inhibitor provided by the present invention has both immunoregulatory and chemotherapeutic functions and can be used for chemoimmunotherapy of tumors.

[0132] The present invention is not limited to the specific embodiments described above. Any modifications made by a person skilled in the art based on the above concepts without creative effort shall fall within the scope of protection of the present invention. In other words, the above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations directly derived or imagined by a person skilled in the art without departing from the spirit and concept of the present invention shall be considered to be within the scope of protection of the present invention.

Claims

1. A tetravalent platinum complex for inhibiting cholesterol metabolism, characterized in that: The tetravalent platinum complex is obtained by connecting and coordinating a cholesterol metabolism inhibitor with a platinum derivative; the platinum derivative includes a cisplatin derivative; The tetravalent platinum complex includes a single R platinum complex containing one cholesterol metabolism inhibitory group or a double R platinum complex containing two cholesterol metabolism inhibitory groups; The general structural formula of the single R platinum complex is: （1）； The general structural formula of the double R platinum complex is: （3）； Wherein, R is a cholesterol metabolism inhibitory group; The structure of the cholesterol metabolism inhibitory group is as follows: 。 2. A method for preparing the tetravalent platinum complex according to claim 1, characterized in that: The synthesis method of the single R platinum complex comprises the following steps: S1-1, mixing a platinum compound with hydrogen peroxide to react to obtain a platinum derivative; S1-2. Add a cholesterol metabolism inhibitor, N-hydroxysuccinimide, dicyclohexylcarbodiimide, and triethylamine to acetonitrile, stir at room temperature for reaction, and remove the acetonitrile by rotary evaporation. Dissolve the rotary evaporation product in dichloromethane, rinse, collect the organic layer, and add anhydrous Na2SO4 to remove water to obtain an intermediate compound; dissolve the intermediate compound in anhydrous dimethyl sulfoxide to obtain an intermediate compound solution; S1-3. Add the platinum derivative obtained in step S1-1 dropwise to the intermediate compound solution obtained in step S1-2, stir the reaction, filter, collect the filtrate, add excess ether to remove dimethyl sulfoxide, extract with methanol, wash, and dry to obtain a mono-R platinum complex that inhibits cholesterol metabolism.

3. The preparation method according to claim 2, characterized in that In step S1-1, the platinum compound is cisplatin; the mass volume ratio of the platinum compound to hydrogen peroxide is 400 g: 10-30 mL; the temperature of the mixing reaction is 30-70° C., and the time is 2-8 h; In step S1-2, the molar volume ratio of the cholesterol metabolism inhibitor, N-hydroxysuccinimide, dicyclohexylcarbodiimide, triethylamine, and acetonitrile is 0.3 mmol: 0.3 mmol: 0.3 mmol: 0.3 mmol: 15-45 mL; the stirring speed is 160-450 r / min for 12-48 h; and the mass volume ratio of the intermediate compound to anhydrous dimethyl sulfoxide is 100 mg: 3-5 mL. In step S1-3, the mass volume ratio of the platinum derivative to the intermediate compound solution is 100 mg: 3-8 mL; the stirring reaction temperature is 30-70°C, the time is 12-72 h, and the stirring speed is 180-450 r / min; and the drying is vacuum drying.

4. A method for preparing the tetravalent platinum complex according to claim 1, characterized in that: The synthesis method of the double R platinum complex comprises the following steps: S2-1, mixing a platinum compound with hydrogen peroxide to react to obtain a platinum derivative; S2-2. A cholesterol metabolism inhibitor is mixed with the platinum derivative obtained in S2-1, O-benzotriazole-N, N, Nʹ, Nʹ-tetramethyluronium tetrafluoroboric acid, and triethylamine in an N, N-dimethylformamide solution, stirred for reaction, filtered, and the filtrate is collected. The N, N-dimethylformamide is removed by vacuum rotary evaporation, and ethanol and water are added. The mixture is centrifuged, the precipitate is collected, and vacuum dried to obtain a bis-R platinum complex that inhibits cholesterol metabolism.

5. The preparation method according to claim 4, characterized in that In step S2-1, the platinum compound is cisplatin; the mass volume ratio of the platinum compound to hydrogen peroxide is 400 g: 10-30 mL; the temperature of the mixing reaction is 30-70° C., and the time is 2-8 h; In step S2-2, the molar volume ratio of the cholesterol metabolism inhibitor, platinum derivative, O-benzotriazole-N, N, Nʹ, Nʹ-tetramethyluronium tetrafluoroboric acid, triethylamine, and N, N-dimethylformamide solution is 0.3 mmol: 0.66 mmol: 0.66 mmol: 0.66 mmol: 6-25 mL; the stirring reaction temperature is room temperature, the time is 24-96 h, and the stirring speed is 200-600 r / min.

6. Use of the tetravalent platinum complex according to claim 1 or the tetravalent platinum complex prepared by the preparation method according to any one of claims 2 to 5 in the preparation of drugs for treating lung cancer, breast cancer, pancreatic cancer and ovarian cancer.

7. A metal small molecule chemoimmunotherapy agent for tumor chemoimmunotherapy, characterized in that: The metal small molecule chemoimmunotherapeutic agent includes the tetravalent platinum complex according to claim 1 or the tetravalent platinum complex prepared by the preparation method according to any one of claims 2 to 5, and the tetravalent platinum complex includes a single R platinum complex or a double R platinum complex.

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