Transplatinum and methods of making and using the same

Abstract

The present application relates to the field of biological medicine, and particularly relates to a transplatinum, a preparation method and application thereof. The present application provides a transplatinum having a structure of formula I; formula I; wherein R1-R5 are independently selected from hydrogen, halogen, CHO, OH, NH2 or CH3. The transplatinum provided by the present application is a kind of bivalent platinum compound with cell pyroptosis induction ability, which has a trans structure, including two chloro ligands in a trans position, one amino ligand in a trans position and one nitrogen-containing heterocyclic ligand in a trans position, and the nitrogen-containing heterocyclic ligand is designed as a pyridine or derivative ligand. Due to the existence of the trans structure and the planar ligand, the transplatinum adopts a different DNA cross-linking mode from classic platinum drugs such as cisplatin, has cell pyroptosis induction ability, can induce immunogenic death of tumor cells, and activate anti-tumor immune response. As a platinum drug, the transplatinum has a different cancer cell killing mode and excellent cancer cell killing ability from cisplatin.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a trans-platinum, its preparation method, and its application. Background Technology

[0002] Chemotherapy is one of the most widely used cancer treatments. Platinum-based drugs are platinum-based metal drugs with anti-cancer effects. Classic platinum-based anti-tumor drugs, represented by cisplatin (cis-dichlorodiammineplatin), carboplatin, and oxaliplatin, are the mainstay of chemotherapy. Currently, more than 50% of cancer patients receive platinum-based drug treatment. Their main mechanism of action is to cross-link the DNA of tumor cells, thereby leading to apoptosis, which is the autonomous and orderly cell death controlled by genes to maintain homeostasis. Although they have achieved some success, classic cis-based platinum-based drugs still face some drawbacks, such as drug resistance, poor efficacy in certain cancer types, and inability to inhibit cancer metastasis and recurrence.

[0003] Developing platinum-based drugs with different killing mechanisms is an effective way to solve the above problems. Unlike apoptosis, pyroptosis is a programmed cell death mechanism with an "inflammatory effect," characterized by its dependence on caspase-1 and the release of a large number of pro-inflammatory factors. As a novel pro-inflammatory programmed cell death mechanism, pyroptosis differs significantly from apoptosis in terms of cellular morphological changes and its mechanism of occurrence. Specifically, during pyroptosis, tumor cells release inflammatory factors (IL-1β and IL-18), thereby killing tumor cells while activating an anti-tumor immune response. Furthermore, pyroptosis is an immunogenic cell death mechanism. Immunogenic cell death is the process by which tumor cells, upon being stimulated by external factors, die and transform from non-immunogenic to immunogenic, mediating an anti-tumor immune response in the body, which can further activate anti-tumor immune action. Therefore, platinum-based drugs with pyroptosis-inducing capabilities are expected to have broader spectrum, enhanced efficacy, and greater ability to inhibit cancer metastasis and recurrence.

[0004] Most classic platinum-based drugs with the cis configuration are apoptosis inducers. Therefore, developing platinum-based drugs with pyroptosis-inducing capabilities requires significant modifications to the cis configuration, which is challenging and the direction of such modifications is unclear. Trans-platinum (trans-dichlorodiammineplatinum) with the trans configuration can also be used as a platinum-based drug. It has a different DNA-binding mechanism than classic platinum drugs like cisplatin and can still exert its effects in some cisplatin-resistant cells. However, there are currently no clear technical guidelines on how trans-platinum molecules achieve pyroptosis-inducing capabilities. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a trans platinum, its preparation method and application. The trans platinum provided by the present invention has the ability to induce pyroptosis, which can cause immunogenic death of tumor cells and activate anti-tumor immune response while killing tumor cells.

[0006] This invention provides a trans-platinum having a structure of Formula I;

[0007] Formula I;

[0008] The R1 to R5 are independently selected from hydrogen, halogen, CHO, OH, NH2 or CH3.

[0009] The halogen is preferably F, Cl, Br or I.

[0010] To address the problems of drug resistance, poor efficacy in certain cancer types, and inability to reduce cancer cell metastasis and recurrence associated with classic platinum drugs, this invention designs the aforementioned trans-platinum. The trans-platinum provided by this invention is a divalent platinum compound with pyroptosis-inducing ability. It has a trans structure comprising two chloride ligands in the trans position, one amino ligand in the trans position, and one nitrogen-containing heterocyclic ligand in the trans position. The nitrogen-containing heterocyclic ligand is designed as a pyridine or its derivative ligand. Due to its trans structure and the presence of planar ligands, the trans-platinum provided by this invention adopts a different DNA cross-linking mechanism than classic platinum drugs such as cisplatin, exhibiting pyroptosis-inducing ability. It can induce immunogenic death of tumor cells, activate anti-tumor immune responses, and has a good cancer cell killing effect.

[0011] Preferably, the trans-platinum has a structure of formula I-1;

[0012] Formula I-1;

[0013] The R3 is selected from hydrogen, halogen, CHO, OH, NH2 or CH3, wherein the halogen is preferably F, Cl, Br or I.

[0014] More preferably, the trans-platinum has a structure of formula I-1, wherein R3 is selected from H, CH3, Br or I.

[0015] The trans-platinum with the structure of Formula I-1 provided by this invention further modifies the DNA binding ability of the trans-platinum molecule by introducing groups with different spatial configurations, hydrophilicity / hydrophobicity, halogen bond and hydrogen bond giving capabilities at the 4 position of the pyridine ligand, ultimately causing pyroptosis and further causing immunogenic death of tumor cells, thereby activating an anti-tumor immune response while killing tumor cells.

[0016] The present invention also provides a method for preparing trans-platinum according to any of the above technical solutions, comprising the following steps:

[0017] Hydrochloric acid, cisplatin, and a compound with the structure shown in formula a are reacted to yield trans-platinum;

[0018] Formula a;

[0019] R1 to R5 are the same as described above, and will not be repeated here.

[0020] Specifically, the method for preparing trans-platinum according to any of the above technical solutions includes the following steps:

[0021] S1) Heat-treat cisplatin and the compound with the structure shown in formula a;

[0022] S2) React hydrochloric acid with the reaction product obtained in step S1) to obtain trans-platinum.

[0023] This invention first involves heat-treating cisplatin and the compound with the structure shown in Formula a. Specifically, this invention first involves hydrothermal treatment of cisplatin and the compound with the structure shown in Formula a. More specifically, this invention first involves mixing and dispersing cisplatin and the compound with the structure shown in Formula a in water, and then heating and stirring to perform hydrothermal treatment. The molar ratio of cisplatin and the compound with the structure shown in Formula a is 1:(2~10), preferably 1:(2.5~4). The heat treatment temperature for cisplatin and the compound with the structure shown in Formula a is 50℃~100℃, preferably 70℃~90℃; the time is 2 h~48 h, preferably 12 h~48 h.

[0024] This invention involves heat-treating cisplatin and a compound with the structure shown in formula a, followed by reacting hydrochloric acid with the reaction product obtained in step S1) to obtain trans-platinum. Specifically, hydrochloric acid is added to the reaction product obtained in step S1), and the mixture is heated and stirred to carry out the reaction, followed by purification to obtain trans-platinum. The purification method of this invention includes recrystallization, precipitation, high-performance liquid chromatography separation, or water washing. The mass concentration of the hydrochloric acid used in this invention is 36%~38%; based on 1 mg of the cisplatin obtained in step S1), the amount of hydrochloric acid used is 5 μL~50 μL, preferably 10 μL~40 μL. The reaction temperature of the hydrochloric acid and the reaction product obtained in step S1) is 50℃~100℃, preferably 70℃~90℃; the reaction time is 2 h~72 h, preferably 24 h~72 h.

[0025] This invention also provides the application of the trans-platinum described in any of the above-described technical solutions, or the trans-platinum obtained by the preparation method described in any of the above-described technical solutions, in the preparation of antitumor drugs. This invention also provides an antitumor method using the trans-platinum described in any of the above-described technical solutions, or the trans-platinum obtained by the preparation method described in any of the above-described technical solutions. The trans-platinum provided by this invention has the ability to induce pyroptosis, immunogenic cell death, and activate antitumor immune responses. As a platinum drug, it exhibits a cancer cell killing mechanism different from cisplatin and superior cancer cell killing ability.

[0026] This invention provides a trans-platinum, its preparation method, and its application. This invention provides a trans-platinum having the structure of Formula I; Formula I; wherein R1~R5 are independently selected from hydrogen, halogen, CHO, OH, NH2 or CH3. The trans-platinum provided by this invention adopts a DNA cross-linking mode different from classic platinum drugs such as cisplatin, which can induce pyroptosis and further cause immunogenic death of tumor cells. While killing tumor cells, it activates an anti-tumor immune response. As a platinum drug, it has a cancer cell killing mode different from cisplatin and excellent cancer cell killing ability. Attached Figure Description

[0027] Figure 1 The 1H NMR spectrum of trans-[PtCl2(Py-H)(NH3)] obtained in Example 1;

[0028] Figure 2 The 1H NMR spectrum of trans-[PtCl2(Py-CHO)(NH3)] obtained in Example 2;

[0029] Figure 3 The 1H NMR spectrum of trans-[PtCl2(Py-OH)(NH3)] obtained in Example 3;

[0030] Figure 4 The 1H NMR spectrum of trans-[PtCl2(Py-CH3)(NH3)] obtained in Example 5;

[0031] Figure 5 The 1H NMR spectrum of trans-[PtCl2(Py-Br)(NH3)] obtained in Example 8;

[0032] Figure 6 The 1H NMR spectrum of trans-[PtCl2(Py-I)(NH3)] obtained in Example 9;

[0033] Figure 7 The figures show the in vitro cytotoxicity test results of the products of Examples 1-9 and cisplatin and antiplatin;

[0034] Figure 8 The figure shows the results of the investigation into the cell death mechanism of trans-[PtCl2(Py-I)(NH3)].

[0035] Figure 9 A graph showing the number of cells with exposed calreticulin on their surface after different drug treatments, quantified by trans-[PtCl2(Py-I)(NH3)] flow cytometry.

[0036] Figure 10 The figure shows the in vivo tumor suppression experiment results of trans-[PtCl2(Py-I)(NH3)]. Detailed Implementation

[0037] This invention discloses a trans-platinum, its preparation method, and its applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0038] The present invention will be further described below with reference to the embodiments:

[0039] Example 1

[0040] Cisplatin (1.00 g, 3.34 mmol), pyridine (Py-H) (0.61 g, 7.69 mmol), and 20 mL of deionized water were added to a round-bottom flask and stirred at 80 °C for 24 h until the solution became clear. Then, 4 mL of concentrated HCl (36%) was added to the solution, and the mixture was stirred at 80 °C for 72 h. After the reaction was complete, the precipitate was collected by filtration and washed three times with deionized water. The precipitate was then dried under vacuum to obtain the yellow product trans-[PtCl2(Py-H)(NH3)]. Figure 1 As shown, Figure 1 The image shows the hydrogen NMR spectrum of trans-[PtCl2(Py-H)(NH3)] obtained in Example 1.

[0041] Example 2

[0042] Cisplatin (50.0 mg, 0.167 mmol), 4-aldehyde pyridine (Py-CHO) (44.8 mg, 0.418 mmol), and 5 mL of deionized water were added to a round-bottom flask and stirred at 70 °C for 24 h until the solution became clear. Then, 0.8 mL of concentrated HCl (36%) was added to the solution, and the mixture was stirred at 90 °C for 72 h. After the reaction was complete, the precipitate was collected by filtration and washed three times each with deionized water, anhydrous ethanol, and diethyl ether. The precipitate was then dried under vacuum to obtain a yellow trans-[PtCl2(Py-CHO)(NH3)]. Figure 2 As shown, Figure 2 The image shows the hydrogen nuclear magnetic resonance spectrum of trans-[PtCl2(Py-CHO)(NH3)] obtained in Example 2.

[0043] Example 3

[0044] Cisplatin (89.7 mg, 0.3 mmol), 4-hydroxypyridine (Py-OH) (42.8 mg, 0.66 mmol), and 5 mL of deionized water were added to a round-bottom flask and stirred at 85 °C for 24 h until the solution became clear. Then, 1 mL of concentrated HCl (36%) was added to the solution, and the mixture was stirred at 90 °C for 72 h. After the reaction was complete, the precipitate was removed by filtration, the reaction solution was concentrated, and the product was obtained by recrystallization. The product was then dried under vacuum to obtain a yellow trans-[PtCl2(Py-OH)(NH3)]. Figure 3 As shown, Figure 3 The image shows the hydrogen NMR spectrum of trans-[PtCl2(Py-OH)(NH3)] obtained in Example 3.

[0045] Example 4

[0046] Cisplatin (89.7 mg, 0.3 mmol), 4-aminopyridine (Py-NH2) (62.0 mg, 7.69 mmol), and 5 mL of deionized water were added to a round-bottom flask and stirred at 70 °C for 24 h until the solution became clear. After the reaction was complete, a small amount of black precipitate appeared. The filtrate was centrifuged and used for the next reaction. Subsequently, 1 mL of concentrated HCl (36%) was added to the solution, and the mixture was stirred at 80 °C for 72 h. After the reaction was complete, the precipitate was removed by filtration, the reaction solution was concentrated, and the product was obtained by recrystallization. The product was then dried under vacuum to obtain a yellow trans-[PtCl2(Py-NH2)(NH3)].

[0047] Example 5

[0048] Cisplatin (89.7 mg, 0.3 mmol), 4-methylpyridine (Py-CH3) (69.8 mg, 0.75 mmol), and 5 mL of deionized water were added to a round-bottom flask and stirred at 70 °C for 24 h until the solution became clear. Then, 1 mL of concentrated HCl (36%) was added to the solution, and the mixture was stirred at 80 °C for 72 h. After the reaction was complete, the precipitate was collected by filtration and washed three times each with deionized water, anhydrous ethanol, and diethyl ether. The precipitate was then dried under vacuum to obtain a yellow trans-[PtCl2(Py-CH3)(NH3)]. Figure 4 As shown, Figure 4 The image shows the hydrogen nuclear magnetic resonance spectrum of the trans-[PtCl2(Py-CH3)(NH3)] obtained in Example 5.

[0049] Example 6

[0050] Cisplatin (89.7 mg, 0.3 mmol), 4-fluoropyridine hydrochloride (Py-F) (100 mg, 0.75 mmol), and 5 mL of deionized water were added to a round-bottom flask and stirred at 70 °C for 24 h until the solution became clear. Then, 1 mL of concentrated HCl (36%) was added to the solution, and the mixture was stirred at 80 °C for 72 h. After the reaction was complete, the solution was recrystallized at 4 °C. The precipitate was collected by filtration and washed three times each with deionized water, anhydrous ethanol, and diethyl ether. The precipitate was then dried under vacuum to obtain a yellow trans-[PtCl2(Py-F)(NH3)].

[0051] Example 7

[0052] Cisplatin (89.7 mg, 0.3 mmol), 4-chloropyridine hydrochloride (Py-Cl) (112.5 mg, 0.75 mmol), and 5 mL of deionized water were added to a round-bottom flask and stirred at 70 °C for 24 h until the solution became clear. Then, 1 mL of concentrated HCl (36%) was added to the solution, and the mixture was stirred at 80 °C for 72 h. After the reaction was complete, the precipitate was collected by filtration and washed three times each with deionized water, anhydrous ethanol, and diethyl ether. The precipitate was then dried under vacuum to obtain a yellow trans-[PtCl2(Py-Cl)(NH3)].

[0053] Example 8

[0054] Cisplatin (89.7 mg, 0.3 mmol), 4-bromopyridine (Py-Br) (118.5 mg, 0.75 mmol), and 5 mL of deionized water were added to a round-bottom flask and stirred at 70 °C for 24 h until the solution became clear. Then, 1 mL of concentrated HCl (36%) was added to the solution, and the mixture was stirred at 80 °C for 72 h. After the reaction was complete, the precipitate was collected by filtration and washed three times each with deionized water, anhydrous ethanol, and diethyl ether. The precipitate was then dried under vacuum to obtain a yellow trans-[PtCl2(Py-Br)(NH3)]. Figure 5 As shown, Figure 5 The image shows the hydrogen NMR spectrum of trans-[PtCl2(Py-Br)(NH3)] obtained in Example 8.

[0055] Example 9

[0056] Cisplatin (89.7 mg, 0.3 mmol), 4-iodopyridine (Py-I) (153.8 mg, 0.75 mmol), and 5 mL of deionized water were added to a round-bottom flask and stirred at 70 °C for 24 h until the solution became clear, with a small amount of wine-red crystals forming. The filtrate was centrifuged and used for the next reaction. Subsequently, 1 mL of concentrated HCl (36%) was added to the solution, and the mixture was stirred at 80 °C for 72 h. After the reaction was complete, the precipitate was collected by filtration and washed three times each with deionized water, anhydrous ethanol, and diethyl ether. The precipitate was then dried under vacuum to obtain a yellow trans-[PtCl2(Py-I)(NH3)]. Figure 6 As shown, Figure 6 The image shows the hydrogen nuclear magnetic resonance spectrum of trans-[PtCl2(Py-I)(NH3)] obtained in Example 9.

[0057] Example 10

[0058] In vitro cytotoxicity assays were used to evaluate the antitumor activity of trans-platinum complexes. Specifically, different cancer cells were seeded into 96-well plates (6000 cells per well) and cultured for 24 h. Fresh cell culture medium was used instead of the old medium, and the cells were incubated with different concentrations of different platinum drugs. Cells were incubated for another 48 h after the addition of each drug. MTT was dissolved in PBS (5 mg / mL) and added to each well (20 μL), followed by incubation for 4 h. Finally, after removing the medium, DMSO (150 μL) was added to each well, and the plates were shaken for 5 min in a microplate reader. The absorbance of the final product at 490 nm was recorded. The experimental results are as follows: Figure 7 As shown, Figure 7The graph shows the in vitro cytotoxicity results of the products from Examples 1-9 and cisplatin and antiplatinum, where the vertical axis represents cell viability and the horizontal axis represents the concentration of platinum drug molecules. The experimental results indicate that most of the synthesized antiplatinum compounds possess strong cancer cell killing capabilities.

[0059] Example 11

[0060] Cell death mode assay: MCF-7 cells were seeded in 6-well plates at a cell density of 5 × 10⁶ cells per well. 5 Cells were collected and cultured for 12 h in an incubator. Fresh culture medium was used instead of the old culture medium, and the cells were incubated with different platinum drugs (cisplatin and trans-[PtCl2(Py-I)(NH3)]) at 40 μM. Cells were further incubated for 12 h or 24 h after the addition of different drugs. Cell morphology was then observed using an optical microscope. The experimental results are as follows: Figure 8 As shown, Figure 8 The figure shows the results of the investigation into the cell death mechanism induced by trans-[PtCl2(Py-I)(NH3)]. In the control group, cells showed no significant changes in morphology; in the cisplatin group, cells first became rounded, then fragmented, characteristic of apoptosis; in the trans-[PtCl2(Py-I)(NH3)] group, cell vesicle extrusion (black arrows) was observed, characteristic of pyroptosis. The experimental results indicate that cisplatin induces apoptosis, while trans-[PtCl2(Py-I)(NH3)] induces pyroptosis.

[0061] Example 12

[0062] Cell immunogenicity death assay: MCF-7 cells were placed in 6-well plates at a density of 1 × 10⁶ cells per well. 6 Cells were incubated for 24 h. The culture medium was then replaced with PBS, cisplatin (40 μM), and trans-[PtCl2(Py-I)(NH3)] (40 μM), and incubated for another 24 h. The culture medium was then discarded, and the cells were incubated with Cy5-labeled calreticulin (CRT) antibody at 4°C for 0.5 h. After washing three times with PBS, the exposure of CRT on the surface of tumor cells was tested by flow cytometry. The experimental results are as follows: Figure 9 As shown, Figure 9 This image shows the number of cells with exposed calreticulin on their surface after treatment with different drugs, quantified by flow cytometry using trans-[PtCl2(Py-I)(NH3)]. The results indicate that trans-[PtCl2(Py-I)(NH3)] induces significant CRT exposure, thus inducing immunogenic cell death in tumor cells.

[0063] Example 13

[0064] In vivo antitumor experiment: 4T1 cells were inoculated into the left axilla of mice at a concentration of 1 million cells / mouse on day -8 to establish a subcutaneous proximal tumor model. On day -2, the same number and concentration of cells were implanted into the right axilla of mice to establish a distal tumor. On day 0, the proximal tumor volume reached 40-100 mm. 3 Balb / c mice were randomly divided into three groups (n = 5), and administered intratumoral drugs (saline, cisplatin, trans-[PtCl2(Py-I)(NH3)]) once on days 0, 3, and 6, at a dose of Pt (3 mg / kg). The experiment ended on day 14, mice were sacrificed, and tumor tissue was removed. The experimental results are as follows: Figure 10 As shown, Figure 10 The figure shows the in vivo tumor inhibition experiment results of trans-[PtCl2(Py-I)(NH3)]. At the end of the experiment, it was found that trans-[PtCl2(Py-I)(NH3)] could significantly inhibit the growth of both proximal and distal tumors.

[0065] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A trans-platinum compound, characterized in that, It has the structure of formula I-1; Equation I-1; R3 is I.

2. The method for preparing trans-platinum according to claim 1, characterized in that, Includes the following steps: Hydrochloric acid, cisplatin, and a compound with the structure shown in formula a are reacted to yield trans-platinum; Formula a; The R1~R2 and R4~R5 are independently selected from hydrogen.

3. The method for preparing trans-platinum according to claim 2, characterized in that, Specifically, the following steps are included: S1) Heat-treat cisplatin and the compound with the structure shown in formula a; S2) React hydrochloric acid with the reaction product obtained in step S1) to obtain trans-platinum.

4. The method for preparing trans-platinum according to claim 3, characterized in that, In step S1), the molar ratio of cisplatin to the compound with the structure shown in formula a is 1:(2~10).

5. The method for preparing trans-platinum according to claim 3, characterized in that, In step S1), the temperature of the heat treatment is 50℃~100℃, and the time of the heat treatment is 2 h~48 h.

6. The method for preparing trans-platinum according to claim 3, characterized in that, In step S2), the mass concentration of the hydrochloric acid is 36%~38%; The amount of hydrochloric acid used is 5 μL to 50 μL, based on 1 mg of cisplatin as described in step S1).

7. The method for preparing trans-platinum according to claim 3, characterized in that, In step S2), the reaction temperature is 50℃~100℃, and the reaction time is 2 h~72 h.

8. The use of the trans-platinum of claim 1 or the trans-platinum obtained by any of the preparation methods of claims 2 to 7 in the preparation of antitumor drugs.

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