A tetravalent platinum hexosamine complex targeting CRPC based on regulating O-GlcNAc glycosylation and its application

Abstract

The present invention belongs to the technical field of pharmaceutical chemistry, and specifically relates to a tetravalent platinum hexosamine complex targeting CRPC based on regulating O-GlcNAc glycosylation and its application. The present invention designs the structure of the compound to synthesize four tetravalent platinum hexosamine complexes R3-R6 with symmetrical structures on both sides of the axis, and eight tetravalent platinum hexosamine complexes R11-R14 and R16-R19 with asymmetric structures on both sides of the axis. By glycosylation modification of platinum drugs with fluoroacetyl hexosamine that can interfere with sugar metabolism, while achieving the targeting of platinum drugs, HBP is regulated to effectively control the concentration of UDP-GlcNAc substrates, thereby reducing the high-branched N-glycan on the cell membrane surface, which can target CRPC and enhance its sensitivity to platinum drugs. The present invention studies the regulation O The ‑GlcNAc-glycosylated tetravalent platinum aminohexose complex targeting CRPC can not only reduce the toxic side effects of the drug but also improve the drug resistance and increase the drug's water accumulation and activity in the body.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal chemistry, and specifically relates to a tetravalent platinum hexosamine complex targeting CRPC based on regulating O-GlcNAc glycosylation and its application. Background Art

[0002] The prostate can secrete a variety of glycoproteins to maintain normal biological functions. The overexpression of glycoproteins in the structure of prostate cancer cells affects the biological processes of cells. These changes in the structure of glycoproteins may become tumor cell biomarkers or potential therapeutic targets.

[0003] It has been reported that compared with healthy subjects, the number of tri- and tetra-antennary N-glycans in the serum and on the surface of tumor cells of patients with castration-resistant prostate cancer (CRPC) is significantly increased. Furthermore, high expression of highly branched N-glycans enhances malignancy, including cell adhesion, proliferation, and differentiation. Therefore, effectively reducing the level of N-glycan branching on the surface of prostate cancer cells will become an important approach for the treatment of androgen-independent prostate cancer.

[0004] Intracellular uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) levels play a key role in the synthesis of highly branched N-glycans, including tri- and tetra-antennary glycans. Increasing intracellular UDP-GlcNAc concentrations increases the expression of highly branched N-glycans on glycoprotein receptors on the surface of tumor cells, enhancing cell invasion and metastasis. Therefore, regulating intracellular UDP-GlcNAc concentrations and reducing the branching level of glycoprotein receptor N-glycans could be an effective approach for enhancing prostate cancer cell sensitivity to cisplatin and for treating CRPC patients. Studies have shown that abnormalities in the hexose biosynthetic pathway (HBP), O-GlcNAc glycosylation, and abnormally high expression of glycosidases UAP1, PGM3, and GFAT are all present in CRPC patients and negatively correlated with survival and disease progression. Therefore, inhibiting and regulating abnormal glycosylation in tumor cells could effectively restore cellular function.

[0005] Platinum complexes play a significant role in cancer chemotherapy, but the clinical application of divalent platinum (Pt(II)) drugs has been limited by their toxic side effects and drug resistance. In recent years, tetravalent platinum (Pt(IV)) anti-tumor drugs have become a research and development focus. Pt(IV) prodrugs are activated upon entry into tumor cells and possess unique DNA binding abilities. Modification of the axial ligands of tetravalent platinum can not only reduce drug toxicity but also improve drug resistance, increase drug accumulation in the body, and enhance drug activity.

[0006] Therefore, the synthesis of novel metal hybrids modified with O-GlcNAc glycosylation that can effectively target proteins is beneficial for reshaping the tumor immune microenvironment and will become an effective strategy to relieve immunosuppression in CRPC. Summary of the Invention

[0007] Based on the deficiencies in the prior art, the present invention aims to propose a tetravalent platinum hexosamine complex targeting CRPC based on regulating O-GlcNAc glycosylation. By designing the structure of the compound, four tetravalent platinum hexosamine complexes R3-R6 with symmetrical structures on both sides of the axial direction and eight tetravalent platinum hexosamine complexes R11-R14 and R16-R19 with asymmetric structures on both sides of the axial direction were synthesized to achieve better anti-tumor activity and targeting ability.

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

[0009] A tetravalent platinum hexosamine complex targeting CRPC based on regulating O-GlcNAc glycosylation, including Pt(IV) hexosamine complexes R3-R6 modified with symmetrical bifunctional glycosyl structures, Pt(IV) hexosamine complexes R11-R14 modified with asymmetrical monofunctional glycosyl structures, and Pt(IV) hexosamine complexes R16-R19 modified with asymmetrical monofunctional glycosyl structures.

[0010] Specifically, the symmetrical bifunctional glycosyl structure-modified Pt(IV) aminohexose complex R3-R6 has the following structural formula:

[0011]

[0012] Specifically, the asymmetric monofunctional glycosyl structure-modified Pt(IV) aminohexose complex R11-R14 has the following structural formula:

[0013]

[0014] Specifically, the asymmetric monofunctional glycosyl structure-modified Pt(IV) aminohexose complex R16-R19 has the following structural formula:

[0015]

[0016] Specifically, the method for preparing the tetravalent platinum hexosamine complex targeting CRPC based on regulating O-GlcNAc glycosylation comprises the following steps:

[0017] The target compounds R3-R6, R11-R14 or R16-R19 can be obtained by using an amino hexose group and a tetravalent platinum skeleton structure through a HATU condensation reaction.

[0018] Specifically, the amino hexose group is L4, L13, L15 or L17.

[0019] Specifically, the tetravalent platinum skeleton structure is 1D, 2D, 2F, 2G, 1C or 2C.

[0020] Specifically, the compounds 1C, 1D, 2C, 2D, 2F, and 2G are synthesized using cisplatin or oxaliplatin as raw materials, and their structures are shown below:

[0021]

[0022] Specifically, the structural formulas of the hexosamine groups L4, L13, L15, and L17 are as follows:

[0023]

[0024] Specifically, in the preparation method, the mass ratio of the amino hexose group to the tetravalent platinum skeleton structure is (1.2-1.8):1, preferably 1.57:1 or 1.85:1.

[0025] Furthermore, the present invention also provides the use of the tetravalent platinum hexosamine complex, or the tetravalent platinum hexosamine complex combined with cisplatin, oxaliplatin or gemcitabine in the preparation of anti-tumor drugs.

[0026] Specifically, the tumor includes but is not limited to human cervical cancer, human breast cancer, human lung adenocarcinoma, human liver cancer, human colon cancer, cisplatin-resistant human lung adenocarcinoma or human prostate cancer.

[0027] Furthermore, the present invention also provides the use of the tetravalent platinum aminohexose complex alone in the preparation of a drug for inhibiting tumor cell proliferation, inducing pyroptosis or promoting apoptosis, wherein the tumor cells are human liver cancer cells Huh-7 and HepG2, human breast cancer cells MDA-MB-231 and MCF-7, human prostate cancer cells DU145, PC3, LNCap, VCap, and RM-1, human lung cancer cells A549, or human colon cancer cells HCT-116 and HT-29.

[0028] Specifically, when used, when the final concentration of the tetravalent platinum hexosamine complex is (5-10) μM and the treatment time is 24-48 hours, it can inhibit the proliferation of tumor cells, induce their pyroptosis or promote their apoptosis.

[0029] Preferably, when used, when the final concentration of the tetravalent platinum hexosamine complex is 5 μM or 10 μM and the treatment time is 24 hours, it can inhibit the proliferation of tumor cells, induce pyroptosis or promote apoptosis.

[0030] Furthermore, the tetravalent platinum hexosamine complex of the present invention and gemcitabine (or cisplatin, oxaliplatin) in combination have a synergistic anti-tumor effect. To this end, the present invention also provides the use of the tetravalent platinum hexosamine complex combined with gemcitabine (or cisplatin, oxaliplatin) in the preparation of a drug for inhibiting tumor cell proliferation, inducing pyroptosis or promoting apoptosis, wherein the tumor cells are human prostate cancer cells DU145, PC3, LNCap, VCap or RM-1, or human lung cancer cells A549, human colon cancer cells HCT-116.

[0031] Furthermore, when the tetravalent platinum hexosamine complex and gemcitabine (or cisplatin, oxaliplatin) are used in combination, the tetravalent platinum hexosamine complex and gemcitabine can be mixed and used or used separately; the tetravalent platinum hexosamine complex and gemcitabine can be added and used at one time or added and used continuously in multiple times.

[0032] Specifically, when the tetravalent platinum hexosamine complex and gemcitabine (or cisplatin, oxaliplatin) are added and used at one time, they can inhibit the proliferation of cancer cells, induce their pyroptosis or promote their apoptosis. The molar ratio of the tetravalent platinum hexosamine complex and gemcitabine (or cisplatin, oxaliplatin) is 1: (2-10), and the treatment time is 24-48h; further preferably, the molar ratio of the tetravalent platinum hexosamine complex and gemcitabine (or cisplatin, or oxaliplatin) is specifically 1:2, 1:4, 1:6, 1:8 or 1:10, and the treatment time is 24h.

[0033] Furthermore, when the concentration of the tetravalent platinum hexosamine complex is (0.25-2) μM, the concentration of gemcitabine (or cisplatin, oxaliplatin) is (0.5-12) μM, and the treatment time is 24-48 h, it can inhibit the proliferation of cancer cells, induce their pyroptosis or promote their apoptosis; further preferably, the concentration of the tetravalent platinum hexosamine complex is specifically, for example, 0.25 μM, 0.5 μM, 1 μM or 2 μM, the concentration of gemcitabine (or cisplatin, oxaliplatin) is specifically, for example, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 4 μM, 5 μM, 6 μM, 8 μM, 10 μM or 12 μM, and the treatment time is 24 h.

[0034] Furthermore, the present invention also provides the use of the tetravalent platinum hexosamine complex in the preparation of a drug for inhibiting tumor growth in a mouse-derived metastasis model of prostate cancer. Specifically, the mouse used is a C57BL / 6 mouse.

[0035] Specifically, the tetravalent platinum hexosamine complex can inhibit the metastasis of tumors in a mouse metastasis model of prostate cancer when the dosage is 0.4-0.95 mg / kg / day and the dosage is administered five times.

[0036] Preferably, the tetravalent platinum hexosamine complex can inhibit the metastasis of tumors in a prostate cancer mouse metastasis model when administered at a dosage of 0.81 mg Pt / kg, 0.93 mg Pt / kg, 1.62 mg Pt / kg or 1.86 mg Pt / kg every two days for a total of five times.

[0037] Compared to existing technologies, this invention enhances the selectivity of platinum drugs for prostate cancer cells by glycosylation-modifying them with fluoroacetylhexosamine, a compound that interferes with glucose metabolism. By glycosylation-modifying the tetravalent platinum backbone, this invention exhibits excellent anti-tumor and anti-metastasis properties with virtually no biotoxicity. The compounds designed in this invention enhance the effective binding of the drug to tumor cell DNA, leveraging the exceptional stability of the tetravalent platinum drug structure over divalent platinum drugs to produce an orally administrable anti-tumor drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 In the wound healing experiment, after 24 hours of incubation, the inhibition of prostate cancer PC3 cell migration by different concentrations of R19, cisplatin, and oxaliplatin; *P<0.05; **P<0.01; ***P<0.001;

[0039] Figure 2 The results show that after 24 hours of incubation, different concentrations of R19, cisplatin, and oxaliplatin inhibited the migration of prostate cancer PC3 cells in the Transwell chamber experiment; *P<0.05; **P<0.01; ***P<0.001;

[0040] Figure 3 Quantitative analysis of apoptosis in PC3 cells induced by different concentrations of R19 and 10 μM cisplatin after 24 hours of incubation; *P<0.05; **P<0.01; ***P<0.001;

[0041] Figure 4 Figure 3. Platinum content in DU145 and PC3 cells after incubation with 10 μM R19, cisplatin, and oxaliplatin for 8 h. ICP-MS was used to detect the platinum content in the cells. A is the platinum uptake of the drugs in DU145 and PC3 cells; B is the platinum uptake in DNA, mitochondria, endoplasmic reticulum, and lysosomes in PC3 cells; C is the platinum uptake in DNA, mitochondria, endoplasmic reticulum, and lysosomes in DU145 cells; D is the platinum uptake in PC3 cells in the presence of the inhibitors phlorizin and EDG. *P<0.05; **P<0.01; ***P<0.001.

[0042] Figure 5Figure 2 shows the in vivo antitumor activity of different doses of R19, cisplatin, and oxaliplatin in RM-1 prostate cancer tumors; A is the weight change curve of mice during treatment; B is the tumor weight of each group at the end of the experiment; C is the tumor image of each group of mice at the end of the experiment; D is the HE staining of the heart, liver, spleen, lung, kidney, and tumor of mice at the end of the experiment. Figure 5 In D, from top to bottom are heart, liver, spleen, lung, kidney and tumor; from left to right are Control, L10, Oxp., Cis., Cis.+L10, R19-1, R19-2; *P<0.05; **P<0.01; ***P<0.001;

[0043] Figure 6 The distribution of platinum content and toxicity evaluation of R19, L10, cisplatin, and oxaliplatin in various tissues of solid tumors; AB represents the distribution of platinum content in heart, liver, spleen, lung, kidney, blood, and tumor tissues; CD represents the effect of the drugs on BUN and SCr levels in the kidney; EF represents the effect of the drugs on AST and ALT activities in the liver; *P < 0.05; **P < 0.01; ***P < 0.001;

[0044] Figure 7 Figure 3 shows the in vivo anti-tumor metastasis activity of different concentrations of R19, cisplatin, and oxaliplatin in prostate cancer tumors; A shows the weight change of mice after 14 days of treatment; B shows the statistics of lung metastasis nodules in mice after 14 days of treatment; C shows the lung weight change of mice after the end of treatment; D shows a representative image of lung metastasis nodules in mice after the end of treatment; E shows the organ index statistics of mice in each group at the end of the experiment; F shows HE staining of lung tissue of mice after the end of treatment; *P<0.05; **P<0.01; ***P<0.001;

[0045] Figure 8 Effects of different concentrations of R19, L10 and cisplatin on O-GlcNAc glycosylation of PC3 protein in prostate cancer cells; *P<0.05; **P<0.01; ***P<0.001;

[0046] Figure 9 HPLC was used to detect the effects of R19 and cisplatin on the UDP-GlcNAc content in prostate cancer PC3 cells; A is the standard UDP-GlcNAc; B is the standard 4F-UDP-GlcNAc; C is the Control group; D is the cisplatin group; E is the R19 group; *P<0.05; **P<0.01; ***P<0.001. DETAILED DESCRIPTION

[0047] 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.

[0048] The abbreviations involved in the embodiments of the present invention include: Ac is acetyl, Bn is benzyl, Me is methyl, Bz is benzoyl, DMSO is dimethyl sulfoxide, DMF is dimethylformamide, DCM is dichloromethane, Boc is tert-butyloxycarbonyl, DAST is diethylaminosulfur trifluoride, Pyridine is pyridine, HATU is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, DIPEA is N,N-diisopropylethylamine, NCS is N-chlorosuccinimide, Cis. is cisplatin, Oxp. is oxaliplatin, and AF is amphotericin. Cisplatin, oxaliplatin, palmitic anhydride, succinic anhydride, 2-acetylglucosamine, and 2-acetylgalactosamine are all commercially available products.

[0049] The main design ideas of the test of the present invention are as follows:

[0050] Different tumor cells can express a variety of sugar transporters on their surfaces and have varying sugar requirements. This study designed and synthesized four symmetrically bifunctional glycosylated Pt(IV) aminohexose complexes, R3-R6. By modifying tetravalent platinum with bifunctional glycosylation, we investigated the differences in cytotoxicity and uptake of different glycosylated products by different tumor cells.

[0051] 2. The reduction potential of tetravalent platinum anti-tumor drugs plays a crucial role in their anti-tumor effects. Different axial ligands of tetravalent platinum significantly influence its reduction potential. Therefore, selective axial ligand design can be used to ensure that the tetravalent platinum prodrug is reduced upon reaching the tumor environment, thereby exerting its anti-tumor effect. This invention also designed and synthesized four Pt(IV) hexosamine complexes R11-R14 modified with asymmetric monofunctional glycosyl structures.

[0052] 3. Furthermore, the present invention also modified the naked sugar of tetravalent platinum by improving the glycosylation ligand, designed and synthesized four asymmetric bifunctional glycosyl structure-modified Pt(IV) aminohexose complexes R16-R19, and studied their differences in cytotoxicity and uptake.

[0053] Example 1 Synthesis of Compound 1C

[0054]

[0055] Dissolve 1 g of cisplatin in 30 mL of distilled water and stir to completely disperse. Add 60 mL of 30% aqueous hydrogen peroxide dropwise at 60°C. Allow to react for 4 hours until completely dissolved and clarified. Then, suspend the solvent to dryness. Add another 100 mL of distilled water and continue stirring to dissolve. Raise the temperature to 80°C until completely dissolved and clarified again. Stop the reaction and store in a refrigerator at 4°C overnight to precipitate yellow crystals. Pour off the solvent and evaporate the crystals to dryness to obtain 1A, a yellow solid, with a yield of 80%.

[0056] 2. Add 0.3 g of compound 1A and 0.31 g of palmitic anhydride to a 100 mL round-bottom flask, seal, and evacuate. Under nitrogen, add 30 mL of anhydrous DMSO and react at room temperature with stirring for 7 days. After the reaction, remove the solvent using an oil pump. Adding a small amount of acetone will precipitate a large amount of light yellow powder. Wash twice with acetone solution and filter with a suction funnel to obtain a light yellow powder 1B with a yield of 87%.

[0057] 3. To a 100 mL round-bottom flask, add 0.35 g of compound 1B and 0.0613 g of succinic anhydride. Under nitrogen, add 30 mL of anhydrous DMSO. Stir at room temperature overnight and evaporate the solvent using an oil pump. Add a small amount of acetone to precipitate a large amount of light yellow powder, which is filtered using a suction funnel to obtain light yellow powder 1C in 87% yield.

[0058] Example 2 Synthesis of Compound 2C

[0059]

[0060] 1. Add 1 g of oxaliplatin to a 250 mL round-bottom flask, add 30 mL of distilled water with stirring, then add 60 mL of 30% aqueous hydrogen peroxide dropwise. React at 60°C for 4 hours to completely dissolve and clarify the solution, then suspend the solvent to dryness. Add 100 mL of distilled water to the round-bottom flask, continue stirring, and raise the temperature to 80°C. Once the solid in the round-bottom flask is completely dissolved and clarified again, stop the reaction and store in a refrigerator at 4°C overnight to precipitate a large amount of white needle-like crystals. Pour off the solvent, and evaporate the crystals to dryness to obtain 2A as a white solid with a yield of 85%.

[0061] 2. To a 100 mL round-bottom flask, add 0.3 g of compound 2A and 0.31 g of palmitic anhydride. Seal the flask and evacuate. Under nitrogen, add 30 mL of anhydrous DMSO. Stir and react at room temperature for 7 days. After the reaction, remove the solvent with an oil pump. Add a small amount of acetone to precipitate a large amount of white powder. Wash the mixture twice with acetone solution and filter it with a suction funnel to obtain 2B, a white powder with a yield of 89%.

[0062] 3. To a 100 mL round-bottom flask, add 0.35 g of compound 2B and 0.063 g of succinic anhydride. Under nitrogen, add 30 mL of anhydrous DMSO. Stir at room temperature overnight and evaporate the solvent using an oil pump. Add a small amount of acetone to precipitate a large amount of light yellow powder, which is filtered using a suction funnel to obtain 2C, a white powder with a yield of 90%.

[0063] Example 3 Synthesis of Compounds 1D and 2D

[0064]

[0065] Compound 1D was prepared using compound 1A prepared in Example 1: 0.15 g of 1A synthesized by the above method and 0.11 g of succinic anhydride were added to a 100-mL round-bottom flask. The mixture was sealed and evacuated. Under nitrogen protection, 30 mL of anhydrous DMSO was added. The mixture was placed in a heating mantle and set to 60°C. The mixture was stirred overnight, and the DMSO was then evaporated using an oil pump. Acetone solution was added to precipitate a large amount of light yellow powder, which was filtered to obtain 0.267 g of compound 1D with a yield of 89%.

[0066] The synthesis method of compound 2D was the same as that of compound 1D, except that 1A was replaced by 2A, and the yield was 50%.

[0067] Example 4 Synthesis of Compounds 2E, 2F, and 2G

[0068]

[0069] To a 250 mL round-bottom flask, 1 g of oxaliplatin was added, followed by 100 mL of distilled water, and stirred to dissolve. Then, 0.4 g of NCS was dissolved in 50 mL of distilled water and added dropwise to the round-bottom flask using a constant pressure dropping funnel. The mixture was stirred overnight at room temperature in the dark. The solvent was evaporated using an oil pump, and the product was repeatedly washed with ethanol and ether solutions to obtain 1.1 g of light yellow powder, Compound 2E, with a yield of 86%.

[0070]

[0071] Compound 2F was prepared from compound 2E: 0.3 g of 2E and 0.15 g of succinic anhydride were added to a 100 mL round-bottom flask, which was sealed and evacuated. 30 mL of anhydrous DMSO was added under nitrogen protection, and the mixture was stirred at room temperature overnight. The solvent was then evaporated, and acetone solution was added to precipitate a large amount of light yellow solid. Filtration gave 0.35 g of compound 2F, with a yield of 93%.

[0072]

[0073] Compound 2G was prepared using compound 2A prepared in Example 2: 0.48 g of 2A and 0.12 g of succinic anhydride were added to a 100 mL round-bottom flask, which was sealed and evacuated. 30 mL of anhydrous DMSO was added under nitrogen protection and stirred overnight. DMSO was then evaporated under reduced pressure, and acetone solution was added to precipitate a large amount of white powder, which was filtered to obtain 0.54 g of compound 2G in a yield of 90%.

[0074] Example 5 Synthesis of Compound L4

[0075]

[0076] 1. Add 25 g of 2-acetylglucosamine, 15 g of sodium acetate, and 82 mL of acetic anhydride to a 250 mL round-bottom flask and heat at 80°C for 8 h. After the reaction, add the reaction solution to ice water, extract and dissolve the product with dichloromethane, wash with saturated NaHCO₃, dry over anhydrous MgSO₄, and concentrate. Recrystallize from ethanol to obtain L1, a white solid, in a 91% yield.

[0077] 2. Place 3 g of L1 in a 100 mL round-bottom flask and dissolve it in 30 mL of 1,2-dichloroethane. Then add 0.87 g of the Lewis acid Yb(CF3SO3)3 and 2.8 mL of 5-chloro-1-pentanol. Insert a condenser, evacuate, and introduce nitrogen. Raise the temperature to 100°C and stir overnight. Monitor the reaction using a plate. The reaction is concentrated to an oily liquid using a water pump. Column chromatography is used to separate L2, a white solid, in 80% yield.

[0078] 3. Take 2 g of L2 and dissolve it in DMF. Use a paper trough to take 0.6 g of NaN3 and add it. Place it in a stirrer and heat it to 70°C to react overnight. Monitor by TLC and wash with DCM / H2O. After concentrating the organic layer, separate it by column chromatography to obtain 1.6 g of L3, with a yield of 87%.

[0079] 4. Take a 100 mL round-bottom flask and dissolve 0.6 g of L3 in 20 mL of methanol solution. Add 0.1 g of Pd / C, evacuate the mixture, add 0.5 mL of dilute hydrochloric acid, introduce hydrogen, and react at room temperature for 2 hours. Monitor by TLC, remove Pd / C by filtration with diatomaceous earth, dissolve the product in methanol, and evaporate it thoroughly to obtain 0.56 g of L4, which is directly used in the next reaction with a yield of 90%.

[0080] Example 6 Synthesis of Compound L15

[0081]

[0082] Synthesis of compound L14

[0083] 5 g of L3 synthesized by the above method was added to a 100 mL round-bottom flask, and 30 mL of methanol was added and stirred to dissolve. After complete dissolution, sodium methoxide was added to adjust the pH until the pH reached 9-10. After reacting for 2 h, a cationic resin was added to terminate the reaction. After evaporating the solvent, 3.5 g of compound L14 was isolated by column chromatography with a yield of 94%.

[0084] Synthesis of compound L15

[0085] 0.5 g of L14 was dissolved in 20 mL of methanol solution, 0.083 g of Pd / C was added, the mixture was vacuumed, 0.5 mL of dilute hydrochloric acid was added, hydrogen was introduced, and the reaction was carried out at room temperature for 2 h. The Pd / C was removed by filtration using diatomaceous earth and the product was dissolved in methanol. After evaporation to dryness, 0.49 g of L15 was obtained, which was used directly in the next reaction with a yield of 89%.

[0086] Example 7 Synthesis of Compound L13

[0087]

[0088] 1. Add 30 g of 2-acetylgalactosamine to a 500 mL round-bottom flask, add 300 mL of benzyl alcohol and stir thoroughly to dissolve, then slowly add 7 mL of hydrochloric acid, raise the temperature to 60°C, stir and react overnight, monitor the reaction by TLC, and after the reaction is complete, cool to room temperature, add ether and wash several times, and filter with a suction funnel to obtain 42 g of white solid L5, with a yield of 91%.

[0089] 2. Add 10 g of L5 to a 500 mL round-bottom flask, add 100 mL of DCM and 10 mL of pyridine, and stir in an ice bath at -20°C. After complete dissolution, add 8.5 mL of benzoyl chloride dropwise. After complete addition, gradually return to room temperature and stir overnight. Monitor the reaction by TLC. After the reaction is complete, add 1 mL of methanol to terminate the reaction. Use a 500 mL separatory funnel to add DCM and H2O for extraction. Add anhydrous Na2SO4 to the upper extract, dry it, concentrate it, and separate and purify it by column chromatography to obtain 9.5 g of light yellow product L6 with a yield of 87%.

[0090] 3. Take 5 g of L6 and place it in a 100 mL round-bottom flask. Under vacuum, add 50 mL of ultra-dry DCM under N2 protection and stir at -20°C for 20 min at a cryogenic pump. After complete dissolution, add 3.85 mL of DAST. After reacting for 30 min, return to room temperature and stir overnight. Monitor by TLC. After the reaction is complete, add 5 mL of methanol to terminate the reaction. Separate and purify by concentrating column chromatography to obtain 6.3 g of light yellow product L7 with a yield of 89%.

[0091] 4. Add 10 g of L7 to a 100 mL round-bottom flask and dissolve it in 60 mL of methanol. After complete dissolution, add sodium methoxide in small amounts and several times to adjust the pH until the pH is 9-10. After reacting for 4 hours, add cationic resin to terminate the reaction. Concentrate and perform column chromatography to obtain 7.2 g of product L8 with a yield of 97%.

[0092] 5. Place 7 g of L8 in a 100 mL round-bottom flask, add palladium carbon and dissolve in methanol solution. Vacuum the mixture, add 1 mL of dilute hydrochloric acid, introduce hydrogen and stir at room temperature for 24 hours. Monitor by TLC. After the reaction is complete, filter through diatomaceous earth to remove palladium carbon, wash with methanol, and evaporate thoroughly to obtain 5.2 g of L9, which is directly used in the next reaction with a yield of 83%.

[0093] 6. Add 5 g of L9 to a 100 mL round-bottom flask and dissolve it in 40 mL of pyridine. Once completely dissolved, add 5 mL of acetic anhydride and stir at room temperature overnight. After the reaction, add the reaction mixture to ice water and extract and dissolve the product with dichloromethane. Separate the organic layer, dry it over anhydrous Na2SO4, and concentrate it. After column chromatography, 6.3 g of L10 as a light yellow oil was obtained, with a yield of 92%.

[0094] 7. Place 5 g of L10 in a 100 mL round-bottom flask and dissolve it in 20 mL of 1,2-dichloroethane. Then add the Lewis acid and 5-chloro-1-pentanol. Insert a condenser, evacuate, and introduce nitrogen. Raise the temperature to 100°C and stir overnight. Check the reaction for completion using a plate. The reaction was concentrated to an oily liquid using a water pump. Column chromatography was used to obtain 4.5 g of L11 as a light yellow oil, yielding 83%.

[0095] 8. Dissolve 3 g of L11 in DMF, add 0.98 g of NaN3 using a paper trough, heat to 70°C and react overnight, monitor by TLC, wash with DCM / H2O, concentrate the organic layer, and separate by column chromatography to obtain 2.2 g of product L12, with a yield of 87%.

[0096] 9. Dissolve 0.5 g of L12 in 20 mL of methanol solution, add 0.1 g of Pd / C, introduce hydrogen, and react at room temperature for 2 hours. Monitor by TLC, filter through diatomaceous earth to remove Pd / C, wash with methanol, and evaporate thoroughly to obtain 0.48 g of L13, which is directly used in the next reaction with a yield of 90%.

[0097] Example 8 Synthesis of Compound L17

[0098]

[0099] Synthesis of compound L16

[0100] 5 g of L12 synthesized by the above method was added to a 100 mL round-bottom flask and dissolved in 30 mL of methanol. After complete dissolution, sodium methoxide was added in small amounts and multiple times to adjust the pH until the pH was 9-10. After reacting for 4 hours, cationic resin was added to terminate the reaction. The product L16 was obtained by concentration column chromatography with a yield of 94%.

[0101] Synthesis of compound L17

[0102] 0.5 g of L16 was dissolved in 20 mL of methanol solution, and 0.1 g of Pd / C was added. After hydrogen was introduced, the reaction was carried out at room temperature for 2 hours and monitored by TLC. The Pd / C was removed by filtration through diatomaceous earth, washed with methanol, and thoroughly evaporated to dryness to obtain 0.48 g of L17, which was directly used in the next reaction with a yield of 90%.

[0103] Example 9 Synthesis of Compound R3

[0104]

[0105] 190 mg of 1D was added to a 100 mL round-bottom flask, which was sealed and equipped with a nitrogen protection device. 20 mL of anhydrous DMF was added to the round-bottom flask and placed on a normal temperature stirrer to completely dissolve it. 400 mg of HATU was dissolved in 5 mL of anhydrous DMF for later use. The dissolved DMF solution was added to the round-bottom flask and reacted for 20 min. 300 mg of L4 was weighed and dissolved in anhydrous DMF. 0.3 mL of DIPEA was added to the dissolved L4 solution. After 20 min, a mixed solution of L4 and DIPEA was added to the round-bottom flask. The reaction was allowed to react for 24 hours. The reaction was monitored by TLC. After the reaction was completed, the solution was concentrated to obtain yellow oily droplets, which were separated by column chromatography to obtain a light yellow product R3 with a yield of 31%. 1 H NMR (300MHz, CDCl3) δ6.37(s,4H),5.47–5.19(t,2H),5.05(t,J=9.1Hz,2H),4.75(s,2H),4.29(d,J=8.4Hz,2H),4.12(d,J=10.9Hz,2 H),3.98–3.73(m,4H),3.48(s,4H),3.15(s,4H),2.55(d,J=42.0Hz,8H),2.08(s,6H),2.02(s,12H),1.96(s,6H),1.77–1.06(m,12H). 13C NMR (75MHz, CDCl3) δ173.41,171.53,170.90,170.73,169.67,169.56,100.79,72.73,71.58,69.67,69.0 4,62.27,54.41,39.51,32.13,29.71,29.33,28.88,23.38,22.70,20.82,20.71,19.13.HRMS:Calcd.for C 46 H 76 Cl2N6O 24 Pt(MH):1361.3855,found:1361.3861.

[0106] Example 10 Synthesis of Compound R4

[0107]

[0108] The target compound R4 is a white solid, and its preparation method is the same as that of the target compound R3, with a yield of 40%.

[0109] 1 H NMR(300MHz, CDCl3)δ6.35(d,J=9.9Hz,2H),5.38–5.24(m,2H),4.81(t,J=3.4Hz,2 H),4.58(t,J=9.3Hz,2H),4.48–4.36(m,2H),4.35–4.21(m,4H),4.00(d,J=6.5Hz, 2H),3.70–3.66(m,2H),3.53(s,2H),3.20(s,4H),2.54(d,J=40.1Hz,8H),2.12(d, J=5.3Hz,12H),1.99(s,6H),1.74–1.46(m,4H),1.42(d,J=6.6Hz,4H),1.26(s,4H). 13 C NMR (75MHz, CDCl3) δ181.98,173.32,171.36,170.88,170.76,97.10,88.18,85.71,71.36,71.11,68.4 2,67.30,67.00,65.59,62.16,50.60,39.55,32.22,30.57,29.69,29.35,23.52,23.13,20.90,20.83. 19 FNMR(376MHz,MeOD)δ-198.37.HRMS:Calcd.forC 42 H 70 Cl2F2N6O20 Pt(MH):1281.3556,found:1281.3552.

[0110] Example 11 Synthesis of Compound R5

[0111]

[0112] The target compound R5 is a white solid, and its preparation method is the same as that of the target compound R3, with a yield of 40%.

[0113] 1 H NMR (300MHz, CDCl3) δ6.66(s,2H),6.17(d,J=9.3Hz,2H),5.22(t,J=10.0Hz,2H),5.09(t,J=9.7Hz,2H),4.8 6(d,J=3.5Hz,2H),4.46–4.17(m,4H),4.10(d,J=10.5Hz,2H),3.96(d,J=8.9Hz,2H),3.68(d,J=9.0Hz,2H),3 .48(d,J=4.8Hz,2H),3.22(s,2H),2.96(s,2H),2.54(s,2H),2.42(m,12H),2.10(s,6H),2.02(d,J=3.8Hz,12 H),1.98(s,6H),,1.73–1.49(m,14H),1.45(d,J=6.6Hz,4H),1.36(d,J=12.1Hz,2H),1.27(d,J=11.1Hz,2H). 13 C NMR (75MHz, CDCl3) δ172.92,171.38,170.94,170.65,169.62,169.49,164.77,97.12,72.79,71.29,68.49,67.58,62.1 5,51.88,39.38,32.08,31.87,31.54,29.13,28.84,28.72,23.99,23.48,23.10,20.83,20.79,20.70.HRMS:Calcd.forC 54 H 84 N6O 28 Pt(MH):1458.4907,found:1458.4915.

[0114] Example 12 Synthesis of Compound R6

[0115]

[0116] The target compound R6 is a white solid, and its preparation method is the same as that of the target compound R3, with a yield of 40%.

[0117] 1 H NMR (300MHz, CDCl3) δ7.01 (s, 2H), 6.41 (d, J = 9.4Hz, 2H), 5.45–5.22 (m, 2H), 4.80 (s, 2H), 4 .62–4.36(m,4H),4.28(dt,J=10.9,5.5Hz,4H),4.00(s,2H),3.77–3.58(m,2H),3.46(s,6H ),3.19(s,4H),2.92(s,2H),2.51(d,J=37.1Hz,8H),2.10(d,J=6.0Hz,12H),1.98(s,6H),1 .61(s,4H),1.59–1.48(m,6H),1.43(d,J=6.4Hz,4H),1.36(d,J=16.3Hz,4H),1.26(s,4H). 13 C NMR (75MHz, CDCl3) δ172.90,171.22,170.77,170.75,164.86,97.09,88.21,85.74,71.36,71.12,68.42,67.27,6 6.96,62.14,62.00,50.43,39.35,32.07,31.91,31.59,29.67,29.08,28.82,23.96,23.49,23.01,20.86,20.78. 19 F NMR(376MHz,CDCl3)δ-196.90.HRMS:Calcd.for C 50 H 78 F2N6O 24 Pt(MH):1378.4609,found:1378.4600.

[0118] Example 13 Synthesis of Compound R11

[0119]

[0120] The target compound R11 is a white solid and is prepared by the same method as the target compound R3 with a yield of 40%. 1H NMR(300MHz,MeOD)δ5.19(t,J=10.5,10.1,3.2Hz,1H),5.03–4.94(m,1H),4.69–4. 52(m,1H),4.32–4.03(m,1H),3.98–3.75(m,2H),3.72–3.61(m,1H),3.61–3.43(m, 2H),3.15(s,2H),2.97–2.78(m,1H),2.73–2.37(m,5H),2.37–2.15(m,2H),2.05(s ,3H),1.99(s,3H),1.97(s,3H),1.90(s,3H),1.79–1.45(m,7H),1.43–1.20(m,5H). 13 C NMR(75MHz,MeOD)δ174.88,174.45,173.43,172.42,172.02,171.90,171.45,171.33,102.11,75.51,72.82,70.61,70.21,63.28,55.48,40.4 2,32.59,32.44,31.63,31.63,30.45,30.21,30.13,29.98,29.52,25.02,24.41,24.34,22.80,20.69,20.66,20.61,20.59.HRMS:Calcd.forC 31 H 49 ClN4O 16 Pt(MH):963.2402,found:963.2419.

[0121] Example 14 Synthesis of Compound R12

[0122]

[0123] The target compound R12 is a white solid and is prepared by the same method as the target compound R3 with a yield of 40%. 1H NMR(300MHz,MeOD)δ5.47–5.27(m,1H),4.82–4.76(m,1H),4.62–4.55(m,1H),4.50–4.36(m,1H),4 .31–4.17(m,2H),4.07(s,1H),3.80–3.66(m,1H),3.56–3.47(m,1H),3.29–3.14(m,2H),2.69(t,J= 6.6Hz,2H),2.51(t,J=8.9,6.4Hz,2H),2.30–2.19(m,2H),2.11(s,3H),2.08(s,3H),1.97(s,3H), 1.76–1.65(m,4H),1.63–1.55(m,2H),1.50–1.44(m,2H),1.42–1.38(m,4H),1.32(d,J=5.4Hz,4H). 13 C NMR(75MHz,MeOD)δ182.00,173.58,172.21,171.06,170.63,165.36,164.41,97.05,88.84,86.38,71.00,70.75,68.18,67.35, 67.04,62.19,54.48,42.44,39.00,31.65,31.37,31.21,31.04,30.75,29.33,29.03,28.78,28.63,23.64,23.24,21.10,19.30. 19 F NMR(376MHz,MeOD)δ-198.53.HRMS:Calcd.forC 29 H 46 ClFN4O 14 Pt(MH):923.2252,found:923.2275.

[0124] Example 15 Synthesis of Compound R13

[0125]

[0126] The target compound R13 is a white solid and is prepared by the same method as the target compound R3 with a yield of 40%. 1H NMR (300MHz, CDCl3) δ5.20(t,J=10.0Hz,1H),5.09(t,J=9.6Hz,1H),4.84(s,1H ),4.38–4.18(m,2H),4.09(d,J=11.1Hz,1H),3.95(d,J=9.7Hz,1H),3.75–3.62 (m,1H),3.43(s,1H),3.19(s,2H),2.52(s,4H),2.19(s,2H),2.09(s,3H),2.03 (s,3H),2.01(s,3H),1.97(s,3H),1.68–1.45(m,10H),1.31(d,J=29.2Hz,6H). 13 C NMR (75MHz, CDCl3) δ172.70,171.31,170.79,170.48,170.45,170.35,169.42,167.29,97.13,71.30,68.47,68.31,67.61,62.13,51. 90,39.34,32.73,31.91,31.89,31.21,29.68,29.51,29.30,28.87,27.21,25.84,23.50,23.14,22.67,20.77,20.66.HRMS:Calcd.for C 31 H 50 N4O 17 Pt(MH):944.2744,found:944.2745.

[0127] Example 16 Synthesis of Compound R14

[0128]

[0129] The target compound R14 is a white solid and is prepared by the same method as the target compound R3 with a yield of 40%. 1H NMR(300MHz, CDCl3) δ6.38(d,J=9.5Hz,1H),5.34–5.28(m,1H),4.79(d,J=3.4Hz,1 H),4.57(t,J=9.3Hz,1H),4.40(dd,J=10.6,8.1Hz,1H),4.31–4.22(m,2H),3.99(s ,1H),3.70–3.64(m,1H),3.18(t,J=7.0Hz,2H),2.97(s,2H),2.51(d,J=19.0Hz,4H ),2.19(s,3H),2.02(s,3H),1.92(s,13H),1.65–1.44(m,12H),1.31–1.09(m,4H). 13 C NMR (75MHz, CDCl3) δ182.17,173.06,171.12,170.68,170.62,165.57,165.41,97.11,88.17,85.70,71.34,71.09,68.43,67.27, 66.96,62.09,53.90,39.28,32.73,32.11,31.87,31.74,29.66,29.26,29.16,28.85,24.02,23.85,23.49,23.03,20.83,20.75. 19 F NMR(376MHz,CDCl3)δ-196.88.HRMS:Calcd.for C 29 H 47 FN4O 15 Pt(MH):904.2594,found:904.2601.

[0130] Example 17 Synthesis of Compound R16

[0131]

[0132] The target compound R16 is a white solid and is prepared by the same method as the target compound R3 with a yield of 40%. 1 H NMR(300MHz,MeOD)δ4.80(d,J=3.5Hz,1H),3.95–3.57(m,6H),3.48–3.36(m,2H),3.25–3.13(m,2H),2.64(t, J=6.1Hz,2H),2.51–2.28(m,6H),2.01(s,3H),1.73–1.49(m,10H),1.45–1.24(m,30H),0.92(t,J=6.6Hz,3H). 13C NMR(75MHz,MeOD)δ183.23,181.38,173.35,172.32,165.47,72.43,71.38,71.06,67.41,61.42,54.16,39.03,35.96,31.69 ,31.09,30.95,29.42,29.38,29.26,29.12,29.09,28.88,28.77,28.73,25.60,23.33,22.35,21.31,13.09.HRMS:Calcd.for C 41 H 74 N4O 15 Pt(MH):1056.4724,found:1056.4740.

[0133] Example 18 Synthesis of Compound R17

[0134]

[0135] The target compound R17 is a white solid and is prepared by the same method as the target compound R3 with a yield of 31%. 1 H NMR (300MHz, MeOD) δ4.85–4.77(m,1H),3.96(t,J=8.5Hz,2H),3.74(t,J=19.1,9.6Hz,4H),3.45(s,2H),3.19(d,J=5.1Hz,2H),2.6 4(t,J=6.3Hz,2H),2.51–2.25(m,6H),2.02(s,3H),1.67(d,J=10.6Hz,4H),1.60–1.49(m,6H),1.30(s,30H),0.92(t,J=6.6Hz,3H). 13 CNMR(75MHz,MeOD)δ184.17,182.33,174.45,174.36,173.44,173.35,166.39,9 7.95,92.24,89.85,71.10,70.78,70.49,70.24,68.72,62.91,62.26,61.42,54. 77,54.66,40.16,40.03,37.00,32.72,32.16,32.03,30.47,30.43,30.32,30.19,30.13,29.94,29.78,29.70,26.64,24.79,24.70,24.26,23.40,22.36,14.19. 19F NMR(376MHz,MeOD)δ-198.53.HRMS:Calcd.for C 41 H 73 FN4O 14 Pt(MH):1058.4680,found:1058.4692.

[0136] Example 19 Synthesis of Compound R18

[0137]

[0138] The target compound R18 is a light yellow solid. Its preparation method is the same as that of the target compound R3, and the yield is 39%. 1 H NMR (300MHz, MeOD) δ4.68 (d, J=3.4Hz, 1H), 3.86–3.66 (m, 2H), 3.59 (dt, J=11. 1,5.6Hz,2H),3.48(dd,J=6.5,4.2Hz,1H),3.43–3.33(m,1H),3.32–3.23(m,2H ),3.17–3.03(m,2H),2.64–2.46(m,2H),2.37m,2H),2.24(q,J=7.5Hz,2H),2. 02–1.83(m,3H),1.58–1.38(m,6H),1.30–1.08(m,26H),0.80(t,J=6.6Hz,3H). 13 C NMR (75MHz, MeOD) δ182.94,181.14,173.89,172.34,97.06,72.40,71.36,71.01,67.40,61.37,54.13,39.05,35. 65,31.70,31.33,29.48,29.43,29.30,29.17,29.14,29.10,28.96,28.72,22.36,17.94,13.09.HRMS:Calcd.for C 33 H 66 Cl2N4O 11 Pt(MH):959.3667,found:959.3661.

[0139] Example 20 Synthesis of Compound R19

[0140]

[0141] The target compound R19 is a light yellow solid. Its preparation method is the same as that of the target compound R3, and the yield is 36%. 1H NMR(300MHz,MeOD)1H NMR (300MHz, Methanol-d4) δ4.81(t,J=7.1,4.4Hz,1H),3.94(t,J=8.7Hz,2H),3.83–3.69(m,4H),3.55–3.38(m,2H),3.21(t,J=13.2,6.8Hz,2H),2.61( d,J=13.0,6.2Hz,2H),2.48(d,J=6.7Hz,2H),2.37(t,J=7.6Hz,2H),2.02(d, J=2.1Hz,3H),1.71–1.53(m,6H),1.40–1.29(m,26H),0.92(t,J=6.7Hz,3H). 13 C NMR(75MHz,MeOD)δ182.93,181.14,173.86,172.31,96.95,91.25,88.87,70.09,69.77,69.45,69.20,67.71,60.40,53.75,53.64,39 .03,35.66,31.69,31.38,31.23,29.42,29.38,29.29,29.16,29.09,28.97,28.78,28.70,25.64,23.24,22.35,21.28,17.97,13.08. 19 F NMR(376MHz,MeOD)δ-198.31.HRMS:Calcd.for C 33 H 65 Cl2FN4O 10 Pt(MH):961.3624,found:961.3630.

[0142] Test Example 1: In vitro antitumor activity test

[0143] The MTT method was used to test the in vitro antitumor activity of the tetravalent platinum aminohexose complexes R3, R4, R5, R6, R11, R12, R13, R14, R16, R17, R18 and R19 synthesized in the above examples in eight human cancer cell lines including HepG2, Huh-7, MDA-MB-231, MCF-7, A549, A549cisR, HCT-116, and HT29, as well as four prostate cancer cells including androgen-independent prostate cancer cells DU145 and PC3, and androgen-dependent prostate cancer cells LNCap and VCap. The test results are shown in Tables 1 and 2. Positive drugs cisplatin and oxaliplatin were selected as controls.

[0144] Table 1 In vitro antitumor activity of tetravalent platinum hexosamine complexes with potential as oral drugs [e]

[0145]

[0146] Note: [a]The RF(resistance factor) is defined as the IC 50 value in PC3divided by the IC 50 value in LNCap.[b]The RF(resistance factor)is defined as the IC 50 value in DU145 divided by the IC 50 value in LNCap.[c]FI(foldincrease)is defined as IC 50 (cisplatin) / IC 50 (R19).[d]FI(fold increase)isdefined as IC 50 (oxaliplatin) / IC 50 (R19).[e]An average of three measurements.ND=not determined.

[0147] Table 2 In vitro antitumor activity of tetravalent platinum hexosamine complexes with potential as oral drugs [d]

[0148]

[0149]

[0150] Note: [a]The RF(resistance factor)is defined as the IC50 value inA549cisR divided by the IC50 value in A549.[b]FI(fold increase)is defined asIC 50 (cisplatin) / IC 50 (R19).[c]FI(fold increase)is defined as IC 50 (oxaliplatin) / IC50 (R19).An average of three measurements.[d]An average of three measurements.ND=not determined.

[0151] The experimental results in Tables 1 and 2 demonstrate that the tetravalent platinum hexosamine complexes of the present invention exhibited superior overall antitumor activity compared to the positive control drugs cisplatin and oxaliplatin across all cancer cells tested. The overall activity of the symmetrically bifunctionally substituted tetravalent platinum hexosamine complexes R3, R4, R5, and R6 was nearly comparable to that of cisplatin and oxaliplatin, but R3 exhibited slightly superior activity to both cisplatin and oxaliplatin in prostate cancer cells.

[0152] Based on the asymmetric bifunctional tetravalent platinum aminohexose complexes R11, R12, R13 and R14, R11 and R12 have good selectivity for toxicity against liver cancer cells Huh-7 and prostate cancer cells PC3 and DU145. In particular, the tetravalent platinum aminohexose complex R12 substituted with fluoroacetylhexose has better activity in prostate cancer cells than R11 with the same tetravalent platinum core, indicating that the introduction of fluoroacetylhexose can increase the cytotoxicity of tetravalent platinum.

[0153] The overall activity of the asymmetric bifunctionally substituted tetravalent platinum aminohexose complexes R16, R17, R18, and R19 was significantly superior to that of the positive drugs cisplatin and oxaliplatin, especially the IC of R19 in prostate cancer cells. 50 It is worth noting that the RF value of R19 (RF=0.74) is much lower than that of cisplatin (RF=1.64), and the IC50 value of R19 in PC3 and DU145 cells reaches the nanomolar level, indicating that it has high selectivity for androgen-independent prostate cancer cells and has the potential to reverse androgen-independent prostate cancer cells, showing that R19, a quadrivalent platinum anti-tumor drug, may have good prospects in the treatment of prostate cancer.

[0154] Experimental Example 2: Effect of R19 on the Migration Ability of Prostate Cancer Cells

[0155] 1. Wound healing experiment

[0156] In order to verify the inhibitory effect of R19 on the migration ability of prostate cancer cells, the wound healing assay was used to detect the effect of R19 on the migration ability of PC3 prostate cancer cells. Figure 1 As shown, compared with the positive control drugs cisplatin and oxaliplatin, with the increase of drug concentration, the inhibitory effect of R19 on prostate cancer cell migration was significantly stronger than cisplatin and oxaliplatin.

[0157] 2. Transwell chamber experiment

[0158] In order to further verify the inhibitory effect of R19 on the migration ability of prostate cancer cells, the Transwell assay was used to detect the effect of R19 on the migration ability of PC3 prostate cancer cells. Figure 2 As shown, compared with the positive control drug cisplatin, as the drug concentration increases, the inhibitory effect of compound R19 on prostate cancer cell migration is significantly stronger than cisplatin and oxaliplatin. Experimental Example 3: R19 induces apoptosis of prostate cancer cells

[0159] In order to study the relationship between the anticancer activity of tetravalent platinum hexosamine complex R19 and cell apoptosis, the present invention uses the Annexin V / PI double staining method to test the apoptosis of R19 and cisplatin in PC3 cells. PC3 cells were incubated with R19 at concentrations of 5μM and 10μM and cisplatin 10μM for 24 hours, and the cells were collected. Flow cytometry was then used to analyze the apoptosis induced by the compounds. The results are as follows: Figure 3 The apoptotic ratio of R19 was positively correlated with its concentration. That is, at a concentration of 10 μM, the ratio of early apoptotic cells was 15.13%, and the ratio of late apoptotic cells was 8.53%, both higher than 9.02% and 2.71% of cisplatin, respectively. This indicates that R19 has stronger in vitro cytotoxicity and apoptosis-inducing abilities than cisplatin.

[0160] Experimental Example 4: R19 and Gem combination drug experiment

[0161] In this study, the in vitro antitumor activity of the combination of R19 and Gem (gemcitabine) is shown in Tables 3 and 4. The MTT assay was used to test the resistance of compound R19 and Gem to two human cancer cell lines, DU145 and PC3, at different concentrations. The treatment time was 24 hours. By combining R19 and Gem, this study utilized the synergistic effect of the two drugs together, achieving an objective therapeutic level at low concentrations and exhibiting significant cytotoxicity.

[0162] Table 3 In vitro antitumor activity of R19 combined with Gem

[0163]

[0164] As can be seen from Table 3, in DU145, when R19 (0.5 μM) + Gem (2 μM) = 1:4, an objective therapeutic level can still be achieved, with significant cytotoxicity.

[0165] Table 4 In vitro antitumor activity of R19 combined with Gem

[0166]

[0167] As can be seen from Table 4, in PC3, when R19 (1 μM) + 5-Fu (8 μM) = 1:8, FA = 0.74, which can still reach an objective therapeutic level and has significant cytotoxicity.

[0168] Experimental Example 5: Platinum Uptake by R19 in Prostate Cancer Cells

[0169] The biological activity of a drug is closely related to its accumulation in cancer cells. To study the platinum accumulation of R19 in cells, the present invention added a 10 μM compound to cells and cultured them for 8 hours. The cells were then collected and the corresponding kits were used to separate the organelles and nuclear DNA. After acidification with nitric acid, the platinum content in the cell DNA and several organelles was determined by ICP-MS. The results are shown in Figure 2. Figure 4 As shown, the platinum accumulation of R19 in DU145 and PC3 cells was 6 times higher than that of cisplatin and oxaliplatin. 5 The DNA platinum element levels of DU145 and PC3 in the individual cells were 37.65ng Pt and 47.58ng Pt, respectively, which were significantly higher than cisplatin and oxaliplatin by 2-3 times. At the same time, the results of the present invention found that R19 was distributed in a considerable amount in the endoplasmic reticulum, lysosomes and mitochondria, and R19 had a higher uptake in the endoplasmic reticulum, indicating that the reason why R19 overcomes androgen-independent prostate cancer is not only because it has a higher binding rate with DNA, but also because the drugs taken in by other organelles also play a role. In addition, in the presence of inhibitors, the cell's uptake of drugs was significantly reduced, which once again proves that the large-scale accumulation of R19 in cells may occur after the glucose transporter crosses the membrane.

[0170] Experimental Example 6: In vivo anti-tumor activity and anti-metastasis effect of R19

[0171] 1. R19 inhibits the growth of orthotopic solid tumors in mice bearing prostate cancer cells

[0172] In vitro experiments have shown that R19 has a good killing effect on prostate cancer cells. In order to verify its efficacy in vivo, the present invention tested the in vivo anti-tumor activity of R19 through tumor-bearing experiments. RM-1 prostate cancer cells were used to implant tumors in the axilla of C57BL / 6 mice to create a solid tumor model. After one week, the average tumor volume of the mice was 80-100 mm 3The mice were randomly divided into the Control group, L10 group (10 mg / kg), oxaliplatin group (2.46 mg Pt / kg), cisplatin group (1.95 mg Pt / kg), cisplatin + L10 group (1.95 mg Pt / kg + 5 mg / kg L10), R19 group (0.93 mg Pt / kg) and R19 group (1.86 mg Pt / kg). The drug was administered intraperitoneally once every two days for a total of five times. The changes in the weight and tumor volume of the mice were recorded every other day. After the last dose, the mice were observed for 5 days. Then, the eye blood was collected from the mice, and the mice were killed by dislocation. The tumors and organ tissues were removed, photographed and the weights were recorded. The results are as follows Figure 5 shown.

[0173] Tumor growth curve ( Figure 5 B) shows that the average tumor volume of the control group on day 13 was 1800 mm 3 , the volume of R19 (1.62 mg Pt / kg) group was 520 mm 3 , which was 75% and 54% lower than that of negative group and oxaliplatin group. Figure 5 C) showed that after treatment with R19 (1.62 mg Pt / kg), the tumor inhibition rate was as high as 80.4%, and the inhibition rate of the R19 (0.81 mg Pt / kg) group was 70.2%, both of which were better than cisplatin (66.9%) and oxaliplatin (53.2%). Figure 5 A) showed that the body weight of mice decreased by 15% after cisplatin treatment, while the body weight of mice after R19 administration was 103% to 106% of that before administration. In addition, the toxicology experiment ( Figure 5 D) shows that R19 has a slight inhibitory effect on the spleen of mice after administration, with no significant effect on other organs. The results show that R19 can effectively inhibit tumor growth in vivo and has little toxicity to mice.

[0174] 2. Distribution of platinum in R19 animal tissues

[0175] In vitro experiments showed that R19 showed good targeting to prostate cancer cells, and the uptake and DNA binding in tumor cells were both high. To verify whether R19 could also target tumor tissues in vivo, the present invention tested the distribution of R19 in the heart, liver, spleen, lung, kidney, tumor tissue and blood. Equal amounts of tissues from an orthotopic solid tumor model of prostate cancer mice were taken 5 days after administration, and their distribution in animal tissues was detected by ICP-MS after acidification with nitric acid. The results are as follows: Figure 6 A and Figure 6 As shown in B, the platinum accumulation of R19 in tumor tissues of mice is twice that of cisplatin.

[0176] In addition, it was found that its content in the liver and kidneys was also very high. In order to evaluate whether it has toxic damage to the liver and kidneys, after the animals were finished, the present invention used corresponding detection kits to measure the blood urea nitrogen (BUN) and creatinine (SCr) content and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in the mice. The results are as follows Figure 6 The levels of BUN, SCr, ALT, and AST in mice treated with cisplatin and oxaliplatin were significantly increased compared with the negative control group, while R19 had almost no effect, indicating that R19 did not cause damage to the liver and kidneys of mice compared with cisplatin and oxaliplatin.

[0177] 3. R19 inhibits lung metastasis in mice bearing prostate cancer cells

[0178] In vitro experiments have shown that R19 has a higher inhibitory effect on the migration of prostate cancer cells. In order to further verify this result, the present invention studied the in vivo anti-tumor metastasis activity of R19. RM-1 prostate cancer cells were implanted into the tail vein of mice to create a lung metastasis mouse model. After seven days, the mice were randomly divided into a control group, an L10 group (10 mg / kg), an oxaliplatin group (2.46 mg Pt / kg), a cisplatin group (1.95 mg Pt / kg), a cisplatin + L10 group (1.95 mg Pt / kg + 5 mg / kg L10), an R19 group (0.93 mg Pt / kg) and an R19 group (1.86 mg Pt / kg). The mice were administered five times every other day by intraperitoneal injection and observed for 5 days after administration.

[0179] The weight of mice was recorded every day. After 5 days of observation, mice in each group were killed by dislocation. The organs were removed, weighed and recorded. Figure 7 As shown. The results showed that the number of lung nodules in the experimental group was significantly reduced compared with the negative control group. After treatment with R19 (0.81 mg Pt / kg), the inhibition rate of lung nodules was 66.37%, and the inhibition rate of R19 (1.62 mg Pt / kg) was 82.01%, both better than the cisplatin group (36.87%) and the oxaliplatin group (17.46%), indicating that R19 inhibited the lung metastasis of mouse prostate cancer to a certain extent. In addition, in the in vivo toxicology experiment, the comparison between the experimental group and the control group showed that there was no significant change in the organ index. Then, HE staining of the lung tissue of the mouse was performed and it was found that ( Figure 7 E), Lung injury was significantly reduced after R19 treatment compared with the positive control drug.

[0180] Experimental Example 7: R19 reduces the expression of O-GlcNAc glycosylation of prostate cancer cell proteins

[0181] In order to study the effect of tetravalent platinum hexosamine complex R19 on O-GlcNAc glycosylation of prostate cancer cell proteins, different concentrations of R19 and positive drugs L10 and cisplatin were used to act on prostate cancer PC3 cells for 24 hours, and Western blotting was used to analyze the O-GlcNAc glycosylation level of proteins in the cells. Figure 8 As shown in the figure, with the increase of R19 concentration, the degree of protein O-GlcNAc glycosylation in PC3 cells decreased significantly in a concentration-dependent manner. The opposite was true for cisplatin. After cisplatin treatment of PC3 prostate cancer cells, the protein O-GlcNAc level was significantly higher than the protein expression level of R19.

[0182] Experimental Example 8: R19 reduces the content of UDP-GlcNAc in prostate cancer cells

[0183] After cellular stress, the degree of protein O-GlcNAc glycosylation changes. This change in O-GlcNAc glycosylation is primarily regulated by OGA and OGT. In addition, the donor UDP-GlcNAc may also contribute to the glycosylation change. The present invention measured the effects of R19 and cisplatin on cellular UDP-GlcNAc concentration and the effect of UDP-GlcNAc concentration on R19's inhibition of anti-tumor cell proliferation.

[0184] The results of Experimental Example 7 showed that R19 significantly reduced O-GlcNAc glycosylation in prostate cancer PC3 cells. R19 may have reduced the content of UDP-GlcNAc in cells through the hexosamine biosynthesis pathway, thereby reducing O-GlcNAc glycosylation in cells. Therefore, Experimental Example 8 set up a blank group, a cisplatin group, and an R19 group. The corresponding drug was added at a concentration of 10 μM during cell culture. After 24 hours, the cells were collected and treated to obtain samples containing UDP-GlcNAc. The samples were quantitatively analyzed by HPLC. The results are shown in Figure 2. Figure 9 As shown in the figure, compared with the blank control group, the UDP-GlcNAc content in the cisplatin group was significantly increased, while the UDP-GlcNAc concentration in the R19 group was significantly decreased, while the concentration of 4F-UDP-GlcNAc was significantly increased. The experimental results show that R19 induces a decrease in the intracellular UDP-GlcNAc content in PC3 cells, thereby reducing the degree of intracellular protein O-GlcNAc glycosylation.

[0185] In addition, the present invention also conducted related experiments such as transporter inhibitor experiments, R19 in vivo anti-tumor immunity evaluation experiments, R19-induced prostate cancer cell apoptosis experiments, R19 expression experiments on phosphorylated histone H2A.X, R19 expression experiments on apoptosis-related proteins, R19 increased p53 stability experiments, and R19-induced prostate cancer cell cycle arrest experiments, which are not listed here one by one. The above experiments lead to the conclusion that the cytotoxicity of the compound R19 synthesized in the present invention relies on the glucose transporter highly expressed on the cell membrane surface to exert its effect across the membrane; R19 enhances the anti-tumor immune effect by reducing the proportion of T-reg cells with immunosuppressive function in mice; R19's in vitro cytotoxicity and ability to induce cell apoptosis are stronger than cisplatin; R19 significantly upregulates the expression of p53, Caspase-3 and phospho-histone H2A-X, enhances DNA damage, and induces apoptosis of prostate cancer cells; R19 increases the stability of p53 by upregulating the protein expression of TXNIP and SGT1, thereby activating the p53 pathway, inducing cell apoptosis and blocking the cell cycle; R19 induces prostate cancer PC3 cells mainly in the G1 / S phase.

[0186] In summary, the present invention modifies platinum drugs with glycosylation using fluoroacetylhexosamine, a compound that interferes with glucose metabolism. This not only achieves platinum drug targeting, but also regulates HBP to effectively reduce the content of UDP-GlcNAc in prostate cancer cells, thereby reducing the presence of highly branched N-glycan on the cell membrane surface, thereby targeting CRPC and enhancing its sensitivity to platinum drugs. The present invention also reaches the following conclusions:

[0187] 1. The present invention synthesized several tetravalent platinum hexosamine complexes with oral potential. Anti-tumor testing revealed that these compounds exhibited excellent selectivity for prostate cancer cells, with R19 in particular demonstrating nanomolar cytotoxicity against androgen-independent prostate cancer cells. 2. R19 exhibited high cellular uptake and DNA binding in prostate cancer cells, and its high platinum accumulation and cytotoxicity were closely related to sugar transporters on the tumor cell membrane surface. 3. R19 exhibited excellent anti-tumor activity and anti-tumor metastasis capabilities in mice with prostate cancer, with virtually no biotoxicity. 4. R19 can reduce the level of UDP-GlcNAc in prostate cancer cells, thereby reducing protein O-GlcNAcylation in prostate cancer cells and inhibiting highly branched N-glycans on the cell membrane surface. 5. R19 increased the expression of key enzymes in the HBP pathway and elevated the level of 4F-UDP-GlcNAc, playing an important role in the fight against prostate cancer.

Claims

1. A tetravalent platinum hexosamine complex targeting CRPC based on regulating O-GlcNAc glycosylation, characterized in that: Specifically, the Pt(IV) aminohexose complexes R3-R6 modified with a symmetrical bifunctional glycosyl structure, the Pt(IV) aminohexose complexes R11-R14 modified with an asymmetrical monofunctional glycosyl structure, or the Pt(IV) aminohexose complexes R16-R19 modified with an asymmetrical monofunctional glycosyl structure; The symmetrical bifunctional glycosyl structure-modified Pt(IV) aminohexose complex R3-R6 has the following structural formula: The asymmetric monofunctional glycosyl structure-modified Pt(IV) aminohexose complex R11-R14 has the following structural formula: The asymmetric monofunctional glycosyl structure-modified Pt(IV) aminohexose complex R16-R19 has the following structural formula:

2. The method for preparing the tetravalent platinum hexosamine complex according to claim 1, characterized in that: The method comprises the following steps: using an amino hexose group and a tetravalent platinum skeleton structure to undergo a HATU condensation reaction to obtain target compounds R3-R6, R11-R14 or R16-R19; The amino hexose group is L4, L13, L15 or L17; The tetravalent platinum skeleton structure is 1D, 2D, 2F, 2G, 1C or 2C; Compounds 1C, 1D, 2C, 2D, 2F, and 2G are synthesized using cisplatin or oxaliplatin as raw materials, and their structures are shown below: The structural formulas of the hexosamine groups L4, L13, L15, and L17 are as follows:

3. The preparation method according to claim 2, characterized in that The mass ratio of the amino hexose group to the tetravalent platinum skeleton structure is (1.2-1.8):

1.

4. Use of the tetravalent platinum hexosamine complex according to claim 1, or the tetravalent platinum hexosamine complex according to claim 1 in combination with cisplatin, oxaliplatin or gemcitabine in the preparation of an anti-tumor drug, characterized in that: The tumor refers to human breast cancer, human lung adenocarcinoma, human liver cancer, human colon cancer, cisplatin-resistant human lung adenocarcinoma or human prostate cancer.

5. Use of the tetravalent platinum hexosamine complex according to claim 1 alone, or in combination with gemcitabine, cisplatin or oxaliplatin, in the preparation of a drug for inhibiting tumor cell proliferation, inducing pyroptosis or promoting apoptosis, characterized in that: The tumor cells are human liver cancer cells Huh-7 and HepG2, human breast cancer cells MDA-MB-231 and MCF-7, human prostate cancer cells DU145, PC3, LNCap, VCap and RM-1, human lung cancer cells A549, or human colon cancer cells HCT-116 and HT-29.

Images