A Pt(Ⅳ)-naphtholactam complex targeting TGF-β signaling pathway based on regulating lysosomal function and its application

Abstract

The present invention belongs to the technical field of pharmaceutical chemistry, and specifically relates to a Pt(IV)-naphtholactam complex and application based on regulating lysosomal function and targeting TGF-β signaling pathway. The present invention first introduces a naphtholactam ligand at the axial position of tetravalent platinum on the basis of the 1,8-naphtholactam mother nucleus, and introduces carbon chains of different lengths at the 1 position or performs amino modification at the 3 position and then condenses with a tetravalent platinum terminally connected to palmitate, thereby synthesizing 9 target compounds Y1-Y9. The Pt(IV)-naphtholactam complex Y1-Y9 mainly plays a dual anti-tumor effect by reducing tetravalent platinum to divalent platinum and the axial ligand naphtholactam with certain optical activity. The synthesis method of the present invention can greatly improve the fat solubility of the drug, promote the absorption of the drug, enhance the binding ability of the drug to human serum protein, reduce the probability of being poisoned by sulfur-containing proteins after entering the blood, improve the stability of the drug, and thus improve its bioavailability.

Description

Technical Field

[0001] The invention belongs to the technical field of medicinal chemistry, and specifically relates to a Pt(IV)-naphtholactam complex targeting a TGF-β signaling pathway based on regulating lysosomal function and an application thereof. Background Art

[0002] Triple-negative breast cancer (TNBC), as a special subtype of breast cancer, is currently the most difficult clinical type of breast cancer to treat, with the characteristics of high malignancy, high mortality, high recurrence rate and high metastasis rate. In clinical treatment, poor anti-tumor metastasis activity and immunosuppression are one of the main challenges faced by chemotherapy combined with immunotherapy in the treatment of triple-negative breast cancer (TNBC) and cancer stem cells (CSCs).

[0003] Among the commonly used anticancer drugs in clinical practice, divalent platinum drugs generally have serious toxic side effects and are prone to drug resistance. However, the axial position of tetravalent platinum is easy to modify and has high bioavailability. In order to overcome the defects of traditional divalent platinum drugs, the modification and transformation of tetravalent platinum prodrugs has always been a hot topic in the research of platinum drugs.

[0004] Based on this, the present invention oxidizes divalent cisplatin or divalent oxaliplatin into tetravalent platinum with higher stability, introduces palmitate at one end to improve its fat solubility, and introduces naphtholactam ligand with the same anti-tumor activity at the other end, so that after the tetravalent platinum is reduced to divalent platinum by reducing substances in the body, the naphtholactam ligand with the same anti-tumor activity can be released. In addition, since naphtholactam itself is a planar three-membered ring structure and has a certain fluorescence, it is more conducive to studying its inherent anti-tumor mechanism. Summary of the invention

[0005] Based on the deficiencies in the prior art, the purpose of the present invention is to propose a Pt(IV)-naphtholactam complex and its application based on regulating lysosomal function and targeting the TGF-β signaling pathway. By introducing palmitate and a naphtholactam ligand with anti-tumor activity, a series of Pt(IV)-naphtholactam complexes are obtained, and their anti-tumor activity is explored.

[0006] To achieve the above object, the technical solution created by the present invention is implemented as follows:

[0007] A Pt(IV)-naphtholactam complex targeting the TGF-β signaling pathway based on regulating lysosomal function, the general structural formula of which is shown in Formula I:

[0008]

[0009] Specifically, in the formula, L1 and L2 are respectively one of the following functional groups, and the two groups L1 and L2 are the same or different:

[0010]

[0011] Specifically, in the Pt(IV)-naphtholactam complex, the parent nucleus Any one of cisplatin, carboplatin, heptaplatin, nedaplatin, oxaliplatin, lobaplatin, miplatin, picoplatin, and NDDP, preferably cisplatin or oxaliplatin:

[0012]

[0013] Specifically, the Pt(IV)-naphtholactam complex targeting the TGF-β signaling pathway based on regulating lysosomal function is a compound with the following structure:

[0014]

[0015] Furthermore, R' is R1 is H or NH2, n=2, 3, 4, 5; specifically, R' is Any of .

[0016] Further preferably, the Pt(IV)-naphtholactam complex targeting the TGF-β signaling pathway based on regulating lysosomal function is a compound with the following structure:

[0017]

[0018]

[0019] Specifically, the preparation method of the Pt(IV)-naphtholactam complex based on regulating lysosomal function and targeting TGF-β signaling pathway comprises the following steps:

[0020] 1) reacting compound K1 or K2 with succinic anhydride at room temperature to generate a carboxyl group at the axial position of tetravalent platinum to obtain compound 72 or 74;

[0021] 2) Compound 72 or 74 is subjected to condensation reaction with the intermediate compound under nitrogen protection at room temperature (20-25° C.) for 12-24 hours. After the reaction, the mixture is separated by column and concentrated to obtain compounds Y1-Y9.

[0022] Specifically, the structural formulas of compounds K1 and K2 are:

[0023]

[0024] Specifically, HATU is used as a catalyst during the condensation reaction of step 2).

[0025] Specifically, the mass ratio of compound 72 or 74 to the intermediate compound is (1.9-2.5):1; preferably 2.5:1, 2.3:1, 2.06:1, 2.1875:1, 1.9:1.

[0026] Specifically, the structural formulas of compounds 72 and 74 are:

[0027]

[0028] Specifically, the intermediate compounds are B1-B5, and the structural formula is:

[0029]

[0030] Furthermore, the present invention also provides the use of the Pt(IV)-naphtholactam complex targeting the TGF-β signaling pathway based on regulating lysosomal function in the preparation of anti-tumor drugs.

[0031] Specifically, the tumor includes, but is not limited to, human cervical cancer, human breast cancer, human lung adenocarcinoma, human liver cancer, human colon cancer, cisplatin-resistant human lung adenocarcinoma or human prostate cancer.

[0032] Furthermore, the present invention also provides the use of the Pt(IV)-naphtholactam complex alone in the preparation of a drug for inhibiting tumor cell proliferation, inducing pyroptosis or promoting apoptosis, wherein the tumor cells are human liver cancer cells Huh-7, HepG2, human breast cancer cells MDA-MB-231, MCF-7, human lung cancer cells A549, or human colon cancer cells HCT116, HT-29.

[0033] Specifically, when used, when the final concentration of the Pt(IV)-naphtholactam complex is (1.5-3) μM and the treatment time is 24-48 hours, it can inhibit the proliferation of tumor cells, induce pyroptosis or promote apoptosis.

[0034] Preferably, when used, when the final concentration of the Pt(IV)-naphtholactam complex is 1.5 μM or 3 μM and the treatment time is 24 h, it can inhibit the proliferation of tumor cells, induce pyroptosis or promote apoptosis.

[0035] Furthermore, the combination of the Pt(IV)-naphtholactam complex of the present invention and gemcitabine (or cisplatin, oxaliplatin) has a synergistic anti-tumor effect. To this end, the present invention also provides the use of the Pt(IV)-naphtholactam complex combined with gemcitabine (or cisplatin, oxaliplatin) in the preparation of anti-tumor drugs, and the tumor cells are human breast cancer cells MDA-MB-231, MCF-7, or human lung cancer cells A549, human colon cancer cells HCT-116.

[0036] Furthermore, when the Pt(IV)-naphtholactam complex and gemcitabine (or cisplatin, oxaliplatin) are used in combination, the Pt(IV)-naphtholactam complex and gemcitabine can be mixed and used or used separately; the Pt(IV)-naphtholactam complex and gemcitabine can be added once or added continuously for multiple times.

[0037] Specifically, when the Pt(IV)-naphtholactam complex and gemcitabine (or cisplatin, oxaliplatin) are added and used at one time, the proliferation of cancer cells can be inhibited, pyroptosis can be induced or apoptosis can be promoted. The molar ratio of the Pt(IV)-naphtholactam complex and gemcitabine (or cisplatin, oxaliplatin) is 1:(2-10), and the treatment time is 24-72h; further preferably, the molar ratio of the Pt(IV)-naphtholactam complex and gemcitabine (or cisplatin, or oxaliplatin) is specifically 1:2, 1:4, 1:6, 1:8 or 1:10, and the treatment time is 72h.

[0038] Further, when the concentration of the Pt(IV)-naphtholactam complex is (0.25-2) μM, the concentration of gemcitabine (or cisplatin, oxaliplatin) is (0.5-12) μM, and the treatment time is 24-72 h, the proliferation of cancer cells can be inhibited, pyroptosis can be induced, or apoptosis can be promoted; further preferably, the concentration of the Pt(IV)-naphtholactam complex is specifically, for example, 0.25 μM, 0.5 μM, 1 μM or 2 μM, the concentration of gemcitabine (or cisplatin, oxaliplatin) is specifically, for example, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 4 μM, 5 μM, 6 μM, 8 μM, 10 μM or 12 μM, and the treatment time is 72 h.

[0039] Furthermore, the present invention also provides the use of the Pt(IV)-naphtholactam complex in the preparation of a drug for inhibiting tumor growth in a mouse-derived breast cancer metastasis model. Specifically, the mouse used as the mouse source is a Balb / C mouse.

[0040] Specifically, the Pt(IV)-naphtholactam complex can inhibit the metastasis of tumors in a triple-negative breast cancer mouse metastasis model when the dosage is 2.5-5 mg / kg / day and the dosage is administered seven times in total.

[0041] Preferably, the Pt(IV)-naphtholactam complex can inhibit the metastasis of a triple-negative breast cancer mouse metastasis model tumor when the dosage is 10 mg / kg or 2.15 mg Pt / kg every two days for a total of seven administrations.

[0042] Compared with the prior art, the beneficial effects of the present invention are:

[0043] The present invention is based on 1,8-naphtholactam mother core, introduces naphtholactam ligand at the axial position of tetravalent platinum, introduces carbon chains of different lengths at position 1 or performs amino modification at position 3, and then condenses with tetravalent platinum with one end connected to palmitate, thereby synthesizing 9 target compounds Y1-Y9. The Pt(IV)-naphtholactam complexes Y1-Y9 mainly play a dual anti-tumor effect by reducing tetravalent platinum to divalent platinum and the axial ligand naphtholactam with certain optical activity.

[0044] The present invention introduces palmitic acid ester at one end of the Pt (IV) mother core to form an alkyl fatty chain, which can greatly improve the fat solubility of the drug, promote the absorption of the drug, enhance the binding ability of the drug with human serum protein, reduce the probability of being poisoned by sulfur-containing proteins after entering the blood, improve the stability of the drug, thereby improving its bioavailability, and at the same time reduce the toxic and side effects of the drug, improve the drug resistance, and increase the enrichment level of the drug in the body, providing a new idea and research direction for the treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The absorption and emission spectra of compound Y4 in H2O (A) and PBS (B) are shown in Table 1. Figure 1 The top two figures are absorption spectra, and the bottom two figures are emission spectra;

[0046] Figure 2 The cellular localization of compound Y4 in A549 cells was detected using confocal microscopy;

[0047] Figure 3 After compound Y4, cisplatin and oxaliplatin at a concentration of 10 μM were treated in MDA-MB-231 cells and MCF-7 cells for 24 hours, ICP-MS was used to detect the total platinum uptake level of drugs in breast cancer cells;

[0048] Figure 4 Compound Y4, cisplatin and oxaliplatin at a concentration of 10 μM were treated in MDA-MB-231 cells (A) and MCF-7 cells (B) for 24 h, and the uptake of platinum in the endoplasmic reticulum (ER), mitochondria (mito), lysosomes (lyso) and cell nuclei was detected by ICP-MS;

[0049] Figure 5 After 24 hours of incubation, the inhibition of MDA-MB-231 cell migration by different concentrations of compound Y4, cisplatin, and oxaliplatin; *P<0.05; **P<0.01; ***P<0.001;

[0050] Figure 6After 24 hours of incubation, the inhibition of MCF-7 cell migration by different concentrations of compound Y4, cisplatin, and oxaliplatin; *P<0.05; **P<0.01; ***P<0.001;

[0051] Figure 7 After 24 hours of incubation, the inhibition of MDA-MB-231 cell migration by different concentrations of compound Y4, cisplatin, and oxaliplatin was evaluated by scratch test; *P<0.05; **P<0.01; ***P<0.001;

[0052] Figure 8 After 24 hours of incubation, the inhibition of MCF-7 cell migration by different concentrations of compound Y4, cisplatin, and oxaliplatin was evaluated by scratch test; *P<0.05; **P<0.01; ***P<0.001;

[0053] Fig. 9 The in vivo antitumor activities of different concentrations of Y4, naphtholide intermediate, cisplatin and oxaliplatin in 4T1 breast cancer; A) The weight change curve of mice during treatment; B) The change curve of tumor growth and time; C) The tumor weight of each group at the end of the experiment; D) The tumor picture at the end of the experiment; Fig. 9 In D, the first row is the PBS group; the second row is the naphtholide ligand group (5 mg / kg); the third row is the oxaliplatin group (5 mg / kg 2.46 mg Pt / kg); the fourth row is the cisplatin group (5 mg / kg 3.26 mg Pt / kg); the fifth row is the cisplatin + ligand group (5 mg / kg, 3.26 mg Pt / kg + 5 mg / kg); the sixth row is the Y4-5 group (5 mg / kg, 1.07 mg Pt / kg); the seventh row is the Y4-10 group (10 mg / kg 2.15 mg Pt / kg); E) Statistics of organ indexes of mice in each group at the end of the experiment; *P<0.05; **P<0.01; ***P<0.001;

[0054] Fig.10 The survival curves of mice after being treated with different concentrations of compound Y4, naphtholide intermediate, cisplatin, and oxaliplatin; Fig.11 The in vivo anti-tumor metastasis activity of different concentrations of Y4, naphtholide ligand, cisplatin and oxaliplatin in 4T1 breast cancer tumors; A) The body weight of mice treated with Y4, intermediate, cisplatin, oxaliplatin and control group for 14 days; B) The statistical data of lung metastasis nodules of mice after treatment with Y4, intermediate, cisplatin, oxaliplatin and control group; C) Representative images of lung metastasis nodules of mice after treatment; Fig.11From left to right in C, they are PBS group (Control group), Y4 ligand group (5 mg / kg), oxaliplatin group (5 mg / kg, 2.46 mg Pt / kg), cisplatin group (5 mg / kg, 3.26 mg Pt / kg), cisplatin+Y4 ligand group (3.26 mg Pt / kg+5 mg / kg), Y4-5 group (5 mg / kg, 1.07 Pt / kg), and Y4-10 group (10 mg / kg, 2.15 mg Pt / kg); *P<0.05; **P<0.01; ***P<0.001; D) Statistics of organ indexes of mice in each group at the end of the experiment;

[0055] Fig.12 is the effect of compound Y4 on mouse immune cells; wherein Fig.12 A and 12B are the statistical diagrams of the proportion of immune cells in the blood and tumors of mice detected by cell flow cytometry at the end of the experiment. Fig.12 C is a graph showing the proportion of Treg cells in mouse blood detected by flow cytometry; Fig.12 D is a representative diagram of the proportion of granulocyte-like MDSC cells in mouse tumor tissue detected by cell flow cytometry; Fig.12 E is a representative figure of the proportion of DC cells in mouse tumor tissue detected by cell flow cytometry; *P<0.05; **P<0.01; ***P<0.001; Fig.13 Quantitative analysis of apoptosis of MDA-MB-231 cells induced by different concentrations of Y4 and 10 μM cisplatin and oxaliplatin after 24 hours of drug incubation;

[0056] Fig.14The study of the mechanism of action of compound Y4 on MDA-MB-231 cells and MCF-7 cells; A) Scatter plot of differentially expressed genes after 24h of treatment with compound Y4 in MDA-MB-231 cells; B) Scatter plot of differentially expressed genes after 24h of treatment with compound Y4 in MCF-7 cells; the X-axis represents the difference fold after log2 conversion, and the Y-axis represents the significance value after log10 conversion; red represents up-regulated differentially expressed genes, blue represents down-regulated differentially expressed genes, and gray represents non-differentially expressed genes; C) Scatter plot of differentially expressed genes after 24h of treatment with compound Y4 in MDA-MB-231 cells and MCF-7 cells Venn diagram of gene expression in F-7 cells after 24 h of action and differential clustering heat map in MDA-MB-231 cells and MCF-7 cells when P<0.001, FDR<0.001, log2(Y4 / control)abs>2.5; D) KEGG pathway enrichment analysis; E) Genes related to the TGF-β signaling pathway screened in MDA-MB-231 cells and MCF-7 cells; F) Lysosome-related genes screened in MDA-MB-231 cells and MCF-7 cells. DETAILED DESCRIPTION

[0057] The technical solution of the present invention is further described in detail below through specific examples, but the protection scope of the present invention is not limited thereto. Unless otherwise specified, all reagents mentioned herein are commercially available high-purity reagents that meet the experimental requirements.

[0058] The abbreviations involved in the embodiments of the present invention include: Me is methyl, Et is ethyl, TEA is triethanolamine, DMSO is dimethyl sulfoxide, DMF is dimethylformamide, DCM is dichloromethane, Boc is tert-butyloxycarbonyl, palmitic anhydride is palmitic anhydride, HATU is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, Cis. is cisplatin, and Oxp. is oxaliplatin. The cisplatin, oxaliplatin, 1,8-naphtholactam, succinic anhydride, 1,3-dibromopropane, acetonitrile, sodium azide, 1,4-dibromobutane, 1,6-dibromohexane, diamine, and palmitic anhydride are all commercially available products.

[0059] Example 1 Synthesis of Compounds K1 and K2

[0060]

[0061] Synthesis of compound 70

[0062] Under light-proof conditions, 1 g of cisplatin was weighed into a 250 mL round-bottom flask, and 30 mL of distilled water was added. The solution was placed in an oil bath and stirred to completely disperse the solution. Then, 50 mL of 30% aqueous hydrogen peroxide solution was added to a constant pressure dropping funnel wrapped in tin foil in advance, and the solution was slowly dripped into the round-bottom flask drop by drop. The solution was reacted at 60° C. for 4 hours. After the solution became a colorless, clear and transparent liquid after 4 hours, the solvent was removed by a rotary evaporator and evaporated to dryness. 100 mL of distilled water was added to the evaporated round-bottom flask and the solution was stirred at 80° C. The solution was taken out from the oil bath, cooled to room temperature and placed in a 4° C. refrigerator overnight. After crystallization, the yellow solid compound 70 (yield 75%) was separated by filtration.

[0063] Synthesis of compound K1

[0064] Weigh 0.35 g of compound 70 and 0.52 g of palmitic anhydride into a 100 mL round-bottom flask, seal it with a rubber stopper, evacuate it and protect it with nitrogen, add 30 mL of anhydrous DMSO, stir and react at room temperature for 7 days, then remove DMSO with an oil pump, drop a small amount of acetone and sonicate it in an ultrasonic instrument to precipitate a light yellow-green powder, then filter it with a suction funnel, add acetone, ether, etc., and filter and wash it repeatedly to remove impurities, to obtain compound K1 with a yield of 91%.

[0065] Synthesis of compound 71

[0066] The synthesis of compound 71 was the same as compound 70, except that the raw material cisplatin was replaced by oxaliplatin, and finally a white solid compound 71 was obtained (yield 65%).

[0067] Synthesis of compound K2

[0068] Weigh 0.35 g of compound 71 and 0.4 g of palmitic anhydride into a 100 mL round-bottom flask, seal it with a rubber stopper, evacuate it and protect it with nitrogen, add 30 mL of anhydrous DMSO, stir and react at room temperature for 7 days, then use an oil pump to remove DMSO, add a small amount of acetone and sonicate it in an ultrasonic instrument to precipitate a large amount of white powder, then use a suction funnel to filter, add acetone, ether, etc., and repeatedly filter and wash to remove impurities to obtain compound K2 with a yield of 85%.

[0069] Example 2 Synthesis of Compound 72

[0070]

[0071] Weigh 0.35 g of compound K1 into a 100 mL round-bottom flask, add 0.061 g of succinic anhydride, seal the reaction bottle with a rubber stopper, evacuate the reaction system to a vacuum state, add 30 mL of anhydrous DMSO, stir overnight at room temperature, then evaporate to remove DMSO, add a small amount of acetone, and a large amount of light yellow powder can be observed to precipitate. Use a suction funnel to filter, and then repeatedly wash with acetone and ether solution to remove excess impurities to obtain light yellow solid compound 72 with a yield of 90%.

[0072] Example 3 Synthesis of Compound 74

[0073]

[0074] Weigh 0.35 g of compound K2 into a 100 mL round-bottom flask, add 0.052 g of succinic anhydride, seal the reaction bottle with a rubber stopper, evacuate the reaction system to a vacuum state, add 30 mL of anhydrous DMSO, stir at room temperature overnight, then evaporate to remove DMSO, add a small amount of acetone, and a large amount of off-white powder can be observed to precipitate. Use a suction funnel to filter, and then repeatedly wash with acetone and ether solution to remove excess impurities to obtain off-white solid compound 74 with a yield of 90%.

[0075] Example 4 Synthesis of intermediate compounds B1, B2, B3

[0076]

[0077] 1. Synthesis of B1

[0078] Synthesis of compound 81

[0079] Weigh 1 g of 1,8-naphtholactam into a 250 mL round-bottom flask, use acetonitrile as solvent, add 1,3-dibromopropane (2 times the equivalent of 1,8-naphtholactam) into the reaction flask, then add 3 times the equivalent of K2CO3 and an appropriate amount of KI, heat at 90°C, reflux overnight, check whether the reaction is complete by spotting, evaporate the acetonitrile to dryness, pass through a column with petroleum ether and ethyl acetate, and concentrate to obtain a yellow solid product 81 with a yield of 83%.

[0080] Synthesis of compound 82

[0081] Weigh 3.76 g of compound 81 into a 250 mL round-bottom flask, carefully weigh 4 equivalents of sodium azide into the round-bottom flask, use DMF as solvent, react at 70°C overnight, evaporate DMF with an oil pump the next day, filter with petroleum ether and ethyl acetate, and concentrate to obtain an orange-yellow oily product 82 with a yield of 74%.

[0082] Synthesis of compound 83

[0083] Weigh 2.0 g of compound 82, add 200 mg of palladium carbon to the reaction bottle at the ratio of product weight to palladium carbon of 10:1, then seal the reaction bottle thoroughly with a rubber stopper, evacuate to remove air, insert a balloon filled with hydrogen below the liquid level with a long needle, react overnight, and check whether the reaction is complete by tapping a plate. After the reaction is complete, remove the palladium carbon by suction filtration with a Buchner funnel, pass through a column with dichloromethane and methanol to remove reaction impurities, and thoroughly concentrate and evaporate to dryness to obtain a red solid, namely compound 83 (intermediate B1) with a yield of 69%.

[0084] 2. Synthesis of B2

[0085] The synthesis steps of B2 are the same as those of B1, except that 1,3-dibromopropane is replaced by 1,4-dibromobutane.

[0086] 3. Synthesis of B3

[0087] The synthesis steps of B3 are the same as those of B1, except that 1,3-dibromopropane is replaced by 1,6-dibromohexane.

[0088] Example 5 Synthesis of intermediate compounds B4 and B5

[0089]

[0090] 1. Synthesis of Compound 84

[0091] 16.9 g of 1,8-naphtholactam was weighed and added into a 500 mL round-bottom flask. With glacial acetic acid as the solvent, 20 mL of concentrated nitric acid was slowly dripped dropwise using a constant pressure dropping funnel under ice bath conditions. After the dripping was completed, the mixture was moved to an oil bath and the temperature was raised to 55° C. The reaction was carried out for 3 h. The reaction was detected by spotting whether the reaction was complete. The reaction solution was poured into ice water while hot to precipitate a yellow precipitate. The precipitate was filtered with a Buchner funnel and washed with water several times until the filtrate was weakly acidic. The filter cake was dried in an oven to obtain an orange-yellow solid product 84 with a yield of 82%.

[0092] Synthesis of compounds 85, 86 and 87

[0093] The synthesis methods of compounds 85, 86 and 87 were the same as those of compounds 81, 82 and 83, using compound 84 as the starting material.

[0094] 2. Synthesis of B4

[0095] The synthesis steps of B4 are the same as those of B2.

[0096] 3. Synthesis of B5

[0097] The synthesis steps of B5 are the same as those of B3.

[0098] Example 6 Synthesis of Compounds Y2, Y4, Y6, and Y9

[0099]

[0100] Weigh 0.35 g of the compound 72 synthesized above into a 100 mL round-bottom flask, add as little anhydrous DMF as possible to dissolve it completely, seal it with a rubber stopper, evacuate it, and start stirring. Weigh 1.25 equivalents of intermediate B1 (0.14 g), B2 (0.15 g), B3 (0.17 g) or B4 (0.16 g) into a 4 mL ep tube and dissolve it completely with anhydrous DMF for later use. Drain HATU in advance to remove all moisture for later use. Then weigh HATU Dissolve 0.45 g of the compound completely with as little anhydrous DMF as possible, inject it into the reaction bottle with a syringe, continue stirring for 15 minutes, then carefully add the pre-dissolved intermediate with a syringe. The whole process should be carried out under nitrogen protection, continue stirring at room temperature for 24 hours, and use a plate to check whether the reaction is complete. After the reaction is complete, use an oil pump to evaporate and remove anhydrous DMF, perform column chromatography, use dichloromethane and methanol to pass through the column, evaporate and concentrate the product to obtain yellow-green solid compounds Y2, Y6, Y9 or reddish-brown solid product Y4.

[0101] Y2 1 H NMR(300MHz,Chloroform-d)δ8.53(s, 1 H),8.19(d,J=8.3Hz,1H),7.97(m, 1 H),

[0102] 7.66(m, 1 H),7.45(dd,J=9.5,5.7Hz, 1 H), 6.91(d, J=6.4Hz, 1 H),3.67(m,2H),3.12(d,J=7.4Hz,4H),2.56(s,4H),1.55–1.21(m,36H),0.88(m,3H). 13 C NMR (75MHz, DMSO) δ181.32,180.46,171.63,167.41,139.36,131.52,129.44,129.14,126.37,124.66,124.50,1 20.50,106.25,36.13,31.89,31.77,31.34,29.54,29.19,27.03,26.15,25.94,22.58,14.44.ESI-MS: (negative ion mode):m / z[MH] - :calcd:893.3139,obsd:893.3148.(yield:68%)

[0103] Y41 H NMR(300MHz,Methanol-d4)δ8.39(dd,J=8.5,1.3Hz,1H),8.29(d,J=8.2Hz,1H),8.03(d,J=7.0Hz,1H),7.70(dd,J=8.1,6.8Hz,1H),7.49(dd,J=8.5,4.4Hz,1H),3.73(p,J=6.7Hz,2H),3.23(d,J=7.4Hz,2H),2.58(d,J=6.7Hz,2H),2.38(dt,J=15.2,7.1Hz,4H),1.41–1.34(m,12H),1.33–1.23(m,24H),0.90(d,J=2.3Hz,3H). 13 C NMR(75MHz,CDCl3)δ186.89,184.98,177.92,171.95,138.89,133.41,132.24,130.76,130.71,128.02,125.51,124.52,112.39,46.39,39.66,35.25,33.29,33.06,33.01,32.91,32.82,30.34,29.95,29.62,26.31,15.79.ESI-MS:(negative ion mode):m / z[M-H] - :calcd:908.3248,obsd:908.3226.(yield:84%)

[0104] Y6 1 H NMR(300MHz,Chloroform-d)δ8.58(s,1H),8.15–7.87(m,1H),7.63(d,J=7.2Hz,

[0105] 1H),7.43(dd,J=14.7,7.0Hz,1H),6.88(d,J=6.3Hz,1H),6.35(s,1H),3.63(dd,J=11.6,6.0Hz,2H),3.37–2.92(m,2H),2.51(s,3H),2.25(s,3H),1.46(d,J=7.2Hz,4H),1.38(d,J=6.7Hz,6H),1.18(s,30H),0.81(s,3H). 13C NMR (75MHz, CDCl3) δ177.24,173.52,168.31,130.94,129.05,128.69,126.52,124.98,124.45,124.30,120.43,105.56,42.39 ,40.01,37.11,35.97,31.95,29.75,29.72,29.68,29.64,29.51,29.39,29.34,26.16,24.88,22.72,14.17.ESI-MS: (negative ion mode):m / z[MH] - :calcd:921.3453,obsd:921.3439.(yield:70%)

[0106] Y9 1 H NMR(300MHz,Chloroform-d)δ8.37–7.80(m,2H),7.82–7.55(m,1H),7.57–7.39(m,2H),6.99(d,J=6.7Hz,1H),3.98(s,2H),3.23(s ,2H),3.07–2.83(m,2H),2.36(s,2H),2.20(s,2H),1.95(s,2H),1.47(dd,J=20.8,7.3Hz,8H),1.24(s,24H),0.87(t,J=6.7Hz,3H). 13 C NMR (75MHz, CDCl3) δ175.42,172.90,167.80,138.61,132.43,131.29,130.92,128.80,128.63,126.06,124.65,120.90,47.48,42 .17,40.63,38.73,37.31,37.11,36.28,31.94,30.95,30.37,28.94,28.01,24.85,23.75,23.00,22.71,14.08.ESI-MS: (negative ion mode):m / z[MH] - :calcd:879.2982,obsd:879.2992.(yield:69%)

[0107] Example 7 Synthesis of Compounds Y1, Y3, Y5, Y7, and Y8

[0108]

[0109] Weigh 0.35 g of the compound 74 synthesized above into a 100 mL round-bottom flask, add as little anhydrous DMF as possible to dissolve it completely, seal it with a rubber stopper, evacuate it, and start stirring. Weigh 1.25 equivalents of intermediate B1 (0.14 g), B2 (0.15 g), B3 (0.17 g), B4 (0.16 g) or B5 (0.18 g) respectively, and dissolve it completely in anhydrous DMF in a 4 mL ep tube. Drain HATU in advance to remove all water and set it aside. Then weigh HATU Dissolve 0.45 g of the compound completely with as little anhydrous DMF as possible, inject it into the reaction bottle with a syringe, continue stirring for 15 minutes, then carefully add the pre-dissolved intermediate with a syringe. The whole process should be carried out under nitrogen protection, continue stirring at room temperature for 24 hours, and use a plate to check whether the reaction is complete. After the reaction is complete, use an oil pump to evaporate and remove anhydrous DMF, perform column chromatography, pass dichloromethane and methanol through the column, and evaporate and concentrate the product to obtain yellow-green solid compounds Y1, Y5, Y8, or reddish-brown solid product Y3, and yellow solid product Y7.

[0110] Y1 1 H NMR(300MHz,Chloroform-d)δ8.51(d,J=4.5Hz,1H),8.21(d,J=8.5Hz,1H),

[0111] 7.95(t,J=7.7Hz,1H),7.63(t,J=7.6Hz,1H),7.44(m,1H),6.93(d,J=6.8Hz,1H),3.88(s,1H),3.71(m,1H),3.16(d q,J=14.9,7.4,6.8Hz,4H),2.61–2.07(m,6H),1.44(dd,J=21.6,6.9Hz,14H),1.21(d,J=7.7Hz,28H),0.84(t,3H). 13 C NMR (75MHz, CDCl3) δ184.23,182.13,173.20,168.29,139.09,134.90,130.95,129.05,128.69,128.61,126.44,124.96,120.45,54 .17,42.38,39.90,39.11,31.92,29.72,29.69,29.66,29.57,29.37,29.20,26.49,26.24,25.73,22.69,14.12.ESI-MS: (negative ion mode):m / z[MH] - :calcd:990.4196,obsd:990.4207.(yield:76%)

[0112] Y3 1 H NMR(300MHz,Chloroform-d)δ8.45(s,1H),8.15(s,1H),7.88(d,J=50.8Hz,1H),

[0113] 7.16(s,1H),6.61(s,1H),5.08(s,2H),3.83–3.46(s,2H),3.12(s,2H),2.70–1.92(d,6H),1.52–1.34(d,10H),1.23(s,28H),0.86(s,3H). 13 C NMR(75MHz,CDCl3)δ173.32,167.53,139.59,134.73,126.26,124.64,120.17,42.25,31.92,29.73,29.67,29.60,29.37,29.24,25.77,22.69,14.12.ESI-MS:(negative ion mode):m / z[M-H] - :calcd:1005.4305,obsd:1005.4319.

[0114] Y5 1 H NMR(300MHz,Chloroform-d)δ7.99(dd,J=11.8,7.8Hz,2H),7.67(t,J=7.6

[0115] Hz,1H),7.47(dd,J=16.9,7.8Hz,2H),6.89(t,J=6.7Hz,1H),3.87(t,J=7.1Hz,2H),3.22–2.97(m,2H),2.70–2.31(m,4H),2.20(t,J=7.6Hz,2H),1.75–1.44(m,12H),1.62–1.29(m,5H),1.15(s,30H),0.85(s,3H). 13C NMR(75MHz,CDCl3)δ184.23,182.10,172.94,168.23,139.28,130.92,129.10,128.68,128.60,126.57,125.03,124.31,120.39,105.41,40.05,31.94,29.75,29.73,29.69,29.61,29.44,29.38,29.25,29.12,28.61,26.35,25.75,24.02,22.71,14.14.ESI-MS:(negative ion mode):m / z[M-H] - :calcd:1018.4509,obsd:1018.4508.

[0116] Y7 1 H NMR(300MHz,DMSO-d6)δ8.73(s,2H),8.53(s,1H),7.50(s,2H),3.87(s,2H),3.62(d,J=145.9Hz,4H),2.51(s,2H),2.32–1.58(m,4H),1.21(s,36H),0.84(s,3H). 13 C NMR(75MHz,CDCl3)δ173.20,173.07,172.37,171.03,167.93,167.89,138.95,130.46,130.00,129.88,126.07,125.86,125.73,125.62,125.25,123.92,121.16,42.29,41.99,40.03,39.95,39.82,35.35,29.66,29.63,29.51,29.48,29.33,26.00,25.78,25.53,25.41,25.34,25.25,25.21,22.66,14.10.ESI-MS:(negative ion mode):m / z[M-H] - :calcd:1033.4618,obsd:1033.4626.

[0117] Y8 1 H NMR(300MHz,Chloroform-d)δ8.01(s,2H),7.70(dt,J=5.9,3.1Hz,2H),7.52(dd,

[0118] J=5.6,3.4Hz,2H),3.98(s,2H),3.69(d,J=4.0Hz,2H),3.29–3.02(m,2H),2.65(m,6H),1.99(s ,2H),1.50(s,3H),1.48(s,3H),1.44(s,2H),1.41(s,2H),1.24(s,28H),0.87(d,J=5.0Hz,3H). 13 C NMR (75MHz, DMSO) δ180.32,172.28,171.45,167.10,134.72,129.44,128.56,127.70,126.33,124.60,124.35,122.20,119 .34,41.61,31.73,31.54,30.06,29.31,29.04,28.96,28.62,28.45,26.33,25.60,22.66,22.34,14.19.ESI-MS: (negative ion mode):m / z[MH] - :calcd:976.4039,obsd:976.4042.

[0119] Test Example 1: In vitro antitumor activity test 1

[0120] The MTT method was used to test the activity of the Pt(IV)-naphtholactam complexes Y1-Y9 synthesized in the above examples in eight human cancer cell lines including liver cancer cells Huh-7, liver cancer HepG2, triple-negative breast cancer cells MDA-MB-231, breast cancer cells MCF-7, lung adenocarcinoma cells A549, cisplatin-resistant lung adenocarcinoma cells A549cisR, colon cancer cells HCT-116, and HT-29 cells. The test results are shown in Table 1. Positive drugs cisplatin and oxaliplatin were selected as controls.

[0121] Table 1 In vitro antitumor activity of Pt(IV)-naphthalene lactam complexes

[0122]

[0123] Note: [c]FI(fold increase)is defined as IC 50 (cisplatin) / IC 50 (Y4).;[d]FI(fold increase)is defined as IC 50 (oxaliplatin) / IC 50 (Y4).

[0124] From the experimental results in Table 1, it can be seen that compound Y4, which uses cisplatin as the core, is connected to palmitate at one end and amino-modified naphtholactam 4-carbon chain at the other end, has the best antitumor activity. 50 The values ​​are generally lower than those of other cancer cells, which indicates that compound Y4 with cisplatin as the core has higher selectivity in the treatment of breast cancer.

[0125] Test Example 2: Optical property test of Pt(IV)-naphthalene lactam complex

[0126] Based on the above anti-tumor activity tests of Y1-Y9, it was found that the IC value of compound Y4, which has cisplatin as the parent core, one end of which is connected to palmitate and the other end of which is connected to amino-modified 4-carbon naphthyl lactam, is 50 The value is the smallest, so the present invention selects Y4 as the lead compound to carry out subsequent experiments. Since naphtholactam, like naphthalene imide, has a planar three-membered ring structure, due to the large conjugated system of naphtholactam, Y4 was also subjected to full-band UV scanning and fluorescence spectral analysis in aqueous solution and PBS solution. The results are as follows Figure 1 As shown, there is no significant difference in the excitation and emission of Y4 in either aqueous solution or PBS solution. The maximum excitation wavelength is 273 nm, and the maximum emission wavelength is 545 nm, showing strong fluorescence properties.

[0127] Experimental Example 3: Localization of Pt(IV)-naphtholactam complex in cells

[0128] In order to further study the potential anti-tumor molecular mechanism of the lead compound Y4 with cisplatin as the parent core, based on the above ultraviolet full-band scanning and fluorescence intensity analysis of the lead compound Y4, the present invention uses the corresponding organelle dyes to dye the cells, and uses laser confocal microscopy to determine the accurate sub-organelle localization of compound Y4 in tumor cells. A549 cells are first treated with Y4 (10 μM) for 2 h, and then stained with mitochondrial red dye Mito-Tracker, endoplasmic reticulum red dye ER-Tracker, lysosome red dye Lyso-Tracker, and Golgi red dye Golgi-Tracker, respectively. The images are taken by confocal microscopy, scale: 25 μm. The results show that after the action of compound Y4, when the lysosomal dye is added, the green fluorescence representing the drug Y4 can overlap well with the red fluorescence of the lysosomal dye. The results are as follows: Figure 2 As shown, this indicates that the lead compound can be well localized in the lysosome.

[0129] Experimental Example 4: ICP-MS measurement of platinum uptake of Pt(IV)-naphthalene lactam complex in cancer cells

[0130] The anti-tumor activity of a drug is closely related to its bioavailability and the degree of accumulation in cancer cells. In order to explore the cellular uptake of a series of Pt(Ⅳ)-naphtholide complexes and the level of drug binding to nuclear DNA after reaching the cells, the present invention selected triple-negative breast cancer cells MDA-MB-231 and breast cancer cells MCF-7 to test the cellular drug uptake and DNA binding levels of the test compounds. Y4 with cisplatin as the parent nucleus, as well as positive control drugs cisplatin and oxaliplatin were selected for testing in triple-negative breast cancer cells MDA-MB-231 and breast cancer cells MCF-7. Compounds at a concentration of 10 μM were added to the test cells and cultured for 24 hours. The cells were collected and washed with PBS, and different subcellular organelle extraction kits were used to separate organelles and nuclear DNA at different centrifugation speeds, and the platinum content in cells and DNA was determined by ICP-MS. The experimental results are as follows: Figure 3 , 4 As shown, from the perspective of platinum uptake of the whole cell, after 24 hours of treatment with Y4, whether in MDA-MB-231 cells or MCF-7 cells, the platinum uptake of the Y4-treated group was higher than that of the positive control drugs cisplatin and oxaliplatin, and the enrichment of platinum in MDA-MB-231 cells was not much different from that in MCF-7 cells, which indicates that compound Y4 can significantly increase the accumulation of platinum drugs in cancer cells compared with positive drugs.

[0131] Subsequently, the same method was used to test the degree of binding of Y4 to nuclear DNA, as well as the uptake of platinum in mitochondria, endoplasmic reticulum, and lysosomes. The results showed that after 24 hours of Y4 action, the distribution of platinum in lysosomes was the highest in both MDA-MB-231 cells and MCF-7 cells, and Y4 had no obvious selectivity for triple-negative breast cancer. In other words, compound Y4 had similar effects on MDA-MB-231 cells and MCF-7 cells, and the uptake of platinum after Y4 action in MDA-MB-231 cells and MCF-7 cells was significantly higher than that of cisplatin and oxaliplatin. The inventors believe that compound Y4 may improve its ability to kill tumor cells by increasing the accumulation of platinum drugs in tumor cells, regulating lysosomal function, and enhancing its binding ability to DNA.

[0132] Experimental Example 5: Transwell chamber assay to detect the effect of drugs on the migration ability of breast cancer cells

[0133] In order to further verify the inhibitory effect of Pt(Ⅳ)-naphtholactam complex on the migration ability of breast cancer cells, the present invention used a Transwell chamber experiment to detect the effect of Pt(Ⅳ)-naphtholactam complex Y4 on the migration ability of breast cancer cells. The experimental results are as follows Figure 5 , 6As shown, compared with the positive control drugs cisplatin and oxaliplatin, as the drug concentration increases, the inhibitory effect of compound Y4 on breast cancer cell migration is significantly stronger than cisplatin, and the effect on MDA-MB-231 cells is more significant.

[0134] Experimental Example 6: Cell scratch test to detect the effect of drugs on the migration ability of breast cancer cells

[0135] On the basis of the transwell chamber, the present invention also conducted a cell scratch experiment in MDA-MB-231 cells and MCF-7 cells to detect the effect of Pt(IV)-naphtholactam complex Y4 on the migration ability of breast cancer cells. The experimental results are as follows Figure 7 , 8 As shown, compared with the positive drug cisplatin, as the drug concentration increases, the inhibitory effect of compound Y4 on breast cancer cell migration is significantly stronger than cisplatin.

[0136] Test Example 7: In vivo activity test and in vivo anti-metastasis test

[0137] (1) Pt(Ⅳ)-naphtholide complex inhibits the growth of solid tumors in situ in mice bearing triple-negative breast cancer cells

[0138] Compound Y4 showed strong ability to inhibit breast cancer cell viability, migration and invasion through in vitro MTT test, transwell chamber test and cell scratch test. In order to understand whether Y4 still has good anti-tumor efficacy in mice, the present invention conducted a subcutaneous tumor-bearing experiment in mice to detect the in vivo anti-tumor activity of Y4. Five-week-old Balb / C mice were implanted with mouse-derived 4T1 triple-negative breast cancer cells under the armpit to construct a solid tumor model. Each mouse required about 3 million 4T1 cells. After the mice grew for one week, the size of the solid tumor was measured, and the average tumor volume was 40-50mm 3 The mice were randomly divided into PBS group, cisplatin group (5mg / kg 3.26mg Pt / kg), oxaliplatin group (5mg / kg 2.46mg Pt / kg), intermediate B4 group (5mg / kg), cisplatin (5mg / kg 3.26mg Pt / kg) + intermediate B4 (5mg / kg) group, Y4 (5mg / kg 1.07mg Pt / kg) group, Y4 (10mg / kg 2.15mg Pt / kg) group, and the drugs were administered once every two days for a total of seven times. The drugs were administered by intraperitoneal injection. The weight and tumor volume of the mice were recorded in time during the administration. The mice were observed for three days after the end of the administration. After three days, the mice were killed by dislocation, and the tumors and organ tissues were removed, and the weights of the organs and tumors were recorded.

[0139] In vivo antitumor activity assay Fig. 9As shown, the growth curve of tumor volume ( Fig. 9 B) shows that on the 14th day of tumor growth, the average tumor volume of the PBS group was 649.3 mm 3 The average tumor volume in the cisplatin group (5 mg / kg 3.26 mg Pt / kg) was 300 mm 3 The average tumor volume in the oxaliplatin group (5 mg / kg, 2.46 mg Pt / kg) was 391 mm 3 The average tumor volume of the lead compound Y4 group (5 mg / kg 1.07 mg Pt / kg) was 290 mm 3 The average tumor volume of the Y4 (10 mg / kg 2.15 mg Pt / kg) group was 220.6 mm 3 , the solid tumor weight statistics show that ( Fig. 9 C), after treatment with compound Y4 (10 mg / kg 2.15 mg Pt / kg), the weight of solid tumors can be reduced to 21% of the negative control group, the tumor inhibition rate of Y4 (5 mg / kg 1.07 mg Pt / kg) group is 68.1%, and the tumor inhibition rate of Y4 (10 mg / kg 2.15 mg Pt / kg)) group is 78.4%, which are better than cisplatin (52.6.0%) and oxaliplatin (51.4%). During the administration period, the weight changes of mice were recorded to reflect the toxic and side effects of Y4. The weight change curve shows ( Fig. 9 A), after the last administration, the average body weight of mice in the Y4 group (10 mg / kg 2.15 mg Pt / kg) recovered to 100.2% of the original body weight, while that in the cisplatin group decreased to 85% of the initial body weight. Fig. 9 E) Analysis found that compared with the negative control, the organ index of compound Y4 did not change significantly. The above results show that Pt(IV)-naphtholide complex can effectively inhibit tumor growth in mice, and its toxic side effects are less than those of the traditional chemotherapy drug cisplatin.

[0140] (2) Pt(Ⅳ)-naphtholactam complex prolongs the survival period of mice

[0141] In order to further explore whether compound Y4 can play a positive role in the survival cycle of mice and improve the survival rate of mice, the present invention carried out a life extension rate experiment on mice. First, 1.5 million 4T1 cells were injected into the tail vein of five-week-old Balb / C female mice to establish a lung metastasis model of mice. One week after the tail vein injection, the mice were intraperitoneally injected. The experiment was divided into six groups, namely, a negative solvent group, a cisplatin group (5 mg / kg 3.26 mg Pt / kg), an oxaliplatin group (5 mg / kg 2.46 mg Pt / kg), an intermediate B4 (ligand) group (5 mg / kg), a Y4 (5 mg / kg 1.07 mg Pt / kg) group, and a Y4 (10 mg / kg 2.15 mg Pt / kg) group. The drugs were administered once every two days for a total of seven times. The drugs were then stopped and the survival of the mice was recorded every day (such as Fig.10 The results showed that compared with the PBS group, the life extension rate of mice in the Y4 low-dose group (5mg / kg 1.07mg Pt / kg) was 35.7%, and the life extension rate of mice in the Y4 high-dose group (10mg / kg 2.15mg Pt / kg) was 50%, compared with the positive drug cisplatin group, the life extension rate was 21%, and compared with the positive drug oxaliplatin group, the life extension rate was 27%. In summary, it can be seen that compound Y4 has good in vivo anti-tumor activity and can significantly extend the life cycle of mice.

[0142] (3) Pt(Ⅳ)-naphtholactam complex inhibits lung metastasis in breast cancer-bearing mice

[0143] According to the chamber experiment and the scratch experiment, it can be seen that the Pt(Ⅳ)-naphtholactam complex Y4 can significantly inhibit the migration of breast cancer cells. In order to further verify this result, the present invention conducted an in vivo tumor-bearing experiment to test the in vivo anti-tumor metastasis activity of compound Y4. Five-week-old Balb / C female mice were injected with 1.5 million 4T1 cells per mouse through the tail vein to establish a mouse lung metastasis model. One week after the tail vein injection, the drugs were administered by intraperitoneal injection. The tumor-bearing mice were randomly divided into seven groups with 5 mice in each group, namely, PBS group, cisplatin group (5 mg / kg 3.26 mg Pt / kg), oxaliplatin group (5 mg / kg 2.46 mg Pt / kg), intermediate B4 group (5 mg / kg), cisplatin + intermediate group (3.26 mg Pt / kg + 5 mg / kg) Y4-5 group (5 mg / kg 1.07 mg Pt / kg), Y4-10 group (10 mg / kg 2.15 mg Pt / kg), the drug was administered once every two days for a total of seven times. The weight changes of the mice were recorded in time during the administration period. After three days of observation, the mice in each group were killed by dislocation. The lungs and other organs of the mice were carefully removed, weighed and recorded. The experimental results are shown in Fig.11 As shown, the number of lung nodules in the drug-treated group was significantly reduced compared with the cisplatin group and the negative solvent group. In addition, in the in vivo toxicology experiment, the comparison between the experimental group and the control group showed that there was no significant change in the organ index, indicating that compound Y4 can significantly inhibit lung metastasis in mice, significantly reduce the number of lung nodules and has less toxic side effects.

[0144] (4) Effects of Pt(Ⅳ)-naphtholactam complexes on mouse immune cells

[0145] In order to further explore the effect of Pt(Ⅳ)-naphtholide compound Y4 on immune cells, that is, to explore whether the compound will initiate immunity, the present invention tested the mouse subcutaneous tumor bearing experiment to detect whether Y4 will initiate mouse autoimmunity. A solid tumor model was established by performing an axillary tumor implantation experiment with mouse-derived 4T1 triple-negative breast cancer cells on five-week-old female Balb / C mice. The experiment was divided into three groups, each with three mice, namely the negative solvent group, the cisplatin group (5mg / kg 3.26mg Pt / kg), and the Y4-10 group (10mg / kg2.15mg Pt / kg). Each mouse required approximately 3 million 4T1 cells. The drug was administered seven days after the axillary tumor was implanted, and the drug was administered once every two days for a total of seven times. Two days after the end of the drug administration, the eye blood and tumor tissue of each group of mice were collected to analyze the immune cells in the mouse blood and tumor tissue. The experimental results are as follows: Fig.12 A(blood), Fig.12 As shown in B (tumor), CD45+ represents white blood cells, CD5+ represents T cells, CD19+ represents B cells, CD4+ represents helper T lymphocytes, CD8+ represents cytotoxic T cells, CD4+CD25+ represents Treg cells, CD11b+ represents myeloid cells, Ly6c-Ly6G+ represents granulocyte-like MDSC, Ly6c+Ly6G- represents monocyte-like MDSC, and CD11C+ represents dendritic cells. Treg cells and MDSC cells both play an immunosuppressive role. Through flow cytometry data analysis, it was found that After Y4 acted on mice, Treg cells in the mouse blood were analyzed and found to be significantly different. After analyzing the data of immune cells in tumor tissues, it was found that after compound Y4 took effect, the CD11C+ flow cytometry data analysis found that dendritic cells were significantly upregulated after Y4 acted. The Ly6C-Ly6G+ flow cytometry data analysis found that Y4 can significantly downregulate MDSC derived from granulocytes, thereby affecting the immunosuppressive effect of MDSC cells. This shows that compound Y4 has a significant effect on Treg cells, dendritic cells and MDSC cells derived from granulocytes. In the future, a more in-depth study will be conducted on the immune effects of compound Y4. Experimental Example 8: Pt(Ⅳ)-naphtholactam complex Y4 induces cell apoptosis

[0146] In order to explore the apoptosis of MDA-MB-231 cells caused by Pt(Ⅳ)-naphtholactam complexes while exerting anti-tumor effects, the present invention uses the Annexin V / PI double staining method to test the apoptosis of triple-negative breast cancer cells MDA-MB-231 caused by Pt(Ⅳ)-naphtholactam complex Y4 and positive drugs cisplatin and oxaliplatin. After adding Y4 1.5μM, 3μM and cisplatin 3μM to MDA-MB-231 cells for 24 hours, the cells were collected and washed with PBS, and then flow cytometry was used to analyze the apoptosis induced by the compounds. The experimental results are shown in the figure. Fig.13 As shown, the proportion of apoptotic cells at different concentrations of Y4 is Y4 (1.5μM, 18.88%), Y4 (3.0μM, 24.49%), among which the apoptotic proportions of Y4 (1.5μM) and Y4 (3.0μM) are higher than that of cisplatin (3μM, 13.16%). The above results indicate that Y4 has stronger in vitro cytotoxicity and ability to induce cell apoptosis compared with cisplatin and oxaliplatin.

[0147] Experimental Example 9: Cancer cell killing mechanism of compound Y4 and RNA sequencing data analysis

[0148] In order to further explore the specific mechanism of compound Y4's killing effect on breast cancer cells, RNA sequencing technology (RNA-seq) was used to analyze the transcriptome changes of MDA-MB-231 cells and MCF-7 cells after 24 hours of treatment with compound Y4. Under the condition of false detection rate (FDR) lower than 0.05, |log2FC|>1 was used as the critical value for judging whether a gene was upregulated or downregulated, log2FC>0 was upregulated, and log2FC<0 was downregulated. The results showed that in MDA-MB-231 cells, the expression levels of 2393 genes were significantly changed ( Fig.14 A), of which 1921 genes were up-regulated, 472 genes were down-regulated, and 2802 genes had significant changes in expression in MCF-7 cells ( Fig.14 B), among which 1901 genes were up-regulated and 901 genes were down-regulated; Venn diagram ( Fig.14 C) shows the correlation between the number of genes with significant changes in expression in MDA-MB-231 cells and MCF-7 cells. Compound Y4 has almost the same tumor killing effect in MDA-MB-231 cells and MCF-7 cells. Therefore, under the conditions of P<0.001, FDR<0.001, |log2FC|>2.5, 45 genes with significant differences were screened from the genes involved in these two cells, and KEGG pathway enrichment analysis was performed on these 45 genes ( Fig.14D), the results showed that after 24 hours of compound Y4 action, phagosome signaling-related pathways, transforming growth factor-β (TGF-β) signaling pathways, hypertrophic cardiomyopathy and other related signaling pathways were significantly enriched. It has been reported that the TGF-β signaling pathway plays an important role in the epithelial-mesenchymal transition (EMT) and metastasis of breast cancer, and can promote the occurrence and metastasis of breast cancer cells by inducing EMT in breast cancer cells. Based on the above processing analysis, the present invention screened 15 genes related to the TGF-β signaling pathway in two breast cancer cells ( Fig.14 E), the results showed that CHRD gene was upregulated most significantly, and there are literature reports that CHRD protein is a highly effective inhibitor of the homodimeric secretory growth factor BMP of the transforming growth factor-β (TGF-β) superfamily, and inhibition of the TGF-β signaling pathway can significantly downregulate the number of peripheral Treg cells and thus downregulate the immune escape caused by the activation of the TGF-β signaling pathway in the tumor microenvironment, which is consistent with the immune data results obtained in the present invention. Therefore, it is speculated that compound Y4 can kill tumor cells and antagonize Treg cell function by upregulating CHRD protein and targeting the TGF-β signaling pathway.

[0149] According to the above ICP-MS and co-localization experimental data, it was found that after the action of compound Y4, the platinum content was accumulated in the lysosomes, which indicates that the Pt(IV)-naphtholactam complex of the present invention may achieve the effect of killing tumor cells by targeting lysosomes and regulating lysosomal function. Through conditional screening, the present invention screened 19 genes related to lysosomal function in MDA-MB-231 and MCF-7 cells ( Fig.14 F), it was found that only the lysosomal membrane protein ABCB9 was related to the TGF-β signaling pathway and ABCB9 showed an up-regulated trend in the sequencing data. As a drug transporter on the lysosomal membrane, ABCB9 protein can significantly up-regulate the lysosomal uptake of platinum drugs. In addition, inhibiting lysosomal function or inhibiting autophagy can reduce the sensitivity of melanoma to platinum drugs by activating the TGF-β signaling pathway and promote drug resistance. Based on the above analysis, it is speculated that compound Y4 may target lysosomes through ABCB9, promote autophagy, and inhibit the TGF-β signaling pathway to play an anti-breast cancer and inhibit Treg cell function.

[0150] Experimental Example 10: Pt(IV)-naphtholactam complex Y4 and gemcitabine (Gem) combined drug experiment

[0151] In this experiment, Y4 and gemcitabine (Gem) were combined to obtain in vitro antitumor activity, as shown in Tables 2 and 3. The MTT method was used to test the resistance of Pt(Ⅳ)-naphtholactam complex Y4 and gemcitabine at different concentrations in two human cancer cell lines, including MDA-MB-231 and MCF-7, with a treatment time of 72 hours. In this experiment, Y4 and gemcitabine were combined to achieve an objective treatment level at low concentrations, with significant cytotoxicity.

[0152] Table 2 In vitro antitumor activity of compound Y4 combined with Gem

[0153]

[0154]

[0155] As can be seen from Table 2, in MCF-7 cells, when Y4 (0.5 μM) + Gem (2 μM) = 1:4, an objective therapeutic level can still be achieved, with significant cytotoxicity.

[0156] Table 3 In vitro antitumor activity of compound Y4 combined with Gem

[0157]

[0158] As can be seen from Table 3, in MDA-MB-231 cells, when Y4 (1 μM) + Gem (8 μM) = 1:8, FA = 0.74, which can still reach an objective therapeutic level and has significant cytotoxicity.

[0159] In summary, the present invention obtains a series of Pt(IV)-naphthalene lactam complexes (compounds Y1-Y9) by oxidizing tetravalent platinum on the basis of cisplatin or oxaliplatin and then condensing it with naphtholactam ligands. The anti-tumor activity of 9 compounds was screened, and the results showed that compound Y4 with cisplatin as the parent core and amino group modified at position 4 generally had better anti-tumor activity and was more selective for breast cancer cells. Compound Y4 was determined as an anti-tumor lead compound, and the following conclusions were obtained:

[0160] 1. In the in vivo anti-tumor experiment, the present invention constructed a mouse breast cancer solid tumor model and a mouse lung metastasis model. The results showed that in the solid tumor model, the inhibition rate of Y4 on tumor cells was 78.4%, and the inhibition rate of the positive control drug cisplatin was 54.9%. In the mouse lung metastasis model, after the action of compound Y4, the number of lung nodules in the mouse was significantly reduced, which shows that compound Y4 has better anti-tumor effect than the positive drug cisplatin in both in vivo solid tumor and lung metastasis experiments. The life extension rate experiment of mice can confirm that compound Y4 can prolong the survival period of mice to a certain extent.

[0161] 2. After Y4 acted on mice, the immune cells in the mouse blood and tumor tissues were extracted and analyzed by flow cytometry. The results showed that compound Y4 had a significant effect on Treg cells, dendritic cells and granulocyte-derived MDSC cells, indicating that the killing effect of Pt(Ⅳ)-naphtholide complex Y4 on tumor cells may be related to the effect on these immune cells and thus enhancing the immunity of mice.

[0162] 3. In the study of the mechanism of action, the present invention found that compound Y4 was obviously located in lysosomes through laser confocal cell imaging, and it can be seen from the transwell chamber experiment and cell scratch experiment that Y4 can significantly inhibit the migration of breast cancer cells. Then, ICP-MS experiments were performed to detect the binding amount of the drug to DNA and the degree of aggregation in lysosomes, endoplasmic reticulum, mitochondria, and Golgi bodies. The results showed that the total platinum uptake in MDA-MB-231 was greater than that in MCF-7, and among the four organelles, lysosomes contained the most platinum, which was consistent with the confocal photography results. The apoptosis of MDA-MB-231 cells was detected by flow cytometry 24 hours after Y4 took effect, and the results showed that compound Y4 can significantly promote apoptosis of breast cancer cells. Through the analysis of RNA sequencing data after Y4 acted for 24 hours, the inventors believe that compound Y4 may target lysosomes through ABCB9, promote autophagy, and inhibit TGF-β signaling pathway to play an anti-breast cancer and inhibit Treg cell function.

Claims

1. A Pt(IV)-naphtholactam complex targeting TGF-β signaling pathway based on regulating lysosomal function, characterized in that: The structural formula is as follows: R' is R1 is H or NH2, n=2, 3, 4, 5.

2. The Pt(IV)-naphtholactam complex targeting TGF-β signaling pathway based on regulating lysosomal function according to claim 1, characterized in that: The structural formula is as follows:

3. The method for preparing the Pt(IV)-naphtholactam complex targeting the TGF-β signaling pathway based on regulating lysosomal function according to claim 1 or 2, characterized in that: The steps include: 1) reacting compound K1 or K2 with succinic anhydride at room temperature to generate a carboxyl group at the axial position of tetravalent platinum to obtain compound 72 or 74; 2) Compound 72 or 74 is subjected to condensation reaction with the intermediate compound under nitrogen protection at room temperature for 12-24 hours. After the reaction, the mixture is separated by column and concentrated to obtain compounds Y1-Y9; The structural formulas of compounds K1 and K2 are: The structural formulas of compounds 72 and 74 are: The intermediate compounds are B1-B5, and the structural formula is:

4. Use of the Pt(IV)-naphtholactam complex targeting the TGF-β signaling pathway based on regulating lysosomal function according to claim 1 or 2, or the Pt(IV)-naphtholactam complex combined with cisplatin, oxaliplatin or gemcitabine in the preparation of anti-tumor drugs.

5. The use according to claim 4, characterized in that: The tumor refers to human cervical cancer, human breast cancer, human lung adenocarcinoma, human liver cancer, human colon cancer or human prostate cancer.

6. The use according to claim 5, characterized in that: The Pt(IV)-naphtholactam complex is used alone in the preparation of a drug for inhibiting tumor cell proliferation, inducing pyroptosis or promoting apoptosis. The tumor cells are human liver cancer cells Huh-7 and HepG2, human breast cancer cells MDA-MB-231 and MCF-7, human lung cancer cells A549, or human colon cancer cells HCT116 and HT-29.

7. The use according to claim 4, characterized in that: When the Pt(IV)-naphtholactam complex is used alone, when the final concentration of the Pt(IV)-naphtholactam complex is 1.5-3 μM and the treatment time is 24-48 hours, the proliferation of tumor cells can be inhibited, pyroptosis can be induced or apoptosis can be promoted.

8. The use according to claim 4, characterized in that: When the concentration of the Pt(IV)-naphtholactam complex targeting the TGF-β signaling pathway based on regulating lysosomal function is 0.25-2 μM, the concentration of gemcitabine or cisplatin or oxaliplatin is 0.5-12 μM, and the treatment time is 24-72 h, the Pt(IV)-naphtholactam complex combined with gemcitabine, cisplatin or oxaliplatin can inhibit the proliferation of cancer cells, induce their pyroptosis or promote their apoptosis.

9. Use of the Pt(IV)-naphtholactam complex according to claim 1 or 2 in the preparation of a drug for inhibiting tumor growth in a mouse-derived metastasis model of breast cancer.

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