A naphthalimide-platinum (IV) complex capable of reversing progression of glioma mediated by Lamp2 and application thereof

Abstract

The application belongs to the technical field of pharmaceutical chemistry, and particularly relates to a naphthalimide-platinum (IV) complex capable of reversing glioma progression based on Lamp2 mediation and application thereof. The application synthesizes a tetravalent platinum-naphthalimide complex with different structures on the two sides of the axis by designing the structure of the compound, so as to realize good activity, low toxicity and antitumor efficacy. The naphthalimide-platinum (IV) complex has high inhibitory activity on tumor cells, and NTCDI-Pt can more effectively inhibit glioma progression compared with cisplatin. The compound NTCDI-Pt has high antitumor activity and anti-metastasis ability in glioma mice, and has high biological safety. The naphthalimide-platinum (IV) complex as a new trend of naphthalimide derivative research has good application prospect by coordinating metal platinum (IV) with naphthalimide to play the mode of its double efficacy.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pharmaceutical chemistry, and particularly relates to a naphthalimide-platinum (IV) complex capable of reversing the progression of glioma based on Lamp2 mediation and application thereof. BACKGROUND

[0002] Glioblastoma multiforme (GBM) is the most common invasive primary brain malignancy, and the median survival of current clinical radiotherapy combined with temozolomide chemotherapy is only 12-16 months. Glioma has the characteristics of diffuse infiltration, incomplete surgical resection and easy drug resistance. Cisplatin is a platinum-based DNA alkylating agent, which is a first-line chemotherapy drug for various cancers including glioma. It can inhibit tumor cell division and induce apoptosis-mediated cell death by inducing DNA damage. However, cisplatin induces protective autophagy, which is prone to produce cisplatin resistance in several cancers such as human lung adenocarcinoma, ovarian cancer and GBM. Autophagy is a lysosome-dependent degradation pathway closely related to the drug resistance of tumor chemotherapy, immunotherapy and targeted therapy, and glioma lacks effective treatment.

[0003] Autophagy promotes the process of immune escape by regulating the PD-1 / PD-L1 checkpoint pathway, MHC class I / II molecules and the proliferation and differentiation of various immune cells in the tumor microenvironment; therefore, inhibiting autophagy can enhance the therapeutic effect of immune checkpoint blockade. It has been reported that inhibiting autophagy can promote glioblastoma cell aging, prevent glioma progression and lead to SASP profile stimulation of anti-tumor immunity. Tumor-associated macrophages (TAM) play an important role in the tumor microenvironment. TAM mainly has two subtypes: M1 and M2; re-polarizing TAM from M2 to M1 is an effective cancer treatment strategy. A study found that the autophagy inhibitor chloroquine (CQ) can re-polarize M2 macrophages to M1 phenotype in a mouse laryngeal cancer model in vivo and in vitro. Therefore, anti-tumor drugs targeting autophagy and TAM have become a hot spot of current research. The 1,8-naphthalimide pharmacophore can be embedded in DNA and produce anti-tumor activity through the p53 pathway, lysosome, mitochondrial pathway and the like. Inspired by these related researches and the continuous interest in developing new platinum (IV) drugs, the application complexes a series of naphthalimide-platinum (IV) complexes by combining the active basic fragment naphthalimide with cisplatin or oxaliplatin. The anti-cancer effect of the naphthalimide-platinum (IV) complex is evaluated, and the influence of the naphthalimide-platinum (IV) complex on autophagy and macrophage polarization state is discussed.

[0004] The present application first synthesizes an antitumor lead compound NTCDI-Pt, and then studies its inhibition of lysosome uptake of platinum ions and macrophage-dependent autophagy based on the regulation of lysosome function, and promotion of extracellular secretion of HMGB1 (high mobility group protein B1) to induce M1 polarization of tumor-associated macrophages to inhibit glioma progression, thereby providing a potential strategy for the treatment of GBM. By exploring a new autophagy inhibitor, it is found that cisplatin promotes autophagy and increases the number of lysosomes by up-regulating the expression of Lamp2, so that the lysosome takes up too much platinum ion from the cytoplasm; and platinum (IV) cannot be combined with nuclear DNA to cause glioma drug resistance. The compound NTCDI-Pt down-regulates the expression of Lamp2 by activating TFEB (transcription factor EB) phosphorylation, thereby regulating the function of lysosomes to inhibit the uptake of platinum ions by lysosomes, and increasing the uptake of platinum ions by the nucleus. NTCDI-Pt also inhibits protective autophagy by regulating the nuclear translocation of TFEB; and promotes extracellular secretion of HMGB1 to activate macrophage function to prevent immune escape of Pt chemotherapy drugs.

[0005] The present application first introduces nitrile or chlorine atom modified naphthalimide into the axial ligand of tetravalent platinum, selects two commonly used divalent platinum drugs cisplatin and oxaliplatin, and then compares the influence of different parent platinum cores on antitumor activity. SUMMARY

[0006] In view of the problems in the prior art, the present application provides a naphthalimide-platinum (IV) complex capable of reversing the progression of glioma based on Lamp2 mediation, which is synthesized by designing the structure of the compound, so as to realize the antitumor effect with good activity and low toxicity.

[0007] The present application also provides application of the naphthalimide-platinum (IV) complex capable of reversing the progression of glioma based on Lamp2 mediation in the preparation of a tumor treatment drug.

[0008] To achieve the above object, the technical scheme created by the present application is as follows:

[0009] A naphthalimide-platinum (IV) complex capable of reversing the progression of glioma based on Lamp2 mediation, the structural general formula of which is shown as formula I:

[0010]

[0011] Specifically, in the formula, L1 is: In the formula, L2 is: Wherein, m is 1, 2 or 3.

[0012] Specifically, in the naphthalimide-platinum (IV) complex, the parent nucleus is any one of cisplatin, carboplatin, heptaplatin, nedaplatin, oxaliplatin, lobaplatin, miriplatin, picoplatin, NDDP, preferably cisplatin or oxaliplatin:

[0013]

[0014] Specifically, the naphthalimide-platinum (IV) complex includes asymmetric functional naphthalimide-platinum (IV) complex and symmetric functional naphthalimide-platinum (IV) complex, and the structural formula of the naphthalimide-platinum (IV) complex is as follows:

[0015]

[0016] Specifically, the asymmetric functional naphthalimide-platinum (IV) complex is compound 7a, 7b, 11a, 14a, 14a, NTCDI-Pt (16a), 16b.

[0017] Specifically, the symmetric functional naphthalimide-platinum (IV) complex is compound 9a, 15a, 15b.

[0018] Further, the preparation method of the naphthalimide-platinum (IV) complex includes the following steps:

[0019] The nitro-modified naphthalimide or chlorine atom-modified naphthalimide is condensed with oxaliplatin, cisplatin, oxaliplatin derivative or cisplatin derivative by TBTU to obtain the target compounds 7a, 7b, 11a, 14a, 14a, NTCDI-Pt (16a), 16b, 9a, 15a, 15b.

[0020] Specifically, the nitro-modified naphthalimide is The chlorine atom-modified naphthalimide is

[0021] Specifically, the oxaliplatin derivative is The cisplatin derivative is

[0022] Specifically, in the preparation of the asymmetric functional naphthalimide-platinum (IV) complex, the mass ratio of each raw material is oxaliplatin, cisplatin, oxaliplatin derivative or cisplatin derivative: nitro-modified naphthalimide or chlorine atom-modified naphthalimide: TBTU = (1.2-1.7):1:(1.5-2).

[0023] Specifically, in the preparation of symmetrical functional naphthalimide-platinum (IV) complex, the mass ratio of each raw material is oxaliplatin, cisplatin, oxaliplatin derivative or cisplatin derivative: nitro-modified naphthalimide or chlorine atom-modified naphthalimide: TBTU = 1: (1.5-2): (1.7-3.5).

[0024] Specifically, in the preparation process, the amount of nitro-modified naphthalimide or chlorine atom-modified naphthalimide is 200 mg-550 mg; the amount of TBTU is 0.3 g-0.9 g; the amount of oxaliplatin, cisplatin, oxaliplatin derivative or cisplatin derivative is 0.3-0.4 g.

[0025] Preferably, in the preparation of compounds 7a and 7b, the nitro-modified naphthalimide is compound 5a or 5b, and the oxaliplatin derivative is compound 6; the mass ratio of nitro-modified naphthalimide to oxaliplatin derivative is 1: (1.2-1.7), preferably 1:1.5.

[0026] Preferably, in the preparation of compound 11a, the nitro-modified naphthalimide is compound 5b, and the cisplatin derivative is compound 10; the mass ratio of nitro-modified naphthalimide to cisplatin derivative is 1: (1.2-1.7), preferably 1:1.5.

[0027] Preferably, in the preparation of compounds 14a and 14b, the chlorine atom-modified naphthalimide is compound 13a or 13b, and the oxaliplatin derivative is compound 6; the mass ratio of chlorine atom-modified naphthalimide to oxaliplatin derivative is 1: (1.2-1.7), preferably 1:1.5.

[0028] Preferably, in the preparation of compounds 16a and 16b, the chlorine atom-modified naphthalimide is compound 13a or 13b, and the cisplatin derivative is compound 10; the mass ratio of chlorine atom-modified naphthalimide to cisplatin derivative is 1: (1.2-1.7), preferably 1:1.5.

[0029] Preferably, in the preparation of compound 9a, the nitro-modified naphthalimide is compound 5b, and the oxaliplatin derivative is compound 8; the mass ratio of nitro-modified naphthalimide to oxaliplatin derivative is 1: (1.5-2), preferably 1:1.73.

[0030] Preferably, in the preparation of compounds 15a and 15b, the chlorine atom-modified naphthalimide is compound 13a or 13b, and the oxaliplatin derivative is compound 8; the mass ratio of chlorine atom-modified naphthalimide to oxaliplatin derivative is 1: (1.5-2), preferably 1:1.73.

[0031] The specific synthesis route of the naphthalimide-platinum (IV) complex is as follows:

[0032]

[0033]

[0034] Further, the preparation method of the nitro-modified naphthalimide or the chlorine atom-modified naphthalimide comprises the following steps:

[0035] The compound 3 or the compound 12 is reacted with the amino acid (compound 4) with different carbon chain lengths to obtain the nitro-modified naphthalimide or the chlorine atom-modified naphthalimide.

[0036] Specifically, the compound 3 has the structural formula as shown in the following formula: The compound 12 has the structural formula as shown in the following formula:

[0037] Specifically, the amino acid (compound 4) with different carbon chain lengths has the structural formula as shown in the following formula: Wherein, m is 1, 2 or 3.

[0038] Specifically, the mass ratio of the compound 3 or the compound 12 to the amino acid (compound 4) with different carbon chain lengths is (1.1-1.5):1.

[0039] Specifically, the synthesis route of the nitro-modified naphthalimide or the chlorine atom-modified naphthalimide is shown in the following formula:

[0040]

[0041] Further, the preparation method of the oxaliplatin derivative or the cisplatin derivative comprises the following steps:

[0042] The oxaliplatin or the cisplatin and the palmitic anhydride are placed in anhydrous DMSO and reacted for 5-10 days (preferably 7 days) to obtain the oxaliplatin derivative compound 6 or the cisplatin derivative compound 10.

[0043] Specifically, in the preparation process, the mass ratio of the cisplatin or the oxaliplatin to the palmitic anhydride is (0.8-3.5):1.

[0044] Specifically, the synthesis route is shown in the following formula:

[0045]

[0046] Further, the present application also provides the use of the naphthalimide-platinum (IV) complex or the naphthalimide-platinum (IV) complex combined with cisplatin or oxaliplatin or 5-fluorouracil in the preparation of an antitumor drug.

[0047] Specifically, the tumor includes, but is not limited to, human colon cancer, human liver cancer, human breast cancer, human lung cancer, cisplatin-resistant human lung adenocarcinoma or human glioblastoma.

[0048] Further, the application also provides the use of the naphthalimide-platinum (IV) complex alone in the preparation of a drug for inhibiting the proliferation of tumor cells, inducing pyroptosis or promoting apoptosis, wherein the tumor cells are human colon cancer cells HCT-116, HT-29, human liver cancer cells Huh-7, HepG-2, human breast cancer cells MDA-MB-231, MCF-7, human lung cancer cells A549 or human glioma cells U251, U87.

[0049] Specifically, when the naphthalimide-platinum (IV) complex is used, the terminal concentration of the naphthalimide-platinum (IV) complex is (0.5-5) μM, and the treatment time is 24-48 h, the proliferation of tumor cells can be inhibited, and apoptosis can be induced.

[0050] Preferably, when the naphthalimide-platinum (IV) complex is used, the terminal concentration of the naphthalimide-platinum (IV) complex is 0.5 μM, 1 μM or 5 μM, and the treatment time is 24 h, the proliferation of tumor cells can be inhibited, and apoptosis can be induced.

[0051] Further, the application also provides the use of the naphthalimide-platinum (IV) complex in the preparation of a drug for inhibiting the growth of a tumor in a glioma mouse-derived orthotopic model.

[0052] Specifically, the naphthalimide-platinum (IV) complex can inhibit the metastasis of a tumor in a prostate cancer mouse-derived metastasis model when the dosage is 1-2.8 mg / kg / day, and the administration is performed five times in total.

[0053] Preferably, the naphthalimide-platinum (IV) complex can inhibit the metastasis of a tumor in a prostate cancer mouse-derived metastasis model when the dosage is 5 mg / kg or 2.4 mg Pt / kg every two days, and the administration is performed five times in total.

[0054] Further, the application also provides an antitumor pharmaceutical composition containing the naphthalimide-platinum (IV) complex.

[0055] Compared with the prior art, the application has the following beneficial effects:

[0056] 1. The naphthalimide-platinum (IV) complex NTCDI-Pt has high selectivity for glioma cells. The compound NTCDI-Pt has high antitumor activity and anti-metastasis ability in a glioma mouse in vivo, and has high biological safety.

[0057] 2. The compound NTCDI-Pt exhibits superior platinum uptake and DNA-binding levels in glioma cells compared to cisplatin.

[0058] 3. The compound NTCDI-Pt reduces lysosomal abundance and increases platinum ion uptake; it can regulate TFEB nuclear translocation and inhibit protective autophagy; the compound NTCDI-Pt promotes HMGB1 extracellular secretion, activates macrophage function, and enables macrophages to enter the pro-inflammatory M1 phenotype.

[0059] 4. As a platinum-based anticancer lead compound, NTCDI-Pt can effectively increase the nuclear accumulation of platinum ions compared to cisplatin, which initially explains why NTCDI-Pt has higher cytotoxicity than cisplatin. This invention analyzed the abundance of lysosomes in glioma cells treated with NTCDI-Pt. The decrease in LAMP2 expression indicates reduced lysosomal uptake of platinum ions. TFEB establishes a link between autophagy and lysosomal biogenesis; when cells are under stress, TFEB nuclear translocation promotes autophagic flux and lysosomal development.

[0060] 5. Further research into the mechanism of action of the compounds described in this invention shows that NTCDI-Pt inhibits protective autophagy by suppressing TFEB nuclear translocation. Furthermore, experiments in this invention demonstrate that HMGB1 extracellular secretion induced by NTCDI-Pt can effectively activate macrophage function, causing them to enter the pro-inflammatory M1 phenotype.

[0061] In summary, the synergistic enhancement of classical DNA damage induced by the compound NTCDI-Pt is due to the increased uptake of platinum ions mediated by decreased lysosomal abundance and the activation of macrophage function. Attached Figure Description

[0062] Figure 1 To illustrate the inhibition of cell migration in glioma cells after 24 hours of incubation with different concentrations of NTCDI-Pt, cisplatin, and the combined treatment group in a wound healing assay; *P<0.05; **P<0.01; ***P<0.001;

[0063] Figure 2 The effect of different concentrations of NTCDI-Pt, cisplatin, and combined drug administration on cell migration inhibition in glioma cells after 24 hours of incubation in Transwell chamber assays; *P<0.05; **P<0.01; ***P<0.001;

[0064] Figure 3Quantitative analysis of glioma cell apoptosis induced by different concentrations of NTCDI-Pt and 10 μM cisplatin after 24 hours of incubation and analysis of mouse model results; C57BL / 6j mouse glioma models were established using GL261-luc cells, and further treated with PBS, ligand, Cis, Cis combined with ligand, TMZ or NTCDI-Pt for 14 days; among them, Figure 3 A is the quantitative analysis of glioma cell apoptosis induced by different concentrations of NTCDI-Pt and 10 μM cisplatin; Figure 3 B is a schematic diagram of the experimental process of the in vivo anti-glioma model animal experiment of NTCDI-Pt; Figure 3 C is a representative fluorescence image of the tumor collected at the beginning or end of the experiment; Figure 3 D is the evaluation of the relative tumor growth inhibition rate at the end of the experiment; Figure 3 E is the mouse body weight recorded every 2 days during the experiment; Figure 3 F is the evaluation of organ index at the end of treatment; *P<0.05; **P<0.01; ***P<0.001;

[0065] Figure 4 NTCDI-Pt increases nuclear uptake of platinum ions by reducing lysosome abundance; Figure 4 A is the detection of the uptake of platinum ions in U87 and U251 organelles by ICP-MS; Figure 4 B is the western blot analysis of the expression of DNA damage marker γ-H2AX in U87 and U251 cells treated with NTCDI-Pt; Figure 4 C is the Western blot analysis of the expression of Lamp2 protein in U87 and U251 cells; *P<0.05; **P<0.01; ***P<0.001;

[0066] Figure 5 NTCDI-Pt inhibits protective autophagy by regulating TFEB nuclear translocation; Figure 5 A is a mechanism diagram illustrating that NTCDI-Pt reduces lysosome abundance by regulating TFEB nuclear translocation; Figure 5 B is the Western blot analysis of the expression of autophagy-related proteins; Figure 5 C is the Western blot detection of the degree of TFEB phosphorylation; Figure 5 D is the Western blot detection of TFEB nuclear translocation; *P<0.05; **P<0.01; ***P<0.001;

[0067] Figure 6 NTCDI-Pt activates tumor-associated macrophages by promoting the extracellular release of HMGB1; Figure 6 A is the immunohistochemical analysis of Arg1+ macrophages; Figure 6 B is the polarization of macrophages co-cultured with GL261 under cisplatin, cisplatin-BafAl or NTCDI-Pt treatment by flow cytometry analysis; Figure 6 C is the translocation of HMGB1 from nucleus to cytoplasm detected by Western blot; Figure 6 D is the concentration of HMGB1 in the culture medium detected by ELISA; Figure 6 E is the mode diagram of NTCDI-Pt triggering the massive release of HMGB1 from nucleus to extracellular environment. DETAILED DESCRIPTION

[0068] The technical solutions of the present application are further described in detail below through specific examples, but the protection scope of the present application is not limited thereto. Except for the specific description, all the reagents mentioned in this paper are commercially available high-purity reagents that meet the experimental requirements.

[0069] The abbreviations involved in the examples of the present application include: Ac is acetyl, Me is methyl, Bz is benzoyl, DMSO is dimethyl sulfoxide, DMF is dimethyl formamide, DCM is dichloromethane, Boc is tert-butoxycarbonyl, TMZ is temozolomide, TBTU is O-benzotriazol-N,N,N',N'-tetramethyluronium tetrafluoroborate, Cis. is cisplatin, Oxp. is oxaliplatin, and AF is amonafide. The cisplatin and oxaliplatin are commercially available products.

[0070] Synthesis of compound 1B in Example 1

[0071]

[0072] (I) 1 g of cisplatin was dissolved in 30 mL of distilled water, stirred to completely disperse, and 60 mL of 30% hydrogen peroxide aqueous solution was added dropwise. The temperature was set to 60°C, and the reaction was allowed to proceed for 4 hours until the solution was completely dissolved and clear. The solvent was then suspended and dried. 100 mL of distilled water was added again and the solution was stirred to dissolve. The temperature was raised to 80°C, and after the solution was completely dissolved and clear again, the reaction was stopped. The solution was left to precipitate yellow crystals in a refrigerator at 4°C overnight. The solvent was poured out, and the crystals were dried by evaporation to obtain yellow solid 1A with a yield of 80%.

[0073] (II) 0.3 g of compound 1A and 0.31 g of palmitic anhydride were added to a 100 mL round-bottom flask, which was sealed and vacuumed. 30 mL of anhydrous DMSO was added under nitrogen protection, and the reaction was stirred at room temperature for 7 days. After the reaction was completed, the solvent was removed by oil pump. A small amount of acetone was added to precipitate a large amount of light yellow powder. The powder was washed with acetone solution twice and filtered by suction to obtain light yellow powder 1B (compound 10) with a yield of 87%.

[0074] Synthesis of compound 2B of example 2

[0075]

[0076] (I) Take 1 g of oxaliplatin into a 250 mL round bottom flask, add 30 mL distilled water and stir, add 60 mL of 30% hydrogen peroxide aqueous solution drop by drop, and allow it to completely dissolve and become clear at 60°C for 4 hours, and then suspend the solvent. Add 100 mL of distilled water to the round bottom flask and continue stirring, and raise the temperature to 80°C, and after the solid in the round bottom flask completely dissolves and becomes clear again, stop the reaction and allow it to precipitate as a large amount of white needle-shaped crystals in a refrigerator at 4°C overnight, pour out the solvent, and evaporate the crystals to dryness to obtain white solid 2A (compound 8) with a yield of 85%.

[0077] (II) Add 0.3 g of compound 2A and 0.31 g of palmitic anhydride to a 100 mL round bottom flask, seal it, and vacuumize it, and then add 30 mL of anhydrous DMSO under nitrogen protection, and allow it to react at room temperature for 7 days while stirring, and then remove the solvent using an oil pump after the reaction is completed. A large amount of white powder is precipitated by adding a small amount of acetone, and the white powder is washed with acetone solution twice, and then filtered using a suction filter to obtain white powder 2B (compound 6) with a yield of 89%.

[0078] Synthesis of naphthalimide modified by a nitro or chlorine atom

[0079]

[0080] Both compound 2 and compound 12 in the present application use commercially available compound raw materials, and the synthesis methods use conventional means in the prior art, and the synthesis methods are not the points of the present application, and thus will not be described in detail.

[0081] (I) Synthesis of compound 3

[0082] Add 10 mmol (19.8 g) of 1,8-naphthalic anhydride (compound 2) to a 250 mL round bottom flask, and then add 100 mL of concentrated sulfuric acid, and slowly stir to dissolve it. Slowly drop the mixed acid consisting of 11.2 mL of concentrated nitric acid (63%-68%) and 50 mL of concentrated sulfuric acid (98%) into the flask using a constant pressure separatory funnel under ice bath conditions. After dropping, continue the reaction under ice bath conditions for 2.5 hours, and then raise the temperature to 50°C and continue the reaction for 1 hour, and the reaction solution is dark red. Immediately precipitate a yellow solid by slowly pouring it into ice water while stirring, and then filter the precipitate using a Buchner funnel to obtain a filter cake, and wash the filter cake until it is neutral, and then dry it in a 50°C oven to obtain a ginger-colored solid (i.e., compound 3) with a yield of 70%.

[0083] (II) Synthesis of compounds 5a, 5b, 13a, and 13b

[0084] Compound 3 (2.3 g) was placed in a 250 mL round bottom flask, 100 mL of absolute ethanol was added, followed by 2 g of amino acid with different carbon chain length (i.e. compound 4); refluxed at 80 °C for 6 h and spot plate monitoring was carried out; after the reaction was completed, the precipitate was cooled at room temperature, filtered under reduced pressure to obtain filter cake, which was dried in an oven at 50 °C to obtain powdery solid 5a, 5b (yield 75%).

[0085] When the structure of compound 4 is , the obtained product is compound 5a; when the structure of compound 4 is , the obtained product is compound 5b.

[0086] The synthesis method of compounds 13a and 13b is the same as that of 5a and 5b, except that compound 3 (2.3 g) is replaced by compound 12 (2.86 g);

[0087] When the structure of compound 4 is , the obtained product is compound 13a; when the structure of compound 4 is , the obtained product is compound 13b.

[0088] Synthesis of target compound naphthalimide-platinum (IV) complex of example 4

[0089] (I) Synthesis of compounds 7a, 7b

[0090]

[0091] 200 mg of compound 5a or 5b was weighed into a 100 ml round bottom flask, placed in a clean magnetic tube, covered with a rubber plug, vacuumed and filled with nitrogen protection. 20 ml of absolute DMF was injected into the round bottom flask with a syringe, and stirred at room temperature until completely dissolved.

[0092] 0.34 g of TBTU was taken and dissolved in absolute DMF for standby. The TBTU dissolved DMF solution was injected into the round bottom flask, and reacted for 15 to 30 minutes. 0.3 g of compound 6 was weighed with an analytical balance and dissolved in absolute DMF. After 15 to 30 min, the DMF solution of compound 6 was added to the round bottom flask, 0.15 mL of triethylamine solution was extracted from the syringe and injected into the reaction solution, vacuumed, and filled with nitrogen protection device for 24 h. The reaction end point was monitored by spot plate after the reaction was completed, and the DMF was removed by vacuum distillation, and column chromatography was used to separate compounds 7a, 7b (yield 52% and 55% respectively).

[0093] (II) Synthesis of compound 11a

[0094]

[0095] The synthetic method is the same as compound 7b, except that compound 6 is replaced by compound 10 to obtain compound 11a (yield 51%).

[0096] The nuclear magnetic resonance data of the product obtained by the above synthetic method are as follows:

[0097] Compound 7a yield: 52%; m.p.: 79.3.0-79.8 °C; 1H NMR (300 MHz, Chloroform-d) δ 9.52 (d, J = 3.0 Hz, 2H), 9.30 (d, J = 3.0 Hz, 2H), 4.39 (t, J = 6.0 Hz, 2H), 2.21 (d, J = 6.0 Hz, 2H), 1.28-1.11 (m, 36H), 0.78 (t, J = 6.0 Hz, 3H); 13C NMR (75 MHz, Chloroform-d) δ 176.47, 163.19, 161.45, 127.43, 126.91, 126.47, 125.83, 116.17, 47.15, 42.40, 41.75, 38.53, 36.69, 34.04, 31.83, 31.47, 29.76, 28.80, 28.31, 25.61, 24.86, 22.60, 14.06, 8.75; ESI-MS m / z: 1009.3162 (M+H)+, obsd: 1009.3145. Calcd for C 39 H 53 N5O 14 Pt: C 46.34%, H 5.28%, N 6.93%. Found: C 46.05%, H 5.10%, N 6.24%.

[0098] Compound 7b yield: 55%; m.p.: 71.0-72.7 °C;1H NMR (300 MHz, Chloroform-d) δ 9.76 (d, J = 3.0 Hz, 2H), 9.19 (d, J = 3.0 Hz, 2H), 4.14 (t, J = 6.0 Hz, 2H), 2.20 (d, J = 6.0 Hz, 2H), 1.95 (d, J = 6.0 Hz, 2H), 1.36-0.95 (m, 36H), 0.86 (t, J = 6.0 Hz, 3H);13C NMR (75 MHz, Chloroform-d) δ 175.95, 172.75, 161.61, 137.44, 130.88, 126.96, 124.97, 117.72, 41.91, 41.44, 40.89, 40.45, 40.19, 39.76, 35.25, 34.26, 32.81, 33.03, 33.03, 31.78, 29.30, 25.43, 24.98, 22.56, 17.09, 14.32, 14.05, 13.03; ESI-MS m / z: 1023.3340 (M+H)+, obsd: 1023.3323. Calcd for C 40 H 55 N5O 14 Pt: C 46.87%, H 5.41%, N 6.83%. Found: C 46.75%, H 5.39%, N 6.84%.

[0099] Compound 11a yield: 51%; m.p.: 181.2-183.4 °C; 1H NMR (300 MHz, Chloroform-d) δ 9.40 (d, J = 3.0 Hz, 2H), 9.34 (d, J = 3.0 Hz, 2H), 4.29 (t, J = 6.0 Hz, 2H), 3.01 (d, J = 6.0 Hz, 2H), 1.66 (d, J = 6.0 Hz, 2H), 1.30-1.17 (m, 28H), 0.88 (t, J = 6.0 Hz, 2H); 13C NMR (75 MHz, Chloroform-d) δ 178.22, 175.74, 173.34, 167.87, 161.73, 147.71, 132.49, 132.08, 131.00, 130.53, 128.88, 127.25, 127.15, 125.34, 46.78, 45.61, 38.80, 34.08, 33.77, 32.03, 30.44, 29.80, 29.01, 24.88, 23.82, 23.09, 22.80, 17.32, 14.24, 14.17, 11.06, 8.81; ESI-MS m / z: 926.2288 (M+H)+, obsd: 926.2261. Calcd for C 32 H 46 C l2 N4O 10 Pt: C 41.46%, H 4.97%, Cl 7.89%, N 6.04%. Found: C 40.95%, H 4.83%, N 6.02%.

[0100] (III) Synthesis of compounds 14a, 14b, 16a (NTCDI-Pt) and 16b

[0101]

[0102] Synthesis method is the same as compound 7a, 7b, except that compound 5a is replaced by compound 13a to obtain compound 14a (yield 53%), compound 5b is replaced by compound 13b to obtain compound 14b (yield 56%);

[0103] Compound 6 is replaced by compound 10, compound 5a is replaced by compound 13a to obtain compound 16a (NTCDI-Pt) (yield 54%);

[0104] Compound 6 is replaced by compound 10, compound 5b is replaced by compound 13b to obtain compound 16b (yield 57%). The obtained product has the following nuclear magnetic data:

[0105] Compound 14a yield: 53%; m.p.: 101.2-102.5 °C;1H NMR (300 MHz, Chloroform-d) δ 8.64 (d, J = 6.0 Hz, 1H), 8.62-8.54 (d, J = 6.0 Hz, 2H), 8.48 (d, J = 9.0 Hz, 1H), 8.21 (t, J = 9.0 Hz, 1H), 4.2 (t, J = 6.0 Hz, 2H), 2.28 (d, J = 6.0 Hz, 2H), 1.23 (m, 36H), 1.09 (t, J = 6.0 Hz, 3H);13C NMR (75 MHz, Chloroform-d) δ 177.41, 175.47, 163.68, 163.42, 159.14, 158.15, 132.09, 131.20, 130.74, 129.33, 127.88, 127.40, 127.01, 123.01, 122.46, 121.51, 41.92, 40.00, 37.23, 33.96, 33.21, 31.95, 31.26, 29.79, 29.72, 29.68, 29.57, 29.52, 29.39, 28.43, 25.71, 25.59, 24.84, 22.72, 14.24, 14.16, 13.05; ESI-MS m / z: 954.3096 (M+H)+, obsd: 955.41. Calcd for C 39 H 54 ClN3O 10 Pt: C 49.03%, H 5.70%, Cl 3.71%, N 4.40%. Found: C 48.75%, H 5.12%, N 3.84%.

[0106] Compound 14b yield: 56%; m.p.: 95.2-96.8 °C;1H NMR (300 MHz, Chloroform-d) δ 8.31 (d, J = 6.0 Hz, 1H), 8.22 (d, J = 9.0 Hz, 1H), 8.14 (d, J = 9.0 Hz, 1H), 7.57 (d, J = 6.0 Hz, 1H), 7.50 (t, J = 6.0 Hz, 1H), 4.02 (t, J = 6.0 Hz, 2H), 2.27 (d, J = 6.0 Hz, 2H), 1.92 (d, J = 6.0 Hz, 2H), 1.14-0.84 (m, 36H), 0.68 (t, J = 6.0 Hz, 3H);13C NMR (75 MHz, Chloroform-d) δ 178.49, 175.05, 164.62, 164.03, 163.76, 159.52, 132.36, 131.50, 131.03, 125.84, 125.04, 122.61, 121.10, 118.11, 110.97, 110.58, 46.60, 39.82, 36.53, 34.29, 33.31, 32.03, 30.11, 28.53, 25.82, 23.80, 22.80, 14.23, 8.81; ESI-MS m / z: 968.3029 (M+H)+, obsd: 968.3209. Calcd for C 40 H 56 ClN3O 10 Pt:C 49.56%, H 5.82%, Cl 3.66%, N 4.33%. Found: C 49.75%, H 5.63%, N 4.52%.

[0107] Compound 16a (NTCDI-Pt) yield: 54%; m.p.: 145.1-146.2 °C;1H NMR (300 MHz, Chloroform-d) δ 8.47 (d, J = 6.0 Hz, 1H), 8.39 (d, J = 6.0 Hz, 1H), 8.30 (d, J = 9.0 Hz, 1H), 7.70 (d, J = 9.0 Hz, 1H), 7.64 (t, J = 6.0 Hz, 1H), 4.38 (t, J = 6.0 Hz, 2H), 2.0 (d, J = 6.0 Hz, 2H), 1.22-0.99 (m, 28H), 0.78 (t, J = 6.0 Hz, 3H);13C NMR (75 MHz, Chloroform-d) δ 172.25, 169.30, 163.33, 163.07, 138.86, 131.78, 130.87, 130.42, 128.98, 128.70, 127.69, 127.19, 122.71, 121.23, 41.91, 41.81, 39.95, 39.86, 37.09, 33.09, 31.84, 31.12, 29.61, 29.46, 29.43, 29.28, 25.45, 22.61, 14.36, 14.16, 14.06; ESI-MS m / z: 856.2007 (M+H)+, obsd: 856.2007. Calcd for C 31 H 42 Cl3N3O6Pt: C 43.46%, H 4.91%, Cl 12.79%, N 4.91%. Found: C 43.46%, H 4.91%, N 4.91%.

[0108] Compound 16b yield: 57%; m.p.: 75.0-75.8 °C;1H NMR (300 MHz, Chloroform-d) δ 8.46 (d, J = 6.0 Hz, 1H), 8.39 (d, J = 6.0 Hz, 1H), 8.30 (d, J = 9.0 Hz, 1H), 7.70 (d, J = 9.0 Hz, 1H), 7.66 (t, J = 6.0 Hz, 1H)), 4.13 (t, J = 6.0 Hz, 2H), 2.38 (d, J = 6.0 Hz, 2H), 2.04 (d, J = 6.0 Hz, 2H), 1.24-0.95 (m, 28H), 0.79 (t, J = 6.0 Hz, 3H);13C NMR (75 MHz, Chloroform-d) δ 172.24, 170.88, 163.27, 138.68, 131.72, 130.82, 130.27, 128.82, 127.66, 127.14, 122.81, 121.33, 41.90, 41.84, 40.02, 39.97, 37.14, 35.30, 33.09, 31.84, 30.64, 29.62, 29.46, 29.29, 25.45, 23.73, 22.62, 14.15, 14.07, 12.99; ESI-MS m / z: 870.2222 (M+H)+, obsd: 870.2161. Calcd for C 32 H 47 Cl3N3O6Pt: C 44.12%, H 5.40%, Cl 12.58%, N 4.83%. Found: C 44.16%, H 5.40%, N 4.81%.

[0109] (iv) Synthesis of compounds 9a, 15a, 15b:

[0110]

[0111] Take 519 mg of compound 5b and place in a 100 mL round bottom flask cleaned magnetic sub, covered with a rubber stopper, vacuumed, and filled with nitrogen protection. Inject 20 mL of anhydrous DMF into the round bottom flask with a syringe and stir at room temperature until completely dissolved. Take 0.89 g of TBTU, dissolve in anhydrous DMF and reserve. Inject the TBTU dissolved DMF solution into the round bottom flask and react for 15 to 30 minutes. Weigh 0.3 g of compound 8 on an analytical balance and dissolve in anhydrous DMF. After 15 to 30 minutes, inject the compound 8 DMF solution into the round bottom flask, extract 0.77 mL of triethylamine solution from a syringe and inject into the reaction solution, vacuum and then fill with nitrogen protection and react for 24 hours. Monitor the reaction endpoint after the reaction is complete with a spot plate. Remove the DMF with vacuum distillation and separate the compound 9a (yield 54%) with column chromatography.

[0112] The synthesis method of compound 15a, 15b is the same as compound 9a, except that compound 5b is replaced by compound 13a, and the symmetrical functional naphthalimide-platinum (IV) complex compound 15a (yield 54%) is synthesized;

[0113] Compound 5b is replaced by compound 13b, and the symmetrical functional naphthalimide-platinum (IV) complex compound 15b (yield 55%) is synthesized. The obtained product has the following nuclear magnetic resonance data:

[0114] Compound 9a yield:54%;m.p.:85.3~86.2℃;1H NMR(300MHz,Chloroform-d)δ9.42(d,J=3.0Hz,4H),9.34(d,J=3.0Hz,4H),4.31(t,J=6.0Hz,4H),3.78(d,J=6.0Hz,2H),2.30(d,J=6.0Hz,4H),1.95(d,J=6.0Hz,4H),1.23-1.16(m,8H);13C NMR(75MHz,Chloroform-d)δ179.67,165.62,163.24,142.70,142.44,141.64,136.74,134.66,132.02,130.77,130.39,127.21,127.12,125.30,46.95,45.53,40.49,38.68,36.61,34.05,33.69,31.53,29.72,22.50,17.25,8.84;ESI-MS m / z:1140.1466(M+H)+,obsd:1140.1462.Calcdfor C 40 H 33 N8O 20Pt: C 41.41%, H 2.63%, N 5.59%. Found: C 41.87%, H 2.71%, N 6.03%.

[0115] Compound 15a yield: 54%; m.p.: 102.3-103.5 °C;1H NMR (300 MHz, Chloroform-d) δ 8.59 (d, J = 6.0 Hz, 2H), 8.52 (d, J = 9.0 Hz, 2H), 8.43 (d, J = 9.0 Hz, 2H), 7.81 (d, J = 6.0 Hz, 2H), 7.76 (t, J = 9.0 Hz, 2H), 4.47 (t, J = 6.0 Hz, 4H), 3.40 (d, J = 6.0 Hz, 2H), 2.18 (d, J = 6.0 Hz, 4H), 1.14-1.09 (m, 8H);13C NMR (75 MHz, Chloroform-d) δ 179.50, 169.12 163.52, 143.21 139.13, 132.06, 131.16, 130.71, 129.16, 128.54, 127.89, 127.41, 122.99, 121.50, 49.69, 41.97, 40.04, 37.27, 31.30, 29.77, 14.31, 13.12; ESI-MS m / z: 1001.0943 (M+H)+, obsd: 999.0801. Calcd for C 38 H 29 Cl2N4O 12 Pt: C 41.41%, H 2.63%, N 5.59%. Found: C 41.87%, H 2.71%, N 6.03%.

[0116] Compound 15b yield: 55%; m.p.: 70.2-71.1 °C;1H NMR (300 MHz, Chloroform-d) δ 8.55 (d, J = 6.0 Hz, 2H), 8.48 (d, J = 9.0 Hz, 2H), 8.39 (d, J = 6.0 Hz, 2H), 7.78 (d, J = 9.0 Hz, 2H), 7.73 (t, J = 9.0 Hz, 2H), 4.19 (t, J = 6.0 Hz, 4H), 3.27 (d, J = 9.0 Hz, 2H), 2.31 (d, J = 6.0 Hz, 4H), 1.98 (d, J = 6.0 Hz, 4H), 1.42-1.23 (m, 8H);13C NMR (75 MHz, Chloroform-d) δ 178.03, 165.02, 163.62, 143.36, 138.88, 131.90, 131.01, 130.48, 129.16, 128.93. 127.79, 127.29, 122.98, 121.50, 41.94, 40.07, 30.72, 23.83, 14.23, 13.06; ESI-MS m / z: 1029.1282 (M+H)+, obsd: 1029.1271. Calcd for C 40 H 35 Cl2N4O 12 Pt: C 46.64%, H 3.40%, N 5.44%. Found: C 46.68%, H 3.51%, N 5.52%.

[0117] Test Example 1: In vitro anti-tumor activity test

[0118] The naphthalimide-Pt(IV) complexes (7a, 7b, 9a, 11a, 14a, 14b, 15a, 15b, 16a, 16b) synthesized in the foregoing examples were tested for in vitro anti-tumor activity in colon cancer cells HT-29, HCT-116; liver cancer cells HepG-2, Huh-7; breast cancer cells MCF-7; triple negative breast cancer cells MDA-MB-231; cisplatin-sensitive lung cancer A549 cells, drug-resistant A549cisR cells; glioma cells U251, U87 by MTT method, and the test results are shown in Table 1, with cisplatin and oxaliplatin as positive controls.

[0119] Table 1 In vitro anti-tumor activity of naphthalimide-Pt(IV) complexes [e]

[0120]

[0121]

[0122] Note: [a] An average of three measurements. [b] FI b (fold increase)isdefined as IC 50 (cisplatin) / IC 50 (NTCDI-Pt),FI c (fold increase)is defined as IC 50 (Oxaliplatin) / IC 50 (NTCDI-Pt),introduced to contrast the antitumor effects withthe positive drugs.;[c]The RF(resistance factor)is defined as the IC 50 valuein A549cisR cells / IC 50 value in A549 cells.

[0123] From the results of Table 1, it can be seen that the present application selects cisplatin and oxaliplatin, two classic antitumor drugs in clinical trials, as reference drugs (Table 1). According to the in vitro biological results, a preliminary structure-activity relationship (SAR) can be obtained. The naphthalimide classic core has stronger synergistic tumor inhibition activity with cisplatin (11a, 16a-16b) than with oxaliplatin (7a-7b, 9a, 14a-14b, 15a-15b). The activity of the oxaliplatin monosubstituted complexes (7a-7b, 11a, 14a-14b) is higher than that of the oxaliplatin disubstituted complexes (9a, 15a-15b). Among them, the activity of the 4-position chloro-substituted complexes is higher than that of the 3-position nitro-substituted complexes. The reason for this result may be related to the spatial potential drug resistance of platinum drugs. The more axial ligand positions connected, the less likely it is to target lysosomes to exert antitumor activity. Among all the drugs, NTCDI-Pt has significantly higher antitumor efficacy on multiple cancer cell lines, with IC 50 values 11 times lower than commercially available cisplatin and oxaliplatin.

[0124] The resistance factor (RF) is 1.24 μM, which is better than that of cisplatin (RF = 1.39 μM) and oxaliplatin (RF = 1.94 μM). It shows great potential in overcoming cisplatin resistance. At the same time, compared with cisplatin, NTCDI-Pt inhibits the IC 50FI were 3.53 and 4.35, respectively. Compared with oxaliplatin, the IC 50 FI values were 3.87 and 4.88, respectively. Based on the above results, the inventors selected GL261-luc cells for in vivo biological detection of NTCDI-Pt. Meanwhile, U87 and U251 cell lines were selected for in vitro mechanism study.

[0125] Test Example 2: Effect of NTCDI-Pt (16a) on migration ability of glioma cells

[0126] (1) Wound healing experiment

[0127] To evaluate the inhibitory effect of NTCDI-Pt on the migration of glioma cells, the inventors detected the effect of NTCDI-Pt on the migration ability of glioma cells by wound healing experiment. The results are shown in Figure 1 As shown, the inhibitory effect of compound NTCDI-Pt on the migration of glioma cells was significantly higher than that of cisplatin and the combination administration group, and the inhibitory effect was in a concentration-dependent manner.

[0128] (2) Transwell chamber experiment

[0129] To further verify the inhibitory effect of NTCDI-Pt on the migration ability of glioma cells, the inventors detected the effect of NTCDI-Pt on the migration ability of glioma cells by Transwell experiment. The results are shown in Figure 2 As shown, compound NTCDI-Pt had an inhibitory effect on glioma cells, and the inhibitory effect was in a dose-dependent manner. The inhibitory effect of compound NTCDI-Pt on the migration of glioma cells was significantly stronger than that of cisplatin and the combination administration group, and the inhibitory effect was in a dose-dependent manner.

[0130] Test Example 3: Induction of glioma cell apoptosis by NTCDI-Pt (16a)

[0131] To study the relationship between the anti-tumor activity of NTCDI-Pt and apoptosis, the inventors tested the induction of glioma cell apoptosis by NTCDI-Pt and positive drug cisplatin by Annexin V / PI double staining method. The inventors incubated glioma cells with NTCDI-Pt at concentrations of 0.5 μM, 1 μM and 5 μM and cisplatin at a concentration of 5 μM for 24 hours, collected the cells, and then analyzed the induction of cell apoptosis by the compounds by flow cytometry. The results are shown in Figure 3As shown in A, the proportion of apoptosis of glioma cells induced by NTCDI-Pt is positively correlated with the concentration, that is, the proportion of early apoptosis cells is 37.57% and the proportion of late apoptosis cells is 9.96% when the concentration of NTCDI-Pt is 5 μM, and the proportion of early apoptosis cells is 25.32% and the proportion of late apoptosis cells is 5.70% when the concentration of NTCDI-Pt is 1 μM.

[0132] Test Example 4: Effect of NTCDI-Pt (16a) on in vivo anti-tumor activity

[0133] (1) NTCDI-Pt inhibits the growth of glioma cell orthotopic tumors in mice

[0134] The in vitro experiment proves that NTCDI-Pt has good killing effect on glioma cells. In order to verify the in vivo anti-tumor activity of NTCDI-Pt, GL261 luc cells (murine glioma cells, 150,000 per mouse) were implanted into the brain of six-week-old C57BL / 6j mice to establish an orthotopic glioma animal model. Before implanting the tumor cells, the mice were raised in the same temperature, humidity, food, and water feeding environment. The mice inoculated with GL261 luc cells were observed every other day, and the tumor formation of all mice was checked by live fluorescence imaging of mice on the third day after inoculation (fluorescence was activated by injecting D-luciferin potassium salt before imaging). The mice that failed to form tumors were re-inoculated. On the seventh day after inoculation, the size of glioma in mice was checked by live fluorescence imaging of mice, and the fluorescence value was recorded by taking pictures. According to the size of the fluorescence value, the mice were evenly divided into six groups (10 mice per group), and labeled as the 0th day of administration, including the PBS group, the naphthalimide ligand group (5 mg / kg), the TMZ group (40 mg / kg), the Cis group (4.5 mg / kg 2.95 mg Pt / kg), the Cis+ligand group (4.5 mg / kg 2.95 mg Pt / kg Cis+5 mg / kg ligand), and the NTCDI-Pt group (10.53 mg / kg 2.4 mg Pt / kg). Intraperitoneal injection was given every two days, a total of 5 times, and the state of the mice was observed and the body weight of the mice was measured during the period. After the administration was completed, the mice were observed for two days, then the mice were sacrificed by cervical dislocation and the brain tissue and internal organs were removed, and the organ index of the mice (the ratio of the weight of each organ to the body weight of the corresponding mouse) was evaluated. The tumor inhibition rate calculation formula is: inhibition rate (%) = [(average fluorescence value of tumor in the PBS control group-average fluorescence value of tumor in the drug treatment group or positive group) / average fluorescence value of tumor in the control group] x 100%).

[0135] The schematic diagram of the in vivo anti-glioma model animal experiment process of NTCDI-Pt is shown in Figure 3 B. From Figure 3The results of in vivo imaging and tumor fluorescence values of C-D can be seen that NTCDI-Pt has the most obvious inhibitory effect on tumor compared with PBS control group, and is slightly better than the positive control drugs TMZ and Cis; followed by Cis+ligand and Cis administration groups. The effect of naphthalimide ligand administration group on inhibiting the progression of glioma is not much different from the control group PBS. It is shown that the main effective structure region of NTCDI-Pt for inhibiting the progression of glioma is not the naphthalimide ligand structure, but closely related to Pt(IV). During the whole animal experiment, except that the body weight of mice in the cisplatin administration group decreased significantly in the later stage, the body weight and organ index of mice in the other groups had no obvious abnormal changes. During the treatment, there was no obvious dehydration, anorexia, movement disorder or other symptoms related to toxicity in the NTCDI-Pt administration group. It is suggested that NTCDI-Pt can inhibit the progression of glioma, and has no strong toxic reaction to mice. Figure 3 C) In the later stage, the body weight and organ index of mice in the other groups had no obvious abnormal changes. During the treatment, there was no obvious dehydration, anorexia, movement disorder or other symptoms related to toxicity in the NTCDI-Pt administration group. It is suggested that NTCDI-Pt can inhibit the progression of glioma, and has no strong toxic reaction to mice. Figure 3 E-F) had no obvious abnormal changes. During the treatment, there was no obvious dehydration, anorexia, movement disorder or other symptoms related to toxicity in the NTCDI-Pt administration group. It is suggested that NTCDI-Pt can inhibit the progression of glioma, and has no strong toxic reaction to mice.

[0136] Test Example 5: NTCDI-Pt (16a) increases nuclear uptake of platinum ions by reducing lysosomal abundance

[0137] To further investigate the reason why NTCDI-Pt shows better activity than cisplatin, the present application measured the absorption and emission wavelengths of NTCDI-Pt, and found that NTCDI-Pt partially accumulates in lysosomes. The role of lysosome function, including autophagy, in GBM cisplatin resistance is crucial for its continued use in the clinic, but how they facilitate antitumor immunity in tumors and macrophages is not clear, and the underlying mechanism is not clear. Therefore, the next step is to analyze the content of platinum in lysosomes and nuclei after incubation with NTCDI-Pt or cisplatin. Compared with cisplatin or cisplatin-BafA1 treatment, NTCDI-Pt has the advantage of rich nuclear accumulation of platinum ions, but the disadvantage of weak lysosomal accumulation is weak( Figure 4 A). The increased nuclear accumulation of platinum may lead to stronger DNA damage effects. Here, the effect of NTCDI-Pt on DNA damage in U87 and U251 cells was analyzed. The results show that, compared with the effects of various ligands, NTCDI-Pt exerts the strongest DNA damage effect( Figure 4 B). Lamp2 is the most abundant component in the lysosomal membrane and is used to determine the abundance of lysosomes. The present application determines the abundance of lysosomes by analyzing the immunofluorescence intensity of Lamp2. The present application observes that Lamp2 significantly increases after cisplatin treatment. Correspondingly, NTCDI-Pt treatment significantly reduces the expression of Lamp2( Figure 4 C). Western blot analysis further confirms that, compared with other drug-treated cells, NTCDI-Pt treatment results in the least expression of Lamp2( Figure 4C). Overall, these observations suggest that NTCDI-Pt promotes accumulation of platinum core by impairing lysosomal abundance.

[0138] Example 6: NTCDI-Pt (16a) inhibits protective autophagy by modulating TFEB nuclear translocation

[0139] In this study, the present application demonstrates that NTCDI-Pt treated glioma cells inhibit lysosomal uptake of platinum ions by modulating TFEB nuclear translocation, reducing lysosomal abundance, and ultimately leading to more platinum ions accumulation in the nucleus. In contrast, cisplatin resistance is caused by increased lysosomal enzyme uptake of platinum ions by cellular protective autophagy. Its possible mechanism is first summarized in Figure 5 A. TFEB links autophagy to lysosomal biogenesis by modulating the expression of genes involved in autophagy. The effect of NTCDI-Pt on autophagic flux in glioblastoma cells was first analyzed. In contrast to the increase in LCII expression after the addition of cisplatin, NTCDI-Pt reduced autophagic flux ( Figure 5 B). To further clarify the inhibitory effect of NTCDI-Pt on autophagy, the present application performed immunohistochemical analysis of LC3II and p62 proteins. Dephosphorylated TFEB, the active form, enters the nucleus and induces autophagy-lysosomal biogenesis. Notably, the subcellular localization of TFEB is strictly controlled by phosphorylation. In addition, the present application also investigated whether the phosphorylation level of TFEB changes ( Figure 5 C). The relative expression level of p-TFEB in glioma tissue was significantly reduced after cisplatin treatment. In contrast, p-TFEB levels were significantly elevated. Further, the present application analyzed the difference in TFEB content between the nucleus and cytoplasm ( Figure 5 D). After cisplatin treatment, more TFEB translocated to the nucleus, and the addition of BafA1 rescued the nuclear accumulation of TFEB. This is consistent with the report. NTCDI-Pt treatment induced the nuclear translocation of TFEB, which explains the inhibition of autophagy and the reduction of lysosomal abundance by NTCDI-Pt.

[0140] Example 7: NTCDI-Pt (16a) activates tumor-associated macrophages by promoting extracellular release of HMGB1

[0141] Glioma is a mixed solid tumor with a large number of infiltrating macrophages. At the end of the in vivo experiment, the present application analyzed Arg1 + macrophage number Figure 6 A). Compared with the untreated group, it was found that NTCDI-Pt could effectively reduce Arg1 +The number of macrophages. Then the present application will be RAW264.7 cells with NTCDI-Pt treated GL261 cells co-cultured, macrophages show obvious M1 polarization Figure 6 B) It is reported that cisplatin can increase the extracellular release of HMGB1 in a dose-dependent manner. Released HMGB1 acts as a damage-associated molecular pattern, sensitizing the immune system to tumor antigens. Therefore, the present application analyzes the changes in the content of HMGB1 in the nucleus and cytoplasm after NTCDI-Pt and cisplatin treatment Figure 6 C) HMGB1 tends to transfer from the nucleus to the cytoplasm. This indicates that HMGB1 can be released more extracellularly. The change in the content of HMGB1 in the culture medium after HMGB1 nuclear translocation is analyzed by enzyme-linked immunosorbent assay Figure 6 D) Glioma cells secrete more HMGB1 in the culture medium. These results indicate that NTCDI-Pt can activate macrophage HMGB1 in a dose-dependent manner. Figure 6 E Briefly describes that NTCDI-Pt triggers the massive release of HMGB1 from the nucleus to the extracellular environment, and the surrounding TAMs are polarized to a pro-inflammatory phenotype.

[0142] In summary, the present application screens the synthesized target compounds for activity to determine the lead compound. Through in vivo and in vitro experiments, the anti-tumor activity of naphthalimide-platinum(IV) complexes is elucidated, and through mechanism research on naphthalimide-platinum(IV) complexes, the anti-tumor mechanism and the influence on the immune metabolism inside the tumor are elucidated. The present application mainly obtains the following conclusions:

[0143] 1. The naphthalimide-platinum(IV) complexes are screened in vitro for activity by MTT method, and the results show that the killing power of the naphthalimide-platinum(IV) complexes on tumor cells is significantly better than that of the positive control drugs cisplatin and oxaliplatin. And it has high selectivity to glioma cells. Through screening, the present application finds the best NTCDI-Pt anti-tumor lead compound.

[0144] 2. Wound healing experiments and Transwell experiments prove that NTCDI-Pt can effectively inhibit the migration of glioma cells.

[0145] 3. By establishing an in vivo glioma solid tumor model, it is found that the compound NTCDI-Pt has a high inhibitory effect on the growth of glioma tumors, and has no effect on the body weight of mice, indicating that NTCDI-Pt has high safety anti-tumor activity.

[0146] 4. In vitro, the molecular mechanism of NTCDI-Pt anti-glioma was explored by cell level. The following conclusions were obtained, (a): NTCDI-Pt reduced lysosome abundance and increased nuclear uptake of platinum ions. (b): The regulation of TFEB nuclear translocation inhibited protective autophagy. (c): NTCDI-Pt promoted the extracellular secretion of HMGB1 to activate macrophage function and make it enter the pro-inflammatory M1 phenotype. The synergistic effect of the increased nuclear uptake of platinum ions mediated by the decrease of lysosome abundance and the activation of macrophage function enhanced the inhibition of glioma progression by NTCDI-Pt.

Claims

1. The application of a Lamp2-mediated naphthalimide-platinum(IV) complex capable of reversing glioma progression in the preparation of antitumor drugs, characterized in that, The tumors involved are human colon cancer cells HCT-116 and HT-29, human liver cancer cells Huh-7 and HepG-2, human breast cancer cells MDA-MB-231 and MCF-7, or human glioma cells U251 and U87. The structural formula of the naphthalimide-platinum(IV) complex is shown below: 。 2. The application according to claim 1, characterized in that, The application of the naphthalimide-platinum (IV) complex in the preparation of drugs that inhibit tumor cell proliferation, induce pyroptosis, or promote apoptosis, wherein the tumor cells are human colon cancer cells HCT-116 and HT-29, human liver cancer cells Huh-7 and HepG-2, human breast cancer cells MDA-MB-231 and MCF-7, or human glioma cells U251 and U87.

3. The application according to claim 2, characterized in that, When applied, the final concentration of the naphthalimide-platinum(IV) complex is (0.5-5) μM and the treatment time is 24-48 h, it can inhibit the proliferation of tumor cells and induce their apoptosis.

4. The use of the naphthalimide-platinum(IV) complex of claim 1 in the preparation of a drug for inhibiting the growth of glioma in a mouse in situ model.

5. The application according to claim 1 or 2, characterized in that, The aforementioned naphthalimide-platinum(IV) complex was prepared by the following method: The target compound can be obtained by reacting nitro-modified or chlorine-modified naphthalimide with oxaliplatin, cisplatin, oxaliplatin derivatives or cisplatin derivatives via a TBTU condensation reaction. The specific synthetic route of the naphthalimide-platinum(IV) complex is shown in the figure below: 。 6. The application according to claim 5, characterized in that, When preparing asymmetric functional naphthalimide-platinum (IV) complexes, the mass ratio of each raw material is: oxaliplatin, cisplatin, oxaliplatin derivative or cisplatin derivative: nitro-modified naphthalimide or chlorine-modified naphthalimide: TBTU = (1.2~1.7):1:(1.5~2).

7. The application according to claim 5, characterized in that, The method for preparing the nitro-modified naphthalimide or the chlorine-modified naphthalimide includes the following steps: Reaction of compound 3 or compound 12 with compound 4 yields nitro-modified naphthalimide or chlorine-modified naphthalimide; The specific synthetic routes for the nitro-modified naphthalimide or chlorine-modified naphthalimide are shown in the figure below: 。 8. The application according to claim 7, characterized in that, In compound 4, m is 1, 2 or 3; the mass ratio of compound 3 or compound 12 to amino acids with different carbon chain lengths is (1.1~1.5):

1.

9. The application according to claim 5, characterized in that, The preparation method of oxaliplatin derivatives or cisplatin derivatives includes the following steps: Using palmitic anhydride as a precursor for the synthesis of conjugates, cisplatin or oxaliplatin and palmitic anhydride are reacted in anhydrous DMSO for 5 to 10 days. Then, the DMSO is removed to obtain a yellow oil. The oil is then washed and dried under vacuum to obtain oxaliplatin derivative compound 6 or cisplatin derivative compound 10. The specific synthesis route is shown in the figure below: 。

Images