A derivative having a tetravalent platinum structure of ciclopirox olamine, and its preparation method and use

Abstract

The present invention provides a derivative having a ciclopirox olamine tetravalent platinum structure, a preparation method, and uses thereof. The compound having a ciclopirox olamine tetravalent platinum tetravalent platinum structure is shown in general formula (I). In view of the synergistic effect of CPX and platinum drugs in damaging mitochondria, the present invention introduces CPX into the platinum (IV) system to construct a series of ciclopirox olamine platinum (IV) anti-tumor complexes. These drugs exert anti-tumor effects by inducing DNA damage, activating mitochondrial autophagy, and activating immunity. The ciclopirox olamine platinum (IV) anti-tumor compounds developed in this research belong to innovative structural systems in the field of pharmaceutical chemistry, aiming to develop new and effective platinum anti-tumor drugs. They are original innovative work and are of great significance.

Description

Technical Field

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

[0002] Cancer is a major cause of unnatural death in humans. Platinum-based anticancer drugs are the most successful antitumor drug examples in the field of pharmaceutical inorganic chemistry. Three platinum (II) drugs, cisplatin (CDDP), carboplatin (CBP) and oxaliplatin (OXP), have been approved worldwide for the treatment of various cancers. However, problems such as poor selectivity, severe side effects, and easy development of drug resistance have severely limited their clinical application. In addition, long-term use of platinum (II) drugs can promote the formation of the tumor microenvironment (TME), inhibit the innate immune response of tumor tissue, and thus inhibit the efficacy of the drug. In recent years, the development of multifunctional platinum (IV) conjugates by introducing a variety of functional ligands into the platinum (IV) system has attracted great attention from researchers and opened up a new path to overcome the shortcomings of classic platinum (II) drugs.

[0003] Autophagy, as an important subcellular process within cells, plays a double-edged sword role in the occurrence and development of tumors. Drug-induced specific autophagy is called type II programmed cell death and is an important target for the development of anti-tumor drugs. Mitochondrial autophagy (Mitophagy), as a special autophagy process, can selectively eliminate mitochondria. It is one of the key steps in maintaining intracellular balance and cell survival, and plays a multifaceted role in the development of cancer. It has been proven that specifically promoting mitochondrial autophagy can disrupt the balance of tumor cells, promote mitochondrial autophagic cell death and apoptosis, and inhibit the development of tumors.

[0004] Immunosuppression is one of the important characteristics of cancer, which helps cancer cells evade immune surveillance and accelerate tumor proliferation and metastasis. Immunotherapy exerts anti-cancer effects by reversing the immunosuppressive TME and has become one of the main means of cancer treatment. Programmed cell death ligand-1 (PD-L1), as one of the most important immune checkpoint proteins (ICP), plays a key role in inducing T cell inactivation and exhaustion in tumor tissues. Downregulating PD-L1 expression during chemotherapy is an effective way to activate anti-tumor immunity. Increasing evidence shows that promoting mitochondrial autophagy has great potential in inhibiting PD-L1 in tumor cells. Therefore, specifically activating mitochondrial autophagy is an ideal anti-tumor strategy that will enhance anti-tumor immunity and promote tumor cell apoptosis.

[0005] Ciclopirox (CPX), as a broad-spectrum fungicide, has significant application potential in the anti-tumor field. It can effectively induce ROS generation and mitochondrial damage, and further promote autophagy and apoptosis of tumor cells. At the same time, in addition to inducing nuclear DNA damage, platinum drugs can also effectively damage mitochondrial DNA, thereby significantly increasing the ROS level of tumor cells. Mitochondrial damage and ROS generation are key initiators of mitochondrial autophagy. Given the synergistic effect of CPX and platinum drugs in damaging mitochondria, we introduced CPX into the platinum (IV) system and constructed a series of ciclopirox platinum (IV) anti-tumor complexes. These drugs exert their anti-tumor effects by inducing DNA damage, activating mitochondrial autophagy, and activating immunity.

[0006] In summary, the ciclopirox olamine platinum (IV) antitumor compound developed in this study represents an innovative structural system in the field of pharmaceutical chemistry, aiming to develop a new and effective platinum-based antitumor drug. This represents original innovation and is of great significance. Summary of the Invention

[0007] In response to the problems existing in the prior art, the present invention proposes a compound having a ciclopirox olamine tetravalent platinum structure. By introducing the ciclopirox olamine molecule into a tetravalent platinum mother nucleus, a series of novel ciclopirox olamine derivative-modified tetravalent platinum compounds are synthesized. This type of ciclopirox olamine tetravalent platinum derivative has significant anti-tumor effects, can effectively damage cell nuclear DNA, promote mitochondrial autophagy, and activate anti-tumor immune responses, providing a new direction for the research and development of anti-tumor drugs.

[0008] To achieve the above-mentioned purpose, the present invention provides a derivative having a tetravalent platinum structure of ciclopirox olamine, the general formula of which is shown as (I):

[0009]

[0010] in, Selected from cisplatin or oxaliplatin; L is Or hydroxy; R3 is selected from propyl, butyl or pentyl.

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

[0012]

[0013] The derivatives of the present invention are selected from:

[0014]

[0015] Another object of the present invention is to provide a method for preparing a derivative represented by general formula (I). The synthetic route of the derivative is as follows:

[0016] Described synthetic route 1 is as follows:

[0017]

[0018] Compound III and compound IV undergo a coupling reaction to obtain a symmetrically disubstituted ciclopirox olamine-modified tetravalent platinum compound I-1; wherein the molar ratio of compound III to compound IV is 1:2.0-3.0.

[0019] Described synthetic route 2 is as follows:

[0020]

[0021] Compound III and compound V undergo a coupling reaction to obtain an asymmetric monosubstituted ciclopirox olamine-modified tetravalent platinum compound I-2; wherein the molar ratio of compound III to compound V is 1:1.0-1.5.

[0022] Furthermore, in the synthetic route 1, the preparation steps of the symmetrically disubstituted ciclopirox olamine modified tetravalent platinum compound I-1 are as follows:

[0023] Under an inert gas atmosphere, compound IV, a condensing agent, and an organic base are dissolved in an anhydrous organic solvent for reaction, compound III is added, and the reaction is carried out in the dark. After post-treatment, the target compound, a symmetrically disubstituted ciclopirox olamine-modified tetravalent platinum compound I-1, is isolated;

[0024] The molar ratio of compound III, compound IV, condensing agent and organic base is 1:2.0-3.0:2.0-3.0:2.0-3.0; the feeding relationship of compound III and organic solvent is 30-80 ml of organic solvent for every 1 g of compound III; the reaction temperature is 10-150° C.; and the reaction time is 12-96 hours.

[0025] In the second synthetic route, the preparation steps of the asymmetric monosubstituted ciclopirox olamine modified tetravalent platinum compound I-2 are as follows:

[0026] Under an inert gas atmosphere, compound III and NHS ester compound V are dissolved in an anhydrous organic solvent for reaction. After the reaction is protected from light, the target compound, asymmetric monosubstituted ciclopirox olamine-modified tetravalent platinum compound I-2, is isolated and obtained through post-treatment.

[0027] The molar ratio of compound III to compound V is 1:1.0-1.5; the feeding relationship between compound III and organic solvent is that 30-80 ml of organic solvent is added for every 1 g of compound III; the reaction temperature is 10-150° C.; and the reaction time is 12-96 hours.

[0028] 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.

[0029] The preparation process can be specifically as follows:

[0030] Synthesis Route 1: TBTU and compound IV (a ciclopirox olamine derivative) are added to a reaction vessel, the air in the system is replaced with nitrogen, anhydrous DMF is added, and the reaction is stirred at room temperature. Anhydrous triethylamine is then added to the reaction system, and the reaction is stirred at room temperature. A tetravalent platinum compound III is then added to the reaction system, and the air in the system is replaced with nitrogen again. The reaction temperature is controlled at 10-150°C, and the reaction is carried out in the dark for 12-96 hours. After the reaction is completed, the solvent is removed under reduced pressure, and column chromatography is performed to obtain a symmetrically disubstituted ciclopirox olamine-modified tetravalent platinum compound I-1.

[0031] Synthesis Route 2: Compound III and compound V are added to a reaction vessel, and anhydrous DMSO is added as a solvent. The air in the system is replaced with nitrogen. The reaction system temperature is controlled at 10-150° C. and the reaction is carried out in the dark for 12-96 hours. After the reaction is completed, the solvent is removed under reduced pressure, and column chromatography is performed to obtain an asymmetric monosubstituted ciclopirox olamine-modified tetravalent platinum compound I-2.

[0032] Further, the compound III is composed of After being oxidized with hydrogen peroxide, the product is prepared. The specific preparation process is as follows:

[0033]

[0034] Divalent platinum compounds II The dihydroxy tetravalent platinum compound III is prepared by oxidizing with hydrogen peroxide at 60-70° C. for 1-8 hours.

[0035] 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.

[0036] 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.

[0037] The ciclopirox olamine 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.

[0038] The ciclopirox olamine 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.

[0039] The administration route of the ciclopirox olamine tetravalent platinum compound of the present invention can be intravenous administration, including intravenous injection, intramuscular injection, intratumoral injection, subcutaneous injection and acupoint injection.

[0040] 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.

[0041] 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 the use in anti-tumor drugs.

[0042] The tetravalent platinum derivative of ciclopirox olamine described in the present invention can have a good therapeutic effect on tumors, induce DNA damage, promote mitochondrial autophagy, effectively activate tumor immune response, and have a good anti-tumor effect.

[0043] Furthermore, the anti-tumor agent is anti-lung cancer, anti-drug-resistant lung cancer, anti-liver cancer or anti-breast cancer, etc.; among them, 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.

[0044] 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 alkaloid or antibody, to achieve a significant anti-tumor effect.

[0045] The ciclopirox olamine 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 combination preparation with anti-tumor activity. The combination 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.

[0046] Compared with the prior art, the compound having a ciclopirox olamine tetravalent platinum structure of the present invention has the following advantages:

[0047] (1) The present invention introduces the ciclopirox olamine molecule into the tetravalent platinum core to synthesize a series of novel ciclopirox olamine derivatives modified with tetravalent platinum compounds, which have been shown to have good anti-tumor and anti-cancer abilities through anti-tumor activity tests.

[0048] (2) This type of ciclopirox olamine tetravalent platinum derivative can induce severe DNA damage in tumor cells and induce cell apoptosis, thereby exerting an anti-tumor effect;

[0049] (3) The mitochondrial autophagy process of tumor cells is closely related to tumor proliferation and immunosuppression in the tumor microenvironment. This type of ciclopirox olamine tetravalent platinum derivative can effectively activate mitochondrial autophagy, thereby exerting an anti-tumor effect;

[0050] (4) Antitumor activity is associated with the activation of immune responses within tumor cells, and this type of ciclopirox olamine tetravalent platinum derivative can effectively activate tumor immune responses;

[0051] (5) The compound of formula I described in the present invention has an innovative structure and 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 platinum (II) drugs, and also opening up new avenues for the modification of tetravalent platinum compounds. In terms of pharmacological activity, the tetravalent platinum compound ciclopirox olamine can effectively damage nuclear DNA, promote mitochondrial autophagy, activate tumor immune response, and thus inhibit tumor proliferation and metastasis. This mechanism of action is significantly different from that of traditional platinum drugs, and can effectively overcome their drug resistance and reduce toxic side effects.

[0052] 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

[0053] Figure 1 Uptake and sub-distribution of different drugs in tumor cells; (a) Platinum content in cell membranes, mitochondria, DNA, and whole cells of 4T1 tumor cells treated with compound 3 (10 μM), CDDP (10 μM), OXP (10 μM), and CDDP+CPX (10 μM / 20 μM) for 24 hours; (b) Platinum content in in vivo tumor tissues, obtained from in vivo anti-tumor experiments. ***P < 0.001.

[0054] Figure 2 To test the stability of compound 3 in biological culture medium, a solution of complex 3 (0.25 mM) was prepared in RPMI1640 and incubated at 37°C for 48 hours. Spectra were recorded at 0, 12, 24, and 48 hours.

[0055] Figure 3 Reducibility of Compound 3 in Reducing Biological Media. A solution containing Compound 3 (0.25 mM) and ascorbic acid (AsA, 1 mM) in RPMI 1640 was prepared and incubated at 37°C for 48 hours. Spectra were measured at 0, 12, 24, and 48 hours.

[0056] Figure 4 DNA-damaging ability of compound 3. A solution containing complex 3 (0.25 mmol), AsA (1 mmol), and 5′-GMP (3 mmol) was prepared in RPMI 1640 and incubated at 37°C for 48 hours. Spectra were measured at 2 and 48 hours. The formation of platinate GMP (an adduct of CDDP and 5′-GMP) was observed.

[0057] Figure 5DNA damage properties of complex 3. Cells were treated with CDDP (10 μM), OXP (10 μM), and complex 3 (10 μM) at 37°C for 24 hours. (a) Bolt image; (b) relative grayscale analysis. ***P < 0.001.

[0058] Figure 6 Flow cytometric analysis of mitochondrial membrane potential in 4T1 cells was performed using JC-1 staining. Cells were treated with CDDP (10 μM), OXP (10 μM), CDDP+CPX (10 μM / 20 μM), and complex 3 (10 μM) at 37°C for 24 hours.

[0059] Figure 7 Flow cytometric analysis of ROS production in 4T1 cells using DCFH-DA staining. Cells were treated with CDDP (10 μM), OXP (10 μM), CDDP+CPX (10 μM / 20 μM), and complex 3 (10 μM) for 24 hours. ***P < 0.001.

[0060] Figure 8 Flow cytometric analysis of 4T1 cell apoptosis using Annexin V-FITC / PI staining. 4T1 cells were incubated with compound 3 (10 μM), CDDP (10 μM), OXP (10 μM), and CDDP + CPX (10 μM / 20 μM) for 24 hours at 37°C. (a) Blank; (b) CDDP (10 μM); (c) OXP (10 μM); (d) CDDP + CPX (10 μM / 20 μM); (e) compound 3; (f) Statistical analysis.

[0061] Figure 9 Western blot analysis of apoptotic protein expression in 4T1 cells after 24 hours of treatment with different compounds. 4T1 cells were incubated with compound 3 (10 μM), CDDP (10 μM), and OXP (10 μM) at 37°C for 24 hours. Western blot analysis was performed to determine the expression of Bcl-2, Bax, Caspase 3, and c-Caspase 3 in 4T1 cells. (a) Bolt plot; (b) Relative grayscale analysis. ***P < 0.001.

[0062] Figure 10 TEM analysis of mitochondrial autophagy. 4T1 cells were incubated with compound 3 (10 μM), CDDP (10 μM), and OXP (10 μM) at 37°C for 24 hours and analyzed by TEM. Mitochondrial damage is indicated by dark arrows. Mitochondrial autophagy is indicated by light arrows.

[0063] Figure 11Western blot analysis of PINK1, Parkin, p62, and LC3 expression in 4T1 cells. 4T1 cells were incubated with compound 3 (10 μM), CDDP (10 μM), and OXP (10 μM) at 37°C for 24 hours, and protein expression was analyzed by Western blot. (a) Bolt image; (b) relative grayscale analysis. ***P < 0.001.

[0064] Figure 12 Immunohistochemical staining of P62 in 4T1 homograft tumor tissue. Tumor tissue was obtained from an in vivo antitumor experiment. (a) Representative images; (b) Quantification of P62 expression. ***P < 0.001.

[0065] Figure 13 Effects of the autophagy inhibitor 3MA on complex 3-induced mitochondrial autophagy in 4T1 tumor cells. (a) MTT assay to assess the effect of 3MA on 4T1 cell survival. 4T1 cells were treated with complex 3 (10 μM), 3 (10 μM) + 3MA (70 μM), CDDP (10 μM), CDDP (10 μM) + 3MA (70 μM), Blank, and 3MA groups for 24 hours, and their survival rates were measured. (b) Western blot analysis of LC3 and p62 expression. 4T1 cells were treated with complex 3 (10 μM), 3 (10 μM) + 3MA (70 μM), and Blank groups for 24 hours. (c) Grayscale analysis. *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significant difference.

[0066] Figure 14 PD-L1 protein and CD4 in 4T1 homograft tumor tissue + and CD8 + Immunohistochemical staining of TIL. Tumor tissue was obtained from an in vivo anti-tumor experiment. (a) Representative photos; (b) PD-L1 expression and CD4 + and CD8 + Quantification data of TIL. ***P<0.001.

[0067] Figure 15 The complexes' inhibitory effects on 4T1 cell migration were evaluated using an in vitro Transwell assay. 4T1 tumor cells were treated with platinum complex 3 (5 μM), CDDP (5 μM), and OXP (5 μM) at 37°C for 24 hours, stained with crystal violet, and photographed for analysis. (a) Representative images. (b) Relative migration rate analysis. ***P < 0.001.

[0068] Figure 16An in vitro scratch wound healing assay was used to evaluate the inhibitory activity of the complexes on 4T1 cell migration. Tumor cells were treated with platinum complex 3 (5 μM), CDDP (5 μM), and OXP (5 μM) at 37°C for 24 hours. Wound healing was assessed by imaging at 0, 12, and 24 hours. (a) Representative images; (b) Wound closure analysis. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant.

[0069] Figure 17 Western blot analysis of MMP protein expression in 4T1 cells after 24 hours of treatment with different compounds. 4T1 cells were incubated with compound 3 (10 μM), CDDP (10 μM), and OXP (10 μM) at 37°C for 24 hours. (a) Bolt image; (b) Relative grayscale analysis. ***P < 0.001.

[0070] Figure 18 .4T1 homograft tumor tissue proteins MMP9 and CD34 + Immunohistochemical staining of microvessels. Tumor tissue was obtained from an in vivo anti-tumor experiment. (a) Representative photos; (b) Quantification of MMP9; (c) CD34 + Quantification data of microvessels. ***P<0.001.

[0071] Figure 19 In vivo antitumor activity of compound 3, CDDP and OXP, and CDDP + CPX in BALB / c mice bearing 4T1 tumors (n = 5). (a) Schematic diagram of experimental design; (b) Changes in mouse body weight during treatment; (c) Tumor growth over time; (d) Tumor weight at the end of the experiment. The TGI of the test drug is given above the bar [TGI = (1 - tumor weight of drug-treated group / tumor weight of control group) × 100%]; (e) Tumor images. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significantly different.

[0072] Figure 20 .H&E staining results of 4T1 tumor tissues in the compound 3, CDDP, OXP and CDDP+CPX treatment groups.

[0073] Figure 21 .H&E staining results of the liver, spleen and kidney of mice in the compound 3, CDDP, OXP and CDDP+CPX treatment groups. DETAILED DESCRIPTION

[0074] The technical solutions of the present invention are further described below with reference to specific examples. However, the 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 techniques or conditions described in the literature in the art or in the product instructions shall be followed. 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 test reagents used, unless otherwise specified, are conventional biochemical reagents. The experimental methods described, unless otherwise specified, are conventional methods.

[0075] Example 1.

[0076] 1. Preparation of tetravalent platinum represented by compound III

[0077] 1. Synthesis of dihydroxycisplatin (IV) III-1

[0078]

[0079] Cisplatin (1.0 g) was added to a stirred solution of 30 mL of distilled water. 50 mL of 30% H₂O₂ was then added dropwise. The mixture was stirred at 60°C for 4 h. The resulting mixture was recrystallized at 4°C and filtered to yield a crude, light-colored solid. Further recrystallization from water afforded III-1 (0.91 g, 81%) as a yellow solid.

[0080] 2. Synthesis of dihydroxyoxaliplatin (IV) III-2

[0081]

[0082] Oxaliplatin (1.0 g) was added to a stirred 30 mL distilled water solution. 50 mL 30% H₂O₂ was then added dropwise. The mixture was stirred at 60°C for 4 h. The resulting mixture was recrystallized at 4°C and filtered to yield a crude, light-colored solid. Further recrystallization from water afforded III-2 (0.83 g, 76%) as a white solid.

[0083] 2. Preparation of Ciclopirox Olamine Derivatives Represented by Compound IV and Compound V

[0084] 1. Preparation of Ciclopirox Olamine Derivative IV-1

[0085]

[0086] Ciclopirox olamine (2.02 g, 10 mmol) and methyl 4-bromobutyrate (2.17 g, 12 mmol) were dissolved in 50 mL of DMF. Anhydrous potassium carbonate (1.66 g, 12 mmol) was then added, and the mixture was stirred for 24 hours. The residue was purified by column chromatography. The CPX ester obtained was a yellow oil (2.46 g, 80%). The ester (2.46 g, 8.0 mmol) was dissolved in 5 mL of ethanol. 8 mL of 5% NaOH / H2O solution was then added, and the mixture was stirred at room temperature for 12 hours. After the reaction was completed, the resulting solution was acidified to pH = 3-4 and extracted with dichloromethane. The organic phase was collected and concentrated to obtain a white ciclopirox olamine derivative IV-1 (1.59 g, 68%).

[0087] 2. Preparation of Ciclopirox Olamine Derivative IV-2

[0088]

[0089] Ciclopirox olamine (2.02g, 10mmol) and methyl 5-bromopentanoate 9 (2.34g, 12mmol) were dissolved in 50mL DMF. Anhydrous potassium carbonate (1.66g, 12mmol) was then added and the mixture was stirred for 24 hours. The residue was purified by column chromatography. The CPX ester obtained was a yellow oil (2.73g, 85%). The ester (2.73g, 8.5mmol) was dissolved in 5mL ethanol. 8mL of 5% NaOH / H2O solution was then added and the mixture was stirred at room temperature for 12 hours. After the reaction was completed, the resulting solution was acidified to pH=3-4 and extracted with dichloromethane. The organic phase was collected and concentrated to give ciclopirox olamine derivative IV-2 (1.93g, 74%).

[0090] 3. Preparation of Ciclopirox Olamine Derivative IV-3

[0091]

[0092] Ciclopirox olamine (2.02g, 10mmol) and methyl 6-bromovalerate (2.51g, 12mmol) were dissolved in 50mL DMF. Anhydrous potassium carbonate (1.66g, 12mmol) was then added and the mixture was stirred for 24 hours. The residue was purified by column chromatography. The CPX ester obtained was a yellow oil (2.71g, 81%). The ester (2.71g, 8.1mmol) was dissolved in 5mL ethanol. 8mL of 5% NaOH / H2O solution was then added and the mixture was stirred at room temperature for 12 hours. After the reaction was completed, the resulting solution was acidified to pH=3-4 and extracted with dichloromethane. The organic phase was collected and concentrated. Ciclopirox olamine derivative IV-3 (2.34g, 90%) was obtained.

[0093] 4. Synthesis of NHS Ester V

[0094]

[0095] EDC (0.59 g, 3.8 mmol) and N-hydroxysuccinimide (NHS) were dissolved in DMF (10 mL) and the mixture was stirred for 15 minutes. IV-1 (1.0 g, 3.4 mmol) was then added, and the reaction was stirred overnight at room temperature under nitrogen. After completion of the reaction, NHS ester V (0.84 g, 64%) was obtained by extraction.

[0096] Example 2.

[0097] Synthesis of Symmetrical Bicyclopyroxolamine Cisplatin Quaternary Platinum Derivative 1

[0098]

[0099] To a solution of IV-1 (220 mg, 0.75 mmol) in anhydrous DMF (5 mL) were added TBTU (240 mg, 0.75 mmol) and triethanolamine (104 μL, 0.75 mmol). The mixture was stirred at room temperature under nitrogen for 15 minutes. Cisplatin (IV) III-1 (100 mg, 0.30 mmol) was then added, and the mixture was stirred at 55°C in the dark for 48 hours. After the reaction, the mixture was concentrated and further purified by column chromatography on silica gel to obtain Compound 1 (53 mg, 20.0%) as a white solid.

[0100] 1 H NMR(500MHz, CDCl3)δ6.44(br,6H,NH3),6.28(s,2H),5.89(s,2H),4.27(t,J=5.9Hz,4H),2.91–2.72(m,6H), 2.15(s,6H),2.03–1.92(m,8H),1.88–1.83(m,4H),1.79–1.75(m,2H),1.43–1.36(m,4H),1.33–1.24(m,6H). 13 C NMR (126MHz, CDCl3) δ181.8,159.5,153.5,150.6,117.4,105.7,74.9,38.2,32.7,31.2,26.5,25.9,24.0,21.5.MS-ESI:calcdfor[M+Na] + :907(M=C 32 H 50 Cl2N4O8Pt),found:907.HRMS:calcd for[M+H] + :884.2726(M=C32 H 50 Cl2N4O8Pt),found:884.2726.

[0101] Example 3.

[0102] Synthesis of Symmetrical Bicyclopyroxolamine Cisplatin Quaternary Platinum Derivative 2

[0103]

[0104] To a solution of IV-2 (230 mg, 0.75 mmol) in anhydrous DMF (5 mL) were added TBTU (240 mg, 0.75 mmol) and triethanolamine (104 μL, 0.75 mmol). The reaction was stirred at room temperature under nitrogen for 15 minutes. Cisplatin (IV) III-1 (100 mg, 0.30 mmol) was then added, and the mixture was stirred at 55°C in the dark for 48 hours. After the reaction, the mixture was concentrated and further purified by column chromatography on silica gel to yield Compound 2 (77 mg, 28.1%) as a white solid.

[0105] 1 H NMR (500MHz, CDCl3) δ6.68–6.37(br,6H),6.33(s,2H),5.84(s,2H),4.12(t,J=6.0Hz,4H),2.76–7.70(m,2H),2 .48(t,J=6.7Hz,4H),2.13(s,6H),2.00–1.89(m,4H),1.87–1.68(m,14H),1.45–1.31(m,4H),1.30–1.16(m,6H). 13 C NMR (126MHz, CDCl3) δ182.7,159.5,153.2,150.4,117.5,105.4,76.1,38.2,35.6,32.6,27.2,26.5,25.9,21.9,21.5.MS-ESI:calcd for[M+Na] + :936(M=C 34 H 54 Cl2N4O8Pt),found:936.HRMS:calcd for[M+H] + :912.3039(M=C 34 H 54 Cl2N4O8Pt),found:912.3043.

[0106] Example 4.

[0107] Synthesis of Symmetrical Bicyclopyroxolamine Cisplatin Quaternary Platinum Derivative 3

[0108]

[0109] To a solution of IV-3 (240 mg, 0.75 mmol) in anhydrous DMF (5 mL) were added TBTU (240 mg, 0.75 mmol) and triethanolamine (104 μL, 0.75 mmol). The reaction was stirred at room temperature under nitrogen for 15 minutes. Cisplatin (IV) III-1 (100 mg, 0.30 mmol) was then added, and the mixture was stirred at 55°C in the dark for 48 hours. After the reaction, the mixture was concentrated and further purified by silica gel column chromatography to yield compound 3 (81 mg, 28.8%) as a white solid.

[0110] 1 H NMR(500MHz, CDCl3)δ6.34(br,8H,NH3,Ph CH),5.87(s,2H),4.21–4.00(s,4H),2.78–.2.70(m 2H),2.42(t,J=7.0Hz,4H),2.14(s,6H),1.99–1.93(m,4H),1.89–1.83(m,4H),1.79–1. 73(m,6H),1.67–1.61(m,4H),1.57–1.50(m,4H),1.45–1.34(m,4H),1.30–1.22(m,6H). 13 C NMR (126MHz, CDCl3) δ183.5,159.8,153.4,150.7,117.5,105.7,65.9,38.4,36.3,32.8,27.6,26.6,26.0,25.5,25.3,21.6.MS-ESI:calcd for[M+Na] + :964(M=C 36 H 58 Cl2N4O8Pt),found:964.HRMS:calcd for[M+H] + :940.3358(M=C 36 H 58 Cl2N4O8Pt),found:940.3372.

[0111] Example 5.

[0112] Synthesis of Symmetrical Bicyclopyrrolamine Oxaliplatin Quaternary Platinum Derivative 4

[0113]

[0114] To a solution of IV-1 (170 mg, 0.58 mmol) in anhydrous DMF (5 mL) were added TBTU (186 mg, 0.58 mmol) and triethanolamine (81 μL, 0.58 mmol). The reaction was stirred at room temperature under nitrogen for 15 minutes. Oxaliplatin (IV) III-2 (100 mg, 0.23 mmol) was then added and stirred at 55°C in the dark for 48 hours. After the reaction, the mixture was concentrated and further purified by silica gel column chromatography to obtain compound 4 (69 mg, 30.6%) as a white solid.

[0115] 1 H NMR(500MHz, CDCl3)δ9.02-8.96(m,2H,NH2),8.40–8.05(m,2H,NH2),6.24(s,2H),5 .88(s,2H),4.40–4.28(m,2H),4.18–4.09(m,2H),3.28–3.22(m,2H),3.01–2.95(m,2 H),2.79–2.73(m,2H),2.62–2.60(m,2H),2.42–2.36(m,2H),2.17(s,6H),2.00–1.91 (m,8H),1.90–1.75(m,6H),1.66–1.56(m,4H),1.41–1.33(m,6H),1.28–1.19(m,6H). 13 C NMR (126MHz, CDCl3) δ182.6,164.4,159.4,153.5,150.7,117.2,105.5,74.7,62.3,47. 0,38.2,32.9,32.4,31.9,31.8,26.5,26.4,25.9,24.1,24.0,21.5,8.8.MS-ESI:calcd for[M+Na] + :1005(M=C 40 H 58 N4O 12 Pt),found:1005.HRMS:calcd for[M] + :981.3699(M=C 40 H 58 N4O 12 Pt),found:981.3770.

[0116] Example 6.

[0117] Synthesis of asymmetric monocyclopyroxolamine cisplatin tetravalent platinum derivative 5

[0118]

[0119] NHS ester V (140 mg, 0.36 mmol) was dissolved in anhydrous dimethyl sulfoxide (5 mL). Cisplatin (IV) III-1 (100 mg, 0.30 mmol) was added, and the mixture was stirred at 75°C under a nitrogen atmosphere for 24 hours. After the reaction, the mixture was concentrated and further purified by silica gel column chromatography to obtain compound 5 (46 mg, 25.4%) as a white solid.

[0120] 1 H NMR(500MHz,DMSO-d6)δ6.16(s,1H),6.14–5.66(br,7H,NH3,Ph CH),4.14(t,J=6.5Hz,2H),2.81–2.75(m,1H),2.35(t,J=7.4Hz,2H),2.10(s,3 H),1.93–1.85(m,4H),1.79–1.69(m,2H),1.44–1.37(m,2H),1.31–1.06(m,4H). 13 C NMR(126MHz,DMSO-d6)δ180.5,158.4,153.6,150.1,117.1,104.1,75.6,37.9,32.9,32.4,26.4,25.9,24.8,21.4.MS-ESI:calcd for[M+Na] + :633(M=C 16 H 29 Cl2N3O5Pt),found:633.HRMS:calcd for[M+H] + :609.1205(M=C 16 H 29 Cl2N3O5Pt),found:609.1203.

[0121] Example 7.

[0122] Synthesis of asymmetric monocyclopyroxolamine oxaliplatin tetravalent platinum derivative 6

[0123]

[0124] NHS ester V (109 mg, 0.28 mmol) was dissolved in anhydrous dimethyl sulfoxide (5 mL). Oxaliplatin (IV) III-2 (100 mg, 0.23 mmol) was added, and the mixture was stirred at 75°C under a nitrogen atmosphere for 24 hours. After the reaction, the mixture was concentrated and further purified by silica gel column chromatography to obtain Compound 6 (47 mg, 29.0%) as a white solid.

[0125] 1H NMR (500MHz, DMSO-d6) δ8.71–8.01(m,2H,NH2),7.50(d,J=360.0Hz,2H,NH2),6.15(s,1H),5.93(s,1H),4.09(t,J=6.2Hz,2H),2.77– 2.71(m,1H),2.61–2.52(m,2H),2.40(t,J=7.4Hz,2H),2.14–2.00(m,5H),1.90–1.81(m,4H),1.78–1.68(m,2H),1.54–1.01(m,12H). 13 C NMR (126MHz, DMSO-d6) δ181.1,163.8,157.9,153.0,149.7,116.6,103.7,74.8,61.4, 60.1,37.4,32.8,31.9,30.9,30.7,25.9,25.5,24.1,23.7,23.6,20.9.MS-ESI:calcd for[M+Na] + :729(M=C 24 H 37 N3O9Pt),found:729.HRMS:calcd for[M+H] + :707.2250(M=C 24 H 37 N3O9Pt),found:707.2253.

[0126] 5. Experimental Test

[0127] 1. In vitro antitumor activity assay

[0128] In this experiment, the MTT method was used to determine the cell viability. The half-inhibitory concentration (IC 50 ) value to measure the in vitro anticancer activity of the complex. 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 it, 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 50Each experiment was repeated at least three times. The cancer cell lines used in this study included: 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. The reference drugs used in this study included: ciclopirox olamine (CPX), cisplatin (CDDP), oxaliplatin (OLP), satraplatin (STP), and a mixture of cisplatin and ciclopirox olamine (CDDP+CPX).

[0129] The experimental results are shown in Table 1: The CDDP-based double CPX platinum (IV) complexes 1-3 showed strong antitumor activity against all tested tumor cells, with IC 50 The value was lower than 14.96 μM. At the same time, the ligand CPX was almost non-toxic to these cell lines. These evidences indicate that the anti-proliferative activity of the CPX-platinum (IV) system is closely related to the platinum core. Compared with the corresponding CDDP derivative 1, the anti-tumor efficacy of complex 4 with OXP as the parent core was significantly reduced, indicating that CDDP is more conducive to the anti-tumor ability of the CPX-platinum (IV) system. The activity of single CPX-platinum (IV) conjugates 5-6 was relatively lower than that of the corresponding double CPX-platinum (IV) mixtures 1 and 4. In addition, the bridging group between CPX and the platinum core has a significant effect on the anti-proliferative activity, among which the complex 3 with a hexanoyl group (symmetrical bicyclopyroxolamine cisplatin tetravalent platinum derivative 3) has the highest activity (IC 50 ≤7.28μM), which was relatively superior to complexes 1 and 2 with butyryl and valeryl bridge chains.

[0130] Drug resistance is a major problem for platinum(II) drugs in the clinic. To determine the ability of CPX platinum(IV) compounds to overcome drug resistance, we conducted a 3-D-PCR-based 50 The resistance factor (RF) was calculated based on the values ​​of the two CPX platinum (IV) mixtures 1-3. The RF values ​​ranged from 0.28 to 0.33, which were all able to effectively overcome the resistance of CDDP (RF = 1.89). The RF values ​​of the single CPX complex 5 and the CDDP + CPX mixture were higher, at 1.24 and 1.85, respectively. In addition, the selectivity index (SI) (IC of normal hepatocytes LO2) was 50 IC values ​​of HepG2 cells 50 The ratio of the values ​​(SI = 2.64) showed that complex 3 was relatively less toxic to normal cells (SI = 2.64) compared with CDDP and OXP (SI = 1.40 and 0.87) and the CDDP+CPX mixture (SI = 0.53).

[0131] Therefore, the CPX-platinum(IV) framework is a promising anti-tumor drug structure. In particular, the double CPX-platinum(IV) complex 3 containing a CDDP core exhibits the most potent anti-tumor efficacy, effectively reducing CDDP toxicity and overcoming its drug resistance.

[0132] Table 1 In vitro antitumor activity of CPX platinum (IV) compounds.

[0133]

[0134]

[0135] 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+CPX: a mixture of cisplatin and ciclopirox olamine, with a molar ratio of 1:2.

[0136] 2. In vitro and in vivo tumor uptake experiments

[0137] The cellular uptake of drugs plays a crucial role in the DNA damage and antitumor activity of platinum drugs. Therefore, detecting the cellular uptake and subcellular distribution of CPX platinum (IV) complexes in tumor cells in vitro and tumor tissues in vivo is of great significance for evaluating drug activity.

[0138] 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.

[0139] The whole cell platinum content in tumor cells treated with complex 3 was higher than that in CDDP, OXP and CDDP+CPX groups (P<0.001). At the same time, the accumulation of compound 3 in mitochondria and DNA was significantly higher than that in CDDP, OXP and CDDP+CPX groups (P<0.001). Figure 1a). The uptake of CPX platinum (IV) complex 3 in tumor tissue in vivo was also significantly higher than that of CDDP, OXP and CDDP+CPX (P<0.001) ( Figure 1 b). This indicates that CPX platinum (IV) complexes readily enter tumor cells and accumulate in the nucleus and mitochondria, further causing DNA and mitochondrial damage in tumor cells.

[0140] 3. DNA Damage Experiment

[0141] DNA damage is the primary mode of action of platinum-based drugs in inducing apoptosis in tumor cells. Platinum(IV) complexes should be reduced to their divalent form before interacting with DNA. Given the high accumulation of CPX-platinum(IV) complex 3 in DNA, we evaluated its DNA-damaging properties using high-performance liquid chromatography and Western blotting.

[0142] In this study, the stability of complex 3 in biological media and its ability to be reduced in a reducing environment were tested by high-performance liquid chromatography. Chromatographic analysis was performed on an Agilent C18 column (250 mm × 4.6 mm, 5 μm) using a ThermoUltimate 3000RS spectrometer. The mobile phases were: phase A was water containing 0.1% trifluoroacetic acid, and phase B was methanol. The gradient was as follows: 10% phase B (0 to 5 minutes); 10% to 60% phase B (linear increase from 5 to 25 minutes); 60% to 90% phase B (linear increase from 25 to 26 minutes); and 90% phase B (26 to 34 minutes). The flow rate was 1.0 mL / min. Spectra were recorded using a UV detector at λ = 240 nm. To test stability, solutions of complex 3 (0.25 mM) were prepared in RPMI1640 biological culture medium and assayed at 0, 12, 24, and 48 hours. To test its reducibility in a reducing environment, a solution of complex 3 (0.25 mM) in RPMI1640 containing AsA (1 mM, a concentration similar to that in TME) was prepared and tested at 0, 12, 24, and 48 hours. To verify its DNA binding properties, a mixed solution of complex 3 (0.25 mM), AsA (1 mM), and 5′-GMP (3 mM) in RPMI1640 was prepared and monitored for 48 hours.

[0143] Figure 2 The HPLC results in Figure 3 showed that complex 3 remained stable in the biological culture medium RPMI1640 for at least 48 hours. Then, complex 3 was easily reduced in RPMI1640 containing reduced ascorbic acid (AsA, 1 mM, similar to the concentration in TME) ( Figure 3Guanosine-5′-monophosphate (5′-GMP, 3 mM) was added to the solution as a DNA base model. HPLC detection revealed that the platinum (II) complex released by platinum (IV) reduction could bind to 5′-GMP to form a platatinate GMP peak ( Figure 4 ), indicating that complex 3 has DNA binding properties. To further verify the DNA damage properties of complex 3, we used Western blot to measure the expression of DNA damage indicator protein γ-H2AX. Figure 5 The Bolt images in the figure confirm that complex 3 significantly increased γ-H2AX expression compared to the blank group (P<0.001). This shows that CPX platinum (IV) complex 3 remains stable in biological media and is easily reduced to release platinum (II) in the TME, causing significant DNA damage and accompanied by upregulation of γ-H2AX expression.

[0144] 4. Mitochondrial Damage Experiment

[0145] Mitochondria are the primary organelles that provide energy for all metabolic processes and play a crucial role in regulating apoptosis. ROS are a key factor in inducing mitochondrial damage, which in turn induces ROS production. Given the high accumulation of CPX platinum(IV) complex 3 in mitochondria, mitochondrial damage was assessed using JC-1 staining, and ROS production in cells was detected by flow cytometry using DCFH-DA staining.

[0146] Figure 6 The results showed that CPX platinum (IV) complex 3 could effectively induce mitochondrial membrane depolarization (ΔΨm) (31.9%), which was even better than CDDP (22.6%) and OXP (16.3%) and CDDP + CPX mixture (24.0%). Compared with the blank group, CPX platinum (IV) also significantly increased the production of ROS in 4T1 cells (P < 0.001), which was higher than that of CDDP, OXP and CDDP + CPX groups (P < 0.001) ( Figure 7 These results indicate that CPX platinum (IV) complex 3 can effectively cause mitochondrial damage by inducing mitochondrial membrane depolarization and ROS generation.

[0147] 5. Apoptosis Induction Experiment

[0148] Apoptosis is the main form of programmed cell death. DNA damage and mitochondrial damage are key events in initiating apoptosis in tumor cells. To determine the apoptosis-inducing properties of complex 3, we used Annexin V-FITC / propidiumiodide (PI) double staining and flow cytometry to detect cell apoptosis. The results showed that complex 3 could effectively induce apoptosis in 4T1 cells (39.6%), which was superior to CDDP (26.2%) and OXP (12.8%) ( Figure 8 Notably, the apoptosis rate (31.5%) of the mixture of CDDP and CPX (CDDP+CPX) was not significantly increased compared to free CDDP. These findings suggest that the construction of the CPX-platinum(IV) system is key to the anti-tumor effects of CPX-platinum(IV) compounds.

[0149] Subsequently, Western Blot was used to detect the changes in apoptotic proteins, including Bcl-2, Bax, Caspase3 and c-Caspase3, in 4T1 cells after treatment with different compounds for 24 h. Figure 9 Results showed that compared with the blank control group, complex 3 significantly reduced the expression of the anti-apoptotic protein Bcl-2 (P < 0.001). Furthermore, the ratio of the apoptosis execution protein c-Caspase3 / Caspase3 was significantly increased (P < 0.001), similar to the results for CDDP and OXP. Therefore, CPX platinum (IV) complex 3 can effectively induce apoptosis in tumor cells through the Bcl-2 / Bax / Caspase3 pathway.

[0150] 6. Induction of Mitochondrial Autophagy Experiment

[0151] It is well established that mitochondrial dysfunction is a key factor in the induction of mitophagy. Given the severe mitochondrial damage induced by CPX-platinum(IV) compound 3, we investigated whether CPX-platinum(IV) complexes could trigger mitophagy using multiple methods.

[0152] Transmission electron microscopy (TEM) is the gold standard for detecting the presence of autophagy, and we used this method to study mitophagy ( Figure 10 The results showed that CPX platinum (IV) compound 3 caused severe damage to mitochondria, destroying the cristae structure (a special structure of mitochondria) in the mitochondria and forming vacuoles. Subsequently, autophagosomes containing mitochondrial degradation remnants were observed in compound 3 cells, indicating the occurrence of mitophagy.

[0153] It is generally believed that the PINK1 / Parkin axis is a typical pathway mediating mitophagy, inducing the conversion of LC3I to LC3II to initiate mitophagy. p62 is a key protein involved in autophagic degradation. Therefore, we measured the expression of PINK1, Parkin, p62, and LC3 in 4T1 cells by Western blotting. Figure 11 The results in the study demonstrated that PINK1 and Parkin increased in cells treated with complex 3, indicating that mitochondria were ubiquitinated. Subsequently, downregulation of p62 and upregulation of LC3-II / I were observed, confirming the initiation of the autophagic degradation process. In addition, the mitophagy-inducing properties of complex 3 were also confirmed by immunohistochemical staining, indicating that complex 3 significantly inhibited the expression of p62 in tumor tissues ( Figure 12 ), which is basically consistent with the in vitro experimental results. In summary, CPX platinum (IV) compound 3 can effectively destroy mitochondria and further induce mitophagy in tumor cells.

[0154] Given the dual role of mitophagy in cancer, whether autophagy induced by CPX platinum (IV) complex 3 promotes apoptosis of tumor cells is a key question. To clarify this issue, we investigated the effect of the mitophagy inhibitor 3-methyladenine (3MA) on the survival rate of 4T1 cells treated with complex 3 (10 μM) and examined the expression changes of p62 and LC3. The results confirmed that the addition of 3MA reduced the killing effect of complex 3 on 4T1 cells (P < 0.001), while 3MA had no significant effect on the survival rate of CDDP and blank groups ( Figure 13 Furthermore, compared to the compound 3-treated group, the 3+3MA group showed increased p62 expression and a decreased LC3II / I ratio. This suggests that 3MA can inhibit compound 3-induced tumor cell apoptosis by inhibiting CPX platinum (IV) complex-induced mitophagy. These findings suggest that the mitophagy triggered by compound 3 in this work is pro-apoptotic autophagy.

[0155] 7. Immune Activation Experiment

[0156] Reversing the immunosuppressive TME plays a key role in inhibiting tumor development. Activation of mitochondrial autophagy is associated with the suppression of PD-L1 expression in tumor cells and further promotes the activation of T cell immunity. Therefore, to evaluate the effect of CPX platinum (IV) complex on tumor immunity, we used immunohistochemistry to detect PD-L1 expression in tumor tissues and CD4 + and CD8 + Tumor-infiltrating T lymphocytes (TILs) were immunohistochemically stained.

[0157] Figure 14The results showed that CPX platinum (IV) complex 3 can effectively inhibit the expression of PD-L1 in tumor tissue in vivo (8.6% of the blank group, P < 0.001), which is significantly better than CDDP (56.8%, P < 0.001) and OXP (38.6%, P < 0.001). At the same time, the CDDP + CPX mixture can also significantly inhibit the expression of PD-L1 (15.6%, P < 0.001). These facts indicate that CPX ligand is the key to CPX platinum (IV) complex activating anti-tumor immunity by inhibiting PD-L1. Subsequently, CD4 + and CD8 + TILs increased by 2.5-fold and 3.9-fold compared to the blank group, respectively (P < 0.001), indicating that T cell immunity was activated in vivo. In summary, CPX platinum (IV) complex 3 effectively activated T cell anti-tumor immunity by blocking the function of PD-L1 in tumors.

[0158] 8. Anti-tumor migration and anti-angiogenesis experiments

[0159] Metastasis and invasion are key to tumor progression, and tumor progression is closely related to the migration ability of tumor cells. Therefore, we used transwell assay and wound healing assay to evaluate the migration inhibition properties of complex 3 (5 μM) on 4T1 cells. Figure 15 ), complex 3 can significantly inhibit tumor cell metastasis, reducing it to 15.3% of the blank group (P<0.001), which is more effective than CDDP (48.6%, P<0.001) and OXP (63.3%, P<0.001). Figure 16 The scratch healing results in further verified the anti-tumor metastasis ability of complex 3.

[0160] The degradation and angiogenesis of extracellular matrix (ECM) proteins are crucial for promoting the metastasis of tumor cells. MMP-9, the enzyme responsible for degrading ECM, can effectively promote tumor angiogenesis, thereby further promoting tumor metastasis. In this work, the expression of MMP-9 in tumor cells was detected by Western Blot, and its expression in tumor tissues was immunohistochemically stained. Then, CD34 immunohistochemical staining was used to detect angiogenesis in tumor tissues. Compared with the blank group and the CDDP and OXP treatment groups, complex 3 can effectively inhibit the expression of MMP-9 ( Figure 17 ), and MMP-9 in the tumor tissue of the complex 3 treatment group was also significantly downregulated (P < 0.001) ( Figure 18 ). Subsequently, CD34 +Microvessel staining results showed that the angiogenesis rate in the compound 3-treated group was reduced to 12.9% of that in the blank group. In summary, CPX platinum (IV) complex 3 can effectively inhibit tumor cell metastasis by inhibiting the expression of MMP-9 and angiogenesis in tumors.

[0161] 9. In vivo anti-tumor experiments

[0162] To evaluate the in vivo antitumor efficacy of CPX-platinum(IV) complexes, we evaluated the antitumor activity of the bis-CPX tetravalent platinum complex 3 in female BALB / c mice bearing 4T1 tumors, using CDDP and OLP as positive controls. 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.

[0163] 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 5 groups, 5 mice in each group: blank group, compound 3 group, CDDP group, OLP group, CDDP+CPX group, and the dosage was 2 mg Pt / kg. The drug was administered on the 6th, 9th, and 12th days, for a total of 3 times, by tail vein injection ( Figure 19 a) During the experiment, changes in tumor volume were recorded to assess tumor growth rate, and changes in mouse body weight were recorded to assess drug toxicity. Mice were sacrificed on day 13, 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.

[0164] The results showed that complex 3 significantly inhibited the growth of tumors in vivo, compared with the blank group (1422 mm 3 , P < 0.001), complex 3 suppressed the tumor volume to 511 mm 3 , TGI = 57.1%, and CDDP (531mm 3 , TGI=56.2%) and CDDP+CPX(577mm 3 , TGI = 44.2%) (P = ns) ( Figure 19 ce). H&E staining of tumor tissues showed that severe nuclear necrosis and dispersion were observed in the tumors of the complex 3 treatment group, indicating that it has a significant tumor killing effect in vivo, which is similar to CDDP and OXP ( Figure 20In terms of toxicity, CPX platinum (IV) complex 3 has significant advantages over the platinum (II) precursor CDDP and CDDP + CPX mixture ( Figure 19 b). Mouse body weight is an indicator of systemic drug toxicity. The effect of complex 3 on body weight was negligible compared with the blank group (P=ns). However, CDDP caused a significant decrease in mouse body weight (P<0.01), and the CDDP+CPX mixture failed to alleviate the in vivo toxicity of CDDP (P=ns). No significant histological differences were found in the liver, spleen, and kidney of the complex 3-treated group compared with the blank group ( Figure 21 These facts indicate that CPX platinum (IV) complex 3 can effectively inhibit tumor growth in vivo, with activity comparable to that of the platinum (II) drug CDDP and lower toxicity than CDDP.

[0165] 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 having a ciclopirox olamine tetravalent platinum structure as shown in the general formula (I): in, is selected from cisplatin; L is Or hydroxy; R3 is selected from propyl, butyl or pentyl.

2. The compound according to claim 1, characterized in that: The compound is selected from:

3. The compound according to claim 2, characterized in that: The compound is selected from:

4. The method for preparing the compound according to any one of claims 1 to 3, characterized in that: (1) The synthetic route of the symmetrically disubstituted ciclopirox olamine modified tetravalent platinum compound I-1 is as follows: Compound III and compound IV undergo a coupling reaction to obtain a symmetrically disubstituted ciclopirox olamine-modified tetravalent platinum compound I-1; wherein the molar ratio of compound III to compound IV is 1:2.0-3.0; (2) The synthetic route of the asymmetric monosubstituted ciclopirox olamine modified tetravalent platinum compound I-2 is as follows: Compound III and compound V undergo a coupling reaction to obtain an asymmetric monosubstituted ciclopirox olamine-modified tetravalent platinum compound I-2; wherein the molar ratio of compound III to compound V is 1:1.0-1.

5.

5. The method for preparing the compound according to claim 4, wherein: The preparation steps of the synthetic route are: (1) The preparation steps of the symmetrically disubstituted ciclopirox olamine modified tetravalent platinum compound I-1 are as follows: Under an inert gas atmosphere, compound IV, a condensing agent, and an organic base are dissolved in an anhydrous organic solvent for reaction, compound III is added, and the reaction is carried out in the dark. After post-treatment, the target compound, a symmetrically disubstituted ciclopirox olamine-modified tetravalent platinum compound I-1, is isolated; The molar ratio of compound III, compound IV, condensing agent, and organic base is 1:2.0-3.0:2.0-3.0:2.0-3.0; the feeding relationship of compound III and organic solvent is 30-80 ml of organic solvent for every 1 g of compound III; the reaction temperature is 10-150° C.; and the reaction time is 12-96 hours. (2) The preparation steps of the asymmetric monosubstituted ciclopirox olamine modified tetravalent platinum compound I-2 are as follows: Under an inert gas atmosphere, compound III and NHS ester compound V are dissolved in an anhydrous organic solvent for reaction. After the reaction is protected from light, the target compound, asymmetric monosubstituted ciclopirox olamine-modified tetravalent platinum compound I-2, is isolated and obtained through post-treatment. The molar ratio of compound III to compound V is 1:1.0-1.5; the ratio of compound III to organic solvent is 30-80 ml of organic solvent per 1 g of compound III; the reaction temperature is 10-150° C.; and the reaction time is 12-96 hours. The compound III is composed of After oxidation with hydrogen peroxide, it is prepared. The preparation process is as follows: Divalent platinum compounds II The compound III is prepared by oxidizing with hydrogen peroxide at 60-70° C. for 1-8 hours.

6. The method for preparing the compound according to claim 5, characterized in that: 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.

7. A pharmaceutical composition, characterized in that: The invention comprises the compound represented by the general formula (I) as claimed in claim 1 or 2, and pharmaceutically acceptable excipients thereof.

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

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

10. A combined preparation comprising the compound of formula (I) according to claim 1 or 2 or the pharmaceutical composition according to claim 7, and an anti-tumor drug of the paclitaxel class, fluorouracil class, gemcitabine class, vinca alkaloid class or antibody class.

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