A compound with a chloroiodoquine tetravalent platinum structure, its preparation method and its uses

Abstract

The present invention provides a compound having a chloroiodoquine tetravalent platinum structure, a preparation method thereof, and uses thereof. The compound having a chloroiodoquine tetravalent platinum structure is represented by the general formula (I), wherein L1 and L2 are or one of L1 and L2 is and the other is a hydroxyl group. The compound having a chloroiodoquine tetravalent platinum structure according to the present invention has a good therapeutic effect on metastatic malignant tumors, can effectively activate autophagy in tumor cells, and effectively activate the tumor immune response, providing a new direction for the research and development of anti-tumor drugs, especially anti-metastatic malignant tumor drugs.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and in particular relates to a compound having a clioquinol tetravalent platinum structure, a preparation method thereof, and uses thereof. Background Art

[0002] Cancer metastasis is the main cause of death in cancer patients, which involves the spread of cancer cells from the primary tumor tissue to distal tissues. Traditional tumor chemotherapy focuses on the diagnosis and treatment of primary tumors, but has a weak effect on metastatic tumor cells. Therefore, the research and development of drugs for the treatment of metastatic cancer is extremely urgent.

[0003] Platinum-based drugs are the "milestones" of modern chemotherapy. In particular, cisplatin (CDDP), oxaliplatin (OLP) and carboplatin (CBP) are widely used in the treatment of various tumors. However, their efficacy is restricted by severe side effects, poor in vivo stability and serious drug resistance. In order to overcome these drawbacks, platinum(IV) complexes, as prodrugs of platinum(II) drugs, have been widely studied, and the development of new structure candidate drugs with different mechanisms of action from clinical drugs has attracted great scientific interest among researchers. It is worth noting that their applications have also been extended to the field of treatment of invasive tumor metastasis. In view of this, the research on novel multifunctional platinum(IV) complexes as anti-tumor proliferation and anti-metastasis drugs is of great significance.

[0004] Autophagy is a highly conserved self-degradation process that plays a key role in maintaining intracellular homeostasis. However, drug-induced autophagy has also been classified as an alternative cell death mechanism, called type II programmed cell death. Recent studies have explored the role of autophagy in tumor metastasis. Autophagy is a "double-edged sword" in tumor growth and metastasis treatment: it protects cancer cells from chemotherapy drugs and participates in tumor development; at the same time, drug-induced autophagy can also activate the apoptotic signaling pathway, reverse multidrug resistance, and inhibit metastasis. Research has confirmed that specific autophagy inducers that induce autophagic cell death are a class of anti-cancer drugs with great development potential.

[0005] Developing platinum complexes with autophagy-inducing properties is an important topic in the development of new platinum-based drugs. Clioquinol (CLQ) is a class of traditional antibacterial drugs and has been a hot topic in the research of old drug new uses in recent years. The latest research found that clioquinol, as an effective autophagy promoter, can induce autophagy by inhibiting the mTOR pathway, has important development potential in the anti-tumor field, and can effectively combat tumor metastasis.

[0006] Combined use of clioquinol with other chemotherapeutic drugs can improve the sensitivity of tumor cells to drugs and enhance the therapeutic effect. The molecules prepared by complexing it with the platinum-based parent nucleus exhibit strong antitumor activity. Thus, introducing clioquinol into the platinum-based drug system would be an effective strategy for developing new antitumor proliferation and anti-metastasis drugs. SUMMARY OF THE INVENTION

[0007] In view of this, the present invention provides a compound with a clioquinol tetravalent platinum structure. By introducing the clioquinol molecule into the tetravalent platinum parent nucleus, a series of novel tetravalent platinum compounds modified with clioquinol derivatives are synthesized. These clioquinol tetravalent platinum derivatives have good therapeutic effects on metastatic malignant tumors, can effectively activate autophagy in tumor cells, and effectively activate the tumor immune response, providing a new direction for the research and development of antitumor drugs, especially anti-metastatic malignant tumor drugs. The present invention also provides a preparation method of the compound with a clioquinol tetravalent platinum structure and its use in antitumor drugs.

[0008] To achieve the above object, the technical solution of the present invention is realized as follows:

[0009] Specifically, on the one hand, the present invention provides a compound with a clioquinol tetravalent platinum structure, and its technical solution is realized as follows:

[0010] A compound represented by the general formula (I):

[0011]

[0012] Wherein, is selected from cisplatin, carboplatin or oxaliplatin;

[0013] L1 and L2 are or one of L1 and L2 is and the other is a hydroxyl group;

[0014] Wherein, R 3 is selected from methylene, propylene or butylene.

[0015] Furthermore, the compound is any one of the following:

[0016]

[0017] On the other hand, the present invention also provides a preparation method of a compound represented by the general formula (I). The compound has two synthetic routes, namely Synthetic Route 1 or Synthetic Route 2 as follows:

[0018] Synthetic Route 1 is as follows:

[0019]

[0020] Compound 3 and compound 4 undergo a coupling reaction to obtain an asymmetric monosubstituted chloroiodoquinoline-modified tetravalent platinum compound 1; wherein, the molar ratio of compound 3 to compound 4 in the feed is 1:1.0 to 1.3;

[0021] The second synthetic route is as follows:

[0022]

[0023] Compound 3 and compound 4 undergo a coupling reaction to obtain a symmetric disubstituted chloroiodoquinoline-modified tetravalent platinum compound 2; wherein, the molar ratio of compound 3 to compound 4 in the feed is 1:2.2 to 3.

[0024] Furthermore, in the first synthetic route, the preparation steps are as follows:

[0025] Under an inert gas atmosphere, compound 4, a condensing agent, and an organic base are dissolved in an anhydrous organic solvent and reacted, compound 3 is added, and after reaction in the dark, through post-treatment, compound 1 is separated out;

[0026] Among them, the molar ratio of compound 3, compound 4, the condensing agent, and the organic base in the feed is 1:1.0 to 1.3:1.0 to 1.3:1.0 to 1.3; the feeding relationship between compound 3 and the organic solvent is that for every 1 g of compound 3, 30 to 80 ml of the organic solvent is added;

[0027] In the second synthetic route, the preparation steps are as follows:

[0028] Under an inert gas atmosphere, compound 4, a condensing agent, and an organic base are dissolved in an anhydrous organic solvent and reacted, compound 3 is added, and after reaction in the dark, through post-treatment, compound 2 is separated out;

[0029] Among them, the molar ratio of compound 3, compound 4, the condensing agent, and the organic base in the feed is 1:2.2 to 3:2.2 to 3:2.2 to 3; the feeding relationship between compound 3 and the organic solvent is that for every 1 g of compound 3, 30 to 80 ml of the organic solvent is added.

[0030] 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.

[0031] The preparation process may specifically be as follows: Add TBTU and compound 4 (chloroiodoquine derivative) into a reaction vessel, displace the air in the system with nitrogen, add anhydrous DMF, stir and react at room temperature, then add anhydrous triethylamine into the reaction system, stir and react at room temperature, then add tetravalent platinum compound 3 into the reaction system, displace the air in the system with nitrogen again, and place the reaction system in the dark at 25 - 120 °C for reaction for 24 - 72 hours. After the reaction is completed, remove the solvent under reduced pressure, and obtain asymmetric monosubstituted chloroiodoquine-modified tetravalent platinum compound 1 or symmetric disubstituted chloroiodoquine-modified tetravalent platinum compound 2 by column chromatography.

[0032] Further, the compound 3 is prepared by being oxidized by hydrogen peroxide.

[0033] The preparation process may specifically be:

[0034]

[0035] divalent platinum compound being oxidized by hydrogen peroxide at 60 - 70 °C for reaction for 1 - 8 hours to prepare dihydroxy tetravalent platinum compound 3.

[0036] On the other hand, the present invention provides a pharmaceutical composition, comprising an effective therapeutically amount of the compound shown in general formula (I), and its pharmaceutically acceptable excipients.

[0037] The pharmaceutically acceptable excipients in the present invention include one or several of carriers, excipients, diluents; such as binders, lubricants, disintegrants, solubilizers, diluents, stabilizers, suspending agents, non-pigments, flavoring agents, preservatives, solubilizing agents and matrices, etc. The pharmaceutically acceptable excipients may be aqueous or non-aqueous. Conventional excipients include gums, such as gelatin; starches, such as corn starch, potato starch; sugars, such as lactose, glucose and sucrose; cellulosic materials and their mixtures, such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate. The pharmaceutically acceptable excipients that can be used include but are not limited to, tragacanth powder, malt, talc powder, 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), buffering agents (such as magnesium hydroxide, aluminum hydroxide, boric acid and sodium borate, and phosphate buffer solution), alginic acid, pyrogen-free water, isotonic saline, Ringer's solution.

[0038] The chloroiodoquine tetravalent platinum compound or pharmaceutical composition described in the present invention has a dosage form of tablets, capsules, aerosols, dispersible tablets, oral liquids, suppositories, dripping pills, large volume injections, small injections, freeze-dried powder injections, ointments or liniments, etc., including various sustained-release, controlled-release dosage forms or nano-formulations prepared by using the commonly recognized pharmaceutical common sense.

[0039] The chloroiodoquine tetravalent platinum compound described in the present invention can be administered in unit dosage form, and the administration routes can be enteral and parenteral, such as oral, intramuscular, subcutaneous, nasal, etc.

[0040] The administration route of the chloroiodoquine tetravalent platinum compound of the present invention can be intravenous administration. Injections include intravenous injection, intramuscular injection, subcutaneous injection and acupoint injection, etc.

[0041] The method for preparing the active ingredient into a drug in the present invention can be prepared by the methods well known to those of ordinary skill in the art. For example: the active ingredient can be diluted with a carrier or encapsulated in a carrier to achieve rapid release, sustained release or delayed release of the active ingredient after being administered to a subject.

[0042] Another aspect of the present invention provides the use of the compound or pharmaceutical composition represented by the general formula (I) in the preparation of anti-tumor proliferation and anti-tumor metastasis drugs.

[0043] The chloroiodoquine tetravalent platinum derivative described in the present invention can effectively activate autophagy in tumor cells and effectively activate the tumor immune response, has excellent anti-tumor metastasis activity, and has a good therapeutic effect on metastatic malignant tumors.

[0044] Further, the anti-tumor proliferation is anti-human ovarian cancer, anti-human cervical cancer, anti-human lung adenocarcinoma, anti-cisplatin-resistant human lung adenocarcinoma, anti-mouse colon cancer.

[0045] Further, the anti-tumor metastasis is anti-mouse breast cancer cells.

[0046] Another aspect of the invention provides a combined preparation, including the compound or pharmaceutical composition represented by the general formula (I), and the combination with other types of anti-tumor drugs.

[0047] The chloroiodoquine derivative tetravalent platinum compound described in the present invention is expected to be used alone or in combination with marketed platinum-based, 5-fluorouracil-based, paclitaxel-based, etc. to prepare a combined preparation with anti-tumor activity. The combined preparation can be in the form of tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, sustained-release tablets, capsules, hard capsules, soft capsules, sustained-release capsules, oral liquids, mixtures, buccal tablets, granules, infusion, pills, powders, ointments, suspensions, solutions, injections, powder injections, freeze-dried powder injections, suppositories, liniments, ointments, plasters, creams, sprays, aerosols, drops, patches, etc.

[0048] Compared with the prior art, the compound with a clioquinol tetravalent platinum structure, its preparation method and its use according to the present invention have the following advantages:

[0049] (1) The present invention introduces the clioquinol molecule into the tetravalent platinum nucleus, synthesizes a series of novel clioquinol derivatives modified tetravalent platinum compounds, and through anti-tumor activity tests, it is proved that they have good anti-tumor and anti-cancer capabilities; this type of clioquinoline tetravalent platinum derivative has a good therapeutic effect on metastatic malignant tumors and has excellent anti-tumor metastasis activity;

[0050] (2) This type of clioquinoline tetravalent platinum derivative can induce severe DNA damage in tumor cells and induce mitochondria-mediated apoptosis, thereby playing an anti-tumor role;

[0051] (3) The anti-tumor activity is related to the activation of pro-apoptotic autophagy in tumor cells, and this type of clioquinoline tetravalent platinum derivative can effectively activate autophagy in tumor cells;

[0052] (4) The anti-tumor activity is related to the activation of the immune response in tumor cells, and this type of clioquinoline tetravalent platinum derivative can effectively activate the tumor immune response;

[0053] (5) The compound of general formula I described in the present invention has an innovative structure, is expected to obtain a variety of lead molecules effective against tumors, provides new candidate drug molecules for solving the defects of traditional divalent platinum drugs, and also opens up a new way for the modification of tetravalent platinum compounds. Such innovative drug research at the source will have important theoretical value and practical significance for the national economy, social development and people's health, etc. Description of the Drawings

[0054] Figure 1 In vivo anti-tumor activities of Compounds 2-5, 1-1, CDDP and OLP against 4T1 tumors in BALB / c mice (n = 5)*P < 0.05, **P < 0.01, ***P < 0.001, ns: no significant difference; a Schematic diagram of the experimental design; b Survival analysis of mice during treatment; c Relative body weight of mice during treatment; d Tumor growth as a function of time; e Tumor weights of each group at the end of the experiment. The TGI of the test drugs is shown on the column as compared with the normal saline group [TGI = (1 - tumor weight of the drug treatment group / tumor weight of the normal saline group) × 100%]; f Tumor images on the 15th day after the mice were sacrificed; g H&E staining pictures of tumor tissues;

[0055] Figure 2Comparison of organ indices of BALB / c mice in the compound 2-5, CDDP, and OLP treatment groups with those in the blank group (n = 5); a heart; b liver; c spleen; d lung; e kidney; organ index = organ weight / body weight × 100% *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significant difference compared with the control group;

[0056] Figure 3 Stability of compounds 2-5 and 1-1 upon incubation in whole blood at 37°C;

[0057] Figure 4 Evaluation of the migration inhibitory properties of compounds 2-5, CDDP, and OLP against 4T1 cells using an in vitro transwell assay. Tumor cells were treated with or without platinum complexes for 24 h; a typical image; b relative migration rate analysis *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significant difference;

[0058] Figure 5 For the migration inhibition of compounds 2-5, CDDP, and OLP against 4T1 cells at 10 μM in vitro, the wound healing degree was observed at 0 h, 12 h, and 24 h; a typical image; b wound closure analysis *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significant difference;

[0059] Figure 6 Inhibitory effect of compounds 2-5, CDDP, and OLP on lung metastasis of 4T1 breast cancer tumors in vivo (n = 5); *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significant difference compared with the control group; a schematic diagram of the experimental design; b representative photos of the front and back of the lungs of each group at the end of the experiment; c lung nodules in each group; d H&E staining of lung metastasis nodules, with nodules indicated by red arrows;

[0060] Figure 7 Platinum accumulation in 4T1 cells and 4T1 tumor tissues in vivo after treatment with platinum complexes (10 μM) in vitro for 24 h; a platinum content in whole cells; b platinum content in tumor tissues; c distribution of platinum in subcellular structures of tumor cells *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significant difference;

[0061] Figure 8 Stability of complex 2-5 in RPMI1640 within 48 h (Prepare a solution of complex 2-5 (0.5 mM) in RPMI640 and store it at 37°C for 48 h. Record spectra at 0 h, 12 h, 24 h, and 48 h);

[0062] Figure 9Reduction and DNA damage ability of Compound 2-5 (Prepare a solution of Compound 2-5 (0.5 mM) in the presence of ascorbic acid (AsA, 1 mM) and 5'-GMP in RPMI 1640, and store at 37 °C for 24 h); a Record spectra at 0 h, 4 h, 8 h, 12 h, and 24 h; b Formation of platinum-GMP (adduct of cisplatin and 5'-GMP);

[0063] Figure 10 Western blot analysis of γ-H2AX and p53 expression (4T1 cells were incubated with platinum compound 5 (2 μM), CDDP (10 μM), and OLP (10 μM) at 37 °C for 24 h).; a Bands; b Relative gray scale analysis [Relative gray scale = (gray scale of the indicated protein) / (gray scale of β-actin)] *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significant difference;

[0064] Figure 11 Quantification of apoptosis of 4T1 cells using annexin V-FITC / PI staining (4T1 cells were incubated with platinum complexes at 37 °C for 24 h); a Blank; b 2-5 (2 μM); c CDDP (10 μM); d OLP (10 μM); e Stacked columns;

[0065] Detection of ROS production in 4T1 cells treated with platinum complexes at 37 °C for 24 h (stain DCFH-DA); a Representative fluorescence microscope images of cells; b Statistical analysis of fluorescence intensity by flow cytometry *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significant difference compared with the blank group;

[0066] Figure 13 Flow cytometry analysis of mitochondrial membrane potential (ΔΨm) (4T1 cells were treated with or without platinum complexes at 37 °C for 24 h and stained with JC-1); a Blank; b CDDP (10 μM); c CDDP (2 μM); d OLP (10 μM); e 2-5 (10 μM); f 2-5 (2 μM);

[0067] Figure 14 Western blot analysis of the expression of Bcl-2, Bax, caspase3, and c-caspase3 (4T1 cells were incubated with platinum compound 5 (2 μM), CDDP (10 μM), and OLP (10 μM) at 37 °C for 24 h.) a Bands; b Relative gray scale analysis *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significant difference;

[0068] Figure 15Western blot analysis of the expression of p62 and LC3II / I (4T1 cells were incubated with platinum compound 5 (2 μM), CDDP (10 μM) and OLP (10 μM) at 37 °C for 24 h). (a) Blots; (b) Relative gray scale analysis *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significantly different;

[0069] Figure 16 Immunohistochemical staining of P62 in tumor tissues of BALB / c mice bearing 4T1 allografts; a Representative micrographs; b Quantification data of P62 expression *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significant difference compared with the blank group;

[0070] Figure 17 Evaluation of the autophagy induction of compounds 2 - 5 (2 μM), CDDP (10 μM) and OLP (10 μM) on 4T1 cells using monodansylcadaverine (MDC) staining method;

[0071] Figure 18 Cell viability of 4T1 cells treated with compounds 2 - 5 (0.6 μM) and CDDP (0.6 μM) with or without 3 - methyladenine (3 - MA) for 48 h using MTT assay;

[0072] Figure 19 Determination of the expression of autophagy proteins LC3I / II, p62 and Beclin1 in tumor cells treated with compounds 2 - 5 in the presence of 3 - MA by Western Blotting (4T1 cells were treated with compounds 2 - 5 (2 μM) in the presence of 3 - MA (70 μM)); a Blots; b Relative gray scale analysis *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significant difference;

[0073] Figure 20 Immunohistochemical staining of PD - L1, CD4 + and CD8 + in tumor tissues of BALB / c mice bearing 4T1 allografts; a Representative micrographs; b Quantification data of PD - L1; c CD4 + TIL quantification data; d Quantification data of CD8+TIL *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significant difference compared with the blank group;

[0074] Figure 21Western blot analysis of the expression of HIF-1α, VEGFA, and MMP-9 (4T1 cells were incubated with platinum compound 5 (2 μM), CDDP (10 μM), and OLP (10 μM) at 37 °C for 24 h). A blot; B relative gray scale analysis *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significant difference;

[0075] Figure 22 Immunohistochemical staining of VEGFA, MMP-9, and CD34 in tumor tissues of BALB / c mice bearing 4T1 xenografts; a representative micrograph; b quantitative data *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significant difference compared with the blank group. Detailed implementation manners

[0076] Unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are all conventional methods unless otherwise specified.

[0077] The present invention will be described in detail below with reference to the examples and the accompanying drawings.

[0078] I. Preparation of tetravalent platinum shown in compound 3

[0079] 1. Synthesis of cis-dihydroxyplatinum(IV) 3-1

[0080]

[0081] Add 1.0 g of cisplatin and 30 mL of distilled water to a 250 mL round-bottom flask, stir to disperse it, slowly add 50 mL of 30% hydrogen peroxide to the reaction system, raise the temperature to 60 °C and stir for 4 h. Stop the reaction, place it at 4 °C for crystallization for 12 hours, filter and separate to obtain a yellow solid, add an appropriate amount of distilled water, heat to 80 °C to dissolve it clearly, place it at 4 °C for crystallization for 12 hours, and filter to obtain yellow crystals of compound 3-1 (0.82 g, 74%).

[0082] 2. Synthesis of oxaliplatin(IV) dihydroxy 3-2

[0083]

[0084] Add 1.0 g of oxaliplatin and 30 mL of distilled water to a 250 mL round-bottom flask, stir to disperse it, slowly add 50 mL of 30% hydrogen peroxide to the reaction system, raise the temperature to 60 °C and stir for 4 h. Stop the reaction, place it at 4 °C to crystallize for 12 hours, filter and separate to obtain a yellow solid, add an appropriate amount of distilled water, heat to 80 °C to make it clear, place it at 4 °C to crystallize for 12 hours, and filter to obtain white crystals of compound 4-2 (0.85 g, 78%).

[0085] 3. Synthesis of dihydroxycarboplatin(IV) 3-3

[0086]

[0087] Add 1.0 g of carboplatin and 30 mL of distilled water to a 250 mL round-bottom flask, stir to disperse it, slowly add 50 mL of 30% hydrogen peroxide to the reaction system, raise the temperature to 60 °C and stir for 4 h. Stop the reaction, place it at 4 °C to crystallize for 12 hours, filter and separate to obtain a yellow solid, add an appropriate amount of distilled water, heat to 80 °C to make it clear, place it at 4 °C to crystallize for 12 hours, and filter to obtain white crystals of compound 3-3 (0.83 g, 76%).

[0088] II. Preparation of the chloroiodoquine derivative shown in Compound 4

[0089] 1. Preparation of chloroiodoquine derivative 4-1

[0090]

[0091] Place chloroiodoquine (3.06 g, 10 mmol) and methyl 2-bromoacetate (1.84 g, 12 mmol) in a reaction flask, add 50 mL of DMF and stir evenly. Then add anhydrous potassium carbonate (1.66 g, 12 mmol) and stir for 24 h. Stop the reaction, remove the solvent under reduced pressure, and obtain chloroiodoquine ester 5-1 as a white solid (2.91 g, 77%) by column chromatography.

[0092]

[0093] Dissolve chloroiodoquine ester 5-1 (2.91 g, 7.7 mmol) in 100 mL of ethanol, then add 10.9 mL of 5% NaOH / H2O solution and stir at room temperature for 12 h. After the reaction is completed, acidify the resulting solution with 1 mmol of hydrochloric acid to pH = 3-4, and then extract with ethyl acetate to obtain chloroiodoquine derivative 4-1 as a white solid (2.24 g, 80%).

[0094] 2. Preparation of chloroiodoquine derivative 4-2

[0095]

[0096] Place clioquinol (3.06 g, 10 mmol) and methyl 4-bromobutyrate (2.17 g, 12 mmol) in a reaction flask, add 50 mL of DMF and stir evenly. Then add anhydrous potassium carbonate (1.66 g, 12 mmol) and stir for 24 h. Stop the reaction, remove the solvent under reduced pressure, and obtain clioquinol ester 5-2 as a pink solid (3.57 g, 88%) by column chromatography.

[0097]

[0098] Dissolve clioquinol ester 5-2 (3.57 g, 8.8 mmol) in 100 mL of ethanol, then add 13.4 mL of 5% NaOH / H₂O solution and stir at room temperature for 12 h. After the reaction is completed, acidify the resulting solution with 1 mmol of hydrochloric acid to pH = 3-4, and then extract with ethyl acetate to obtain clioquinol derivative 4-2 as a white solid (2.79 g, 81%).

[0099] 3. Preparation of clioquinol derivative 4-3

[0100]

[0101] Place clioquinol (3.06 g, 10 mmol) and methyl 5-bromovalerate (2.34 g, 12 mmol) in a reaction flask, add 50 mL of DMF and stir evenly. Then add anhydrous potassium carbonate (1.66 g, 12 mmol) and stir for 24 h. Stop the reaction, remove the solvent under reduced pressure, and obtain clioquinol ester 5-3 as a white solid (3.51 g, 87%) by column chromatography.

[0102]

[0103] Dissolve clioquinol ester 5-3 (3.51 g, 8.8 mmol) in 100 mL of ethanol, then add 13.1 mL of 5% NaOH / H₂O solution and stir at room temperature for 12 h. After the reaction is completed, acidify the resulting solution with 1 mmol of hydrochloric acid to pH = 3-4, and then extract with ethyl acetate to obtain clioquinol derivative 4-3 as a white solid (2.92 g, 82%).

[0104] To make the objectives, technical solutions and advantages of the present invention clearer, the representative embodiments of the present invention will be described in detail below, without being limited thereto.

[0105] III. Preparation of Asymmetric Monochloroiodoquinol Derivative Tetravalent Platinum Compound 1 (Examples 1-2)

[0106] Synthesis Route 1:

[0107]

[0108] Add TBTU and Compound 4 (chloroiodoquine derivative) into the reaction vessel, displace the air in the system with nitrogen, add anhydrous DMF, stir the reaction at room temperature, then add anhydrous triethylamine into the reaction system, stir the reaction at room temperature, then add the tetravalent platinum compound 3 into the reaction system, displace the air in the system with nitrogen again, and place the reaction system in the dark at 25-120 °C for reaction for 24-72 hours. After the reaction is completed, remove the solvent under reduced pressure, and obtain the asymmetric monosubstituted chloroiodoquine-modified tetravalent platinum compound 1 by column chromatography;

[0109] Among them, the molar ratio of the feed of Compound 3, Compound 4, TBTU, and triethylamine is 1:2.2-3:2.2-3:2.2-3; the feeding relationship between Compound 3 and DMF is that 30-80 mL of DMF is added for every 1 g of Compound 3.

[0110] Example 1

[0111] Synthesis of Asymmetric Monochloroiodoquine Derivative Cisplatin Tetravalent Platinum Compound 1-1

[0112]

[0113] Dissolve chloroiodoquine derivative 4-3 (110 mg, 0.27 mmol), TBTU (87 mg, 0.27 mmol) and TEA (38 μL, 0.27 mmol) in 5 mL of DMF and stir at room temperature for 15 min. Then add Compound 3-1 (100 mg, 0.25 mmol), and stir the system in the dark at 50 °C for 48 h. After the reaction is completed, remove the solvent by distillation under reduced pressure, and obtain white solid Compound 1-1 (48 mg, 26.6%) by column chromatography. Determined by HPLC (eluent: MeOH / H2O = 80 / 20), the purity is 99.6%.

[0114] Compound 1-1: 1 H NMR (500 MHz, DMSO-d6) δ 9.03 (dd, J = 4.1, 1.7 Hz, 1H), 8.54 (dd, J = 8.6, 1.7 Hz, 1H), 8.13 (s, 1H), 7.75 (dd, J = 8.6, 4.1 Hz, 1H), 6.45–5.52 (t, J = 51.4 Hz, 6H), 4.31 (t, J = 6.3 Hz, 2H), 2.30 (t, J = 7.4 Hz, 2H), 1.97–1.84 (m, 2H), 1.80–1.70 (m, 2H). 13CNMR(126MHz, DMSO-d6) δ 181.42, 155.58, 151.44, 142.23, 135.15, 133.57, 127.17, 125.69, 123.60, 92.05, 74.99, 36.59, 30.08, 22.81. MS-ESI: calcd for [M+H] + : 722 (M = C 14 H 19 Cl3IN3O4Pt), found: 722. HRMS: calcd for [M+H] + : 720.9206 (M = C 14 H 19 Cl3IN3O4Pt), found: 720.9207.

[0115] Example 2

[0116] Synthesis of the asymmetric monochloroiodoquinoline derivative oxaliplatin tetravalent platinum compound 1-2

[0117]

[0118] Dissolve the chloroiodoquinoline derivative 4-3 (102 mg, 0.25 mmol), TBTU (81 mg, 0.25 mmol) and TEA (35 μL, 0.25 mmol) in 5 mL of DMF and stir at room temperature for 15 min. Then add oxaliplatin 3-2 (100 mg, 0.23 mmol), and the system is stirred in the dark at 50 °C for 48 h. After the reaction is completed, the solvent is evaporated under reduced pressure, and the white solid compound 1-2 (70 mg, 34.1%) is obtained by column chromatography. The purity is 99.6% as determined by HPLC (eluent: MeOH / H2O = 80 / 20).

[0119] Compound 1-2: 1 H NMR (500 MHz, DMSO-d6) δ 9.01 (dd, J = 4.1, 1.5 Hz, 1H), 8.53 (dd, J = 8.6, 1.5 Hz, 1H), 8.12 (s, 1H), 7.75 (dd, J = 8.6, 4.2 Hz, 1H), 4.29 (t, J = 6.0 Hz, 2H), 2.65–2.55 (m, 1H), 2.41–2.27 (m, 2H), 2.15–1.96 (m, 2H), 1.87–1.72 (m, 4H), 1.56–1.31 (m, 4H), 1.16–1.00 (t, J = 7.0 Hz, 3H). 13CNMR(126MHz, DMSO-d6) δ 182.6, 164.3, 155.5, 151.4, 142.2, 135.1, 133.6, 127.2, 125.7, 123.6, 91.8, 74.7, 65.4, 62.0, 60.5, 36.9, 31.2, 29.9, 24.1, 22.8, 15.6. MS-ESI: calcd for [M+H] + : 820 (M = C 22 H 27 ClIN3O8Pt), found: 820. HRMS: calcd for [M+H] + : 819.0252 (M = C 22 H 27 ClIN3O8Pt), found: 819.0259. HPLC purity: 99.6%.

[0120] IV. Preparation of Symmetric Dichloroiodoquine Derivative Tetravalent Platinum Compound 2 (Examples 3 - 7)

[0121] Synthetic Route 2:

[0122]

[0123] Add TBTU and Compound 4 (chloroiodoquine derivative) into the reaction vessel, displace the air in the system with nitrogen, add anhydrous DMF, stir the reaction at room temperature, then add anhydrous triethylamine into the reaction system, stir the reaction at room temperature, then add tetravalent platinum compound 3 into the reaction system, displace the air in the system with nitrogen again, and place the reaction system in the dark at 25 - 120 °C for 24 - 72 hours. After the reaction is completed, remove the solvent under reduced pressure, and obtain symmetrically disubstituted chloroiodoquine-modified tetravalent platinum compound 2 by column chromatography;

[0124] Among them, the molar ratio of Compound 3, Compound 4, TBTU, and triethylamine is 1:2.2 - 3:2.2 - 3:2.2 - 3; the feeding relationship between Compound 3 and DMF is that for every 1 g of Compound 3, 30 - 80 ml of DMF is added.

[0125] Example 3

[0126] Synthesis of Symmetric Dichloroiodoquine Derivative Cisplatin Tetravalent Platinum Compound 2 - 1

[0127]

[0128] Dissolve chloroiodoquine derivative 4-2 (352 mg, 0.90 mmol), TBTU (289 mg, 0.90 mmol) and TEA (126 μL, 0.90 mmol) in 5 mL of DMF, and stir at room temperature for 15 min. Then add compound 3-1 (100 mg, 0.30 mmol), and stir the system in the dark at 50 °C for 48 h. After the reaction is completed, evaporate the solvent under reduced pressure, and obtain white solid compound 2-1 (65.0 mg, 20.0%) by column chromatography. The purity is 96.2% as determined by HPLC (eluent: MeOH / H2O = 80 / 20).

[0129] Compound 2-1: 1 H NMR (500 MHz, DMSO-d6) δ 9.03 (dd, J = 4.2, 1.6 Hz, 2H), 8.53 (dd, J = 8.6, 1.6 Hz, 2H), 8.13 (s, 2H), 7.76 (dd, J = 8.6, 4.2 Hz, 2H), 6.58 (s, 6H), 4.34 (t, J = 6.4 Hz, 4H), 2.60 (t, J = 7.6 Hz, 4H), 2.11–2.00 (m, 4H). 13 C NMR (126 MHz, DMSO) δ 180.82, 155.33, 151.43, 142.17, 135.10, 133.55, 127.16, 125.76, 123.60, 92.04, 74.62, 32.92, 26.89. MS-ESI: calcd for [M] + : 1081 (M = C 26 H 26 Cl4I2N4O6Pt), found: 1081. HRMS: calcd for [M + H] + : 1079.8416 (M = C 26 H 26 Cl4I2N4O6Pt), found: 1079.8418.

[0130] Example 4

[0131] Synthesis of symmetric dichloroiodoquine derivative oxaliplatin tetravalent platinum compound 2-2

[0132]

[0133] The chloroiodoquine derivative 4-2 (209 mg, 0.90 mmol), TBTU (172 mg, 0.90 mmol) and TEA (75 μL, 0.90 mmol) were dissolved in 5 mL of DMF and stirred at room temperature for 15 min. Then compound 3-2 (100 mg, 0.23 mmol) was added, and the system was stirred under dark at 50 °C for 48 h. After the reaction was completed, the solvent was removed by evaporation under reduced pressure, and the white solid compound 2-2 (96.0 mg, 35.4%) was obtained by column chromatography. The purity was determined to be 99.9% by HPLC (eluent: MeOH / H2O = 80 / 20).

[0134] Compound 2-2: 1 H NMR (500 MHz, DMSO-d6) δ 9.02 (dd, J = 4.1, 1.6 Hz, 2H), 8.54 (dd, J = 8.6, 1.7 Hz, 2H), 8.42 (d, J = 8.4 Hz, 2H, NH2), 8.36–8.23 (m, 2H, NH2), 8.14 (s, 2H), 7.75 (dd, J = 8.6, 4.2 Hz, 2H), 4.31 (t, J = 6.4 Hz, 4H), 2.66 (t, J = 7.6 Hz, 4H), 2.62–2.53 (m, 2H), 2.14–1.98 (m, 6H), 1.44 (dd, J = 36.3, 9.6 Hz, 4H), 1.10 (d, J = 6.5 Hz, 2H). 13 C NMR (126 MHz, DMSO-d6) δ 181.01, 163.91, 155.28, 151.46, 142.16, 135.09, 133.58, 127.19, 125.84, 123.64, 91.90, 74.39, 61.61, 33.03, 31.42, 26.73, 23.97. MS-ESI: calcd for [M+H] + : 1179 (M = C 34 H 34 Cl2I2N4O 10 Pt), found: 1179. HRMS: calcd for [M+H] + : 1177.9462 (M = C 34 H 34 Cl2I2N4O 10 Pt), found: 1177.9462.

[0135] Example 5

[0136] Synthesis of symmetric dichloroiodoquine derivative carboplatin tetravalent platinum compound 2-3

[0137]

[0138] Dissolve the chloroiodoquine derivative 4-2 (290 mg, 0.90 mmol), TBTU (238 mg, 0.90 mmol) and TEA (104 μL, 0.90 mmol) in 5 mL of DMF, and stir at room temperature for 15 min. Then add compound 3-3 (100 mg, 0.25 mmol), and the system is stirred under dark conditions at 50 °C for 48 h. After the reaction is completed, the solvent is removed by distillation under reduced pressure, and the white solid compound 2-3 (92.0 mg, 31.9%) is obtained by column chromatography. The purity is 96.2% as determined by HPLC (eluent: MeOH / H2O = 80 / 20).

[0139] Compound 2-3: 1 H NMR (500 MHz, DMSO-d6) δ 8.98 (t, J = 5.2 Hz, 2H), 8.47 (dd, J = 14.1, 8.5 Hz, 2H), 8.06 (d, J = 11.0 Hz, 2H), 7.70 (ddd, J = 8.8, 6.5, 4.2 Hz, 2H), 6.69–6.10 (m, 6H, NH3), 4.26 (dt, J = 14.3, 6.4 Hz, 4H), 2.74–2.52 (m, 6H), 2.46–2.33 (t, J = 9.1 Hz, 1H), 2.08–1.93 (m, 4H), 1.86–1.65 (m, 2H). 13 C NMR (126 MHz, DMSO-d6) δ 180.4, 179.8, 176.5, 176.2, 155.3, 155.3, 151.3, 151.3, 142.1, 142.0, 135.1, 133.5, 133.42, 127.1, 127.1, 125.7, 125.5, 123.5, 123.5, 91.9, 91.6, 74.6, 55.7, 40.6, 40.5, 40.4, 40.3, 40.3, 40.2, 40.1, 40.0, 39.9, 39.8, 39.7, 39.5, 35.4, 33.2, 32.4, 28.0, 26.9, 26.8, 16.1. MS-ESI: calcd for [M+Na] + : 1175 (M = C 32 H 32 Cl2I2N4O 10 Pt), found: 1175. HRMS: calcd for [M+H] + : 1151.9305 (M = C 32 H 32 Cl2I2N4O 10Pt), found: 1151.9316.

[0140] Example 6

[0141] Synthesis of symmetric dichloroiodoquinoline derivative cisplatin tetravalent platinum compound 2-4

[0142]

[0143] Dissolve the chloroiodoquinoline derivative 4-1 (327 mg, 0.90 mmol), TBTU (289 mg, 0.90 mmol) and TEA (125 μL, 0.90 mmol) in 5 mL of DMF and stir at room temperature for 15 min. Then add compound 3-1 (100 mg, 0.30 mmol), and stir the system in the dark at 50 °C for 48 h. After the reaction is completed, evaporate the solvent under reduced pressure, and obtain the white solid compound 2-4 (90.0 mg, 29.9%) by column chromatography. The purity is 98.4% determined by HPLC (eluent: MeOH / H2O = 80 / 20).

[0144] Compound 2-4: 1 H NMR (500 MHz, DMSO-d6) δ 9.01 (dd, J = 4.1, 1.7 Hz, 2H), 8.54 (dt, J = 8.6, 1.3 Hz, 2H), 8.13 (s, 2H), 7.76 (dd, J = 8.6, 4.1 Hz, 2H), 6.63 (s, 6H, NH3), 5.14 (s, 4H). 13 C NMR (126 MHz, DMSO-d6) δ 180.87, 158.85, 155.63, 146.09, 140.14, 138.39, 131.81, 129.96, 128.27, 95.59, 75.07. MS-ESI: calcd for [M+H] + : 1025 (M = C 22 H 18 Cl4I2N4O6Pt), found: 1025. HRMS: calcd for [M+H] + : 1023.7790 (M = C 22 H 18 Cl4I2N4O6Pt), found: 1023.7797.

[0145] Example 7

[0146] Synthesis of symmetric dichloroiodoquinoline derivative cisplatin tetravalent platinum compound 2-5

[0147]

[0148] Dissolve chloroiodoquine derivative 4-3 (364 mg, 0.90 mmol), TBTU (288 mg, 0.90 mmol) and TEA (126 μL, 0.90 mmol) in 5 mL of DMF, and stir at room temperature for 15 min. Then add compound 3-1 (100 mg, 0.30 mmol), and stir the system under light protection at 50 °C for 48 h. After the reaction is completed, evaporate the solvent under reduced pressure, and obtain white solid compound 2-5 (100 mg, 30.1%) by column chromatography. The purity is 99.9% determined by HPLC (eluent: MeOH / H2O = 80 / 20).

[0149] Compound 2-5: 1 H NMR (500 MHz, DMSO-d6) δ 9.03 (dd, J = 4.2, 1.7 Hz, 2H), 8.54 (dt, J = 8.6, 2.1 Hz, 2H), 8.13 (d, J = 2.9 Hz, 2H), 7.75 (dd, J = 8.6, 4.2 Hz, 2H), 6.59 (br, 6H, NH3), 4.31 (t, J = 6.3 Hz, 4H), 2.39 (t, J = 7.4 Hz, 4H), 1.89 (dd, J = 8.7, 6.0 Hz, 4H), 1.78 (q, J = 7.4 Hz, 4H). 13 C NMR (126 MHz, DMSO-d6) δ 181.24, 155.52, 151.41, 142.17, 135.15, 133.62, 127.17, 125.72, 123.60, 91.98, 74.87, 35.79, 29.92, 22.64. MS-ESI: calcd for [M+Na] + : 1132 (M = C 28 H 30 Cl4I2N4O6Pt), found: 1132. HRMS: calcd for [M+H] + : 1107.8729 (M = C 28 H 30 Cl4I2N4O6Pt), found: 1107.8729.

[0150] V. Experimental Tests

[0151] To better understand the essence of the present invention, the following pharmacological experimental results of the inhibitory effects of compounds on tumors in in vivo and in vitro experiments are used to illustrate the potential uses of these compounds in the pharmaceutical field. The pharmacological experiments give partial activity data of some compounds. It must be noted that the pharmacological experiments of the present invention are used to illustrate the present invention rather than limit the present invention. Any simple improvement made to the present invention according to the essence of the present invention falls within the protection scope of the present invention.

[0152] 1. In vitro anti-tumor proliferation activity experiment

[0153] Test method:

[0154] In this experiment, the MTT method was used to determine the cell survival rate, and the in vitro anti-cancer activity of the complex was measured according to the half inhibitory concentration (IC 50 ) value of each test sample on cell growth.

[0155] 100 μL of tumor cells in the logarithmic growth phase were inoculated into a 96-well plate, and the inoculation cell density was 3000 - 5000 cells / well. The last column was reserved as the zero-adjustment well. It was placed in a 37 °C cell incubator for culture for 12 h, and then 100 μL of the compound culture medium solution with gradient concentrations was added to the 96-well plate, and it was continued to be placed in a 37 °C cell incubator for culture for 48 h. 20 μL of a 5 mg / mL MTT solution was added to each well of the 96-well plate, taken out after culturing in a 37 °C cell incubator for 4 h, the culture medium was aspirated, 150 μL of DMSO was added, and it was shaken in the dark on a shaker at 37 °C for 20 min. The absorbance OD value of each well was measured with an enzyme-linked immunosorbent assay reader at 570 nm, and its IC 50 value was calculated. Each group of experiments was repeated at least three times.

[0156] The cancer cell lines selected 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.

[0157] Discussion on anti-tumor activity:

[0158] Table 1

[0159]

[0160]

[0161] Table 1 a RF: Drug resistance coefficient, 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 + CLQ (1:2): A mixture of CDDP and CLQ with a molar ratio of 1:2.

[0162] The test results are shown in Table 1: Compounds 2-1 to 2-3 with different platinum parent nuclei have distinct anti-tumor properties. Compound 2-1 with a CDDP parent nucleus has better activity than 2-2 with an OLP parent nucleus and 2-3 (CBP) with a CBP parent nucleus. The linking group between CLQ and the platinum nucleus significantly affects the anti-tumor efficacy of the target product. Compound 2-5 with a valeryl group has the most significant anti-tumor effect, and its activity is stronger than that of compounds 2-4 and 2-1 with acetyl and butyryl groups. The anti-tumor activity of the single CLQ-substituted platinum(IV) compounds 1-1 and 1-2 is lower than that of the double CLQ platinum compound 2-5. At the same time, compared with compounds 2-5 and 1-1 with a CDDP nucleus, the anti-tumor activity of compound 1-2 with an OLP parent nucleus is relatively weak.

[0163] The calculation of the resistance coefficient is an important method to investigate the drug's ability to overcome drug resistance. The ratio of the IC50 value of A549R to A549 is the resistance coefficient. The RF values of the double CLQ platinum(IV) compounds 2-1, 2-4, and 2-5 are 2.42, 2.31, and 0.61 respectively, which are significantly lower than that of CDDP (RF = 3.78), indicating that this type of drug can effectively overcome the drug resistance of CDDP.

[0164] To further evaluate the toxicity of the CLQ platinum(IV) complex, we tested its toxicity to normal liver cells LO2 and calculated the selectivity index of the drug (SI = IC 50 (LO2) / IC 50 (HepG2)). The SI values of the double CLQ platinum(IV) complexes 2-1 and 2-5 (1.41, 1.21) are higher than those of the reference drugs CDDP, OLP, and JM216 (0.72, 0.15, 0.47), indicating that this type of drug can effectively reduce the toxicity of the drug to normal cells.

[0165] Conclusion on in vitro anti-tumor activity:

[0166] The chloroquine iodohydroxyquinoline tetravalent platinum compound has good in vitro anti-tumor activity. The structure of the platinum parent nucleus will have a significant impact on the activity. The chloroquine iodohydroxyquinoline tetravalent platinum compound with a cisplatin parent nucleus has significant activity, which is higher than that of cisplatin, oxaliplatin, and the platinum(IV) reference drug JM216; the linking group between chloroquine iodohydroxyquinoline and the platinum nucleus also significantly affects the anti-tumor efficacy of the chloroquine iodohydroxyquinoline tetravalent platinum complex. The double CLQ tetravalent platinum complex 2-5 with a valeryl group has the most significant anti-tumor effect, and at the same time can overcome the drug resistance of divalent platinum drugs and reduce toxicity.

[0167] 2. In Vivo Antitumor Proliferation Activity Experiment

[0168] To evaluate the antitumor efficacy of CLQ-platinum(IV) complexes in vivo, we evaluated the antitumor activities of tetravalent platinum complexes 2-5 and 1-1 with dual and single CLQ ligands in female BALB / c mice bearing 4T1 tumors. CDDP and OLP were used as positive control drugs, and the normal saline treatment group served as the blank control.

[0169] Test method:

[0170] Female BALB / c mice (18 - 20 g) were purchased from Shandong Pengyue Experimental Animal Breeding Co., Ltd. All animals were fed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

[0171] After in vitro expansion of 4T-1 cells, the cells were digested and collected, and washed 3 times with normal saline. The cells were resuspended in normal saline solution. Tumor cells were inoculated into the right dorsal side of female BALB / c mice at an inoculation density of 5×10 5 . On the 4th day after inoculation, the tumor was palpable. The mice were randomly divided into 5 groups of 5 mice each: normal saline group, compound 1-1 group, compound 2-5 group, OLP group, and CDDP group. The dosing dose was 2 mg Pt / kg. The drugs were administered on the 4th, 7th, and 10th days, for a total of 3 administrations, and the administration method was tail vein injection. During the experiment, the changes in tumor volume of the mice were recorded to evaluate the tumor growth rate; the changes in body weight of the mice were recorded to evaluate the drug toxicity. On the 15th day, the mice were sacrificed, and the serum, tumor tissues, and visceral tissues (heart, lung, liver, spleen, kidney) of the mice were collected. The tumor tissues were weighed. The tissue samples were fixed with formalin and evaluated by hematoxylin and eosin (HE) staining and immunohistochemical analysis, etc.

[0172] Result analysis:

[0173] Figure 1 The results in b and c showed that the body weight of the mice in the single CLQ-platinum(IV) complex 1-1 treatment group decreased severely (12.5%) after two administrations (on the 8th day), and the survival rate of the mice decreased to 20% on the 8th day and to 0% on the 9th day. This demonstrated the obvious toxicity of complex 1-1. Due to the toxicity of compound 1-1, it was not further investigated in subsequent experiments. The platinum(II) reference drug CDDP caused a severe decrease in body weight to 85.8%. Notably, compared with the blank group, the dual CLQ-platinum(IV) complex 2-5 had no significant effect on body weight (p = ns). Subsequently, the organ index (the ratio of organ mass to body weight) was calculated to further study the toxicity of platinum drugs ( Figure 2) The spleens of the CDDP and OLP treatment groups were significantly inhibited. Platinum(IV) compounds 2-5 could significantly relieve the inhibition of platinum drugs on the spleen, and their spleen indices were comparable to those of the blank group (P<0.001). To better study the reasons for the different in vivo toxicities of compounds 2-5 and 1-1, we determined their stabilities in mouse whole blood. Figure 3 The results in 3 showed that the reduction rate of the single CLQ platinum(IV) complex 1-1 in blood was significantly faster than that of the double CLQ platinum complex 2-5, indicating that the blood stability of the single CLQ platinum(IV) compound was poor, which might be an important reason for its high toxicity. Then, the tumor volume changes and tumor mass data showed that compared with the blank group (2162 mm 3 ), the tumor volume of the compound 2-5 treatment group was 760 mm

[0174] Conclusion on in vivo anti-tumor activity:

[0175] The in vivo experimental results showed that the double CLQ tetravalent platinum compound 2-5 could effectively inhibit tumor growth, had significant in vivo anti-tumor activity, was comparable to the reference drug cisplatin, superior to oxaliplatin, and had significantly lower toxicity than divalent platinum drugs and the single CLQ tetravalent platinum compound 1-1, showing great development potential.

[0176] 3. In vivo and in vitro anti-tumor metastasis experiments

[0177] Metastasis is the main cause of death in patients with malignant tumors, and about 90% of cancer patients die from tumor metastasis. This patent investigated the in vivo and in vitro anti-tumor metastasis activities of CLQ tetravalent platinum compounds.

[0178] Experimental method:

[0179] Transwell experiment: The experiment was carried out in a Transwell chamber with micropores (8 μm pores). First, 4T1 cells (5×10 in 0.2 mL) 4The cells were resuspended in RPMI 1640 medium and seeded in the upper chamber. 10% FBS-RPMI 1640 medium containing different compounds (2-5: 2 μM, CDDP and OLP: 10 μM) was added to the lower chamber. Secondly, the cells were incubated in a 37 °C incubator with 5% CO2 for 24 hours. Then, the cells were fixed with 4% paraformaldehyde for 20 minutes and stained with 0.1% crystal violet for 20 minutes. The non-migrating cells in the upper chamber were gently scraped off with a cotton swab. The migrating cells on the lower surface of the chamber were photographed in five random fields with an inverted microscope to examine the anti-migration effect of the drugs.

[0180] Wound healing assay: 4T1 cells (8×10 5 ) were seeded in 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 scratch was made in each well. Then, the cells were treated with 1% FBS-RPMI 1640 medium containing the compounds (2-5: 2 μM; CDDP, OLP: 10 μM), and the degree of scratch healing was photographed and recorded at 0, 12, and 24 hours respectively.

[0181] In vivo anti-metastasis experiment: BALB / c female mice (18-20 g) were used as experimental subjects, and a lung metastasis model was established by injecting 4T1 cells (2×10 5 ) via the tail vein. The mice were randomly divided into four groups (n = 5): treated with 2-5, CDDP, OLP, and normal saline (blank group). Three times of tail vein injections were given on the 4th, 7th, and 10th days after cell inoculation, and the dosage was 2 mg Pt / kg. The mice were sacrificed on the 19th day, and the lung tissues were dissected. Then, after fixation with 4% formaldehyde solution for 24 h, the lung metastasis nodules of each mouse were counted and analyzed, and the lung tissues were stained with HE for observation.

[0182] Result analysis:

[0183] As Figure 4 shown, the results of the Transwell experiment showed that the anti-metastasis ability of bis-CLQ platinum(IV) complex 2-5 was stronger than that of CDDP and OLP. The wound healing assay ( Figure 5 ) further verified the anti-metastasis ability of compound 2-5. Even at a low concentration of 2 μM, compound 2-5 still had a strong ability to inhibit wound healing.

[0184] The results of in vivo anti-tumor metastasis activity were as Figure 6As shown, CLQ platinum(IV) compounds 2-5 can significantly inhibit the formation of pulmonary tumor nodules in vivo. The number of nodules is 24.0% of that in the blank group, and is significantly lower than those in the CDDP and OLP treatment groups (72.1% and 51.1%, P<0.001). The results of H&E staining further confirmed this result. Compared with the blank group, as well as the CDDP and OLP treatment groups, the number of nodules in the lung tissue of the compound 2-5 treatment group was less and smaller.

[0185] Conclusion on anti-tumor metastasis:

[0186] In summary, chloroquine iodine hydroxyquinoline platinum(IV) complexes 2-5 exhibit strong anti-tumor migration activity both in vivo and in vitro. In vitro Transwell and scratch experiments confirmed its anti-tumor cell migration and metastasis ability. More importantly, this compound still shows strong anti-tumor metastasis ability in the in vivo lung metastasis model, significantly stronger than the divalent platinum reference drugs CDDP and OLP. The above results confirm its great development potential as a new anti-tumor metastasis drug.

[0187] 4. In vitro and in vivo tumor uptake experiments

[0188] Uptake is a key factor affecting the efficacy of chemotherapeutic drugs, especially for platinum complexes. Therefore, detecting the cellular accumulation and distribution of platinum in 4T1 cells is of great significance for evaluating drug activity.

[0189] Experimental method:

[0190] Logarithmic growth phase 4T1 cells were placed in a 6-well plate (10 6 / well), cultured in a 37°C, 5% carbon dioxide incubator for about 12 hours, treated with drugs, and then cultured for another 24 hours. Cells were digested with trypsin without EDTA and collected. Cells were washed twice with PBS, digested with concentrated nitric acid, and the platinum element content in the cells was detected by atomic absorption method, and then the uptake of the drug in tumor cells was measured. Tumor tissues of different experimental groups were weighed and digested, and the platinum element was quantified by atomic absorption method to calculate the uptake of the drug.

[0191] Conclusion analysis:

[0192] Figure 7 The results in b show that compared with the platinum(II) drugs CDDP and OLP, the uptake of CLQ platinum(IV) complexes 2-1, 2-4, 2-5 and 1-1 in tumor cells is higher than that of divalent platinum drugs. Figure 7The results in [[a]] and [[7c]] show that the platinum content of compounds 2-5 in whole tumor cells is 5.4 times and 8.1 times that of platinum(II) drugs CDDP and OLP, respectively, and the binding amounts to DNA are 14.0 times and 27.5 times that of CDDP and OLP, respectively. In in vivo experiments, the drug accumulation of highly active compounds 2-5 in tumor tissues is also higher than that of CDDP and OLP. These facts indicate that after the CLQ platinum(IV) complex enters tumor cells, it can effectively bind to DNA and induce DNA damage. The conclusion of this part is consistent with the results of in vivo and in vitro activity experiments.

[0193] 5. DNA Damage Experiments

[0194] Platinum drugs mainly exert their anti-tumor activity by binding to the DNA of tumor cells and causing DNA damage. Therefore, it is crucial to detect the binding ability of platinum drugs to DNA, which is of great significance for exploring their relationship with biological activity.

[0195] Experimental Methods:

[0196] The reduction ability of platinum(IV) compounds 2-5 in ascorbic acid ASA was determined by HPLC to investigate their reduction ability; the binding ability of the released divalent platinum fragment to 5-GMP after the reduction of 2-5 in ASA was tested to investigate their binding characteristics to DNA; the expression levels of DNA damage-related proteins γ-H2AX and P53 were detected by Western Blot to examine the DNA damage characteristics.

[0197] Conclusion Analysis:

[0198] As Figure 8 shown, compounds 2-5 remained stable in biological medium (RPMI1640) within 48 hours. As Figure 9 shown, in the presence of reducing ascorbic acid (AsA, 1 mM, similar to the concentration in TME) and 5'-GMP, the absorption peak of complex 2-5 decreased significantly, and at the same time, the peak of acid 4-3 gradually increased. In addition, a sum peak of platinum(II) and GMP appeared, which indicated the binding potential of the released platinum(II) fragment to DNA bases (5'-GMP was used as a DNA base model), as well as the DNA damage ability of the CLQ platinum(IV) compound 2-5. By Western Blot analysis to evaluate the proteins related to DNA damage ( Figure 10 ), compound 2-5 significantly up-regulated the expression of the DNA double-strand break detection index protein γ-H2AX, and at the same time promoted the high expression of the DNA damage-related protein P53. The up-regulation of P53 expression also has a positive effect on inducing apoptosis and overcoming cisplatin resistance. These facts reveal the DNA damage ability of the CLQ platinum(IV) complex.

[0199] 6. Apoptosis Experiment

[0200] Mitochondria play a key role in regulating the apoptotic pathway. Platinum drugs can target mitochondria by inducing a decrease in mitochondrial membrane potential (ΔΨm) and increasing ROS production, which helps overcome drug resistance. The Bcl-2 protein plays a key role in inhibiting cell apoptosis, while the Bcl-2 / Bax / caspase3 pathway plays an important role in promoting mitochondria-mediated apoptosis. Therefore, we used JC-1 and DCFHDA staining to detect the effects of compounds 2-5 on the mitochondrial membrane potential (ΔΨm) and ROS production in tumor cells, and Western Blot to detect the effects of the drugs on apoptotic proteins.

[0201] Experimental Method:

[0202] Take 4T1 cells in the logarithmic growth phase with good status and place them in a 6-well plate (10 6 / well). After culturing in a 37°C, 5% carbon dioxide incubator for about 12 hours, add drugs to them and continue culturing for 24 hours. Digest the cells with trypsin without EDTA, collect them, and divide them into three parts. For the first part, wash the cells twice with PBS, add 5 μL of Annexin V-FITC staining agent and 5 μL of PI staining agent respectively, mix well, react at room temperature in the dark for 5-15 minutes, and test the samples with a flow cytometer within 1 hour. For the second part, stain with DCFHDA and detect with a flow cytometer to detect the production of ROS in the cells. For the third part, stain with JC-1 and detect with a flow cytometer to examine the changes in mitochondrial membrane potential.

[0203] Treat and collect cells by the above method, extract proteins, separate the proteins by SDS-Page electrophoresis, transfer the membrane to a PVDF membrane, block it in 5% skim milk for 1 hour, incubate with the primary antibody overnight at 4°C, incubate with the secondary antibody for 1 hour, then develop the color with an ECL chemiluminescence developing solution, and image the membrane using a Tanon 46000SF scanning system.

[0204] Result Analysis:

[0205] Figure 11 The Annexin V-FITC results in [[ ]] show that even at a low concentration (2 μM), compounds 2-5 can effectively induce apoptosis in tumor cells, comparable to the high-concentration (10 μM) platinum (II) drugs CDDP and OLP, and this trend is consistent with the in vitro anti-tumor activity. Figure 12 The ROS production results shown in [[ ]] indicate that compared with the blank group, the low-concentration 2 μM bis-CLQ platinum (IV) compounds 2-5 lead to a significant increase in the ROS production level. Figure 13The JC-1 staining results in Figure 14 showed that after treatment with compounds 2-5 (2 μM), the ΔΨm of tumor cells decreased significantly, indicating that the bis-CLQ platinum(IV) compounds 2-5 could cause severe mitochondrial damage. The results of Western Blot detection of proteins Bcl-2, Bax, caspase3, and c-caspase3 related to the mitochondrial apoptosis pathway (

[0206] 7. Autophagy experiments

[0207] Drug-induced apoptosis is an important target for anti-tumor drugs. Existing studies have demonstrated that CLQ can induce pro-death autophagy in tumor cells. Therefore, we investigated the autophagy-inducing ability of CLQ tetravalent platinum.

[0208] Experimental method:

[0209] This experiment was carried out according to the operation guide of the kit (MDC method for autophagy staining detection kit, Beyotime). Take 4T1 cells in the logarithmic growth phase with good status and place them in a 6-well plate (10 6 / well), culture them in an incubator at 37 °C with 5% carbon dioxide for about 12 hours, perform drug treatment on them, and continue to culture for 24 hours. Divide them into a blank group, a compound 2-5 group (2 μM), a positive control CDDP group (10 μM), and an OLP group (10 μM). Then, add 1 ml of MDC staining solution to each well, incubate in the cell incubator at 37 °C in the dark for 30 min, observe under a fluorescence microscope, and select 5 fields of view to take pictures and count the results. At the same time, detect the expression of autophagy-related proteins p62, LC3I / II, and Beclin-1 by Western Blot, and the method is the same as above.

[0210] Result analysis:

[0211] Figure 15 The Western Blot result analysis showed that after treatment with compound 2-5, the expression levels of autophagy-related proteins p62 and LC3I / II changed significantly. Compared with the CDDP and OLP groups, compound 2-5 down-regulated the expression of p62 and increased the ratio of LC3II / I. Immunohistochemical staining of P62 in 4T1 tumor tissues ( Figure 16) It also shows that Compounds 2-5 have a significant down-regulating effect on p62. These facts indicate that CLQ platinum(IV) can effectively induce autophagy, and this result is also confirmed by the MDC staining results ( Figure 17 ). Autophagy is a double-edged sword in apoptosis: it is involved in the activation of the apoptosis pathway and can also protect cancer cells from chemotherapy. Therefore, to further clarify the relationship between autophagy induced by Compounds 2-5 and apoptosis, we detected the effect of the autophagy inhibitor 3-methyladenine (3-MA) on the cell viability of 4T1 cells treated with Compound 2-5 (0.6 μM). Figure 18 The results showed that the addition of the autophagy inhibitor 3-MA could effectively increase the viability of 4T1 cells treated with Compound 2-5, indicating that inhibiting autophagy could reduce the anti-tumor activity of Compound 2-5. Then, we investigated the effect of 3-MA on the expression of autophagy-related proteins LC3I / II, p62, and Beclin1. Figure 19 The results showed that the addition of the autophagy inhibitor 3-MA in tumor cells treated with Compound 2-5 decreased the LC3II / I ratio (P<0.01), increased p62 (P<0.001), and down-regulated Beclin1 (P<0.001). In summary, the CLQ platinum(IV) complex induced pro-apoptotic autophagy in this study.

[0212] 8. Immune activation experiment

[0213] Immune suppression, as a hallmark of tumors, is conducive to the formation of the tumor microenvironment, survival, and promotes the immune escape and metastasis of tumor cells. PD-L1 is an important immune checkpoint protein that can cause T cell dysfunction and immune suppression. Enhanced autophagy is related to the inhibition of PD-L1 expression in tumor cells. In addition, it is now increasingly recognized that targeting the PI3K / AKT / mTOR pathway can affect host inhibitory immunity by increasing the number of T cells in tumor tissues.

[0214] Test method:

[0215] Using immunohistochemistry, the expression of PD-L1 and CD4 + and CD8 + tumor-infiltrating lymphocytes (TILs) in tumor tissues were detected to examine the effect of the CLQ platinum(IV) complex on tumor immunity.

[0216] Result analysis:

[0217] Figure 20The results in [specific context] showed that compounds 2 - 5 significantly inhibited the secretion of PD - L1 in tumor tissues, with the expression level being 20.5% of that in the blank group, significantly lower than that of platinum(II) drugs CDDP (44.9%, P < 0.001) and OLP (55.8%, P < 0.0001). Meanwhile, in the tumor tissues of the compound 2 - 5 group, the density of CD4 + and CD8 + TILs increased significantly, reaching 2.2 times and 1.5 times that of the blank group, indicating its ability to enhance T - cell immunity, while CDDP and OLP had little effect on TILs. In summary, the CLQ platinum(IV) complex can stimulate T - cell immunity by inhibiting the expression of PD - L1. The anti - tumor proliferation activity and anti - tumor metastasis activity of CLQ are related to immune activation.

[0218] 9. Inhibition of angiogenesis and metastasis experiments

[0219] Angiogenesis is the key to the metastasis and growth of invasive tumors and an important link in controlling tumor development. Vascular endothelial growth factor (VEGF) is a key factor related to angiogenesis. In addition, MMP - 9 promotes tumor metastasis by degrading extracellular matrix proteins. At the same time, studies have confirmed that MMP - 9 and VEGFA show a synergistic effect in increasing blood vessel density and promoting tumor metastasis.

[0220] Test method:

[0221] The expression of VEGFA and MMP - 9 proteins was monitored by Western Blot of in vitro tumor cells and immunohistochemical staining of in vivo tumor tissues. CD34 is a biomarker of angiogenesis, and we detected the expression of CD34 by immunohistochemistry to determine the microvessel density in tumor tissues.

[0222] Conclusion analysis:

[0223] Figures 21 - 22 The results showed that whether in vitro cell models or in vivo tumor models, the CLQ platinum(IV) compound 2 - 5 could effectively inhibit the expression of proteins VEGFA and MMP - 9, while the platinum(II) drugs CDDP and OLP had a weaker effect on their expression, indicating that the anti - angiogenesis property of compound 2 - 5 is related to the introduction of CLQ. At the same time, the microvessel density of CD34 in the tumors of the compound 2 - 5 treatment group decreased significantly, significantly lower than that of the blank group, CDDP, and OLP treatment groups. Thus, it can be seen that the CLQ tetravalent platinum compound 2 - 5 can synergistically inhibit angiogenesis in tumor tissues and inhibit the occurrence and development of tumor metastasis by inhibiting the expression of VEGFA and MMP - 9.

[0224] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A compound represented by the general formula (I): Among them, Selected from cisplatin; L1 and L2 are R 3 Selected from methylene, propylene or butylene.

2. The compound according to claim 1, wherein: The compound is any one of the following:

3. A process for preparing the compound according to claim 1 or 2, characterized in that: The synthetic route of the compound is as follows: Compound 3 and compound 4 undergo a coupling reaction to obtain a symmetrically disubstituted chloroiodoquine-modified tetravalent platinum compound 2; wherein, the molar ratio of the feed of compound 3 to compound 4 is 1:2.2 - 3.

4. The preparation method of the compound according to claim 3, characterized in that: In the synthetic route, the preparation steps are: Under an inert gas atmosphere, compound 4, a condensing agent, and an organic base are dissolved in an anhydrous organic solvent and reacted, compound 3 is added, and after reaction in the dark, after post-treatment, compound 2 is separated; Wherein, the molar ratio of the feed of compound 3, compound 4, the condensing agent, and the organic base is 1:2.2 - 3:2.2 - 3:2.2 - 3; the feeding relationship between compound 3 and the organic solvent is that for every 1 g of compound 3, 30 - 80 ml of the organic solvent is added.

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

6. A pharmaceutical composition, characterized in that: Comprising a therapeutically effective amount of the compound represented by the general formula (I) according to claim 1 or 2, and a pharmaceutically acceptable excipient thereof.

7. Use of the compound represented by the general formula (I) according to claim 1 or 2 or the pharmaceutical composition according to claim 6 in the preparation of an anti - tumor proliferation and anti - tumor metastasis drug; the anti - tumor proliferation is against the proliferation of lung adenocarcinoma, cisplatin - resistant lung adenocarcinoma, breast cancer, and liver cancer; the anti - tumor metastasis is against the lung metastasis of breast cancer tumors.

8. A combined preparation, comprising the compound represented by the general formula (I) according to claim 1 or 2 or the pharmaceutical composition according to claim 6, and the combined use with other types of anti - tumor drugs.

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