A derivative having a tetravalent platinum structure of desloratadine and its preparation method and use

Abstract

The present invention provides a derivative having a desloratadine tetravalent platinum structure, a preparation method, and a use thereof. The compound having a desloratadine tetravalent platinum structure is shown in general formula (I), #imgabs0# wherein #imgabs1# is selected from cisplatin or oxaliplatin; L is hydroxyl or #imgabs2#. The desloratadine tetravalent platinum compound of the present invention has good anti-tumor proliferation activity and a good therapeutic effect on metastatic malignant tumors; it can effectively inhibit the tumor epithelial-mesenchymal transition (EMT) process and effectively activate tumor immune response, and can be used to prepare anti-tumor drugs, especially providing a new direction for the research and development of anti-metastatic malignant tumor drugs.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a desloratadine tetravalent platinum derivative having a structure, a preparation method thereof, and an application thereof in anti-tumor drugs. Background Art

[0002] Cancer metastasis is the leading cause of cancer-related mortality. It involves the spread of cancer cells from the primary tumor to distant tissues. Traditional cancer chemotherapy focuses on the diagnosis and treatment of primary tumors but is less effective against metastatic tumor cells. Therefore, the development of therapeutics for metastatic cancer is urgent.

[0003] Epithelial-mesenchymal transition (EMT) and immunosuppression are key factors promoting tumor metastasis. As a key process in cancer progression, EMT promotes tumor cells to lose characteristics such as polarity and tight junctions inherent in epithelial cells, and is considered a hallmark of tumor migration. In addition, EMT can effectively stimulate angiogenesis by promoting hypoxia and increasing the secretion of VEGF, thereby leading to tumor cell metastasis. At the same time, immunosuppression in the tumor microenvironment (TME), as a hallmark of cancer, helps cancer cells evade immune surveillance and accelerate their metastasis. In addition, the feedback loop between EMT and immunosuppression has been verified in tumors. EMT can promote the expression of immunosuppressive checkpoint molecules such as PD-L1 to suppress immunity. At the same time, immunosuppressive factors will further promote the EMT process of tumor cells.

[0004] Platinum (II) drugs are an important cornerstone of cancer chemotherapy drugs, exerting their anti-tumor effects by causing intrachain DNA cross-link damage. However, their clinical efficacy in the treatment of metastatic cancer is weak. Increasing evidence shows that long-term use of platinum (II) drugs can promote the occurrence of EMT in tumor cells. At the same time, the treatment process of platinum (II) drugs is often accompanied by immunosuppression characterized by T cell exhaustion and macrophage repolarization, which is also a factor that cannot be ignored in affecting its chemotherapy effect. Therefore, the development of new platinum drugs with EMT inhibition and immunomodulatory properties is expected to overcome the inherent defects of traditional platinum drugs and enhance their anti-tumor metastasis ability. Platinum (IV) compounds are prodrugs of platinum (II) drugs and are easy to modify in structure. Introducing different functional groups into their structure to obtain new drugs provides an effective strategy for the development of multifunctional platinum drugs.

[0005] Desloratadine (DLT), an FDA-approved antihistamine targeting HRH1, has great potential in reversing immune evasion and EMT in the TME, and exhibits anti-tumor activity against a variety of solid tumors, including bladder, liver, and gastrointestinal tumors. Literature has confirmed that antihistamines can stimulate anti-tumor immunity by inhibiting histamine secretion in tumor tissues, promoting M1 polarization of macrophages, and activating T cell immunity. At the same time, DLT can inhibit the EMT process by blocking the N-myristoyltransferase 1 (NMT1) and hippocampal calcineurin-like protein (HPCAL1 / VILIP3) pathways, thereby inhibiting the β-catenin signaling pathway (β-Catenin, cyclin D1, and c-Myc).

[0006] Therefore, this study incorporated DLT into the platinum (IV) system, which is currently an innovative structural system in the field of pharmaceutical chemistry, and aimed to explore effective anti-tumor proliferation and anti-metastasis platinum drugs with EMT inhibition and immunomodulatory properties. Summary of the Invention

[0007] In response to the problems existing in the prior art, the present invention proposes a compound having a desloratadine tetravalent platinum structure. By introducing the desloratadine molecule into the tetravalent platinum mother nucleus, a series of novel desloratadine derivative-modified tetravalent platinum compounds are synthesized. This type of desloratadine tetravalent platinum derivative has significant anti-tumor proliferation ability, has a good therapeutic effect on metastatic malignant tumors, can effectively inhibit the tumor epithelial-mesenchymal transition process, and effectively activate the tumor immune response, providing a new direction for the research and development of anti-tumor drugs, especially anti-metastatic malignant tumor drugs.

[0008] To achieve the above object, the present invention provides a compound having a desloratadine tetravalent platinum structure,

[0009] The general formula is shown in (I): in, Selected from cisplatin or oxaliplatin;

[0010] L is Or hydroxyl; wherein, R3 is selected from propyl, butyl or pentyl.

[0011] Furthermore, the derivative is any one of the following:

[0012] The derivatives of the present invention are selected from:

[0013]

[0014] Another object of the present invention is to provide a method for preparing a compound represented by general formula (I). The synthetic route of the compound is as follows:

[0015] Described synthetic route 1 is as follows:

[0016]

[0017] Compound 3 and compound 4 undergo a coupling reaction to obtain an asymmetric monosubstituted desloratadine-modified tetravalent platinum compound 1; wherein the molar ratio of compound 3 to compound 4 is 1:1.0-1.3;

[0018] Described synthetic route 2 is as follows:

[0019]

[0020] Compound 3 and compound 4 undergo a coupling reaction to obtain a symmetrically disubstituted desloratadine-modified tetravalent platinum compound 2; wherein the molar ratio of compound 3 to compound 4 is 1:2.2-3.

[0021] Furthermore, in the first synthetic route, the preparation steps of the asymmetric monodesloratadine tetravalent platinum derivative are as follows:

[0022] Under an inert gas atmosphere, compound 4, a condensing agent, and an organic base are dissolved in an anhydrous organic solvent for reaction, compound 3 is added, and the reaction is carried out in the dark. After post-treatment, compound 1 is isolated;

[0023] The molar ratio of compound 3, compound 4, condensing agent, and organic base is 1:1.0-1.3:1.0-1.3:1.0-1.3; the feeding relationship of compound 3 and organic solvent is that 30-80 ml of organic solvent is added for every 1 g of compound 3;

[0024] In the second synthetic route, the preparation steps of the symmetrical tetravalent platinum derivative of bis-desloratadine are as follows:

[0025] Under an inert gas atmosphere, compound 4, a condensing agent, and an organic base are dissolved in an anhydrous organic solvent for reaction, compound 3 is added, and the reaction is carried out in the dark. After post-treatment, compound 2 is isolated;

[0026] The molar ratio of compound 3, compound 4, condensing agent and organic base is 1:2.2-3:2.2-3:2.2-3; the feeding relationship between compound 3 and organic solvent is that 30-80 ml of organic solvent is added for every 1 g of compound 3.

[0027] Furthermore, the inert gas is nitrogen, helium or argon; the condensing agent is TBTU, HATU or EDCI; the organic base is triethylamine, N,N-diisopropylethylamine or 4-dimethylaminopyridine; and the organic solvent is DMF or DMSO.

[0028] The preparation process can be specifically as follows: TBTU and compound 4 (desloratadine derivative) are added to a reaction vessel, the air in the system is replaced with nitrogen, anhydrous DMF is added, the reaction is stirred at room temperature, anhydrous triethylamine is added to the reaction system, the reaction is stirred at room temperature, the tetravalent platinum compound 3 is added to the reaction system, the air in the system is replaced with nitrogen again, and the reaction system is placed at 25 to 120° C. in the dark for 24 to 72 hours. After the reaction is completed, the solvent is removed under reduced pressure, and column chromatography is performed to obtain an asymmetric monosubstituted desloratadine-modified tetravalent platinum compound 1 or a symmetric disubstituted desloratadine-modified tetravalent platinum compound 2.

[0029] Furthermore, the compound 3 is composed of After being oxidized with hydrogen peroxide, the product is prepared. The specific preparation process is as follows:

[0030]

[0031] Divalent platinum compounds The bis(hydroxy) tetravalent platinum compound 3 is prepared by oxidation with hydrogen peroxide at 60-70°C for 1-8 hours.

[0032] In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound represented by formula (I) and pharmaceutically acceptable excipients thereof.

[0033] Pharmaceutically acceptable excipients in the present invention include one or more of carriers, excipients, and diluents; such as binders, lubricants, disintegrants, solubilizers, diluents, stabilizers, suspending agents, pigments, flavoring agents, preservatives, solubilizers, and bases. Pharmaceutically acceptable excipients can be aqueous or non-aqueous. Conventional excipients include colloids, such as gelatin; starches, such as corn starch and potato starch; sugars, such as lactose, glucose, and sucrose; and cellulosic materials and mixtures thereof, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate. Pharmaceutically acceptable excipients include, but are not limited to, tragacanth powder, malt, talc, oils (such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil, etc.), alcohols (such as propylene glycol, ethanol, glycerol, sorbitol, mannitol, polyethylene glycol, etc.), esters (such as ethyl oleate, ethyl laurate, agar), buffers (such as magnesium hydroxide, aluminum hydroxide, boric acid and sodium borate and phosphate buffer), alginic acid, pyrogen-free water, isotonic saline, and Ringer's solution.

[0034] The desloratadine tetravalent platinum compound or pharmaceutical composition of the present invention is in the form of tablets, capsules, aerosols, dispersible tablets, oral liquids, suppositories, pills, large infusions, small injections, lyophilized powder injections, ointments or liniments, including various sustained-release, controlled-release dosage forms or nanoformulations prepared using conventional methods recognized in pharmaceutical science.

[0035] The desloratadine tetravalent platinum compound of the present invention can be administered in the form of a unit dose, and the administration route can be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal, etc.

[0036] The administration route of the desloratadine tetravalent platinum compound of the present invention can be intravenous administration, including intravenous injection, intramuscular injection, intratumoral injection, subcutaneous injection and acupoint injection.

[0037] The method of preparing the active ingredient into a medicine in the present invention can be prepared by methods known to those skilled in the art. For example, the active ingredient can be diluted with a carrier or encapsulated in a carrier so that it can be quickly released, slowly released, or delayed released after administration to a subject.

[0038] Another object of the present invention is to provide the use of the compound or pharmaceutical composition represented by general formula (I) in the preparation of anti-tumor drugs, specifically in the preparation of anti-tumor proliferation and anti-tumor metastasis drugs.

[0039] The desloratadine tetravalent platinum derivatives of the present invention can have a good therapeutic effect on metastatic malignant tumors, can effectively inhibit the tumor epithelial-mesenchymal transition process, and effectively activate the tumor immune response, and have a good therapeutic effect on metastatic malignant tumors.

[0040] Furthermore, the anti-tumor agent is anti-lung cancer, anti-drug-resistant lung cancer, anti-liver cancer or anti-breast cancer, etc.; wherein, the anti-tumor proliferation agent is specifically anti-human lung adenocarcinoma, anti-cisplatin-resistant human lung adenocarcinoma, anti-human liver cancer or anti-mouse breast cancer; the anti-tumor metastasis agent is anti-mouse breast cancer cell.

[0041] The invention also provides a combined preparation comprising a compound or pharmaceutical composition as represented by general formula (I) and an anti-tumor drug such as paclitaxel, fluorouracil, gemcitabine, vinca alkaloids, or antibodies.

[0042] The desloratadine derivative tetravalent platinum compound described in the present invention is expected to be used alone or in combination with marketed platinum drugs, paclitaxels, fluorouracils, gemcitabines, vinca alkaloids, antibodies, and the like to prepare a combined preparation with anti-tumor activity. The combined preparation can be in the form of tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, sustained-release tablets, capsules, hard capsules, soft capsules, sustained-release capsules, oral liquids, mixtures, lozenges, granules, electuary preparations, pills, powders, ointments, suspensions, solutions, injections, powder injections, freeze-dried powder injections, suppositories, liniments, ointments, plasters, creams, sprays, aerosols, drops, patches, and the like.

[0043] Compared with the prior art, the compound having the desloratadine tetravalent platinum structure of the present invention has the following advantages:

[0044] (1) The present invention introduces the desloratadine molecule into the tetravalent platinum core to synthesize a series of novel desloratadine derivatives modified with tetravalent platinum compounds. Antitumor activity tests have shown that these compounds have good anti-tumor and anti-cancer abilities. These desloratadine tetravalent platinum derivatives have good therapeutic effects on metastatic malignant tumors and have excellent anti-tumor metastasis activity.

[0045] (2) This type of desloratadine tetravalent platinum derivative can induce severe DNA damage in tumor cells and induce mitochondrial-mediated apoptosis, thereby exerting anti-tumor effects;

[0046] (3) Tumor proliferation and metastasis are related to the epithelial-mesenchymal transition (EMT) process of tumors, and this type of desloratadine tetravalent platinum derivative can effectively inhibit the epithelial-mesenchymal transition (EMT) process of tumors;

[0047] (4) Antitumor activity is related to the activation of immune responses in tumor cells, and this type of desloratadine tetravalent platinum derivative can effectively activate tumor immune responses;

[0048] (5) The innovative structure of the compound of general formula I described in the present invention is expected to obtain a variety of lead molecules that are effective against tumors, providing new candidate drug molecules to solve the defects of traditional divalent platinum drugs, and also opening up new avenues for the modification of tetravalent platinum compounds; in terms of pharmacological activity, the tetravalent platinum compound desloratadine can inhibit the EMT process in tumors, activate anti-tumor immunity, cause DNA damage, and thus inhibit tumor proliferation and metastasis. This mechanism of action is significantly different from that of traditional platinum drugs, can effectively overcome their drug resistance, reduce toxic side effects, and effectively expand the application of platinum drugs in the treatment of malignant metastatic tumors.

[0049] This type of innovative drug research at the source will have important theoretical value and practical significance for national economic and social development and people's health. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1In vivo antitumor activity of compounds 2-4, CDDP, and OLP against 4T1 tumors in female BALB / c mice (n = 5). **P < 0.01, ***P < 0.001, ns: not statistically significant. (a) Schematic diagram of experimental design; (b) Tumor growth over time; (c) Tumor weight at the end of the experiment. The TGI of each test drug compared with the saline group is shown above the bars [TGI = (1 - tumor weight in drug-treated group / tumor weight in saline group) × 100%]; (d) Relative body weight of mice during treatment; (e) Tumor images after mouse sacrifice; (f) H&E-stained images of tumor tissue.

[0051] Figure 2 Comparison of organ indices of BALB / c mice treated with compound 2-4, CDDP, and OLP compared with the blank control group (n=5); a heart; b liver; c spleen; d lung; e kidney; Organ index = organ weight / body weight × 100%. **P<0.01, ***P<0.001, ns: no significant difference compared with the control group.

[0052] Figure 3 H&E staining of the liver, spleen, and kidney of mice in the compound 2-4, CDDP, and OLP treatment groups.

[0053] Figure 4 In vitro transwell assay to evaluate the inhibitory effect of complex 2-4, CDDP, and OLP (10 μM) on 4T1 cell migration. Tumor cells were treated with or without platinum complexes for 24 h. (a) Representative images. (b) Relative migration rate analysis. ***P < 0.001.

[0054] Figure 5 Inhibitory effects of complexes 2-4, CDDP, and OLP on 4T1 cell migration at 10 μM; wound healing was observed at 0, 12, and 24 hours. (a) Representative images; (b) Wound closure analysis; *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significant difference.

[0055] Figure 6 Inhibitory effects of CDDP, OLP, and compounds 2-4 on 4T1 breast cancer tumor lung metastasis in vivo (n=5). ***P<0.001. (a) Schematic diagram of the experimental design; (b) Representative photographs of the anterior and posterior lung surfaces of each group at the end of the experiment; (c) Pulmonary nodules in each group, with the inhibition rates of the test drugs compared to the blank group shown above; (d) H&E staining of pulmonary metastatic nodules; nodules are indicated by red arrows.

[0056] Figure 7Platinum accumulation in 4T1 cells and 4T1 tumor tissues in vivo after 24 hours of treatment with platinum complexes (10 μM). (a) Platinum content in whole cells; (b) Platinum content in tumor tissue; (c) Distribution of platinum in tumor cell substructures. **P < 0.01, ***P < 0.001.

[0057] Figure 8 Western blot analysis of γ-H2AX and P53 expression (4T1 cells were incubated at 37°C for 24 hours with or without compound 2-4 (10 μM), CDDP (10 μM), and OLP (10 μM). (a) Western blot; (b) Relative grayscale analysis. Relative grayscale intensity = (grayscale intensity of the indicated protein) / (grayscale intensity of β-actin). ***P < 0.001.

[0058] Figure 9 Apoptosis of 4T1 cells was quantified using Annexin V-FITC / PI staining (4T1 cells were incubated with platinum complexes at 37°C for 24 h); (a) blank; (b) CDDP (10 μM); (c) OLP (10 μM); (d) 2-4 (10 μM); (e) stacked columns.

[0059] Figure 10 ROS and DCFH-DA staining in 4T1 cells treated with platinum complexes for 24 h at 37°C; (a) Representative images; (b) Statistical analysis of fluorescence intensity *P<0.05, ***P<0.001.

[0060] Figure 11 Mitochondrial membrane potential (ΔΨm) analyzed by flow cytometry (4T1 cells were treated with or without platinum complexes at 37°C for 24 h and stained with JC-1); (a) blank; (b) CDDP (10 μM); (c) OLP (10 μM); (d) 2-4 (10 μM).

[0061] Figure 12 Western blot analysis of Bcl-2, Bax, caspase3, and c-caspase3 expression in 4T1 cells incubated with platinum compounds 2-4 (10 μM), CDDP (10 μM), and OLP (10 μM) at 37°C for 24 h. (a) Western blot; (b) relative grayscale analysis; ***P < 0.001.

[0062] Figure 13Western blot analysis of E-cadherin, N-cadherin, Vimentin, and Snail1 expression (4T1 cells were incubated with platinum compound 2-4 (10 μM), CDDP (10 μM), and OLP (10 μM) at 37°C for 24 h). (a) Western blot; (b) Relative grayscale analysis; ***P < 0.001.

[0063] Figure 14 Immunohistochemical staining of E-cadherin and N-cadherin in tumor tissues. (a) Representative micrographs, (b) Quantitative data, ***P < 0.001.

[0064] Figure 15 Western blot analysis of NMT1 and HPCAL1 expression (4T1 cells were incubated with platinum compound 2-4 (10 μM), CDDP (10 μM), and OLP (10 μM) at 37°C for 24 h). (a) Western blot; (b) relative grayscale analysis; ***P < 0.001.

[0065] Figure 16 Western blot analysis of β-Catenin, cyclin D1, and c-Myc expression (4T1 cells were incubated with platinum compounds 2-4 (10 μM), CDDP (10 μM), and OLP (10 μM) at 37°C for 24 h). (a) Western blot; (b) relative grayscale analysis; ***P < 0.001.

[0066] Figure 17 Western blot analysis of HIF-1α, VEGFA, and MMP-9 expression (4T1 cells were incubated with platinum compound 2-4 (10 μM), CDDP (10 μM), and OLP (10 μM) at 37°C for 24 h). (a) Western blot; (b) relative grayscale analysis; ***P < 0.001.

[0067] Figure 18 Immunohistochemical staining of CD34 in tumor tissues. (a) Representative micrographs. (b) Quantitative data. ***P < 0.001.

[0068] Figure 19In vivo antitumor immunity of compounds 2-4, CDDP, and OLP against 4T1 tumors in BALB / c female mice (n=5). *P<0.05, **P<0.01, ***P<0.001. (a) Schematic diagram of the experimental design; (b) Relative body weight of mice during treatment; (c) Survival analysis of mice during treatment; (d) Changes in primary and distant tumor volumes over time; (e) Weights of primary and distant tumors at the end of the experiment; (f) Images of mouse tumors at the end of the treatment period.

[0069] Figure 20 M1 / M2 macrophages and CD4 + 、CD8 + Immunohistochemical staining of T cells. (a) Representative micrographs. (b) Quantitative data of M1 macrophage marker CD86 and M2 macrophage marker CD206. (c) CD4 + and CD8 + Quantification data of T cells. **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0070] The technical solutions of the present invention are further described with reference to specific examples. However, these examples are intended to explain the present invention and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the examples, the experiments were performed in accordance with the techniques or conditions described in the literature in the art or in accordance with the product instructions. Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The experimental reagents used, unless otherwise specified, are conventional biochemical reagents. The experimental methods described, unless otherwise specified, are conventional methods.

[0071] In order to make the objectives, technical solutions and advantages of the present invention more clear, representative embodiments of the present invention are described in detail below, but are not limited thereto.

[0072] Example 1.

[0073] 1. Preparation of tetravalent platinum represented by compound 3

[0074] 1. Synthesis of dihydroxycisplatin (IV) 3-1

[0075]

[0076] Add 1.0 g of cisplatin and 30 mL of distilled water to a 250 mL round-bottom flask and stir to disperse. Slowly add 50 mL of 30% hydrogen peroxide to the reaction system, raise the temperature to 60 ° C and stir to react for 4 h; stop the reaction, let it stand at 4 ° C for crystallization for 12 hours, filter and separate to obtain a yellow solid, add an appropriate amount of distilled water, heat to 80 ° C to dissolve it, let it stand at 4 ° C for crystallization for 12 hours, and filter to obtain compound 3-1 yellow crystals (0.82 g, 74%).

[0077] 2. Synthesis of dihydroxyoxaliplatin (IV) 3-2

[0078]

[0079] To a 250 mL round-bottom flask, 1.0 g of oxaliplatin and 30 mL of distilled water were added and stirred to disperse. 50 mL of 30% hydrogen peroxide was slowly added dropwise to the reaction system, and the temperature was raised to 60 ° C. and stirred for 4 h. The reaction was stopped and the mixture was allowed to crystallize at 4 ° C for 12 hours. The yellow solid was separated by filtration. An appropriate amount of distilled water was added and the mixture was heated to 80 ° C to dissolve it. The mixture was allowed to crystallize at 4 ° C for 12 hours and filtered to obtain compound 3-2 as a white crystal (0.85 g, 78%).

[0080] 2. Preparation of Desloratadine Derivatives Shown as Compound 4

[0081] 1. Preparation of Desloratadine Derivative 4-1

[0082]

[0083] Desloratadine (3.11 g, 10 mmol) and methyl 4-bromobutyrate (2.17 g, 12 mmol) were dissolved in 50 mL of DMF, and anhydrous potassium carbonate (1.66 g, 12 mmol) was added and stirred for 24 h. After the reaction, the solvent was removed in vacuo and the residue was purified by column chromatography to obtain desloratadine ester 5-1 as a pink oily substance (3.31 g, 81%).

[0084] Desloratadine ester 5-1 (3.31 g, 8.1 mmol) was dissolved in 5 mL of ethanol, and 81 mL of 5% NaOH / H2O solution was added, and the mixture was stirred at room temperature for 12 h. After the reaction was completed, the resulting solution was acidified to pH 3-4 and back-extracted with dichloromethane. The aqueous phase was collected and the solvent was removed under vacuum. The product was redissolved in DMF, the supernatant was collected, and the DMF was removed under vacuum to obtain desloratadine derivative 4-1 as a white solid (2.83 g, 89%).

[0085] 2. Preparation of Desloratadine Derivative 4-2

[0086]

[0087] Desloratadine (3.11 g, 10 mmol) and methyl 5-bromovalerate (2.34 g, 12 mmol) were dissolved in 50 mL of DMF. Anhydrous potassium carbonate (1.66 g, 12 mmol) was added and stirred for 24 hours. After the reaction, the solvent was removed in vacuo, and the residue was purified by column chromatography to obtain desloratadine ester 5-2 as a pink oil (3.26 g, 77%).

[0088] Desloratadine ester 5-2 (3.26 g, 7.7 mmol) was dissolved in 5 mL of ethanol, followed by the addition of 77 mL of 5% NaOH / H₂O solution and stirring at room temperature for 12 h. After completion of the reaction, the resulting solution was acidified to a pH of 3-4 and back-extracted with dichloromethane. The aqueous phase was collected and the solvent removed under vacuum. The product was redissolved in DMF, the supernatant was removed, and the DMF was removed under vacuum to yield desloratadine derivative 4-2 as a white solid (2.92 g, 93%).

[0089] 3. Preparation of Desloratadine Derivative 4-3

[0090]

[0091] Desloratadine (3.11 g, 10 mmol) and methyl 6-bromohexanoate (2.51 g, 12 mmol) were dissolved in 50 mL of DMF. Anhydrous potassium carbonate (1.66 g, 12 mmol) was added and stirred for 24 hours. After the reaction, the solvent was removed in vacuo, and the residue was purified by column chromatography to obtain desloratadine ester 5-3 as a pink oil (3.51 g, 81%).

[0092] Desloratadine ester 5-3 (3.51 g, 8.0 mmol) was dissolved in 5 mL of ethanol, followed by the addition of 80 mL of 5% NaOH / H₂O solution and stirring at room temperature for 12 h. After completion of the reaction, the resulting solution was acidified to a pH of 3-4 and back-extracted with dichloromethane. The aqueous phase was collected and the solvent removed under vacuum. The product was redissolved in DMF, the supernatant was removed, and the DMF was removed under vacuum to yield desloratadine derivative 4-3 as a white solid (3.03 g, 89%).

[0093] Example 2.

[0094] Synthesis of Asymmetric Monodesloratadine-Oxaliplatin Quaternary Platinum Derivative 1-1

[0095]

[0096] Desloratadine derivative 4-2 (104 mg, 0.25 mmol), TBTU (82 mg, 0.25 mmol) and TEA (35 μL, 0.25 mmol) were dissolved in 5 mL of DMF and stirred at room temperature for 15 min. Then, oxaliplatin derivative 3-2 (100 mg, 0.23 mmol) was added and vigorously stirred at 50°C in the dark for 48 h. After the reaction was completed, the solvent was evaporated under reduced pressure and the residue was purified by basic alumina silica gel column chromatography. White solid compound 1-1 (57.0 mg, 23.1%) was obtained by column chromatography. The purity of the obtained compound was 97.51% as determined by high performance liquid chromatography (eluent: MeOH / H2O=85:15, T R =4.226min).

[0097] Compound 1-1: 1 H NMR (500MHz, Methanol-d4) δ8.32 (dd, J=4.9, 1.5Hz, 1H), 7.66 (dd, J=7.7, 1.4Hz, 1H), 7.2 6(dd,J=7.7,4.9Hz,1H),7.23(d,J=2.0Hz,1H),7.18(dd,J=8.2,2.1Hz,1H),7.13(d,J=8.2 Hz,1H),3.46-3.38(m,2H),2.90-2.79(m,4H),2.78-2.69(m,2H),2.52-2.43(m,2H),2.43 -2.34(m,4H),2.32-2.17(m,6H),1.67-1.60(m,2H),1.59-1.46(m,6H),1.31-1.21(m,2H). 13 C NMR(126MHz,MeOH-d4)δ183.7,165.5,165.4,157.2,145.6,139.8,138.1,138.0,137.1,134.5,132.7,132.4,130.4,128.9, 125.6,122.7,62.0,60.6,57.6,54.1,54.0,36.3,31.3,31.2,30.9,30.6,29.9,29.8,25.6,23.7,23.6,23.6.MS-ESI:calcd for[M+Na] + :847(M=C 32 H 41 ClN4O7Pt),found:847.HRMS:calcd for[M+H] + :824.2390(M=C 32 H 41ClN4O7Pt),found:824.2408.

[0098] Example 3.

[0099] Synthesis of Symmetrical Quaternary Platinum Derivative 2-1 with Didesloratadine and Oxaliplatin

[0100]

[0101] Desloratadine derivative 4-2 (238 mg, 0.58 mmol), TBTU (186 mg, 0.58 mmol) and TEA (81 μL, 0.58 mmol) were dissolved in 5 mL of DMF and stirred at room temperature for 15 min. Then, oxaliplatin derivative 3-2 (100 mg, 0.23 mmol) was added and vigorously stirred at 50°C in the dark for 48 h. After the reaction was completed, the solvent was evaporated under reduced pressure and the residue was purified by basic alumina silica gel column chromatography. White solid compound 2-1 (95.0 mg, 26.1%) was obtained by column chromatography. The purity of the obtained compound was 96.5% as determined by high performance liquid chromatography (eluent: MeOH / H2O=85:15, T R =5.823min).

[0102] Compound 2-1: 1 H NMR(500MHz,MeOH-d4)δ8.29(dd,J=4.9,1.3Hz,2H),7.62(dd,J=7.7,1.1Hz,2H),7.22(dd ,J=7.7,4.9Hz,2H),7.19(d,J=1.8Hz,2H),7.14(dd,J=8.2,2.0Hz,2H),7.10(d,J=8.2Hz, 2H),3.43-3.33(m,4H),2.87-2.76(m,8H),2.75-2.69(m,2H),2.49-2.40(m,4H),2.40-2. 30(m,10H),2.27-2.13(m,8H),1.60-1.57(m,2H),1.56-1.42(m,10H),1.28-1.19(m,2H). 13 C NMR(126MHz,MeOH-d4)δ182.8,165.2,157.2,145.6,139.8,138.1,137.9,137.1,134.5,132.7,132.4,130.4,1 28.9,125.6,122.7,61.7,57.5,54.0,54.0,35.6,31.3,31.2,30.6,29.9,29.8,25.5,23.7,23.4.MS-ESI:calcd for[M] +:1217(M=C 56 H 66 Cl2N6O8Pt),found:1217.HRMS:calcd for[M+H] + :1216.4040(M=C 56 H 66 Cl2N6O8Pt),found:1216.4030.

[0103] Example 4.

[0104] Synthesis of Symmetrical Quaternary Platinum Derivative 2-2 Containing Didesloratadine and Cisplatin

[0105]

[0106] Desloratadine derivative 4-1 (297 mg, 0.75 mmol), TBTU (241 mg, 0.75 mmol) and TEA (104 μL, 0.75 mmol) were dissolved in 5 mL of DMF and stirred at room temperature for 15 min. Then, cisplatin derivative 3-1 (100 mg, 0.30 mmol) was added and stirred vigorously at 50°C in the dark for 48 h. After the reaction was completed, the solvent was evaporated under reduced pressure and the residue was purified by basic alumina silica gel column chromatography. White solid compound 2-2 (65.0 mg, 20.0%) was obtained by column chromatography. The purity of the obtained compound was 95.3% as determined by high performance liquid chromatography (eluent: MeOH / H2O=85:15, T R =6.424min).

[0107] Compound 2-2: 1 H NMR(500MHz,MeOH-d4)δ8.30(dd,J=4.8,1.2Hz,2H),7.63(d,J=6.9Hz,2H),7.23(dd,J=7.7,4.9Hz,2H),7.19(d,J=1.6Hz,2H),7.1 7-7.09(m,4H),3.44-3.33(m,4H),2.91-2.77(m,8H),2.54-2.41(m,8H),2.40-2.33(m,6H),2.32-2.17(m,6H),1.83-1.72(m,4H). 13C NMR(126MHz,MeOH-d4)δ182.0,157.2,145.6,139.7,138.1,137.9,137.1,134.5,132.7,132.4, 130.4,128.9,125.6,122.7,57.1,54.0,53.9,33.4,31.3,30.6,29.8,29.7,22.3.MS-ESI:calcd for[M] + :1092(M=C 46 H 54 Cl4N6O4Pt),found:1092.HRMS:calcd for[M+H] + :1090.2687(M=C 46 H 54 Cl4N6O4Pt),found:1090.2708.

[0108] Example 5.

[0109] Synthesis of Symmetrical Quaternary Platinum Derivatives 2-3 Containing Didesloratadine and Cisplatin

[0110]

[0111] Desloratadine derivative 4-2 (318 mg, 0.75 mmol), TBTU (240 mg, 0.75 mmol) and TEA (104 μL, 0.75 mmol) were dissolved in 5 mL of DMF and stirred at room temperature for 15 min. Then, cisplatin derivative 3-1 (100 mg, 0.30 mmol) was added and stirred vigorously at 50°C in the dark for 48 h. After the reaction was completed, the solvent was evaporated under reduced pressure and the residue was purified by basic alumina silica gel column chromatography. White solid compound 2-3 (80.0 mg, 23.2%) was obtained by column chromatography. The purity of the obtained compound was 96.9% as determined by high performance liquid chromatography (eluent: MeOH / H2O=85:15, T R =6.961min).

[0112] Compound 2-3: 1H NMR (500MHz, MeOH-d4) δ8.32-8.28(m,2H),7.62(d,J=7.7Hz,2H),7.23(dd,J=7.7,4.9Hz,2H),7.19(d,J=1.7Hz,2H),7.14(dd,J=8.2,2.0Hz,2H),7.10( d,J=8.2Hz,2H),3.44-3.33(m,4H),2.94-2.87(m,4H),2.87-2.78(m,4H),2 .52-2.43(m,8H),2.42-2.31(m,10H),2.30-2.23(m,2H),1.65-1.51(m,8H). 13 C NMR(126MHz,MeOH-d4)δ181.6,156.2,144.8,139.0,137.4,136.3,136.2,133.7,132.0,132.0, 129.6,128.1,124.9,122.0,56.6,53.1,34.5,30.5,29.8,28.8,28.7,24.3,22.6.MS-ESI:calcd for[M+Na] + :1143(M=C 48 H 58 Cl4N6O4Pt),found:1143.HRMS:calcd for[M+H] + :1118.3000(M=C 48 H 58 Cl4N6O4Pt),found:1118.3022.

[0113] Example 6.

[0114] Synthesis of Symmetrical Quaternary Platinum Derivatives 2-4 Containing Didesloratadine and Cisplatin

[0115]

[0116] Desloratadine derivative 4-3 (307 mg, 0.75 mmol), TBTU (241 mg, 0.75 mmol) and TEA (104 μL, 0.75 mmol) were dissolved in 5 mL of DMF and stirred at room temperature for 15 min. Then, cisplatin derivative 3-1 (100 mg, 0.30 mmol) was added and stirred vigorously at 50°C in the dark for 48 h. After the reaction was completed, the solvent was evaporated under reduced pressure and the residue was purified by basic alumina silica gel column chromatography. White solid compound 2-4 (75.0 mg, 22.4%) was obtained by column chromatography. The purity of the obtained compound was 98.9% as determined by HPLC (eluent: MeOH / H2O=85:15, TR =7.119min).

[0117] Compound 2-4: 1 H NMR (500MHz, DMSO-d6) δ8.37-8.31(m,2H),7.57(d,J=6.7Hz,2H),7.29(d,J=2.0Hz,2H),7.24-7.15(m,4H),7.07(d,J=8.2Hz,2H),6.78 -6.22(br,6H,NH3),3.42-3.23(m,6H),2.86-2.63(m,8H),2.43-2.27(m,8H),2.25-2.15(m,10H),1.51-1.34(m,8H),1.29-1.21(m,4H). 13 C NMR (126MHz, DMSO-d6) δ180.7,157.0,146.2,140.0,137.8,137.2,133.1,132.1,131.4,130.7, 128.8,125.5,122.2,57.2,54.0,35.6,30.9,30.4,30.0,29.9,26.4,25.8,25.2.MS-ESI:calcd for[M] + :1148(M=C 50 H 62 Cl4N6O4Pt),found:1148.HRMS:calcd for[M+H] + :1146.3313(M=C 50 H 62 Cl4N6O4Pt),found:1146.3347.

[0118] 3. Experimental Test

[0119] To better understand the essence of the present invention, the following pharmacological experimental results demonstrating the tumor inhibition effects of the compounds in vivo and in vitro are used to illustrate the potential applications of these compounds in the pharmaceutical field. The pharmacological experiments provide partial activity data for some of the compounds. It should be noted that the pharmacological experiments herein are intended to illustrate the present invention and are not intended to limit it. Simple modifications to the present invention based on its essence fall within the scope of protection of the present invention.

[0120] 1. In vitro antitumor activity assay

[0121] In this experiment, the MTT method was used to determine the cell viability. The half-inhibitory concentration (IC 50 ) value, which measures the in vitro anticancer activity of the complex.

[0122] 100 μL of tumor cells in the logarithmic growth phase were inoculated into a 96-well plate with a cell density of 5000-8000 / well, and the last column was reserved as a zero well. Place in a 37°C cell culture incubator for 12 hours, then add 100 μL of compound culture medium solution with gradient concentrations to the 96-well plate, and continue to culture in a 37°C cell culture incubator for 48 hours. Add 20 μL of 5 mg / mL MTT solution to each well of the 96-well plate, culture in a 37°C cell culture incubator for 4 hours, remove the plate, remove the culture medium, add 150 μL of DMSO, and shake in a 37°C shaker in the dark for 20 minutes. Measure the absorbance OD value of each well at 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader to calculate its IC 50 Each set of experiments was repeated at least three times.

[0123] The cancer cell lines used in this experiment include: human lung adenocarcinoma cell line A549, cisplatin-resistant lung adenocarcinoma cell line A549R, mouse breast cancer cell line 4T1, human liver cancer cell line HepG2, and human normal liver cell line LO-2.

[0124] The reference drugs used in this experiment include: desloratadine (DLT), cisplatin (CDDP), oxaliplatin (OLP), satraplatin (STP), and a mixture of cisplatin and desloratadine (CDDP-DLT).

[0125] Discussion of Antitumor Activity:

[0126] Table 1 In vitro antitumor activity of DLT platinum (IV) compounds.

[0127]

[0128] Table 1 a RF: resistance factor, RF = IC 50 (A549R) / IC 50 (A549); b SI: Selectivity index, SI = IC 50 (LO2) / IC 50 (HepG2); c ND: not tested or not calculated; d CDDP-DLT: a mixture of cisplatin and desloratadine in a molar ratio of 1:2.

[0129] The experimental results are shown in Table 1: DLT platinum (IV) complexes 1-1—2-4 have strong anti-proliferative activity against all tumor cell lines, while the anti-tumor activity of hydroxycisplatin tetravalent platinum derivatives 3-1, DLT and DLT derivatives 4-3 is low. These facts indicate that the coupling of DLT with the platinum (IV) system significantly improves the anti-tumor efficacy. Complex 2-1 with a double DLT ligand exhibits better activity than single ligand 1-1. Therefore, we selected the double DLT tetravalent platinum complex for further structural optimization. Compared with the corresponding OLP-derived complex 2-1, complex 2-3 with a CDDP core has higher anti-tumor ability, which indicates that the CDDP core is more conducive to enhancing the anti-tumor effect of the DLT platinum (IV) complex. Subsequently, compounds 2-2—2-4 with different linking groups were designed and synthesized, and the effect of the linking group on the anti-tumor activity was investigated. The results showed that complex 2-4 with a hexanoyl group as the linking group had strong anti-tumor activity, IC 50 The value was lower than 3.04 μM, which was comparable to the antitumor activity of CDDP and better than that of OLP and STP.

[0130] The calculation of drug resistance coefficient is an important method to examine the ability of drugs to overcome drug resistance. 50 The ratio of these values ​​is the resistance factor (RF). The RF value of complex 2-4 (RF = 0.36) is 8.9 times lower than that of CDDP (RF = 3.19) and superior to that of OLP (RF = 1.19), STP (RF = 1.80), and the CDDP-DLT mixture (RF = 1.79). This suggests that DLT platinum (IV) complexes 2-4 have the potential to overcome CDDP resistance.

[0131] To further evaluate the toxicity of DLT platinum (IV) complexes, we tested their toxicity to normal hepatocytes LO2 and calculated the drug selectivity index SI = IC 50 (LO2) / IC 50 (HepG2). Encouragingly, the SI values ​​of DLT platinum (IV) complexes 2-4 (2.15) were higher than those of the control drugs (SI = 0.85-1.37), indicating that this class of drugs can effectively reduce the toxicity of drugs to normal cells.

[0132] In summary, desloratadine tetravalent platinum compounds have good in vitro antitumor activity. The structure of the platinum core has a significant effect on the activity. Desloratadine tetravalent platinum compounds with a CDDP core have significant activity. The connecting group between desloratadine and the platinum core also significantly affects the antitumor effect of desloratadine tetravalent platinum complexes. The bis-desloratadine oxaliplatin tetravalent platinum complex 2-4 with a hexanoyl group has the most significant antitumor effect and can overcome the drug resistance of divalent platinum drugs and reduce toxicity.

[0133] 2. In vivo antitumor activity experiments

[0134] To evaluate the antitumor effects of DLT platinum (IV) complexes in vivo, we evaluated the antitumor activity of dual DLT tetravalent platinum complexes 2-4 in female BALB / c mice bearing 4T1 tumors, using CDDP and OLP as positive control drugs and a saline-treated group as blank.

[0135] BALB / c female mice (18-20 g) were purchased from Shandong Pengyue Laboratory Animal Breeding Co., Ltd. All animals were housed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

[0136] After in vitro expansion of 4T1 cells, the cells were digested and collected, washed three times with saline, and resuspended in saline. Tumor cells were inoculated on the right side of the back of female BALB / c mice at a density of 5 × 10 5 On the third day after inoculation, the tumor was palpable. The mice were randomly divided into 4 groups, 5 mice in each group: saline group, compound 2-4 group, CDDP group, and OLP group, and the dose was 2 mg Pt / kg. The drug was administered on the 3rd, 6th, and 9th days, for a total of 3 times, by intraperitoneal injection ( Figure 1 a) During the experiment, changes in tumor volume were recorded to assess tumor growth rate, and changes in mouse body weight were recorded to evaluate drug toxicity. Mice were sacrificed on day 12, and serum, tumor tissue, and organ tissues (heart, lung, liver, spleen, and kidney) were collected. Tumor and visceral tissues were weighed. Tissue samples were fixed in formalin and evaluated by hematoxylin and eosin (H&E) staining and immunohistochemical analysis.

[0137] Figure 1 The results in b show that compared with the blank group (1034 mm 3 ), the tumor volume was significantly suppressed to 379 mm after treatment with complex 2-4. 3 (P<0.001), and CDDP (456 mm 3 ,P=ns) is equivalent and significantly better than OLP(838mm 3 , P < 0.001). Then, the tumor growth inhibition rate calculated based on tumor weight (TGI = (1-tumor weight of drug-treated group / tumor weight of blank group) × 100%) further verified the anti-tumor efficacy of complex 2-4 ( Figure 1 c and 1e), compared with the blank group, the TGI of complex 2-4 was 61.2% (P < 0.001), which was comparable to that of CDDP (TGI = 58.8%, P = ns) and significantly higher than that of OLP (TGI = 19.6%, P < 0.001). Figure 1The H&E tumor tissue staining image in f shows that the tumor cells in the complex 2-4 treatment group showed apoptosis, with severe degeneration, necrosis, and diffusion of cell nuclei. It is worth noting that Figure 1 The results in d showed that compared with the blank group, the effect of complex 2-4 on the body weight of mice was negligible, while CDDP had a significant effect on the body weight of mice (P<0.01), indicating that the systemic toxicity of compound 2-4 to mice was significantly lower than that of CDDP.

[0138] Subsequently, the organ index (ratio of organ mass to body weight) was calculated to further study the toxicity of platinum drugs ( Figure 2 Compared with the CDDP and OLP treatment groups, complex 2-4 alleviated the drug-induced spleen suppression in mice (P<0.001). Figure 3 H&E staining also confirmed its low toxicity. There were no significant histological differences in the liver, spleen, and kidney tissues of the 2-4 treatment group compared with the blank group. These results further confirmed the low toxicity of 2-4 to mice.

[0139] In vivo experimental results showed that DLT platinum (IV) compounds 2-4 had anti-tumor activity comparable to CDDP and superior to OLP, while also exhibiting reduced toxicity compared to CDDP. These findings demonstrate the potential of desloratadine platinum (IV) compounds 2-4 as anti-tumor drugs.

[0140] 3. In vitro and in vivo metastasis inhibition experiments

[0141] Metastasis is the main cause of death in patients with malignant tumors, and approximately 90% of cancer patients die from tumor metastasis. The present invention investigates the in vivo and in vitro anti-tumor metastasis activities of DLT tetravalent platinum compounds.

[0142] Transwell experiment: The experiment was carried out in a Transwell chamber containing micropores (8 μm pores). First, 4T1 cells (5×10 4 Cells) were resuspended in RPMI1640 (serum-free) medium and seeded in the upper chamber, and 10% FBS-RPMI1640 medium with different compounds (2-4, CDDP and OLP: 10 μM) was added to the lower chamber. Secondly, the cells were incubated in a 37°C incubator containing 5% CO2 for 24 hours. Then, the cells were fixed with 4% paraformaldehyde for 20 minutes and stained with 0.1% crystal violet for 20 minutes. The non-migrating cells in the upper chamber were gently scraped off with a cotton swab. The migrating cells on the lower surface of the chamber were photographed in five random fields of view using an inverted microscope to examine the anti-migratory effect of the drug.

[0143] Scratch test: 4T1 cells (8×10 5) were seeded into six-well cell culture plates and incubated in a 37°C incubator with 5% CO2 for 12 hours. After the cell density reached 90%, a wound was scratched in each well. The cells were then treated with 1% FBS-RPMI1640 medium containing the compounds (2-4, CDDP, and OLP: 10 μM). The degree of wound healing was recorded at 0, 12, and 24 hours to assess the anti-migratory effects of the drugs.

[0144] In vivo anti-metastasis experiment: BALB / c female mice (18-20 g) were used as experimental subjects and 4T1 cells (2×10 5 A lung metastasis model was established. Mice were randomly divided into four groups (n=5): 2-4, CDDP, OLP, and saline (blank group) treatment. Pt was administered three times via intraperitoneal injection on days 4, 7, and 10 after cell inoculation, at a dose of 2 mg Pt / kg. Mice were sacrificed on day 11, and lung tissues were dissected. After fixation with 4% formaldehyde for 24 hours, lung metastases were counted and analyzed in each mouse, and lung tissues were stained with H&E.

[0145] like Figure 4 As shown in the results of Transwell assay, the anti-metastatic ability of double DLT platinum (IV) complexes 2-4 was stronger than that of CDDP and OLP. Figure 5 ) further verified the anti-metastatic ability of compound 5 in vitro.

[0146] In vivo anti-tumor metastasis activity results Figure 6 As shown, compared with the blank group, the inhibition rate of lung nodules after treatment with complexes 2-4 reached 92.4%, while the inhibition rate of platinum (II) drugs was lower, with the inhibition rates of CDDP and OLP being 68.7% and 65.2%, respectively (P<0.001). In addition, Figure 6 The H&E staining image in d showed that the lung metastatic nodules in the complex 2-4 treatment group were fewer and smaller than those in the blank group and the CDDP and OLP treatment groups, further verifying the in vivo anti-tumor metastasis ability of complex 2-4.

[0147] In summary, desloratadine tetravalent platinum complex 2-4 exhibited potent anti-metastatic activity both in vitro and in vivo. In vitro Transwell and wound wound healing assays confirmed its ability to inhibit tumor cell invasion and metastasis. More importantly, in an in vivo lung metastasis model, the compound also demonstrated potent anti-metastatic activity, significantly exceeding that of the divalent platinum reference drugs CDDP and OLP. These results demonstrate its significant potential as a novel anti-metastatic agent.

[0148] 4. In vitro and in vivo tumor uptake experiments

[0149] Uptake is a key factor influencing the efficacy of chemotherapeutic drugs. For platinum complexes, their subcellular distribution within the cell nucleus is crucial for their DNA-damaging anti-tumor mechanism. Therefore, examining the cellular uptake and subcellular distribution of DLT platinum(IV) complexes in tumor cells in vitro and tumor tissues in vivo is crucial for evaluating drug activity.

[0150] 4T1 cells in logarithmic growth phase were placed in 6-well plates (10 6 The cells were cultured in a 37°C, 5% CO2 incubator for approximately 12 hours. The cells were then treated with the drug and cultured for an additional 24 hours. The cells were then digested with EDTA-free trypsin and harvested. The cells were washed twice with PBS, nitrolyzed with concentrated nitric acid, and the platinum content in the cells was determined by atomic absorption spectroscopy (AAS). Drug uptake in tumor cells was then determined. Tumor tissue from different experimental groups was weighed and nitrolyzed, and then platinum content was quantified by AAS to calculate drug uptake.

[0151] Lipophilicity is often a significant factor influencing drug uptake. The lipophilicity of DLT platinum (IV) complexes was determined using log K' calculated based on HPLC results. The lipophilicity of DLT platinum (IV) complexes 1-1-2-4 was determined by the logarithm K' of their retention time in HPLC (85 / 15, MeOH / H2O, flow rate 0.5 ml / min). Column dead time (T0 = 2.896 min) was determined using the KCl method. Retention times and log K' for the compounds are shown in Table 2.

[0152] Table 2. Retention times and log K' of compounds.

[0153]

[0154] Conclusion analysis:

[0155] Figure 7 The results of a and Table 2 show that the lipophilicity of the drug significantly affects the uptake level of tumor cells. The accumulation of DLT platinum (IV) complexes 1-1-2-4 was significantly higher than that of CDDP and OLP (P < 0.001). The drug uptake level of the hexanoyl-containing bis-DLT platinum (IV) complex 2-4 was higher than that of the complexes 2-1, 2-2 and 2-3 containing butyryl and valeryl groups and the complex 1-1 containing a single DLT ligand. In addition, the accumulation of complex 2-4 in tumor tissue was also higher than that of CDDP and OLP ( Figure 7b). The distribution of drugs in tumor cells has a significant impact on their anti-tumor activity and mechanism of action. The enrichment of platinum drugs in DNA is considered a key factor affecting their anti-tumor activity. This experiment examined the intracellular subcellular distribution of conjugates 2-4 in the cytoplasm, cell membrane, and DNA. The results showed that the accumulation of complex 2-4 in DNA was 12.6 times and 22.6 times that of the platinum (II) drugs CDDP and OLP ( Figure 7 c), which would promote its induction of strong DNA damage.

[0156] In summary, DLT platinum (IV) complexes have high uptake levels in tumor cells both in vitro and in vivo and are easily accumulated in DNA, leading to severe DNA damage.

[0157] 5. DNA Damage Experiment

[0158] Platinum drugs primarily exert their anti-tumor activity by binding to the DNA of tumor cells, causing DNA damage. Therefore, detecting the ability of platinum drugs to bind to DNA is crucial and has important implications for exploring its relationship to biological activity.

[0159] The expression of DNA damage-related proteins γ-H2AX and P53 was detected by Western Blot to examine the characteristics of DNA damage.

[0160] Figure 8 Western blot results showed that treatment of 4T1 cells with DLT platinum (IV) complexes 2-4 significantly upregulated the expression of phosphorylated histone γ-H2AX, a protein associated with DNA damage (P<0.001). The expression of P53, another protein associated with DNA damage, was also significantly increased (P<0.001). Upregulation of P53 promotes tumor cell apoptosis and overcomes drug resistance. Therefore, DLT platinum (IV) complexes 2-4 can effectively induce DNA damage in tumor cells.

[0161] 6. Cell Apoptosis Experiment

[0162] To further characterize the cell death pattern induced by the dual DLT platinum(IV) compound 2-4, we used Annexin V-FITC / PI dual staining to examine the drug-induced apoptosis. As apoptosis often involves mitochondria and is accompanied by the production of ROS, we used JC-1 staining to measure changes in mitochondrial membrane potential; DCFH-DA staining to measure intracellular ROS levels; and Western blot analysis to examine the drug's effects on apoptotic proteins. This explored the mechanism by which compound 2-4 induces apoptosis in tumor cells.

[0163] Take 4T1 cells in good logarithmic growth phase and place them in 6-well plates (106 / well), cultured in a 37°C, 5% CO2 incubator for approximately 12 hours, then treated with drugs and cultured for another 24 hours. The cells were digested with EDTA-free trypsin, collected, and divided into three portions. In the first portion, the cells were washed twice with PBS, and 5 μL of Annexin V-FITC stain and 5 μL of PI stain were added, mixed, and reacted at room temperature in the dark for 5-15 minutes. The samples were tested by flow cytometry within 1 hour. The second portion was stained with DCFHDA to detect the production of ROS in the cells, and examined using an inverted fluorescence microscope. The third portion was stained with JC-1 and examined by flow cytometry to examine changes in mitochondrial membrane potential.

[0164] The cells were collected and treated using the above method, proteins were extracted, and proteins were separated by electrophoresis using SDS-Page. The membranes were transferred to PVDF membranes and blocked in 5% skim milk for 1 hour. The membranes were incubated with primary antibodies at 4°C overnight and with secondary antibodies for 1 hour. ECL chemiluminescence developer was then used for color development, and the membranes were imaged using a Tanon 46000SF scanning system.

[0165] Figure 9 The results showed that 4T1 cells underwent significant apoptosis (51.8%) after 24 hours of treatment with complex 2-4, a higher apoptosis rate than CDDP (36.6%) and OLP (27.8%). Mitochondria, as important organelles regulating apoptosis, are closely related to the therapeutic effects of platinum-based drugs. Mitochondrial damage is often accompanied by mitochondrial membrane depolarization (ΔΨm) and the production of ROS, which are key events in initiating apoptosis. Subsequently, JC-1 and DCFHDA staining were used to detect mitochondrial ROS generation and the reduction of ΔΨm in 4T1 cells, respectively. Figure 10 and Figure 11 The results showed that complex 2-4 caused severe mitochondrial damage. Compared with the blank group, complex 2-4 led to severe ΔΨm loss, and its effect was even stronger than CDDP and OLP. At the same time, after treatment with complex 2-4, the amount of ROS generated in tumor cells was significantly increased. Western Blot analysis of proteins related to the mitochondrial apoptosis pathway, such as Bcl-2, Bax, caspase3, and c-caspase3 ( Figure 12 ) showed that complex 2-4 significantly inhibited the expression of the anti-apoptotic protein Bcl-2 (P < 0.001) while enhancing the expression of the pro-apoptotic protein Bax (P < 0.001). The secretion of the apoptosis-executing proteins caspase 3 and c-caspase 3 was also significantly increased (P < 0.001). These results suggest that DLT platinum (IV) complex 2-4 can induce severe mitochondrial-mediated apoptosis through the Bcl-2 / Bax / caspase 3 pathway.

[0166] 7. Inhibit metastasis by inhibiting EMT

[0167] The EMT process can effectively promote tumor cell proliferation and metastasis. EMT is usually manifested by the loss of the epithelial marker E-cadherin, the upregulation of the mesenchymal marker N-cadherin, and the enhancement of the key proteins Vimentin and Snail.

[0168] Western blot was used to detect the expression of EMT-related proteins E-cadherin, N-cadherin, Vimentin, and Snail1 in 4T1 tumor cells using the same detection method as above. Simultaneously, immunohistochemistry was used to detect the expression of E-cadherin and N-cadherin in tumor tissues to examine the effect of DLT platinum (IV) complex on the EMT process.

[0169] The brief steps of immunohistochemistry are: paraffin embedding of formaldehyde-fixed tumor tissue, sectioning, dewaxing, primary antibody incubation, secondary antibody incubation, staining to obtain immunohistochemical sections, and using an inverted microscope to photograph any five areas for analysis.

[0170] like Figure 13 The Western Blot results showed that compared with the blank group, compound 2-4 induced a significant upregulation of E-cadherin expression and a significant downregulation of N-cadherin expression (P<0.001), indicating that the EMT phenotype of tumor cells had changed. At the same time, Vimentin and Snail1 proteins were downregulated (P<0.001). In addition, the immunohistochemical staining results of tumors in vivo further verified the inhibitory effect of compound 2-4 on EMT ( Figure 14 ), compared with the blank group, the expression of E-cadherin in the tumor tissue of the compound 2-4 treatment group was significantly increased (P<0.001), while the expression of N-cadherin was inhibited (P<0.001).

[0171] In summary, DLT platinum (IV) complexes 2-4 have the effect of inhibiting the EMT process in tumor cells and tumor tissues, and thus play a positive role in inhibiting tumor cell metastasis in vitro and in vivo.

[0172] 8. Inhibition of EMT through NMT-1 / HPCAL1 and β-Catenin pathways

[0173] The β-catenin pathway, a highly conserved signaling pathway, plays a key role in the EMT process. Its aberrant activation is involved in the initiation and progression of tumor progression and controls a wide range of cancer hallmarks. β-catenin accumulation, a key event in the β-catenin pathway, can further upregulate the expression of cyclin D1 and c-Myc proteins, promoting tumor cell proliferation, immunosuppression, and metastasis. Recently, it has been reported that DLT can block NMT-1, thereby inhibiting the expression of HPCAL1 and blocking the β-catenin signaling pathway. Furthermore, inhibited HPCAL1 can effectively regulate the Bcl-2 apoptosis pathway, thereby inhibiting cancer progression.

[0174] The expressions of NMT-1 and HPCAL1 in tumor cells 4T1 were detected by Western Blot, and the expressions of β-Catenin pathway-related proteins β-Catenin, cycin D1, and c-Myc were detected using the same detection methods as above.

[0175] like Figure 15 As shown in Figure 2, complex 2-4 significantly inhibited the expression of HPCAL1 in 4T1 cells (P<0.001), and the expression of NMT-1 was also reduced compared with the blank group. At the same time, the expression of β-Catenin, cyclin D1 and c-Myc were all downregulated ( Figure 16 ), indicating that the β-Catenin pathway was inhibited.

[0176] In summary, DLT platinum (IV) complexes 2-4 are expected to further significantly inhibit the EMT process by blocking NMT1 / HPCAL1 signaling and inhibiting the β-Catenin pathway in tumor cells.

[0177] 9. Inhibition of angiogenesis and metastasis experiments

[0178] Angiogenesis is key to the metastasis and growth of invasive tumors and is an important step in controlling tumor development. Vascular endothelial growth factor (VEGF) is a key factor associated with angiogenesis. Furthermore, MMP-9 promotes tumor metastasis by degrading extracellular matrix proteins. Furthermore, studies have demonstrated that MMP-9 and VEGFA exhibit synergistic effects in increasing vascular density and promoting tumor metastasis. EMT can influence angiogenesis by inducing the expression of invasion-related factors such as VEGF, HIF1α, and MMP-9, making it an important factor promoting tumor angiogenesis.

[0179] Western blotting of tumor cells in vitro was used to monitor the expression of VEGFA, MMP-9, and HIF-1α proteins. CD34, a biomarker of angiogenesis, was detected by immunohistochemistry to determine the microvessel density in tumor tissues.

[0180] like Figure 17 As shown in the results, compared with the blank group, the compound 2-4 treatment group significantly reduced the expression of HIF-1α, VEGFA and MMP9 in tumor cells (P<0.001). Immunohistochemistry results showed that compared with the blank group and the CDDP and OLP treatment groups, the angiogenesis marker CD34 in the tumor tissues of the compound 2-4 treatment group was significantly down-regulated. + Significantly decreased (P<0.001) ( Figure 18 ).

[0181] In summary, DLT platinum (IV) complex 2-4 can effectively inhibit tumor angiogenesis by inhibiting the expression of HIF-1α, VEGFA, and MMP9, and has a significant inhibitory effect on the metastasis of tumor cells.

[0182] 10. Activate anti-tumor immunity in the body

[0183] Immunosuppression is an important hallmark of tumors, which is conducive to the formation of TME and the survival of tumor cells, and promotes the immune escape and metastasis of tumor cells. The EMT process in tumor cells is the core driving factor of the immunosuppressive network. Among them, M2 macrophage polarization is closely related to the increase of histamine levels and EMT process, and can further trigger tumor-infiltrating lymphocytes such as CD4 by increasing PD-L1 expression. + and CD8 + T cell exhaustion plays a key role in promoting the immune escape of tumor cells.

[0184] To investigate the efficacy of DLT platinum (IV) complex 2-4 in stimulating anti-tumor immunity, a bilateral 4T1 tumor model was established in immunocompetent female BALB / c mice. 1×10 6 On days 5, 8, 11, and 14, mice were injected intratumorally (it) with a dose of 2 mg Pt / kg; on day 6, mice were subcutaneously injected with 1×10 6 cells to form distal tumors. The experimental process is as follows Figure 19 The growth of tumors on both sides was monitored during the experiment. The inhibitory effect of the drug on untreated distant tumors was attributed to the drug's anti-tumor immune effect. In addition, immunohistochemical staining was used to investigate the effects of the drug on the phenotype of M2 macrophages and CD4 + 、CD8 + The impact of T cells.

[0185] Figure 19The results of b showed that the effect of complexes 2-4 on the body weight of mice was less than that of CDDP (12th day, ***P<0.001) and OLP (15th day, *P<0.001), and thus its toxicity was lower than that of platinum (II) drugs CDDP and OLP. After the third administration, a large number of mice in the CDDP treatment group died, and the survival rate dropped to 20% on day 15 ( Figure 19 c) Therefore, due to the severe toxicity of CDDP, its activity was not further studied.

[0186] Figure 19 df results showed that complex 2-4 exhibited superior in vivo activity against both primary and distal tumors compared to divalent platinum. Complex 2-4's inhibition of primary tumors further validated its superior anti-tumor growth ability. Results showed that complex 2-4 exhibited a stronger inhibitory effect on primary tumor growth than CDDP on day 12 (***P < 0.001). At the end of the experiment, complex 2-4 (TGI = 75.8%) exhibited a significantly greater inhibitory effect on primary tumors than OLP (TGI = 19.0%, ***P < 0.001), a trend consistent with its in vivo anti-tumor activity.

[0187] The volume of distal tumors was monitored and TGI was calculated as an indicator for evaluating antitumor immunity. Figure 19 Results in e and f showed that distal tumor growth was significantly inhibited in the complex 2-4 treatment group, with a TGI of 76.6%. Although OLP has been shown to have the potential to stimulate tumor immunity, its TGI was only 36.3% (***P<0.001).

[0188] M2 macrophages and CD4 + 、CD8 + T cell immunohistochemical staining results ( Figure 20 ) showed that DLT platinum (IV) complexes 2-4 have an inhibitory effect on tumor-promoting M2 macrophages (CD206 + The inhibitory effect of the complex 2-4 treatment group was significantly higher than that of the blank group (P<0.01) and better than that of OLP. + and CD8 + The density of T cells was also significantly increased (P<0.01).

[0189] In summary, DLT platinum (IV) complex 2-4 can regulate macrophage polarization and increase CD4 + and CD8 + The density of T cells can effectively activate anti-tumor immunity, and its ability to activate anti-tumor immunity is stronger than that of divalent platinum OLP.

[0190] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A compound represented by the general formula (I): (I); among them, Selected from cisplatin or oxaliplatin; L is hydroxyl or ; The R3 is selected from propylene, butylene or pentylene.

2. The compound according to claim 1, characterized in that: The compound is selected from: 。 3. The method for preparing the compound according to claim 1 or 2, characterized in that: The synthetic route of the compound is as follows: ， in: When the molar ratio of compound 3 to compound 4 is 1:1.0-1.3, compound 3 and compound 4 undergo a coupling reaction to obtain an asymmetric monosubstituted desloratadine-modified tetravalent platinum compound, wherein L is a hydroxyl group; When the molar ratio of compound 3 to compound 4 is 1:2.2-3, compound 3 and compound 4 undergo a coupling reaction to obtain a symmetrical disubstituted desloratadine modified tetravalent platinum compound, and L is .

4. The preparation method according to claim 3, wherein: The preparation steps of the synthetic route are: Under an inert gas atmosphere, compound 4, a condensing agent, and an organic base are dissolved in an anhydrous organic solvent for reaction, compound 3 is added, and the reaction is carried out in the dark, followed by post-treatment to separate and obtain the target compound; wherein, When the molar ratio of compound 3, compound 4, condensing agent, and organic base is 1:1.0-1.3:1.0-1.3:1.0-1.3; the feeding relationship of compound 3 and organic solvent is that 30-80 ml of organic solvent is added for every 1 g of compound 3, an asymmetric monosubstituted desloratadine-modified tetravalent platinum compound is obtained; When the molar ratio of compound 3, compound 4, condensing agent and organic base is 1:2.2~3:2.2~3:2.2~3; the feeding relationship of compound 3 and organic solvent is that 30~80ml of organic solvent is added for every 1g of compound 3, a symmetrical disubstituted desloratadine modified tetravalent platinum compound is obtained.

5. The preparation method according to claim 4, characterized in that: The inert gas is nitrogen, helium or argon; the condensing agent is TBTU, HATU or EDCI; the organic base is triethylamine, N,N-diisopropylethylamine or 4-dimethylaminopyridine; and the organic solvent is DMF or DMSO.

6. A pharmaceutical composition, characterized in that: The invention comprises the compound according to claim 1 or 2, and pharmaceutically acceptable excipients thereof.

7. Use of the compound according to claim 1 or 2 or the pharmaceutical composition according to claim 6 in the preparation of anti-tumor drugs.

8. The use according to claim 7, characterized in that Application in the preparation of anti-tumor proliferation and anti-tumor metastasis drugs.

9. The use according to claim 8, characterized in that: The anti-tumor proliferation is anti-proliferation of human lung adenocarcinoma, anti-human liver cancer or anti-mouse breast cancer; the anti-tumor metastasis is anti-mouse breast cancer cell metastasis.

10. The use according to claim 8, characterized in that: The anti-tumor proliferation is cisplatin-resistant human lung adenocarcinoma.

11. A combined preparation comprising the compound according to claim 1 or 2 or the pharmaceutical composition according to claim 6, and an anti-tumor drug of the platinum type, paclitaxel type, fluorouracil type, gemcitabine type, vinca alkaloid type or antibody type.

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