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

Abstract

The present invention provides a derivative having a tetravalent platinum structure of cetirizine, as well as its preparation method and use. The compound having a tetravalent platinum structure of cetirizine is represented by general formula (I), wherein, #imgabs0#, #imgabs1# is selected from cisplatin or oxaliplatin; L is a hydroxyl group or #imgabs2#. The tetravalent platinum structure of cetirizine described in the present invention inhibits histamine (HA) secretion in tumors through the functional group cetirizine, blocks the HA / HRH1 signaling axis, inhibits the PI3K / AKT / mTOR pathway, and thereby inhibits tumor angiogenesis; induces DNA damage through the platinum nucleus; and the two synergistically exert anti-tumor effects. The compound has significant anti-tumor ability, especially excellent therapeutic effects on metastatic malignant tumors. It is expected to provide a new drug candidate for the clinical treatment of tumors and a new direction for the research and development of new platinum drugs and anti-metastatic malignant tumor drugs.

Description

Technical Field

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

[0002] Despite continuous advances in surgical, chemotherapy, and radiotherapy techniques, malignant tumors remain a major threat to human health. Chemotherapy, as the cornerstone of cancer treatment, is the primary modality. Among them, divalent platinum drugs such as cisplatin (CDDP), carboplatin (CBP), and oxaliplatin (OXP) have advantages such as high efficacy and a broad anti-tumor spectrum against solid tumors such as testicular, ovarian, bladder, colorectal, lung, and head and neck cancers, and thus occupy a key position in clinical practice. However, with the widespread use of platinum drugs, their efficacy is severely hampered by dose-dependent toxicity and the acquisition of drug resistance. Metastatic tumors are a key cause of cancer mortality, accounting for approximately 90% of cancer deaths, yet divalent platinum drugs have very low efficacy against metastatic cancers. Overcoming the inherent drawbacks of divalent platinum drugs and further improving their anti-metastatic properties are expected to bring new hope to the development of platinum drugs. Tetravalent platinum complexes serve as prodrugs for divalent platinum drugs. Designing multifunctional tetravalent platinum complexes by incorporating different functional ligands at the axial position provides an effective strategy for improving platinum drugs. The development of novel multifunctional tetravalent platinum complexes is an effective way to explore new anti-proliferative and anti-metastatic platinum drugs.

[0003] Tumor angiogenesis is the primary pathway for delivering nutrients, oxygen, metabolites, and chemical mediators to tumor tissues, and is a crucial step in tumor progression. More importantly, angiogenesis is a key factor in tumor metastasis, providing a pathway for tumor cells to spread from the primary tumor to distant organs. The PI3K / AKT / mTOR pathway plays a crucial role in multiple cellular processes, and its aberrant activation within the tumor microenvironment (TME) can promote tumor angiogenesis and metastasis. Furthermore, a hypoxic TME is a key factor in activating PI3K / AKT / mTOR signaling, accelerating angiogenesis through the hypoxia-inducible factor (HIF) / vascular endothelial growth factor (VEGF) signaling axis. An immunosuppressive TME is a key hallmark of cancer, significantly impacting tumor growth, metastasis, and treatment by promoting tumor cell evasion of immune surveillance within the TME. Reversing a "cold" TME to a "hot" one has become a promising approach for cancer therapy, offering new hope. As one of the major immune checkpoints (ICPs), the PD-1 / PD-L1 axis promotes cancer immune escape by promoting T cell exhaustion and triggering macrophage polarization toward the M2 subtype, which favors tumor progression. Growing evidence suggests that a positive feedback loop exists between the PI3K / AKT / mTOR and PD-L1 in the TME. Abnormal activation of PI3K / AKT / mTOR increases PD-L1 protein expression, while overexpression of PD-L1 further stimulates the PI3K / AKT / mTOR pathway. Therefore, regulating PI3K / AKT / mTOR to further inhibit tumor angiogenesis and activate anti-tumor immune responses is an effective strategy for preventing tumor spread and metastasis.

[0004] Histamine (HA) is a multifunctional biogenic amine that typically mediates allergic and inflammatory responses by binding to histamine receptors, such as histamine receptor H1 (HRH1). Clinical evidence shows that histamine secretion levels are significantly increased in many tumors, and the HA-HRH1 signaling axis can activate the phosphorylation of PI3K / AKT / mTOR signaling, thereby promoting cancer development and metastasis. Histamine is widely involved in stimulating angiogenesis through the HA-HRH1 axis, which is closely related to the COX-2 and HIF / VEGF pathways. In addition, the HA-HRH1 axis is closely related to the M2 polarization of macrophages and T cell dysfunction, and plays an important role in regulating immune responses. Increasing evidence shows that the use of antihistamines during cancer treatment can effectively inhibit tumor angiogenesis, activate anti-tumor immunity, and further inhibit tumor metastasis. HA-HRH1 has been considered a new target for cancer treatment.

[0005] Cetirizine (CTZ), a second-generation antihistamine, is a selective inhibitor of HRH1. Recent studies have confirmed that CTZ has great potential for development as an anti-tumor drug. Mechanistic studies have revealed that CTZ is a potent PI3K regulator. More importantly, it has significant potential in inhibiting angiogenesis and activating anti-tumor immune responses.

[0006] In light of this, the present invention pioneered the introduction of cetirizine (CTZ) into a tetravalent platinum system to prepare a novel cetirizine tetravalent platinum target compound. This work represents an innovative structural system in the fields of pharmacy, chemistry, and medical technology. This compound can damage DNA through the platinum nucleus; through the cetirizine ligand, it inhibits histamine (HA) secretion, blocks the HA / HRH1 signaling axis, and inhibits the PI3K / AKT / mTOR pathway, thereby inhibiting tumor angiogenesis. The two synergistically exert anti-tumor proliferation and anti-metastasis effects. Summary of the Invention

[0007] To address the challenges of the existing technology, the present invention proposes a compound with a tetravalent platinum structure similar to cetirizine, as well as its preparation method and application. The target compound was tested for its anti-tumor proliferation and anti-metastasis effects through in vivo and in vitro testing, and its anti-tumor mechanism was investigated. The results confirmed that the compound has significant anti-tumor activity, particularly against metastatic malignant tumors. This compound is expected to provide a new drug candidate for the clinical treatment of tumors and offer new directions for the research and development of new platinum-based drugs and anti-metastatic malignant tumor drugs.

[0008] The general formula is shown in (I):

[0009]

[0010] in, Selected from cisplatin or oxaliplatin; L is hydroxyl or

[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 compound represented by general formula (I). The synthetic route of the compound is as follows:

[0016] Described synthetic route 1 is as follows:

[0017]

[0018] Compound II and cetirizine CTZ undergo a coupling reaction to obtain an asymmetric monosubstituted cetirizine-modified tetravalent platinum compound Ia; wherein the molar ratio of compound II to cetirizine CTZ is 1:1.0-1.5;

[0019] Described synthetic route 2 is as follows:

[0020]

[0021] Compound II and cetirizine CTZ undergo a coupling reaction to obtain a symmetrical disubstituted cetirizine-modified tetravalent platinum compound Ib; wherein the molar ratio of compound II to cetirizine CTZ is 1:2.0-5.0.

[0022] Furthermore, in the synthetic route 1, the preparation steps of the asymmetric monocetirizine tetravalent platinum derivative are as follows:

[0023] Under an inert gas atmosphere, cetirizine CTZ, a condensing agent, and an organic base are dissolved in an anhydrous organic solvent for reaction, compound II is added, and the reaction is carried out in the dark. After post-treatment, the monocetirizine tetravalent platinum derivative Ia is isolated;

[0024] The molar ratio of compound II, cetirizine CTZ, condensing agent, and organic base is 1:1.0-1.5:1.0-1.5:1.0-1.5; the feeding relationship of compound II and organic solvent is that 10-100 ml of organic solvent is added for every 1 g of compound II;

[0025] In the second synthetic route, the preparation steps of the symmetrical tetravalent platinum derivative of dicetirizine are as follows:

[0026] Under an inert gas atmosphere, cetirizine CTZ, a condensing agent, and an organic base are dissolved in an anhydrous organic solvent for reaction, compound II is added, and the reaction is carried out in the dark. After post-treatment, the tetravalent platinum derivative Ib of dicetirizine is isolated;

[0027] The molar ratio of compound II, cetirizine CTZ, condensing agent and organic base is 1:2.0-5.0:2.0-5.0:2.0-5.0; the feeding relationship between compound II and organic solvent is that 10-100 ml of organic solvent is added for every 1 g of compound II.

[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: TBTU and cetirizine CTZ are added to a reaction vessel, the air in the system is replaced with nitrogen, anhydrous DMSO 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 II 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 cetirizine-modified tetravalent platinum compound Ia or a symmetric disubstituted cetirizine-modified tetravalent platinum compound Ib.

[0030] Further, the compound II is composed of After being oxidized with hydrogen peroxide, the product is prepared. The specific preparation process is as follows:

[0031]

[0032] Divalent platinum compounds The dihydroxy tetravalent platinum compound II is prepared by oxidizing with hydrogen peroxide at 60-70° C. for 1-8 hours.

[0033] 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.

[0034] 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.

[0035] The cetirizine 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, freeze-dried powder injections, ointments or liniments, including various sustained-release, controlled-release dosage forms or nanoformulations prepared using conventional methods recognized in pharmaceutical science.

[0036] The cetirizine 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.

[0037] The administration route of the tetravalent platinum cetirizine compound of the present invention can be intravenous administration, including intravenous injection, intramuscular injection, intratumoral injection, subcutaneous injection and acupuncture injection.

[0038] 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.

[0039] 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.

[0040] The tetravalent platinum cetirizine derivatives of the present invention can have a good therapeutic effect on metastatic malignant tumors, can effectively inhibit tumor angiogenesis, and effectively activate tumor immune response, and have a good therapeutic effect on metastatic malignant tumors.

[0041] 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.

[0042] 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.

[0043] The cetirizine 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.

[0044] Compared with the prior art, the compound having the structure of tetravalent platinum cetirizine described in the present invention has the following advantages:

[0045] (1) Conventional chemotherapy is ineffective against metastatic tumors, which is the main reason for chemotherapy failure. The present invention successfully synthesized a series of novel cetirizine tetravalent platinum compounds by combining the cetirizine molecule with a tetravalent platinum system. These compounds have shown significant anti-tumor and anti-cancer effects in anti-tumor activity experiments, especially for the treatment of metastatic malignant tumors, and have great development prospects for development into anti-tumor metastasis drugs.

[0046] (2) Angiogenesis is a key step in promoting tumor development and metastasis. The introduction of the cetirizine group in the target compound inhibits histamine (HA) secretion, blocks the HA / HRH1 signaling axis, inhibits the PI3K / AKT / mTOR pathway, inhibits chronic inflammation, hypoxia, and immunosuppressive tumor microenvironment, and thus inhibits tumor angiogenesis; the platinum-based nucleus induces DNA damage. The two synergistically exert effective anti-tumor proliferation and anti-metastasis effects.

[0047] (3) Strong toxicity and side effects are one of the main reasons for the failure of platinum-based chemotherapy. While the tetravalent platinum compound cetirizine exerts excellent anti-tumor activity, its toxicity and side effects are significantly lower than those of cisplatin, and it can effectively overcome clinical resistance to divalent platinum drugs, showing significant therapeutic advantages.

[0048] (5) The innovative structure of the compound of Formula I described in this invention is expected to yield a variety of lead molecules that are effective against tumors, opening up new avenues for the development of platinum compounds. This type of original innovative drug research has important theoretical value and practical significance for national economic and social development and people's health. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Drug accumulation in 4T1 cells in vitro. 4T1 cells were incubated with complexes 1-4 (5 μM), CDDP (5 μM), and a mixture of CDDP-CTZ (5 μM / 10 μM) for 24 hours. Pt was detected by AAS, thereby measuring drug uptake. ***P < 0.001

[0050] Figure 2 Drug subdistribution in 4T1 cells in vitro. 4T1 cells were incubated with complex 1 (5 μM), CDDP (5 μM), and a mixture of CDDP-CTZ (5 μM / 10 μM) for 24 hours. Cell nuclei, cytoplasm, and cell membranes were separated and Pt was detected by AAS, thereby determining the cellular subdistribution of the drug. ***P < 0.001

[0051] Figure 3Western blot analysis of γ-H2AX and P53 expression. 4T1 cells were incubated with cetirizine tetravalent platinum complex 1 (5 μM), CDDP (5 μM), and a mixture of CDDP-CTZ (5 μM / 10 μM) for 24 hours.

[0052] (a) Western blot results; (b) Relative grayscale intensity analysis. Relative grayscale = (grayscale of specific protein) / (grayscale of GAPDH). ***P < 0.001.

[0053] Figure 4 Drug-induced apoptosis activity assay. 4T1 cells were treated with drugs (5 μM) for 24 hours, stained with Annexin V-FITC / PI, and analyzed by flow cytometry. (a) Blank; (b) CDDP (5 μM); (c) CDDP-CTZ (5 μM / 10 μM); (d) Cetirizine platinum complex 1 (5 μM).

[0054] Figure 5 Drug-induced impairment of mitochondrial membrane potential (ΔΨm) activity was assessed. 4T1 cells were treated with platinum complexes at 37°C for 24 hours, stained with JC-1, and analyzed by flow cytometry. (a) Blank; (b) CDDP (5 μM); (c) CDDP / CTZ (5 μM / 10 μM); (d) Cetirizine tetravalent platinum complex 1 (5 μM).

[0055] Figure 6 Drug-induced ROS production activity assay. DCFH-DA staining was used to detect ROS production in 4T1 cells treated with CDDP (10 μM), OXP (10 μM), and cetirizine platinum complex 1 (10 μM) for 24 hours. (a) Representative micrographs; (b) Statistical analysis of fluorescence intensity by flow cytometry. ***P < 0.001.

[0056] Figure 7 Western blot analysis of Bcl-2, Bax, caspase3, and c-caspase3 expression. 4T1 cells were incubated with cetirizine tetravalent platinum complex 1 (5 μM), CDDP (5 μM), and a CDDP-CTZ mixture (5 μM / 10 μM) for 24 hours. (a) Western blot analysis; (b) relative grayscale intensity analysis. ***P < 0.001.

[0057] Figure 8ELISA assay for HRH1. (a) HRH1 levels in the serum of mice treated with blank, CDDP, CDDP-CTZ, and cetirizine-platinum complex 1; (b) HRH1 levels in tumors of mice treated with blank, CDDP, CDDP-CTZ, and cetirizine-platinum complex 1. Both serum and tumor samples were obtained from in vivo antitumor experiments. Data are based on triplicate experiments. ***P < 0.001.

[0058] Figure 9 HA was detected by ELISA. (a) HA in the serum of mice treated with blank, CDDP, CDDP-CTZ, and cetirizine-platinum complex 1; (b) HA in the tumors of mice treated with blank, CDDP, CDDP-CTZ, and cetirizine-platinum complex 1. Serum and tumors were obtained from in vivo antitumor experiments. Data are based on triplicate experiments. ***P < 0.001.

[0059] Figure 10 Western blot analysis of PI3K, p-PI3K, AKT, p-AKT, mTOR, and p-mTOR expression. 4T1 cells were incubated with cetirizine tetravalent platinum complex 1 (5 μM), CDDP (5 μM), and a CDDP-CTZ mixture (5 μM / 10 μM) for 24 hours. (a) Western blot analysis; (b) relative grayscale intensity analysis. ***P < 0.001.

[0060] Figure 11 The anti-angiogenic properties of cetirizine tetravalent platinum complex 1 were assessed in vitro in HUVEC cells. HUVEC cells were treated with CDDP (5 μM), a mixture of CDDP-CTZ (5 μM / 10 μM), a mixture of CDDP-CTZ-HA (5 μM / 10 μM / 10 μM), cetirizine tetravalent platinum complex 1 (5 μM), and a mixture of 1-HA (5 μM / 10 μM) for 24 hours. (a) Representative images; (b) Relative angiogenesis in each group compared to the blank control. Data are based on triplicate experiments. ***P < 0.001.

[0061] Figure 12 Cetirizine tetravalent platinum complex 1 inhibits angiogenesis by suppressing tumor inflammation and hypoxic microenvironment. (a, b) CD34 in tumor tissues in in vivo anti-tumor experiments +Immunohistochemical staining of microvessels; (c) Elisa assay for relative levels of the inflammatory cytokines IL-6 and TNF-α in serum. Serum was obtained from in vivo antitumor experiments; (d, e) Western blot analysis of COX-2, MMP9, HIF-1α, and VEGFA expression. 4T1 cells were incubated with cetirizine tetravalent platinum complex 1 (5 μM), CDDP (5 μM), and a CDDP-CTZ mixture (5 μM / 10 μM) for 24 hours, followed by Western blot analysis. **P < 0.01, ***P < 0.001.

[0062] Figure 13 Immunohistochemical staining of HIF-1α, VEGFA, and MMP9 in in vivo tumor tissue. Tissues were obtained from in vivo anti-tumor experiments. (a) Representative images; (b) Quantitative data. *** P<0.001.

[0063] Figure 14 Effects of histamine on angiogenesis. 4T1 cells were treated with cetirizine tetravalent platinum complex 1 (5 μM), a mixture of CDDP-CTZ (5 μM / 10 μM), a mixture of 1-HA (5 μM / 10 μM), and a mixture of CDDP-CTZ (5 μM / 10 μM / 10 μM). (a) Western blot analysis of COX-2, HIF-1α, and VEGFA levels; (b) relative grayscale intensity analysis. ***P < 0.001.

[0064] Figure 15 Effects of Cetirizine Tetravalent Platinum Complex 1 on Tumor Immunity. (a, b) Western blot analysis of PD-L1 expression in 4T1 cells treated with Cetirizine Tetravalent Platinum Complex 1 (5 μM), CDDP (5 μM), and a mixture of CDDP-CTZ (5 μM / 10 μM) for 24 hours. (c, d) Expression of PD-L1 and CD3 in tumor tissues in in vivo anti-tumor experiments. + and CD8 + T cells and CD86 + -M1 and CD206 + -Immunohistochemical staining of M2 macrophages. ***P<0.001.

[0065] Figure 16In vivo antitumor activity of cetirizine tetravalent platinum complex 1, CDDP, and CDDP-CTZ in female BALB / c mice (n=6) bearing 4T1 tumors. ***P<0.001. (a) Schematic diagram of the experimental design; (b) Tumor growth as a function of time; (c) Tumor weight of mice in each group at the end of the experiment; TGI of the test drug compared with the control group is indicated [TGI = (1-tumor weight of the drug-treated group / tumor weight of the control group) × 100%]; (d) Platinum accumulation in tumor tissues was assessed by AAS; (e) Tumor images after mouse sacrifice; (f) Relative body weight of mice during treatment; (g) H&E-stained tumor images.

[0066] Figure 17 Organ indexes of BALB / c mice in the blank group, cetirizine platinum complex 1 group, CDDP group, and CDDP-CTZ-treated groups (n=6). Tissues were obtained from in vivo antitumor experiments. (a) Heart; (b) Liver; (c) Spleen; (d) Lung; (e) Kidney; Organ index = organ weight / body weight × 100%. ***P < 0.001; ns: not significantly different.

[0067] Figure 18 H&E staining of liver, spleen, and kidney tissues from mice treated with control, CDDP, CDDP-CTZ, and cetirizine platinum complex 1. Tissues were obtained from in vivo antitumor experiments.

[0068] Figure 19 An in vitro Transwell assay was used to evaluate the inhibitory effects of cetirizine tetravalent platinum complex 1 (5 μM), CDDP (5 μM), and CDDP-CTZ (5 μM / 10 μM) on 4T1 cell migration. 4T1 cells were treated with platinum-containing drugs for 24 hours and observed, while untreated cells served as blank controls. (a) Representative images; (b) Relative migration rate analysis. ***P < 0.001.

[0069] Figure 20 The inhibitory effects of cetirizine platinum complex 1 (5 μM), CDDP (5 μM), and CDDP-CTZ (5 μM / 10 μM) on 4T1 cell migration in vitro were assessed using a wound healing assay. The extent of wound healing was observed at 0, 12, and 24 hours. (a) Representative images; (b) Scratch wound healing assay. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant.

[0070] Figure 21Inhibitory effects of cetirizine tetravalent platinum complex 1 and CDDP on the in vivo 4T1 tumor lung metastasis model (n = 5). ***P < 0.001. (a) Schematic diagram of the experimental design; (b) Representative photographs of the front and back of the lungs in each group at the end of the experiment; (c) Pulmonary nodule counts in each group. Inhibition rates are given above the bars; (d) H&E staining of lung metastatic nodules; nodules are indicated by arrows. DETAILED DESCRIPTION

[0071] 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.

[0072] 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.

[0073] Example 1.

[0074] 1. Preparation of tetravalent platinum represented by structural formula II

[0075] 1. Synthesis of Dihydroxycisplatin Tetravalent Platinum IIa

[0076]

[0077] In a 250 mL round-bottom flask, add 1.0 g of cisplatin and 30 mL of distilled water. Slowly add 50 mL of 30% hydrogen peroxide dropwise to the reaction system while stirring. Raise the temperature to 60°C and continue stirring for 4 hours. After the reaction is complete, crystallize at 4°C for 12 hours. Filter and isolate the crude yellow solid. Recrystallize from pure water to obtain 0.78 g of compound IIa as yellow crystals, with a yield of 70%.

[0078] 2. Synthesis of dihydroxyoxaliplatin tetravalent platinum IIb

[0079]

[0080] In a 250 mL round-bottom flask, add 1.0 g of oxaliplatin and 30 mL of distilled water. Slowly add 50 mL of 30% hydrogen peroxide dropwise to the reaction system while stirring. Raise the temperature to 60°C and continue stirring for 4 hours. After the reaction, crystallize at 4°C for 12 hours. Filter and isolate the crude white solid. Recrystallize from pure water to obtain 0.68 g of IIb as white crystals, with a yield of 63%.

[0081] 2. Preparation of Cetirizine Tetravalent Platinum as Shown in Structural Formula I

[0082] 1. Preparation of symmetrical dicetirizine tetravalent platinum derivative 1.

[0083]

[0084] To 5 mL of dry DMSO were added CTZ (350 mg, 0.90 mmol) and HATU (342 mg, 0.90 mmol), and the mixture was stirred at room temperature for 15 min. Then, triethylamine (125 μL, 0.90 mmol) was added, and stirring was continued for 15 min. Subsequently, IIa (100 mg, 0.30 mmol) was added, the atmosphere was replaced with nitrogen, and the reaction was stirred vigorously at 55°C in the dark for 48 hours. After completion of the reaction, the solvent was evaporated, and the crude product was purified by silica gel column chromatography to obtain complex 1 (83.7 mg, 22.3%) as a white solid. Its purity was determined by HPLC (75% methanol / 25% water) to be 97.0%.

[0085] 1 H NMR (500MHz, DMSO-d6) δ7.47–7.44(m,4H),7.42–7.40(m,4H),7.37(d,J=8.5Hz,4H),7.31(t,J=7.6Hz,4H),7.21(t,J= 7.3Hz,2H),4.80(br,6H,NH3),4.41(s,2H),3.89(s,4H),3.65(t,J=5.4Hz,4H),3.01–2.67(m,16H),2.58–2.52(m,4H). 13 C NMR (126MHz, DMSO-d6) δ172.0,141.8,141.4,132.2,129.8,129.3,129.2,128.0,127.8,73.1,67.8,65.2,55.2,51.8,48.3.MS-ESI:calcd for[M] + :1076(M=C 42 H 54 Cl4N6O6Pt),found:1076.HRMS:calcd for[M] + :1074.2585(M=C 42 H 54 Cl4N6O6Pt),found:1074.2579.

[0086] 2. Preparation of symmetrical dicetirizine tetravalent platinum derivative 2.

[0087]

[0088] CTZ (272 mg, 0.7 mmol) and TBTU (224 mg, 0.7 mmol) were added to 5 mL of DMF, and the mixture was stirred at room temperature for 15 min. Triethylamine (97 μL, 0.7 mmol) was then added, and the mixture was stirred for a further 15 min. Compound IIb (100 mg, 0.23 mmol) was then added, the atmosphere was purged with nitrogen, and the reaction was stirred vigorously at 55° C. in the dark for 48 hours. After completion, the solvent was evaporated, and the crude product was purified by silica gel column chromatography to yield complex 2 (111.6 mg, 27.6%) as a white solid. The purity was determined by HPLC (75% methanol / 25% water) to be 95.4%.

[0089] 1 H NMR(500MHz,DMSO-d6)δ8.48–8.30(m,2H,NH2),8.14–7.96(m,2H,NH2),7.49–7.42(m, 4H),7.41–7.35(m,8H),7.33–7.27(m,4H),7.24–7.19(m,2H),4.39(s,2H),4.04(s,4H) ,3.64–3.56(m,4H),3.12–3.06(m,2H),2.87–2.72(m,8H),2.60–2.54(m,2H),2.48–2. 36(m,8H),2.10–2.04(m,1H),1.52–1.40(m,3H),1.28–1.17(m,4H),1.14–1.06(m,2H). 13 C NMR(126MHz,DMSO-d6)δ177.8,163.9,142.4,142.0,131.9,129.9,129.1,129.0, 128.0,127.6,74.0,68.5,61.4,53.0,50.4,46.1,31.3,24.0,9.1.MS-ESI:calcd for[M] + :1173(M=C 50 H 62 Cl2N6O 10 Pt),found:1173.HRMS:calcd for[M+H] + :1172.3630(M=C 50 H 62 Cl2N6O 10 Pt),found:1172.3654.

[0090] 3. Preparation of asymmetric monocetirizine tetravalent platinum derivative 3.

[0091]

[0092] CTZ (117 mg, 0.3 mmol) and HATU (114 mg, 0.30 mmol) were added to 5 mL of DMSO, and the mixture was stirred at room temperature for 15 minutes. Triethylamine (42 μL, 0.30 mmol) was then added, and the mixture was stirred for a further 15 minutes. Compound IIa (100 mg, 0.30 mmol) was then added, the atmosphere was replaced with nitrogen, and the reaction was stirred vigorously at 55°C in the dark for 48 hours. After completion of the reaction, the solvent was evaporated, and the crude product was purified by silica gel column chromatography to yield complex 3 (51.4 mg, 21.5%) as a yellow solid. The purity was determined to be 98.0% by HPLC (75% methanol / 25% water).

[0093] 1 H NMR(500MHz,DMSO-d6)δ7.46(d,J=8.2Hz,2H),7.44–7.36(m,4H),7.32(t,J=7.5Hz,2H),7.23(d ,J=7.4Hz,1H),4.47(s,1H),3.97(s,2H),3.73(t,J=5.1Hz,2H),3.14–2.87(m,8H),2.54(s,2H). 13 C NMR (126MHz, DMSO) δ172.7,142.0,141.7,132.0,129.8,129.2,129.1,128.0,127.8,73.5,68.6,66.1,55.8,52.1,49.2.MS-ESI:calcd for[M+2HCl+H] + :779(M=C 21 H 31 Cl3N4O4Pt),found:779.

[0094] 4. Preparation of asymmetric monocetirizine tetravalent platinum derivative 4.

[0095]

[0096] CTZ (89 mg, 0.23 mmol) and TBTU (74 mg, 0.23 mmol) were added to 5 mL of DMF, and the mixture was stirred at room temperature for 15 min. Triethylamine (32 μL, 0.23 mmol) was then added, and the mixture was stirred for a further 15 min. Compound IIb (100 mg, 0.23 mmol) was then added, the atmosphere was replaced with nitrogen, and the reaction was stirred vigorously at 55°C in the dark for 48 hours. After completion of the reaction, the solvent was evaporated, and the crude product was purified by silica gel column chromatography to yield complex 4 (44.2 mg, 21.6%) as a white solid. The purity was determined by HPLC (75% methanol / 25% water) to be 97.9%.

[0097] 1 H NMR (500MHz, DMSO-d6) δ7.44(d,J=8.2Hz,2H),7.39(d,J=7.7Hz,2H),7.35(d,J=8.3Hz,2H),7.29(t,J=7.6Hz,2H),7.20(d,J=7.3Hz,1H),4.34(s, 1H),4.02(s,2H),3.56(s,2H),2.80–2.56(m,6H),2.38–2.34(m,4H),2.1 4–2.08(m,2H),1.48–1.40(m,2H),1.25–1.21(m,2H),1.18–0.78(m,2H). 13 CNMR(126MHz,MeOD)δ186.7,166.3,141.3,140.8,132.6,129.2,128.6,128.5,1 27.6,127.3,73.8,68.0,64.8,61.2,56.2,52.5,52.5,34.0,7.9.MS-ESI:calcd for[M+H] + :803(M=C 29 H 39 ClN4O8Pt),found:803.HRMS:calcd for[M+H] + :802.2182(M=C 29 H 39 ClN4O8Pt),found:802.2190.

[0098] 3. Experimental Test

[0099] 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 uses 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 of the present invention are intended to illustrate the present invention and not to limit it. Simple modifications to the present invention based on its essence fall within the scope of protection of the present invention.

[0100] 1. In vitro antitumor activity assay

[0101] In this experiment, the MTT method was used to determine the cell viability. The half-inhibitory concentration (IC 50 ) values ​​were used to evaluate the in vitro anticancer activity of the complexes.

[0102] 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 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 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader to calculate its IC 50 Each set of experiments was repeated at least three times.

[0103] 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.

[0104] The reference drugs used in this experiment include: cetirizine CTZ, cisplatin CDDP, oxaliplatin OXP, a mixture of cisplatin and CTZ CDDP-CTZ, and satrapal STP.

[0105] Discussion of Antitumor Activity:

[0106] Table 1 In vitro antitumor activity of CTZ tetravalent platinum compounds.

[0107]

[0108]

[0109] Table 1 a RF: resistance factor, RF = IC 50 (A549R) / IC 50 (A549); bSI: Selectivity index, SI = IC 50 (LO2) / IC 50 (HepG2); c ND: not tested or not calculated; d CDDP-CTZ: a mixture of cisplatin and CTZ with a molar ratio of 1:2.

[0110] The results are shown in Table 1: The ligands in the CTZ tetravalent platinum complexes significantly affect drug uptake, which further affects the anti-tumor efficacy. Compared with the single CTZ tetravalent platinum complexes 3 and 4, the complexes 1 and 2 with dual CTZ ligands showed higher drug accumulation in tumor cells (P<0.001) ( Figure 1 ) and has better anti-tumor properties. In addition, the platinum core plays a key role in affecting the anti-tumor efficacy of cetirizine tetravalent platinum complexes. Complexes 1 and 3 with CDDP as the core are more active than the corresponding OXP-derived complexes 2 and 4. In particular, cetirizine tetravalent platinum complex 1 (i.e., symmetrical dual cetirizine tetravalent platinum derivative 1) has the strongest anti-tumor activity, with an IC 50 The value was less than 4.61 μM, which was stronger than cisplatin, 1.63-7.13 times the activity of the reference drug CDDP, and significantly stronger than OXP and the tetravalent platinum drug STP. It is worth noting that the ligand CTZ has a very low killing effect on tumor cells. Compared with free CDDP, the anti-tumor ability of the mixture of CDDP and CTZ (CDDP-CTZ) was not significantly enhanced. These facts indicate that fusing CTZ with the tetravalent platinum mother core to construct the cetirizine tetravalent platinum compound is a key step in improving the anti-tumor performance.

[0111] Drug resistance is a major obstacle to the clinical effectiveness of platinum drugs. To evaluate the ability of CTZ tetravalent platinum complexes to overcome drug resistance, the resistance factor (RF) (IC 50 Value and A549 IC 50 The results showed that cetirizine tetravalent platinum complex 1 effectively overcame the drug resistance of CDDP, with an RF value of 0.72, which was significantly lower than that of CDDP (RF = 3.86), while the CDDP-CTZ mixture had a weaker effect on it (RF = 3.19). Then, we calculated the selectivity index (SI) of the drug to determine the in vitro toxicity of CTZ tetravalent platinum complex (IC 50 Value and LO2 IC 50 The results showed that cetirizine tetravalent platinum complex 1 could reduce the toxicity to normal cells, with an SI value of 2.77, which was higher than CDDP (SI = 0.81), OXP (SI = 0.60), JM216 (SI = 0.90) and CDDP-CTZ mixture (SI = 0.98).

[0112] Therefore, the symmetrically disubstituted cetirizine tetravalent platinum derivative 1 exhibited satisfactory antitumor activity in vitro and showed great potential in overcoming CDDP resistance and reducing toxicity. Therefore, the complex was screened and further activity and mechanism studies were conducted as an antiproliferative and anti-metastatic drug candidate.

[0113] 2. DNA Damage Mechanism

[0114] DNA damage is the primary anti-tumor mechanism of platinum-based drugs. We used Western blotting to detect the expression of DNA damage marker proteins. AAS was used to detect the Pt element and to examine the drug's cellular distribution.

[0115] 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, 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, lysed with concentrated nitric acid, and the platinum content in each cell was determined by atomic absorption spectroscopy to measure drug uptake in the tumor cells. Drug-treated cells were obtained using the same method, and the cell nucleus, cytoplasm, and cell membrane were separated using a kit and lysed with concentrated nitric acid. The platinum content in each fraction was determined by atomic absorption spectroscopy to measure drug sub-distribution in the tumor cells.

[0116] Cells were collected and treated using the above method, proteins were extracted, and electrophoretic protein separation was performed using SDS-Page. The membranes were transferred to PVDF membranes, blocked in 5% skim milk for 1 hour, incubated with primary antibodies overnight at 4°C, and incubated with secondary antibodies for 1 hour. ECL chemiluminescent color development was then used, and the membranes were imaged using a Tanon 46000SF scanning system. This patent examined the expression of DNA damage marker proteins γ-H2AX and P53 by Western blotting to examine their ability to damage DNA.

[0117] The accumulation of tetravalent platinum complexes in the cell nucleus is considered a key step in exerting their DNA-damaging effects. Here, we used AAS to investigate the distribution of cetirizine tetravalent platinum complex 1 in tumor cells. Figure 1 The results in the study showed that the intracellular uptake level of cetirizine tetravalent platinum complex 1 was higher than that of CDDP and CDDP-CTZ. Figure 2 The accumulation of cetirizine tetravalent platinum complex 1 in nuclear DNA was also significantly higher than that of CDDP and CDDP-CTZ. This suggests that CTZ tetravalent platinum complex 1 can effectively enter tumor cells, accumulate in the nucleus, and affect its anti-tumor activity. This may be one of the reasons why the MTT anti-tumor activity of cetirizine tetravalent platinum complex 1 is stronger than that of CDDP and CDDP-CTZ.

[0118] To further prove that cetirizine tetravalent platinum complex 1 can cause DNA damage, we used Western Blot to test the expression of DNA damage indicator proteins γ-H2AX and P53. Figure 3 As shown in the figure, the expression of γ-H2AX and P53 in 4T1 cells was significantly increased after treatment with cetirizine tetravalent platinum complex 1 (P<0.001). These results confirm the strong DNA damage ability of CTZ tetravalent platinum complex 1.

[0119] 3. Induce mitochondria-mediated cell apoptosis.

[0120] To further validate the mechanism by which CTZ (quaternary platinum) compounds induce cell death, we used Annexin V-FITC / PI double staining to examine the characteristics of drug-induced apoptosis. Since apoptosis often involves mitochondria and is accompanied by mitochondrial membrane potential collapse and ROS production, we used JC-1 staining to examine changes in mitochondrial membrane potential; DCFH-DA staining to measure intracellular ROS levels; and Western blot analysis to examine the effects of the drug on apoptotic proteins. This explored the mechanism by which CTZ (quaternary platinum) compounds induce apoptosis in tumor cells.

[0121] Take 4T1 cells in good logarithmic growth phase and place them in 6-well plates (10 6 / well), cultured in a 37°C, 5% carbon dioxide 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 parts. In the first part, the cells were washed twice with PBS, and 5μL of Annexin V-FITC stain and 5μL of PI stain were added respectively, 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 part was stained with DCFH-DA to detect the production of ROS in the cells, and the results were detected using an inverted fluorescence microscope and flow cytometer. The third part was stained with JC-1 and detected by flow cytometry to examine the changes in mitochondrial membrane potential.

[0122] like Figure 4 As shown in Figure 2, cetirizine tetravalent platinum complex 1 can significantly induce apoptosis of 4T1 tumor cells (29.8%), which is relatively higher than CDDP (23.8%) and CDDP-CTZ mixture (19.6%). This trend is consistent with the IC 50 The values ​​are consistent. Mitochondrial-dependent apoptosis is one of the most important pathways of cell apoptosis. m ) collapse and ROS generation are the characteristics of mitochondrial damage. The results showed that cetirizine tetravalent platinum complex 1 induced ΔΨ in 4T1 cells m The apparent collapse of Figure 5 ), and then the production of ROS was significantly increased compared with the blank group (P<0.001) ( Figure 6 ). Obviously, severe mitochondrial damage was induced during complex 1-induced apoptosis.

[0123] The Bcl-2 signaling pathway is a key pathway that controls mitochondrial apoptosis. This patent uses Western Blot to determine the expression of Bcl-2 pathway proteins ( Figure 7 Cetirizine tetravalent platinum complex 1 inhibited the expression of the anti-apoptotic protein Bcl-2 (P < 0.001), while significantly upregulated the expression of the pro-apoptotic protein Bax and the apoptotic protein c-caspase3 / caspase3 (P < 0.001). These facts indicate that CTZ tetravalent platinum complex can effectively promote tumor cell apoptosis through the mitochondrial-mediated Bcl-2 / Bax / caspase3 pathway.

[0124] 4. Inhibit the HA-HRH1 signaling axis.

[0125] High histamine expression in tumor tissues accelerates cancer-related biological processes by activating the HA-HRH1 axis. Cetirizine (CTZ), a second-generation antihistamine, selectively inhibits the HA-HRH1 axis, significantly impacting immunosuppression, angiogenesis, and tumor metastasis. CTZ tetravalent platinum complexes, upon reduction and release of CTZ, are expected to inhibit HA secretion and HRH1 activity. We used an ELISA assay to investigate the effects of cetirizine tetravalent platinum complex 1 on the HA-HRH1 signaling axis.

[0126] In vivo histamine levels and HRH1 expression in serum and tumor tissue were measured using ELISA kits (Jiangsu Enzyme Immunity Biotechnology Co., Ltd.). Serum and tumor tissue were obtained for in vivo antitumor activity experiments. Experiments were performed according to the manufacturer's protocol. Results are calculated based on triplicate experiments.

[0127] Figure 8 The results showed that HRH1 in the serum and tumor tissues of the group treated with cetirizine tetravalent platinum complex 1 was significantly inhibited (P < 0.001), and its inhibitory effect was better than that of the CDDP-CTZ mixture (P < 0.01), while the effect of CDDP on HRH1 activity was negligible. Subsequently, the HA secretion in the serum and tumor tissues of the group treated with cetirizine tetravalent platinum complex 1 was also reduced (P < 0.001), which was lower than that of the CDDP-CTZ mixture (P < 0.001) ( Figure 9 ). This shows that cetirizine tetravalent platinum complex 1 can exert anti-tumor activity in vivo by inhibiting the HA-HRH1 signaling axis.

[0128] 5. Inhibit the PI3K / AKT / mTOR signaling pathway

[0129] The PI3K / AKT / mTOR pathway is a key convergence point for multiple signaling cascades, playing a crucial role in promoting angiogenesis and inducing immunosuppression within tumor tissues. Overactivated HA-HRH1 signaling in tumors has been reported to effectively activate the PI3K / AKT / mTOR pathway, and CTZ is an inhibitor of both the HA-HRH1 and PI3K pathways. This study evaluated the inhibitory effect of cetirizine tetravalent platinum complex 1 on PI3K / AKT / mTOR signaling using Western blot analysis.

[0130] Figure 10 Blots in the Figure 1 showed that p-PI3K, p-AKT, and p-mTOR proteins were simultaneously downregulated in tumor cells treated with cetirizine tetravalent platinum complex 1. The ratios of p-PI3K / PI3K, p-AKT / AKT, and p-mTOR / mTOR decreased significantly, becoming significantly lower than those in the blank group (P < 0.001). Notably, the CDDP-CTZ mixture also significantly inhibited p-PI3K / PI3K, p-AKT / AKT, and p-mTOR / mTOR (P < 0.001), while CDDP had a minimal effect on these proteins. Therefore, it is reasonable to speculate that the CTZ ligand is involved in the inhibition of PI3K / AKT / mTOR, and that the inhibitory effect of CTZ tetravalent platinum complex 1 is primarily attributable to the CTZ ligand. The inhibitory mechanism of PI3K / AKT / mTOR signaling by cetirizine tetravalent platinum complex 1 further impacts tumor angiogenesis and immunoregulation.

[0131] 6. Inhibit angiogenesis by inhibiting tumor inflammation and hypoxia.

[0132] Inhibiting tumor angiogenesis is considered a key therapeutic target for inhibiting tumor proliferation and metastasis. Cetirizine platinum complex 1 has the ability to inhibit HA and is therefore expected to inhibit angiogenesis. To further verify this hypothesis, the researchers evaluated the anti-angiogenic properties of cetirizine platinum complex 1 in vitro using HUVEC cells and examined its in vivo anti-angiogenic activity in tumor tissues using immunohistochemical staining using CD34 as an angiogenesis marker. Figure 11 The results showed that cetirizine tetravalent platinum complex 1 could effectively inhibit angiogenesis in vitro, which was more significant than the blank group and CDDP group (P<0.001). Figure 12 As shown in ab, CD34 +Microvessels were also significantly inhibited, indicating that angiogenesis in tumors treated with cetirizine platinum complex 1 was significantly reduced compared with the control and CDDP groups, and even lower than that in the CDDP-CPZ combination group. These findings indicate that cetirizine platinum complex 1 can effectively inhibit angiogenesis both in vitro and in vivo.

[0133] Angiogenesis is closely related to the TME. HA in the TME can enhance inflammation and hypoxia in tumors, which plays a crucial role in promoting angiogenesis. To investigate the relationship between angiogenesis inhibition and inflammation and hypoxia in the TME, the researchers used Western blotting analysis and immunohistochemistry to assess the expression of COX-2 and MMP-9, which are associated with inflammation, and the expression of the HIF-1α / VEGFA axis, which is associated with hypoxia. Elisa was used to detect the inflammatory cytokines TNF-α and IL-6 in the TME.

[0134] The study found that cetirizine tetravalent platinum complex 1 effectively inhibited the inflammatory response in the TME compared with the control group, as evidenced by the downregulation of COX-2 and MMP-9 in tumor cells (P<0.001)( Figure 12 de). At the same time, after treatment with cetirizine tetravalent platinum complex 1, the levels of TNF-α and IL-6 in the TME were significantly reduced (P<0.001) ( Figure 12 c). In addition, compared with the control group, cetirizine tetravalent platinum complex 1 reversed hypoxia by reducing the expression of HIF-1α (P<0.01) and VEGFA (P<0.001). These results were further confirmed by immunohistochemical staining results, which showed that the accumulation of MMP9, HIF-1α, and VEGFA in tumor tissues was also significantly reduced compared with the blank group and CDDP group (P<0.001) ( Figure 13 These results suggest that cetirizine platinum complex 1 effectively inhibits angiogenesis by targeting inflammation and hypoxia in the TME.

[0135] To further investigate whether the inhibitory effects of cetirizine platinum complex 1 on inflammation and hypoxia were mediated by histamine inhibition, we introduced HA into tumor cells treated with cetirizine platinum complex 1 and assessed the levels of COX-2, HIF-1α, and VEGFA using Western blot. Figure 14The results showed that compared with cells treated with cetirizine tetravalent platinum complex 1, the expression of COX-2, HIF-1α and VEGFA in 4T1 tumor cells treated with a mixture of cetirizine tetravalent platinum complex 1 and HA (1-HA) was significantly increased (P<0.001), indicating that inflammation and hypoxia were aggravated. Subsequently, compared with the cetirizine tetravalent platinum complex 1 group, HUVEC angiogenesis in the 1-HA group was significantly increased (P<0.001) ( Figure 11 These trends were similar to those observed for CDDP-CTZ and CDDP-CTZ-HA mixtures. These results suggest that the anti-angiogenic properties of cetirizine platinum complex 1 are influenced by histamine-mediated inflammatory and hypoxic microenvironment.

[0136] In conclusion, cetirizine tetravalent platinum complex 1 effectively inhibited tumor angiogenesis by suppressing histamine-mediated inflammation and hypoxic TME, and inhibition of COX-2 / MMP-9 and HIF-1α / VEGFA signaling was the key to the anti-angiogenic effect.

[0137] 7. Activate immunity by inducing T cell activation and macrophage polarization

[0138] HA-HRH1 is a key player in regulating immune responses. Furthermore, aberrant phosphorylation of PI3K / AKT / mTOR in the TME can significantly promote immune escape of tumor cells through multiple signaling pathways. The immune checkpoint PD-L1 is closely associated with HA secretion and the PI3K pathway, participating in T cell exhaustion, leading to M2 macrophage polarization and promoting tumor progression. Therefore, inhibiting histamine and the PI3K / AKT / mTOR signaling network can effectively enhance anti-tumor immunity. Given the antihistamine and PI3K pathway inhibitory properties of cetirizine tetravalent platinum complex 1, we examined the immunomodulatory properties of cetirizine tetravalent platinum complex 1 by Western blotting and immunohistochemistry.

[0139] The results are as follows Figure 15 As shown in the results, cetirizine tetravalent platinum complex 1 inhibited the expression of PD-L1 in tumor cells, and its expression level was significantly lower than that in the blank group and CDDP group (P < 0.001). Subsequently, in vivo tumor tissue immunohistochemistry also confirmed the inhibitory effect of compound 1 on PD-L1 (P < 0.001). Next, T cell immunity in tumor tissue was activated, and in the complex 1 treatment group, CD3 + and CD8 + The number of T cells increased by 8.7 times and 12.8 times compared with the blank group (P<0.001), and was also higher than that of the CDDP group (P<0.001). Macrophage staining results showed that cetirizine tetravalent platinum complex 1 can effectively activate macrophage immunity and promote the polarization of macrophages from M2 type to M1 type.+ The number of M1 macrophages increased (P < 0.001), while the number of CD206+M2 macrophages decreased (P < 0.001). In addition, the immune activation effect of cetirizine tetravalent platinum complex 1 was significantly better than that of the CDDP group and CDDP-CTZ group.

[0140] Therefore, cetirizine tetravalent platinum complex 1 can enhance CD3 + and CD8 + Cetirizine tetravalent platinum complex 1 can reverse the immunosuppressive TME and enhance its anti-proliferative and anti-tumor metastasis properties.

[0141] 8. In vivo antitumor activity

[0142] To evaluate the antitumor effect of CTZ tetravalent platinum complex in vivo, we assessed the antitumor activity of cetirizine tetravalent platinum complex 1 in female BALB / c mice bearing 4T1 tumors, CDDP and its mixture CDDP-CTZ were used as positive control drugs, and the saline-treated group served as blank.

[0143] 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.

[0144] 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 / mouse. On the 3rd day after inoculation, the tumor was palpable. The mice were randomly divided into 4 groups, 6 in each group: normal saline group, cetirizine tetravalent platinum complex 1 group, CDDP group, CDDP-CTZ group, and the dosage was 2mg Pt / kg. The drug was administered on the 3rd, 6th, and 9th days, for a total of 3 times, by tail vein injection ( Figure 16 a). The changes in mouse tumor volume were recorded during the experiment to evaluate the tumor growth rate ( Figure 16 b); Record the changes in mouse body weight to evaluate drug toxicity ( Figure 16 f). On the 12th day, the mice were killed and their serum, tumor tissues and organ tissues including heart, lung, liver, spleen and kidney were collected. Tumor and organ tissues were weighed ( Figure 16 c). Tissue samples were fixed with formalin and evaluated by hematoxylin and eosin (H&E) staining and immunohistochemical analysis ( Figure 16 g).

[0145] Figure 16 The results in bc and 16e showed that the candidate cetirizine tetravalent platinum complex 1 can effectively inhibit 4T1 tumors (323mm in vivo 3 ) growth, compared with the blank group (833mm 3 ) compared with the reference drug CDDP (529mm 3 , TGI = 41.0%, P < 0.001) and CDDP-CTZ mixture (515 mm 3 , TGI=56.8%, P<0.001). Subsequently, H&E staining images of the tumor ( Figure 16 g) It was further confirmed that compared with CDDP and CDDP-CTZ, cetirizine tetravalent platinum complex 1 can induce more tumor cell apoptosis, causing severe degeneration, necrosis, and nuclear dispersion. Uptake is considered a key factor affecting the anti-tumor effect of chemotherapy drugs. Therefore, we used AAS to detect drug uptake in tumors ( Figure 16 d) Similar to the drug uptake trend in tumor cells in vitro, the accumulation level of cetirizine tetravalent platinum complex 1 in vivo is also higher than that of the divalent platinum drug CDDP and the CDDP-CTZ mixture, which is a key factor affecting the drug's in vivo anti-tumor activity.

[0146] Systemic toxicity is an important factor affecting the future development and application prospects of drugs. Cetirizine tetravalent platinum complex 1 did not cause significant weight loss in mice at a dose of 2 mg Pt / kg ( Figure 16 f), there was no difference compared with the blank group (P = ns); while CDDP and CDDP-CTZ mixture caused a significant decrease in body weight, which was significantly different from the blank group (P < 0.001). We further investigated the damage of drugs to internal organs by calculating the visceral index (ratio of visceral mass to body weight). Figure 17 The results showed that CDDP and CDDP-CTZ mixtures caused significant spleen suppression (P<0.001), while compound 1 did not show this phenomenon. This confirmed that compound 1 was less toxic to the spleen than CDDP and CDDP-CTZ, which would further affect the immune response of mice. H&E staining cell morphology studies further proved that cetirizine tetravalent platinum complex 1 had low toxicity. No obvious morphological changes were observed in the liver, spleen and kidney tissues of the cetirizine tetravalent platinum complex 1 treatment group ( Figure 18 In summary, cetirizine tetravalent platinum complex 1 has significant anti-tumor activity, its anti-tumor ability is stronger than cisplatin, and its toxicity is significantly lower than cisplatin.

[0147] 9. In vitro and in vivo metastasis inhibition experiments

[0148] 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 activity of a tetravalent platinum cetirizine lead compound 1.

[0149] 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. 10% FBS-RPMI1640 medium containing different compounds was added to the lower chamber. Next, 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. Non-migrated cells in the upper chamber were gently scraped off with a cotton swab. Migrating cells on the lower surface of the chamber were photographed in five random fields using an inverted microscope to examine the anti-migratory effect of the drug.

[0150] 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 compound. The degree of wound healing was recorded at 0, 12, and 24 hours to assess the anti-migratory effect of the drug.

[0151] In vivo anti-metastasis experiment: BALB / c female mice (18-20 g) were used as experimental subjects and 4T1 cells (2×10 5 ) to establish a lung metastasis model. Mice were randomly divided into three groups (n=5): 1, CDDP and saline (Blank group) treatment. Pt was administered three times via tail vein injection on days 3, 6, and 9 after cell inoculation at a dose of 2 mg Pt / kg. Mice were sacrificed on day 12, and lung tissues were dissected ( Figure 21 a) After fixation with 4% formaldehyde solution for 24 hours, the lung metastatic nodules of each mouse were counted and the lung tissues were stained with H&E for observation.

[0152] Figure 19 and 20 The results of both the Transwell and wound healing assays showed that cetirizine tetravalent platinum complex 1 effectively inhibited tumor cell migration in vitro compared with the blank group (P<0.001). In addition, the anti-metastatic ability of cetirizine tetravalent platinum complex 1 was significantly superior to that of the reference drug CDDP (P<0.001) and the mixture CDDP-CTZ (P<0.001).

[0153] The lungs are the main site of tumor metastasis. Figure 21 The activity in the lung metastasis model shown in bc indicates that the candidate drug cetirizine tetravalent platinum complex 1 also exhibits significant anti-metastatic activity in vivo. Compared with the blank group, the inhibition rate of cetirizine tetravalent platinum complex 1 on lung nodules was 85.6% (P < 0.001), which was significantly higher than that of CDDP (51.4%, P < 0.001). Subsequently, the H&E staining results of the tumor sections further showed that ( Figure 21 d) The group treated with cetirizine tetravalent platinum complex 1 had fewer and smaller tumor nodules. This indicates that candidate cetirizine tetravalent platinum complex 1 not only has good anti-proliferative activity but also has strong anti-tumor metastasis activity, and has great development potential as an anti-metastatic drug.

[0154] 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 cetirizine tetravalent platinum structure, the general formula of which is shown in (I): in, Selected from cisplatin or oxaliplatin; L is hydroxyl or 2. The method for preparing the compound having the structure of tetravalent platinum cetirizine according to claim 1, characterized in that: The synthetic route of the compound is as follows: in: When the molar ratio of compound II to cetirizine CTZ is 1:1.0-1.5, compound II and cetirizine CTZ undergo a coupling reaction to obtain an asymmetric monosubstituted cetirizine-modified tetravalent platinum compound, wherein L is a hydroxyl group; When the molar ratio of compound II to cetirizine CTZ is 1:2.0-5.0, compound II and cetirizine CTZ undergo a coupling reaction to obtain a symmetrical disubstituted cetirizine-modified tetravalent platinum compound, that is, L is 3. The method for preparing a compound having a cetirizine tetravalent platinum structure according to claim 2, wherein: The specific preparation steps of the synthetic route are: Under an inert gas atmosphere, cetirizine CTZ, a condensing agent, and an organic base are dissolved in an anhydrous organic solvent for reaction, compound II is added, the reaction is carried out in the dark, and the target compound is isolated through post-treatment; wherein, When the molar ratio of compound II, cetirizine CTZ, condensing agent, and organic base is 1:1.0-1.5:1.0-1.5:1.0-1.5; and the feeding relationship of compound II and organic solvent is that 10-100 ml of organic solvent is added for every 1 g of compound II, an asymmetric monosubstituted cetirizine-modified tetravalent platinum compound is obtained; When the feeding molar ratio of compound II, cetirizine CTZ, condensing agent and organic base is 1:2.0-5.0:2.0-5.0:2.0-5.0; the feeding relationship of compound II and organic solvent is that 10-100 ml of organic solvent is added for every 1 g of compound II, a symmetrical disubstituted cetirizine-modified tetravalent platinum compound is obtained.

4. The method for preparing a compound having a cetirizine tetravalent platinum structure according to claim 3, wherein: 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.

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

6. Use of the compound according to claim 1 or the pharmaceutical composition according to claim 5 in the preparation of anti-tumor drugs.

7. The use according to claim 6, characterized in that Application in the preparation of anti-tumor proliferation and anti-tumor metastasis drugs.

8. The use according to claim 7, 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.

9. A combined preparation comprising the compound of formula (I) according to claim 1 or the pharmaceutical composition according to claim 5, 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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