Naphthalimide-platinum (IV) complex based on Lamp2 mediation and capable of reversing glioma progress and application of naphthalimide-platinum (IV) complex

Abstract

The invention belongs to the technical field of medicinal chemistry, and particularly relates to a Lamp2-mediated naphthalimide-platinum (IV) complex capable of reversing glioma progress and application of the Lamp2-mediated naphthalimide-platinum (IV) complex. The structure of the compound is designed, and the tetravalent platinum-naphthalimide complex with different structures on two axial sides is synthesized, so that the anti-tumor curative effects of good activity and low toxicity are achieved. The naphthalimide-platinum (IV) complex disclosed by the invention has a relatively high inhibitory activity effect on tumor cells, and particularly, compared with cis-platinum, NTCDI-Pt can more effectively inhibit glioma progress. The compound NTCDI-Pt has relatively high anti-tumor activity and anti-metastasis capability in a glioma mouse body, and is high in biological safety. The naphthalimide-platinum (IV) complex is used as a new trend of naphthalimide derivative research, metal platinum (IV) is matched with naphthalimide, a dual-effect mode of the metal platinum (IV) and the naphthalimide is exerted, and the naphthalimide-platinum (IV) complex has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemistry, and specifically relates to a naphthylimide-platinum (IV) complex capable of reversing glioma progression mediated by Lamp2 and an application thereof. Background Art

[0002] Glioblastoma multiforme (GBM) is the most common invasive primary brain malignancy. Currently, the median survival of patients receiving radiotherapy combined with temozolomide chemotherapy is only 12-16 months. Glioma is characterized by diffuse infiltration, incomplete surgical resection, and easy development of drug resistance. Cisplatin is a platinum-based DNA alkylating agent and a first-line chemotherapy drug for the treatment of various cancers including gliomas. 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 cisplatin resistance in several cancers such as human lung adenocarcinoma, ovarian cancer, and GBM. Autophagy is a lysosome-dependent degradation pathway that is associated with resistance to tumor chemotherapy, immunotherapy, and targeted therapy, and the lack of effective treatment for gliomas is closely related to them.

[0003] Autophagy promotes the immune escape process 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 senescence, prevent glioma progression, and lead to SASP spectrum stimulation of antitumor immunity. Tumor-associated macrophages (TAMs) play an important role in the tumor microenvironment. There are two main subtypes of TAMs: M1 and M2; repolarizing TAMs from M2 to M1 is an effective cancer treatment strategy. A study found that the autophagy inhibitor chloroquine (CQ) can repolarize M2 macrophages to M1 phenotype in in vitro and in vivo mouse laryngeal cancer models. Therefore, anti-tumor drugs targeting autophagy and TAMs have become a hot topic in current research. The 1,8-naphthalimide pharmacophore can be embedded in DNA and produce anti-tumor activity through the p53 pathway, lysosome, mitochondrial pathway, etc. Inspired by these related studies and the continued interest in developing new platinum (IV) drugs, the present invention combines the active basic fragment naphthalimide with cisplatin or oxaliplatin to synthesize a series of naphthalimide-platinum (IV) complexes. The anticancer effects of the naphthalimide-platinum (IV) complexes were evaluated, and their effects on autophagy and macrophage polarization were explored.

[0004] The present invention first synthesizes the anti-tumor lead compound NTCDI-Pt, and then studies its inhibition of lysosomal uptake of platinum ions and macrophage-dependent autophagy based on regulating lysosomal function, and promotes HMGB1 (high mobility group protein B1) extracellular secretion to induce tumor-associated macrophage M1 polarization to inhibit glioma progression, providing a potential strategy for the treatment of GBM. The present invention explores a new type of autophagy inhibitor and finds that cisplatin promotes autophagy and increases the number of lysosomes by upregulating the expression of Lamp2, so that the lysosomes take in too many platinum ions from the cytoplasm; resulting in platinum (IV) being unable to bind to nuclear DNA, leading to glioma resistance. The compound NTCDI-Pt downregulates the expression of Lamp2 by activating TFEB (transcription factor EB) phosphorylation, thereby regulating lysosomal function to inhibit lysosomal uptake of platinum ions and increase the uptake of platinum ions by the cell nucleus. NTCDI-Pt also inhibits protective autophagy by regulating TFEB nuclear translocation; promotes HMGB1 extracellular secretion to activate macrophage function to prevent the immune escape of Pt chemotherapy drugs.

[0005] The present invention introduces naphthalene imide modified with nitro or chlorine atoms into the tetravalent platinum axial ligand for the first time, selects two divalent platinum drugs, cisplatin and oxaliplatin, which are widely used in clinical practice, and then compares the effects of different parent platinum cores on the antitumor activity. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention proposes a Lamp2-mediated naphthalimide-platinum (IV) complex capable of reversing the progression of glioma. By designing the structure of the compound, a tetravalent platinum-naphthalimide complex with different structures on both sides of the axis is synthesized to achieve its anti-tumor efficacy with good activity and low toxicity.

[0007] The present invention also provides the use of the Lamp2-mediated naphthalimide-platinum (IV) complex capable of reversing the progression of glioma in the preparation of tumor therapeutic drugs.

[0008] To achieve the above object, the technical solution created by the present invention is implemented as follows:

[0009] A Lamp2-mediated naphthalimide-platinum (IV) complex capable of reversing glioma progression, the general structural formula of which is shown in Formula I:

[0010]

[0011] Specifically, where L1 is: Where L2 is: Here, m is 1, 2 or 3.

[0012] Specifically, in the naphthalimide-platinum (IV) complex, the parent core Any one of cisplatin, carboplatin, heptaplatin, nedaplatin, oxaliplatin, lobaplatin, miplatin, picoplatin, and NDDP, preferably cisplatin or oxaliplatin:

[0013]

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

[0015]

[0016] Specifically, the asymmetric functional naphthalene imide-platinum (IV) complexes are compounds 7a, 7b, 11a, 14a, 14a, NTCDI-Pt (16a), 16b.

[0017] Specifically, the symmetrically functionalized naphthaleneimide-platinum (IV) complexes are compounds 9a, 15a, and 15b.

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

[0019] The target compounds 7a, 7b, 11a, 14a, 14a, NTCDI-Pt (16a), 16b, 9a, 15a, and 15b can be obtained by reacting nitro-modified naphthaleneimide or chlorine-modified naphthaleneimide with oxaliplatin, cisplatin, oxaliplatin derivatives, or cisplatin derivatives via TBTU condensation reaction.

[0020] Specifically, the nitro-modified naphthalene imide is Chlorine-modified naphthalene imide is

[0021] Specifically, the oxaliplatin derivative is Cisplatin derivatives are

[0022] Specifically, when preparing an 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, when preparing a symmetrically functionalized naphthalimide-platinum (IV) complex, the mass ratio of each raw material is oxaliplatin, cisplatin, oxaliplatin derivatives or cisplatin derivatives: nitro-modified naphthalimide or chlorine-modified naphthalimide: TBTU = 1: (1.5-2): (1.7-3.5).

[0024] Specifically, during the preparation process, the amount of nitro-modified naphthalene imide or chlorine-modified naphthalene imide is 200 mg to 550 mg; the amount of TBTU is 0.3 g to 0.9 g; and the amount of oxaliplatin, cisplatin, oxaliplatin derivatives or cisplatin derivatives is 0.3 to 0.4 g.

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

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

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

[0028] Preferably, when preparing compounds 16a and 16b, the chlorine-modified naphthalene imide is compound 13a and 13b, and the cisplatin derivative is compound 10; the mass ratio of the chlorine-modified naphthalene imide to the cisplatin derivative is 1:(1.2-1.7), preferably 1:1.5.

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

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

[0031] The specific synthesis route of the naphthaleneimide-platinum (IV) complex is shown in the following formula:

[0032]

[0033]

[0034] Furthermore, the preparation method of the nitro-modified naphthalene imide or the chlorine-modified naphthalene imide comprises the following steps:

[0035] Compound 3 or compound 12 is reacted with an amino acid (compound 4) of different carbon chain lengths to obtain a nitro-modified naphthalene imide or a chlorine atom-modified naphthalene imide.

[0036] Specifically, the structural formula of compound 3 is The structural formula of compound 12 is

[0037] Specifically, amino acids with different carbon chain lengths (compound 4) are Here, m is 1, 2 or 3.

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

[0039] The specific synthesis route of the nitro-modified naphthalene imide or the chlorine-modified naphthalene imide is shown in the following formula:

[0040]

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

[0042] Palmitic anhydride is used as a precursor for synthesizing the conjugate, cisplatin or oxaliplatin and palmitic anhydride are allowed to react in anhydrous DMSO for 5 to 10 days (preferably 7 days), then the DMSO is removed, the product is washed, and vacuum dried to obtain oxaliplatin derivative compound 6 or cisplatin derivative compound 10.

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

[0044] The specific synthesis route is shown below:

[0045]

[0046] Furthermore, the present invention also provides the use of the naphthylimide-platinum (IV) complex, or the naphthylimide-platinum (IV) complex combined with cisplatin or oxaliplatin or 5-fluorouracil in the preparation of anti-tumor drugs.

[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] Furthermore, the present invention also provides the use of the naphthylimide-platinum (IV) complex alone in the preparation of a drug for inhibiting tumor cell proliferation, 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 used, when the final concentration of the naphthylimide-platinum (IV) complex is (0.5-5) μM and the treatment time is 24-48 hours, it can inhibit the proliferation of tumor cells and induce their apoptosis.

[0050] Preferably, when used, when the final concentration of the naphthylimide-platinum (IV) complex is 0.5 μM, 1 μM or 5 μM and the treatment time is 24 hours, it can inhibit the proliferation of tumor cells and induce their apoptosis.

[0051] Furthermore, the present invention also provides the use of the naphthylimide-platinum (IV) complex in the preparation of a drug for inhibiting tumor growth in a mouse orthotopic model of glioma. Specifically, the mouse used in the mouse source is C57BL / 6j mouse.

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

[0053] Preferably, the naphthylimide-platinum (IV) complex can inhibit the metastasis of tumors in a prostate cancer mouse metastasis model when the dosage is 5 mg / kg or 2.4 mg Pt / kg every two days for a total of five administrations.

[0054] Furthermore, the present invention also provides an anti-tumor pharmaceutical composition containing the naphthylimide-platinum (IV) complex.

[0055] Compared with the prior art, the beneficial effects of the present invention are:

[0056] 1. The naphthylimide-platinum (IV) complex NTCDI-Pt of the present invention has high selectivity for glioma cells. The compound NTCDI-Pt has high anti-tumor activity and anti-metastasis ability in glioma mice and has high biological safety.

[0057] 2. The compound NTCDI-Pt is superior to cisplatin in terms of platinum uptake and platinum binding level to DNA in glioma cells.

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

[0059] 4. The compound NTCDI-Pt is a platinum anticancer lead compound. Compared with cisplatin, NTCDI-Pt can effectively increase the nuclear accumulation of platinum ions, which preliminarily explains why NTCDI-Pt has higher cytotoxicity than cisplatin. The present invention analyzes the abundance of lysosomes in glioma cells treated with NTCDI-Pt, and the reduction in LAMP2 expression indicates that the uptake of platinum ions by lysosomes is reduced. TFEB establishes a connection between autophagy and lysosomal biogenesis. When cells are under stress, TFEB nuclear translocation promotes the occurrence of autophagic flow and lysosomes.

[0060] 5. Further studies on the mechanism of action of the compounds of the present invention show that the compound NTCDI-Pt inhibits protective autophagy by inhibiting TFEB nuclear translocation. In addition, the experiments of the present invention show that the extracellular secretion of HMGB1 triggered by the compound NTCDI-Pt can effectively activate the function of macrophages and make them enter the pro-inflammatory M1 phenotype.

[0061] In summary, the increased nuclear uptake of platinum ions mediated by decreased lysosomal abundance and the activation of macrophage function synergistically enhanced the classical DNA damage of the compound NTCDI-Pt. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 The inhibition of cell migration in glioma cells after 24 hours of incubation with different concentrations of NTCDI-Pt, cisplatin and combined administration groups in the wound healing experiment; *P<0.05; **P<0.01; ***P<0.001;

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

[0064] Figure 3The quantitative analysis of glioma cell apoptosis induced by different concentrations of NTCDI-Pt and 10 μM cisplatin after 24 hours of incubation and the results of mouse model analysis; C57BL / 6j mouse glioma model was established by GL261-luc cells, and further treated with PBS, ligand, Cis, Cis combined with ligand, TMZ or NTCDI-Pt for 14 days; 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 animal experiment process of NTCDI-Pt anti-glioma model in vivo; Figure 3 C: Representative fluorescence images of tumors collected at the beginning or end of the experiment; Figure 3 D is the relative tumor growth inhibition rate evaluated at the end of the experiment; Figure 3 E is the weight of mice recorded every 2 days during the experiment; Figure 3 F is the organ index evaluated at the end of treatment; *P<0.05; **P<0.01; ***P<0.001;

[0065] Figure 4 NTCDI-Pt increases the nuclear uptake of platinum ions by reducing lysosomal abundance; Figure 4 A is the uptake of platinum ions in the organelles of U87 and U251 detected by ICP-MS; Figure 4 B is western blot analysis of the expression of DNA damage marker γ-H2AX in U87 and U251 cells after NTCDI-Pt treatment; Figure 4 C is Western blot analysis of Lamp2 protein expression 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 lysosomal abundance by regulating TFEB nuclear translocation; Figure 5 B is Western blot analysis of the expression of autophagy-related proteins; Figure 5 C is the degree of TFEB phosphorylation detected by Western blot; Figure 5 D: 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: Immunohistochemical analysis of Arg1+ Macrophages; Figure 6 B is flow cytometry analysis of the polarization of macrophages co-cultured with GL261 under the treatment of cisplatin, cisplatin-BafA1 or NTCDI-Pt; Figure 6 C is Western blot detection of HMGB1 translocation from the nucleus to the cytoplasm; Figure 6 D is the concentration of HMGB1 in the culture medium measured by enzyme-like immunosorbent assay; Figure 6 E is a schematic diagram of NTCDI-Pt triggering the massive release of HMGB1 from the cell nucleus to the extracellular environment. DETAILED DESCRIPTION

[0068] 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.

[0069] The abbreviations involved in the embodiments of the present invention include: Ac is acetyl, Me is methyl, Bz is benzoyl, DMSO is dimethyl sulfoxide, DMF is dimethylformamide, DCM is dichloromethane, Boc is tert-butyloxycarbonyl, TMZ is temozolomide, TBTU is O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate, Cis. is cisplatin, Oxp. is oxaliplatin, and AF is aminafide. The cisplatin and oxaliplatin are both commercially available products.

[0070] Example 1 Synthesis of Compound 1B

[0071]

[0072] (i) Dissolve 1 g of cisplatin in 30 mL of distilled water, stir to disperse completely, add 60 mL of 30% aqueous hydrogen peroxide dropwise, set the temperature to 60°C, react for 4 hours to completely dissolve and clarify, and then suspend the solvent. Add 100 mL of distilled water again and continue to stir and dissolve, raise the temperature to 80°C, and stop the reaction after it is completely dissolved and clarified again. Place in a refrigerator at 4°C overnight to precipitate yellow crystals, pour out the solvent, and evaporate the crystals to obtain a yellow solid 1A with a yield of 80%.

[0073] (ii) Add 0.3 g of compound 1A and 0.31 g of palmitic anhydride to a 100 mL round-bottom flask, seal and evacuate, add 30 mL of anhydrous DMSO under nitrogen protection, stir and react at room temperature for 7 days, and remove the solvent with an oil pump after the reaction. Add a small amount of acetone to precipitate a large amount of light yellow powder, wash it repeatedly with acetone solution twice, and filter it with a suction funnel to obtain light yellow powder 1B (compound 10), with a yield of 87%.

[0074] Example 2 Synthesis of Compound 2B

[0075]

[0076] (i) Take 1 g of oxaliplatin into a 250 mL round-bottom flask, add 30 mL of distilled water and stir, add 60 mL of 30% aqueous hydrogen peroxide solution dropwise, react at 60° C. for 4 hours to completely dissolve and clarify, and then suspend the solvent. Add 100 mL of distilled water to the round-bottom flask and continue stirring, and heat to 80° C. After the solid in the round-bottom flask is completely dissolved and clarified again, stop the reaction, and place in a refrigerator at 4° C. overnight to precipitate a large amount of white needle-like crystals, pour out the solvent, evaporate the crystals, and 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 and evacuate, add 30 mL of anhydrous DMSO under nitrogen protection, stir and react at room temperature for 7 days, and remove the solvent with an oil pump after the reaction. Add a small amount of acetone to precipitate a large amount of white powder, wash it repeatedly with acetone solution twice, and filter it with a suction funnel to obtain white powder 2B (compound 6), with a yield of 89%.

[0078] Example 3 Synthesis of nitro or chlorine atom modified naphthalene imide

[0079]

[0080] In the present invention, both Compound 2 and Compound 12 are made from commercially available raw materials, and the synthesis methods are conventional means in the prior art. The synthesis methods are not the inventive point of the present invention, so they will not be described in detail.

[0081] (I) Synthesis of compound 3

[0082] In a 250mL round-bottom flask, add 10mmol (19.8g) 1,8-naphthoic anhydride (Compound 2), then add 100mL concentrated sulfuric acid, and slowly stir to dissolve it. Under ice bath conditions, use a constant pressure separatory funnel to slowly drip a mixed acid consisting of 11.2mL concentrated nitric acid (63%-68%) and 50mL concentrated sulfuric acid (98%). After dripping, continue ice bath reaction for 2.5 hours, heat to 50℃ and continue to react for 1 hour. The reaction solution is dark red. Stir and slowly pour into ice water. Yellow solid precipitates immediately. Filter with a Buchner funnel to obtain a filter cake. Wash the filter cake until it is neutral and dry it in an oven at 50℃ to obtain a ginger-yellow 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 anhydrous ethanol was added, and then 2 g of amino acids with different carbon chain lengths (i.e., compound 4) were added; the mixture was refluxed at 80 ° C for 6 h, and spot plate monitoring was performed; after the reaction was completed, the mixture was cooled at room temperature to produce a precipitate, and a filter cake was obtained by vacuum filtration using a Buchner funnel, which was then dried in an oven at 50 ° C to obtain powdered solids 5a and 5b (yield 75%).

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

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

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

[0088] Example 4 Synthesis of target compound naphthalene imide-platinum (IV) complex

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

[0090]

[0091] Weigh 200 mg of compound 5a or 5b, place in a 100 ml round-bottom flask, put in a clean magnetic tube, cover with a rubber stopper, evacuate and fill with nitrogen protection. Use a syringe to inject 20 ml of anhydrous DMF into the round-bottom flask and stir at room temperature until completely dissolved.

[0092] Take 0.34g TBTU and dissolve it in anhydrous DMF for later use. Inject the DMF solution containing TBTU into a round-bottom flask and react for 15 to 30 minutes. Weigh 0.3g of compound 6 using an analytical balance and dissolve it in anhydrous DMF. After 15 to 30 minutes, add the DMF solution of compound 6 into a round-bottom flask, extract 0.15mL of triethylamine solution from a syringe, inject it into the reaction solution, evacuate, and place it in a nitrogen protection device for 24 hours. After the reaction is completed, monitor the reaction endpoint with a spot plate, remove DMF by vacuum distillation, and separate by column chromatography to obtain compounds 7a and 7b (yields are 52% and 55%, respectively).

[0093] (II) Synthesis of Compound 11a

[0094]

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

[0096] The NMR data of the product obtained by the above synthesis method are as follows:

[0097] Compound 7a yield: 52%; mp: 79.3.0~79.8℃; 1H NMR(300MHz,Chloroform-d)δ9.52(d,J=3.0Hz,2H),9.30(d,J=3.0Hz,2H),4.39(t, J=6.0Hz,2H),2.21(d,J=6.0Hz,2H),1.28-1.11(m,36H),0.78(t,J=6.0Hz,3H);13C NMR(75MHz,Chloroform-d)δ176.47,163.19,161.45,127.43,126.91,126.47,125.83,116.17,47.15,42.40,41.75,38.53,36.69, 34.04,31.83,31.47,29.76,28.80,28.31,25.61,24.86,22.60,14.06,8.75;ESI-MSm / z:1009.3162(M+H)+,obsd:1009.3145.Calcd for C 39 H 53 N5O 14 Pt: C 46.34%, H 5.28%, N6.93%. 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%; mp: 181.2~183.4℃; 1H NMR(300MHz,Chloroform-d)δ9.40(d,J=3.0Hz,2H),9.34(d,J=3.0Hz,2H),4.29(t,J=6.0Hz,2 13C NMR(75MHz,Chloroform-d)δ178.22,175.74,173.34,167.87,161.73,147.71,132.49,132.08,131.00,130.53,128.88,127.25,127.15,12 5.34,46.78,45.61,38.80,34.08,33.77,32.03,30.44,29.80,29.01, 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] The synthesis method is the same as that of compounds 7a and 7b, except that compound 5a is replaced by compound 13a to obtain compound 14a (yield 53%), and compound 5b is replaced by compound 13b to obtain compound 14b (yield 56%);

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

[0104] Compound 6 was replaced by compound 10, and compound 5b was replaced by compound 13b to obtain compound 16b (yield 57%). The NMR data of the obtained product are as follows:

[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%; mp: 75.0~75.8℃; 1H NMR(300MHz,Chloroform-d)δ8.46(d,J=6.0Hz,1H),8.39(d,J=6.0Hz,1H),8.30(d,J=9.0Hz,1H),7.70(d,J=9.0Hz,1H),7.66(t, 13C NMR (75MHz, Chloroform-d) δ172.24,170.88,163.27,138.68,131.72,130.82,130.27,128.82,127.66,127.14,122.81,121.33,41. 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, and 15b:

[0110]

[0111] Weigh 519 mg of compound 5b and place it in a 100 ml round-bottom flask with a clean magnetic rod, cover with a rubber stopper, vacuum, and fill with nitrogen protection. Use a syringe to inject 20 ml of anhydrous DMF into the round-bottom flask and stir at room temperature until completely dissolved. Take 0.89 g of TBTU and dissolve it in anhydrous DMF for later use. Inject the DMF solution containing TBTU into the round-bottom flask and react for 15 to 30 minutes. Weigh 0.3 g of compound 8 with an analytical balance and dissolve it in anhydrous DMF. After 15-30 minutes, add the DMF solution of compound 8 to the round-bottom flask, extract 0.77 mL of triethylamine solution from the syringe, inject it into the reaction solution, evacuate, and add it to the nitrogen protection device to react for 24 hours. After the reaction is completed, use a spot plate to monitor the reaction end point. Remove DMF by vacuum distillation and separate compound 9a by column chromatography (yield 54%).

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

[0113] Compound 5b was replaced with compound 13b to synthesize the symmetrically functionalized naphthaleneimide-platinum (IV) complex compound 15b (yield 55%). The NMR data of the obtained product are as follows:

[0114] Compound 9a yield: 54%; mp: 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,4 H),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)δ179.67,165.62,163.24,142.70,142.44,141.64,136.74,134.66,132.02,130.77,130.3 9,127.21,127.12,125.30,46.95,45.53,40.49,38.68,36.61,34.05,33.69,31.53,29.72,22.50,17.25,8.84; ESI-MS m / z:1140.1466(M+H)+,obsd:1140.1462.Calcdfor C 40 H 33 N8O 20Pt: C 42.10%, H 2.90%, N 9.82%. Found: C 42.05%, H 2.91%, N 9.80%.

[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%; mp: 70.2~71.1℃; 1H NMR(300MHz,Chloroform-d)δ8.55(d,J=6.0Hz,2H),8.48(d,J=9.0Hz,2H),8.39(d,J=6.0Hz,2H),7.78(d,J=9.0Hz,2H),7.73(t ,J=9.0Hz,2H),4.19(t,J=6.0Hz,4H),3.27(d,J=9.0Hz,2H),2.31(d,J=6.0Hz,4H),1.98(d,J=6.0Hz,4H),1.42-1.23(m,8H); 13C NMR(75MHz,Chloroform-d)δ178.03,165.02,163.62,143.36,138.88,131.90,131.01,130.48,12 9.16,128.93.127.79,127.29,122.98,121.50,41.94,40.07,30.72,23.83,14.23,13.06; 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 antitumor activity test

[0118] The in vitro antitumor activity of the naphthylimide-platinum (IV) complexes (7a, 7b, 9a, 11a, 14a, 14b, 15a, 15b, 16a, 16b) synthesized in the aforementioned examples was tested by the MTT method in colon cancer cells HT-29 and HCT-116; liver cancer cells HepG-2 and Huh-7; breast cancer cells MCF-7; triple-negative breast cancer cells MDA-MB-231; cisplatin-sensitive lung cancer A549 cells and resistant A549cisR cells; and glioma cells U251 and U87. The test results are shown in Table 1. Positive drugs cisplatin and oxaliplatin were selected as controls.

[0119] Table 1 In vitro antitumor activity of naphthalimide-platinum (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 with the positive drugs.;[c]The RF(resistance factor)is defined as the IC 50 valuein A549cisR cells / IC 50 value in A549 cells.

[0123] It can be seen from the experimental results in Table 1 that the present invention selects cisplatin and oxaliplatin, two classic anti-tumor drugs in clinical trials, as reference drugs (Table 1). A preliminary structure-activity relationship (SAR) can be obtained based on the in vitro biological results. The tumor inhibitory activity of the synergistic naphthalimide classic core and cisplatin (11a, 16a-16b) is stronger than the synergistic tumor inhibitory activity of oxaliplatin (7a-7b, 9a, 14a-14b, 15a-15b). The activity of oxaliplatin monosubstituted complexes (7a-7b, 11a, 14a-14b) is higher than that of oxaliplatin disubstituted complexes (9a, 15a-15b). Among them, the activity of the chlorine-substituted complex at the 4th position is higher than that of the nitro-substituted complex at the 3rd position. The reason for this result may be related to the potential spatial resistance of platinum drugs. The more axial ligand positions of platinum are connected, the less conducive it is to target lysosomes to exert anti-tumor activity. Among all drugs, NTCDI-Pt has significantly higher anti-tumor efficacy against multiple cancer cell lines, and IC 50 The value is more than 11 times lower than that of commercially available cisplatin and oxaliplatin.

[0124] The resistance index (RF) was 1.24 μM, which was superior to cisplatin (RF = 1.39 μM) and oxaliplatin (RF = 1.94 μM). It showed great potential in overcoming cisplatin resistance. At the same time, compared with cisplatin, NTCDI-Pt inhibited IC 50The FIs were 3.53 and 4.35, respectively. Compared with oxaliplatin, the ICs of U251 and U87 cells 50 The FI values ​​were 3.87 and 4.88, respectively. Based on the above results, the present invention selected GL261-luc cells for in vivo biological detection of NTCDI-Pt. At the same time, U87 and U251 cell lines were selected for in vitro mechanism studies.

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

[0126] (1) Wound healing experiment

[0127] In order to evaluate the inhibitory effect of NTCDI-Pt on glioma cell migration, the present invention used a wound healing experiment to detect the effect of NTCDI-Pt on the migration ability of glioma cells. Figure 1 As shown, compared with cisplatin, the inhibitory effect of the compound NTCDI-Pt on glioma cell migration was significantly higher than that of the cisplatin and combined administration groups, and the inhibitory effect was concentration-dependent.

[0128] (2) Transwell chamber experiment

[0129] In order to further verify the ability of NTCDI-Pt to inhibit the migration of glioma cells, the present invention used Transwell assay to detect the effect of NTCDI-Pt on the migration ability of glioma cells. Figure 2 As shown. The compound NTCDI-Pt has an inhibitory effect on glioma cells, and the effect is enhanced in a dose-dependent manner. The inhibitory effect of the compound NTCDI-Pt on glioma cell migration is significantly stronger than that of cisplatin and the combined administration group, and the effect is enhanced in a dose-dependent manner.

[0130] Experimental Example 3: NTCDI-Pt(16a) induces apoptosis of glioma cells

[0131] In order to study the relationship between the anti-tumor activity of NTCDI-Pt and cell apoptosis, the present invention uses the Annexin V / PI double staining method to test the apoptosis of glioma cells induced by NTCDI-Pt and positive drug cisplatin. The present invention incubated glioma cells with NTCDI-Pt at concentrations of 0.5μM, 1μM and 5μM and 5μM cisplatin for 24 hours, collected the cells, and then used flow cytometry to analyze the apoptosis induced by the compounds. The results are as follows Figure 3As shown in A, the apoptosis ratio of glioma cells induced by NTCDI-Pt is positively correlated with its concentration, that is, the ratio of early apoptotic cells is 37.57% and the ratio of late apoptotic cells is 9.96% at a concentration of 5 μM NTCDI-Pt, and the ratio of early apoptotic cells is 25.32% and the ratio of late apoptotic cells is 5.70% at a concentration of 1 μM NTCDI-Pt.

[0132] Experimental Example 4: Antitumor activity of NTCDI-Pt(16a) in vivo

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

[0134] In vitro experiments have shown that NTCDI-Pt has a good killing effect on glioma cells, in order to verify the in vivo anti-tumor activity of NTCDI-Pt. The present invention implants GL261luc cells (mouse-derived glioma cells, 150,000 / only) into the brain of C57BL / 6j mice at six weeks of age to establish an in situ glioma animal model. Before tumor cells are not inoculated, mice are placed in the same temperature, humidity, food, drinking water and other feeding environments for feeding. The mice inoculated with GL261luc cells observe the state of mice every other day, and the tumor formation of all mice is checked by mouse live fluorescence imaging on the third day after inoculation (D-fluorescein potassium salt is injected to activate fluorescence before imaging), and the mice that have not successfully formed tumors are re-inoculated with tumors. On the seventh day after inoculation, the size of mouse glioma was checked by in vivo fluorescence imaging of mice and the fluorescence value was recorded by taking photos. The mice were evenly divided into six groups (10 in each group) according to the size of the fluorescence value, and marked as the 0th day of administration, including PBS group, naphthylimide ligand group (5 mg / kg), TMZ (40 mg / kg), Cis group (4.5 mg / kg 2.95 mg Pt / kg), Cis+ligand group (4.5 mg / kg 2.95 mg Pt / kg Cis+5 mg / kg ligand), NTCDI-Pt (10.53 mg / kg 2.4 mg Pt / kg). The drug was administered intraperitoneally once every two days for a total of 5 times, during which the state of the mice was observed and the weight of the mice was measured. After two days of observation after the end of the administration, the mice were killed by cervical dislocation and the brain tissue and internal organs were removed to evaluate the organ index of the mice (the ratio of the weight of each organ to the weight of the corresponding mouse). The formula for calculating the tumor inhibition rate is: inhibition rate (%) = [(average fluorescence value of tumor in PBS control group - average fluorescence value of tumor in drug treatment group or positive group) / average fluorescence value of tumor in control group] × 100%).

[0135] Schematic diagram of the NTCDI-Pt in vivo anti-glioma model animal experiment process Figure 3 As shown in B. Figure 3The in vivo imaging results of CD and the tumor fluorescence value show that compared with the control group PBS, the NTCDI-Pt administration group has the most obvious inhibitory effect on tumors, and is slightly better than the positive control drugs TMZ and Cis; followed by the inhibitory effects of the Cis+ligand and Cis administration groups. The effect of the naphthylimide ligand administration group in inhibiting the progression of glioma is not much different from that of the control group PBS. This shows that the main effective structural region of NTCDI-Pt in inhibiting the progression of glioma is not the naphthylimide ligand structure, but is closely related to Pt(IV). During the entire animal experiment, except for the weight of the mice in the cisplatin administration group ( Figure 3 C) Except for the significant decrease in the late stage, the body weight and organ indexes of the mice in the other groups ( Figure 3 There were no obvious abnormal changes in EF. During the treatment period, the NTCDI-Pt group did not show obvious dehydration, anorexia, movement disorders or other toxicity-related symptoms. This suggests that NTCDI-Pt can inhibit the progression of glioma and has no strong toxic reaction to mice.

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

[0137] To further investigate why NTCDI-Pt exhibited better activity than cisplatin, the present invention measured the absorption and emission wavelengths of NTCDI-Pt and found that NTCDI-Pt partially accumulated in lysosomes. The role of lysosomal function, including autophagy, in GBM cisplatin resistance is critical for its continued clinical application, but how they promote anti-tumor immunity of tumors and macrophages is unclear, and the underlying mechanism is still unclear. Therefore, the platinum content in lysosomes and cell nuclei after incubation with NTCDI-Pt or cisplatin was next analyzed. Compared with cisplatin or cisplatin-BafA1 treatment, NTCDI-Pt has the advantage of abundant intranuclear accumulation of platinum ions, but the disadvantage of weak lysosomal accumulation ( Figure 4 A). Increased accumulation of platinum nuclei may lead to stronger DNA damage. Here, the effect of NTCDI-Pt on DNA damage in U87 and U251 cells was analyzed. The results showed that compared with the effects of various ligands, NTCDI-Pt exerted the strongest DNA damage effect ( Figure 4 B). Lamp2 is the most abundant component in the lysosomal membrane and is used to measure the abundance of lysosomes. The present invention measures the abundance of lysosomes by analyzing the immunofluorescence intensity of Lamp2. The present invention observed a significant increase in Lamp2 after cisplatin treatment. Correspondingly, NTCDI-Pt treatment significantly reduced the expression of Lamp2 ( Figure 4 C). Western blot analysis further confirmed that NTCDI-Pt treatment resulted in minimal Lamp2 expression compared with cells treated with other drugs ( Figure 4C) Collectively, these observations suggest that NTCDI-Pt promotes accumulation of Pt cores by impairing lysosomal abundance.

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

[0139] In this study, the present invention confirmed that NTCDI-Pt-treated glioma cells inhibited the uptake of platinum ions by lysosomes by regulating 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 abnormal uptake of platinum iron by lysosomes by cell-protective autophagy. The possible mechanism diagram is first summarized in Figure 5 A. TFEB links autophagy to lysosomal biogenesis by regulating the expression of genes involved in autophagy. We first analyzed the effect of NTCDI-Pt on autophagic flux in glioblastoma cells. Compared with the increase in LCII expression after the addition of cisplatin, NTCDI-Pt decreased autophagic flux ( Figure 5 B). In order to further clarify the inhibitory effect of NTCDI-Pt on autophagy, the present invention performed immunohistochemical analysis on LC3II and p62 proteins. Dephosphorylated TFEB, the active form, enters the cell nucleus and induces autophagy-lysosome biogenesis. It is worth noting that the subcellular localization of TFEB is strictly controlled by phosphorylation. In addition, the present invention also studied whether the phosphorylation level of TFEB changes ( Figure 5 C). After cisplatin treatment, the relative expression level of p-TFEB in glioma tissues was significantly reduced. In contrast, the p-TFEB level was significantly increased. Further, the present invention 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 that NTCDI-Pt inhibited autophagy and reduced lysosomal abundance.

[0140] Experimental Example 7: NTCDI-Pt(16a) activates tumor-associated macrophages by promoting the 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 invention analyzed the Arg1 + The number of macrophages ( Figure 6 A). Compared with the untreated group, NTCDI-Pt was found to effectively reduce Arg1 +Then the present invention co-cultured RAW264.7 cells with GL261 cells treated with NTCDI-Pt, and the macrophages showed obvious M1 polarization ( Figure 6 B). It has been reported that cisplatin can increase the extracellular release of HMGB1 in a dose-dependent manner. The released HMGB1 acts as a damage-associated molecular pattern, sensitizing the immune system to the recognition of tumor antigens. Therefore, the present invention 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 translocate from the nucleus to the cytoplasm. This suggests that HMGB1 may be released more extracellularly. The changes in HMGB1 content in the culture medium after HMGB1 nuclear translocation were 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 illustrates that NTCDI-Pt triggers a 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 invention screens the activity of the synthesized target compound and determines the lead compound. Through in vivo and in vitro experiments, the anti-tumor activity of the naphthalimide-platinum (IV) complex is clarified. Through the mechanism study of the naphthalimide-platinum (IV) complex, its anti-tumor mechanism and its effect on the immune metabolism inside the tumor are clarified. The present invention mainly obtains the following conclusions:

[0143] 1. The MTT method was used to screen the in vitro activity of the naphthalimide-platinum (IV) complex. The results showed that the naphthalimide-platinum (IV) complex had a significantly better killing effect on tumor cells than the positive control drugs cisplatin and oxaliplatin. It also had a higher selectivity for glioma cells. Through screening, the present invention discovered the best NTCDI-Pt anti-tumor lead compound.

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

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

[0146] 4. The molecular mechanism of NTCDI-Pt against glioma was investigated at the cellular level in vitro. The following conclusions were obtained: (a): NTCDI-Pt reduced lysosomal abundance and increased platinum ion nuclear uptake. (b): Regulated TFEB nuclear translocation and inhibited protective autophagy. (c): NTCDI-Pt promoted HMGB1 extracellular secretion and activated macrophage function, causing it to enter the pro-inflammatory M1 phenotype. The increased platinum ion nuclear uptake mediated by the reduced lysosomal abundance and the activation of macrophage function synergistically enhanced the inhibition of glioma progression by NTCDI-Pt.

Claims

1. A Lamp2-mediated naphthalimide-platinum (IV) complex capable of reversing glioma progression, characterized in that: The naphthaleneimide-platinum (IV) complex includes an asymmetric functional naphthaleneimide-platinum (IV) complex and a symmetric functional naphthaleneimide-platinum (IV) complex. The structural formula of the naphthaleneimide-platinum (IV) complex is as follows:

2. The method for preparing the naphthalimide-platinum (IV) complex according to claim 1, characterized in that: The method comprises the following steps: subjecting nitro-modified naphthalene imide or chlorine-modified naphthalene imide to TBTU condensation reaction with oxaliplatin, cisplatin, oxaliplatin derivatives or cisplatin derivatives to obtain the target compound; The specific synthesis route of the naphthaleneimide-platinum (IV) complex is as follows:

3. The preparation method according to claim 2, characterized in that: When preparing an asymmetric functional naphthalene imide-platinum (IV) complex, the mass ratio of each raw material is oxaliplatin, cisplatin, oxaliplatin derivative or cisplatin derivative: nitro-modified naphthalene imide or chlorine-modified naphthalene imide: TBTU=(1.2-1.7):1:(1.5-2); When preparing a symmetrically functionalized naphthalimide-platinum (IV) complex, the mass ratio of the raw materials is oxaliplatin, cisplatin, an oxaliplatin derivative or a cisplatin derivative: nitro-modified naphthalimide or chlorine-modified naphthalimide: TBTU = 1: (1.5-2): (1.7-3.5).

4. The preparation method according to claim 2, characterized in that: The preparation method of the nitro-modified naphthalene imide or the chlorine-atom-modified naphthalene imide comprises the following steps: Reacting compound 3 or compound 12 with compound 4 to obtain nitro-modified naphthalene imide or chlorine-modified naphthalene imide; The specific synthesis route of the nitro-modified naphthalene imide or the chlorine-modified naphthalene imide is as follows:

5. The preparation method according to claim 4, characterized in that: In compound 4, m is 1, 2 or 3; the mass ratio of compound 3 or compound 12 to compound 4 is (1.1-1.5):

1.

6. The preparation method according to claim 2, characterized in that: The preparation method of oxaliplatin derivatives or cisplatin derivatives comprises the following steps: Palmitic anhydride is used as a precursor for synthesizing a conjugate, cisplatin or oxaliplatin and palmitic anhydride are allowed to react in anhydrous DMSO for 5 to 10 days, and then the DMSO is removed, the product is washed, and vacuum dried to obtain an oxaliplatin derivative compound 6 or a cisplatin derivative compound 10; The specific synthesis route is as follows:

7. Use of the naphthalimide-platinum (IV) complex according to claim 1, or the naphthalimide-platinum (IV) complex combined with cisplatin or oxaliplatin or 5-fluorouracil in the preparation of an anti-tumor drug, characterized in that: The tumor refers to human colon cancer, human liver cancer, human breast cancer, human lung cancer, cisplatin-resistant human lung adenocarcinoma or human glioblastoma.

8. The use according to claim 7, characterized in that: The naphthylimide-platinum (IV) complex is used alone in the preparation of a drug for inhibiting tumor cell proliferation, inducing pyroptosis or promoting 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, human lung cancer cells A549, or human glioma cells U251 and U87.

9. The use according to claim 8, characterized in that: When used, when the final concentration of the naphthylimide-platinum (IV) complex is (0.5-5) μM and the treatment time is 24-48 hours, it can inhibit the proliferation of tumor cells and induce their apoptosis.

10. Use of the naphthylimide-platinum (IV) complex of claim 1 in the preparation of a drug for inhibiting tumor growth in a mouse orthotopic model of glioma.

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