Trans-platinum as well as preparation method and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to trans-platinum and a preparation method and application thereof. The invention provides trans-platinum. The trans-platinum has a structure as shown in a formula I, # imgabs0 # is shown as a formula I; wherein R1 to R5 are independently selected from hydrogen, halogen, CHO, OH, NH2 or CH3. The trans-platinum provided by the invention is a divalent platinum compound with pyroptosis induction capability, has a trans structure, and comprises two chlorine ligands at the trans position, an ammonia ligand at the trans position and a nitrogen-containing heterocyclic ring ligand at the trans position, and the nitrogen-containing heterocyclic ring ligand is designed to be pyridine or a derivative ligand thereof. Due to the existence of a trans structure and a planar ligand, a DNA cross-linking mode different from classical platinum drugs such as cis-platinum is adopted, and the compound has pyroptosis induction capacity, can induce tumor cell immunogenic death and activate anti-tumor immune response, and has a cancer cell killing mode different from that of cis-platinum and excellent cancer cell killing capacity as a platinum drug.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to trans-platinum and a preparation method and application thereof. Background Art

[0002] Chemotherapy is one of the most widely used cancer treatments. Platinum drugs are platinum metal drugs with anti-cancer effects. Classic platinum anti-tumor drugs represented by cisplatin (cis-dichlorodiammineplatin), carboplatin and oxaliplatin are the mainstay of chemotherapy drugs. Currently, more than 50% of cancer patients receive treatment with platinum drugs. Their main mechanism of action is to cross-link the DNA of tumor cells, thereby causing cell apoptosis, where cell apoptosis refers to the autonomous and orderly death of cells controlled by genes to maintain the stability of the internal environment. Although some success has been achieved, classic platinum drugs with cis structures still face some shortcomings, such as drug resistance, poor effect on certain types of cancer, and inability to inhibit cancer metastasis and recurrence.

[0003] Developing platinum drugs with different killing mechanisms is an effective way to solve the above problems. Unlike apoptosis, pyroptosis is a programmed cell death method with an "inflammatory effect", which is characterized by dependence on caspase-1 and the release of a large number of pro-inflammatory factors. As a new pro-inflammatory programmed cell death method, pyroptosis is significantly different from apoptosis in terms of cell morphological changes and occurrence mechanism. Specifically, during pyroptosis, tumor cells release some inflammatory factors (IL-1β and IL-18), thereby activating anti-tumor immune responses while killing tumor cells. In addition, pyroptosis is an immunogenic cell death method. Immunogenic cell death is a process in which tumor cells are transformed from non-immunogenic to immunogenic to mediate the body's anti-tumor immune response when they die under external stimuli, which can further activate anti-tumor immune effects. Therefore, platinum drugs with the ability to induce pyroptosis are expected to have a higher spectrum, enhanced efficacy, and higher ability to inhibit cancer metastasis and recurrence.

[0004] Most of the classical platinum drugs with cis structures are apoptosis inducers. Therefore, if you want to develop platinum drugs with the ability to induce cell pyroptosis, you need to make major modifications based on the cis structure, which is difficult and the direction of modification is unclear. The trans-structured anti-platinum (trans-dichlorodiammineplatinum) can also be used as a platinum drug. It has a different DNA binding mode from classical platinum drugs such as cisplatin, and can still exert its efficacy in some cisplatin-resistant cells. However, there is currently no clear existing technical guidance on how anti-platinum molecules can achieve the ability to induce cell pyroptosis. 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 and a preparation method and application thereof. The trans-platinum provided by the present invention has the ability to induce cell pyroptosis, can cause immunogenic death of tumor cells, and activate anti-tumor immune response while killing tumor cells.

[0006] The present invention provides a trans-platinum having a structure of formula I;

[0007] Formula I;

[0008] Wherein R 1 ~R 5 independently selected from hydrogen, halogen, CHO, OH, NH 2 or CH 3 .

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

[0010] In order to solve the problems of drug resistance of classical platinum drugs, poor effects on certain types of cancer, inability to reduce the metastasis of cancer cells and the recurrence of cancer, the present invention designs the above-mentioned trans-platinum. The trans-platinum provided by the present invention is a class of divalent platinum compounds with cell pyroptosis induction ability, which has a trans structure, including two chlorine ligands in trans positions, an ammonia ligand in trans positions and a nitrogen-containing heterocyclic ligand in trans positions, and 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 the present invention adopts a DNA cross-linking method different from classical platinum drugs such as cisplatin, has cell pyroptosis induction ability, can induce immunogenic death of tumor cells, activate anti-tumor immune response, 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 R 3 Selected from hydrogen, halogen, CHO, OH, NH 2 or CH 3 wherein the halogen is preferably F, Cl, Br or I.

[0014] More preferably, the trans-platinum has a structure of formula I-1, wherein R 3 Selected from H, CH 3 , Br or I.

[0015] The trans-platinum having the structure of formula I-1 provided by the present invention further adjusts the DNA binding ability of the trans-platinum molecule by introducing groups with different spatial configurations, hydrophilicity, halogen bond and hydrogen bond giving abilities at position 4 of the pyridine ligand, ultimately causing cell pyroptosis, further causing immunogenic death of tumor cells, and activating anti-tumor immune response while killing tumor cells.

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

[0017] React hydrochloric acid, cisplatin and the compound of the structure shown in formula a to obtain trans-platinum;

[0018] Formula a;

[0019] The R 1 ~R 5 Same as above, no further elaboration.

[0020] Specifically, the method for preparing trans-platinum described in any of the above technical solutions comprises the following steps:

[0021] S1) heat-treating cisplatin and the compound represented by the structure of formula a;

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

[0023] The present invention first heat-treats cisplatin and the compound of the structure shown in formula a. Specifically, the present invention first hydrothermally treats cisplatin and the compound of the structure shown in formula a. More specifically, the present invention first mixes cisplatin and the compound of the structure shown in formula a and disperses them in water, and heats and stirs to perform hydrothermal treatment. The molar ratio of cisplatin and the compound of the structure shown in formula a of the present invention is 1:(2~10), preferably 1:(2.5~4). The temperature of heat-treating cisplatin and the compound of the structure shown in formula a of the present invention is 50℃~100℃, preferably 70℃~90℃; the time is 2h~48h, preferably 12h~48h.

[0024] In the present invention, after heat-treating cisplatin and the compound of the structure shown in formula a, hydrochloric acid and the reaction product obtained in step S1) are reacted to obtain trans-platinum. Specifically, hydrochloric acid is added to the reaction product obtained in step S1), heated and stirred to react, purified, and trans-platinum is obtained. The purification method of the present invention includes recrystallization, sedimentation, high-efficiency liquid phase separation or water washing. The mass concentration of the hydrochloric acid of the present invention is 36%~38%; based on 1 mg of the cisplatin in step S1), the amount of the hydrochloric acid is 5 μL~50 μL, preferably 10 μL~40 μL. The temperature of the reaction of hydrochloric acid and the reaction product obtained in step S1) is 50℃~100℃, preferably 70℃~90℃; the time is 2 h~72 h, preferably 24 h~72 h.

[0025] The present invention also provides the use of the trans-platinum described in any of the above technical solutions or the trans-platinum obtained by the preparation method described in any of the above technical solutions in the preparation of anti-tumor drugs. The present invention also provides an anti-tumor method using the trans-platinum described in any of the above technical solutions or the trans-platinum obtained by the preparation method described in any of the above technical solutions. The trans-platinum provided by the present invention has the ability to induce cell pyroptosis, immunogenic death, and activate anti-tumor immune response. As a platinum drug, it has a cancer cell killing method different from cisplatin and excellent cancer cell killing ability.

[0026] The present invention provides a trans-platinum and a preparation method and application thereof. The present invention provides a trans-platinum having a structure of formula I; Formula I; wherein R 1 ~R 5 independently selected from hydrogen, halogen, CHO, OH, NH 2 or CH 3 The trans-platinum provided by the present invention adopts a DNA cross-linking method different from that of classic platinum drugs such as cisplatin, which can cause cell pyroptosis and further cause immunogenic death of tumor cells. While killing tumor cells, it activates anti-tumor immune response. As a platinum drug, it has a cancer cell killing method different from cisplatin and excellent cancer cell killing ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The trans-[PtCl 2 (Py-H)(NH 3 )] H NMR spectrum;

[0028] Figure 2 The trans-[PtCl 2 (Py-CHO)(NH 3 )] H NMR spectrum;

[0029] Figure 3 The trans-[PtCl 2 (Py-OH)(NH 3 )] H NMR spectrum;

[0030] Figure 4 The trans-[PtCl 2 (Py-CH 3 )(NH 3 )] H NMR spectrum;

[0031] Figure 5 The trans-[PtCl 2 (Py-Br)(NH 3 )] H NMR spectrum;

[0032] Figure 6 The trans-[PtCl 2 (Py-I)(NH 3 )] H NMR spectrum;

[0033] Figure 7 The results of in vitro cytotoxicity experiments of the products of Examples 1 to 9 and cisplatin and trans-platinum are shown;

[0034] Figure 8 For trans-[PtCl 2 (Py-I)(NH 3 )] The results of the cell death mode exploration;

[0035] Fig. 9 For trans-[PtCl 2 (Py-I)(NH 3 )] Flow cytometry quantification of the number of cells with surface exposed calreticulin after treatment with different drugs;

[0036] Fig.10 For trans-[PtCl 2 (Py-I)(NH 3 )] in vivo tumor inhibition experiment results. DETAILED DESCRIPTION

[0037] The present invention discloses a trans-platinum and a preparation method and application thereof. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0038] The present invention will be further described below in conjunction with 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 to make the solution clear. Subsequently, 4 mL of concentrated HCl (36%) was added to the solution and stirred at 80 °C for 72 h. After the reaction was completed, the precipitate was collected by filtration and washed with deionized water three times. It was then dried in vacuum to obtain the yellow product trans-[PtCl 2 (Py-H)(NH 3 )].like Figure 1 As shown, Figure 1 The trans-[PtCl 2 (Py-H)(NH 3 )]’s H NMR spectrum.

[0041] Example 2

[0042] Cisplatin (50.0 mg, 0.167 mmol), 4-formylpyridine (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 to make the solution clear. Subsequently, 0.8 mL of concentrated HCl (36%) was added to the solution and stirred at 90 °C for 72 h. After the reaction was completed, the precipitate was collected by filtration and washed with deionized water, anhydrous ethanol and ether three times respectively. Then it was dried in vacuum to obtain yellow trans-[PtCl 2 (Py-CHO)(NH 3 )].like Figure 2 As shown, Figure 2 The trans-[PtCl 2 (Py-CHO)(NH 3 )]’s H NMR spectrum.

[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 to make the solution clear. Subsequently, 1 mL of concentrated HCl (36%) was added to the solution and stirred at 90 °C for 72 h. After the reaction was completed, the precipitate was removed by filtration, the reaction solution was concentrated, and the product was obtained by recrystallization. It was then dried in a vacuum to obtain a yellow trans-[PtCl 2 (Py-OH)(NH 3 )].like Figure 3 As shown, Figure 3 The trans-[PtCl 2 (Py-OH)(NH 3 )]’s H NMR spectrum.

[0045] Example 4

[0046] Cisplatin (89.7 mg, 0.3 mmol), 4-aminopyridine (Py-NH 2 ) (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 to make the solution clear. After the reaction was completed, a small amount of black precipitate appeared, and the filtrate was centrifuged for the next reaction. Subsequently, 1 mL of concentrated HCl (36%) was added to the solution and stirred at 80 °C for 72 h. After the reaction was completed, the precipitate was removed by filtration, the reaction solution was concentrated, and the product was obtained by recrystallization. It was then dried in a vacuum to obtain a yellow trans-[PtCl 2 (Py-NH 2 )(NH 3 )].

[0047] Example 5

[0048] Cisplatin (89.7 mg, 0.3 mmol), 4-methylpyridine (Py-CH 3 ) (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 to make the solution clear. Subsequently, 1 mL of concentrated HCl (36%) was added to the solution and stirred at 80 °C for 72 h. After the reaction was completed, the precipitate was collected by filtration and washed with deionized water, anhydrous ethanol and ether three times respectively. Then it was dried in vacuum to obtain yellow trans-[PtCl 2 (Py-CH 3 )(NH 3 )].like Figure 4 As shown, Figure 4 The trans-[PtCl 2(Py-CH 3 )(NH 3 )]’s H NMR spectrum.

[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 to make the solution clear. Subsequently, 1 mL of concentrated HCl (36%) was added to the solution and stirred at 80 °C for 72 h. After the reaction was completed, the solution was placed in a 4 °C refrigerator for recrystallization, and the precipitate was collected by filtration and washed three times with deionized water, anhydrous ethanol and ether respectively. Then it was dried in a vacuum to obtain yellow trans-[PtCl 2 (Py-F)(NH 3 )].

[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 to make the solution clear. Subsequently, 1 mL of concentrated HCl (36%) was added to the solution and stirred at 80 °C for 72 h. After the reaction was completed, the precipitate was collected by filtration and washed with deionized water, anhydrous ethanol and ether three times respectively. Then it was dried in vacuum to obtain yellow trans-[PtCl 2 (Py-Cl)(NH 3 )].

[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 to make the solution clear. Subsequently, 1 mL of concentrated HCl (36%) was added to the solution and stirred at 80 °C for 72 h. After the reaction was completed, the precipitate was collected by filtration and washed with deionized water, anhydrous ethanol and ether three times respectively. Then it was dried in vacuum to obtain yellow trans-[PtCl 2 (Py-Br)(NH 3 )].like Figure 5 As shown, Figure 5 The trans-[PtCl 2 (Py-Br)(NH 3 )]’s H NMR spectrum.

[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 to make the solution clear. At the same time, a small amount of wine-red crystals were produced. The filtrate was centrifuged for the next reaction. Subsequently, 1 mL of concentrated HCl (36%) was added to the solution and stirred at 80 °C for 72 h. After the reaction was completed, the precipitate was collected by filtration and washed three times with deionized water, anhydrous ethanol and ether respectively. Then it was dried in a vacuum to obtain yellow trans-[PtCl 2 (Py-I)(NH 3 )].like Figure 6 As shown, Figure 6 The trans-[PtCl 2 (Py-I)(NH 3 )]’s H NMR spectrum.

[0057] Example 10

[0058] In vitro cytotoxicity experiments were used to evaluate the antitumor activity of trans-platinum complexes. Specifically, different cancer cells were inoculated in 96-well plates (6,000 cells per well) and further cultured in an incubator for 24 h. The old cell culture medium was replaced with fresh culture medium and incubated with different platinum drugs at different concentrations. The cells were further incubated in an incubator for 48 h after the addition of different drugs. MTT was dissolved in PBS (5 mg / mL) and then added to each well (20 μL), and then incubated in an incubator for 4 h. Finally, after removing the culture medium, DMSO (150 μL) was added to each well, and after shaking in a microplate reader for another 5 min, the absorbance of the final product at 490 nm was recorded. The experimental results are shown in the following figure. Figure 7 As shown, Figure 7 The results of the in vitro cytotoxicity test of the products of Examples 1 to 9 and cisplatin and trans-platinum are shown in the figure, wherein the ordinate is the cell survival rate and the abscissa is the concentration of the platinum drug molecule. The experimental results show that most of the synthesized trans-platinum compounds have a strong ability to kill cancer cells.

[0059] Embodiment 11

[0060] Cell death mode experiment: MCF-7 cells were seeded in 6-well plates with a cell density of 5×10 per well. 5 The cells were further cultured in an incubator for 12 h. The old cell culture medium was replaced with fresh culture medium and 40 μM of different platinum drugs (cisplatin and trans-[PtCl 2(Py-I)(NH 3 After adding different drugs, the cells were further incubated in the incubator for 12 h or 24 h. Subsequently, the cell morphology was observed using an optical microscope. The experimental results are shown in Figure 8 As shown, Figure 8 For trans-[PtCl 2 (Py-I)(NH 3 )] The results of the cell death mode exploration. The cells in the control group showed no obvious changes in appearance; the cells in the cisplatin group first became rounded and then showed cell fragments, which are the characteristics of cell apoptosis; trans-[PtCl 2 (Py-I)(NH 3 )] group showed cell bubbling phenomenon (black arrow), which is a characteristic of cell pyroptosis. The experimental results showed that the cell death mode caused by cisplatin was apoptosis, and trans-[PtCl 2 (Py-I)(NH 3 )] The mode of cell death caused by this is pyroptosis.

[0061] Example 12

[0062] Cell immunogenicity death experiment: MCF-7 cells were placed in a 6-well plate at a density of 1×10 6 After 24 h of incubation, the cell culture medium was replaced with PBS, cisplatin (40 μM), and trans-[PtCl 2 (Py-I)(NH 3 )] (40 μM) medium was added and incubated for 24 h. The medium was discarded and then incubated with Cy5-labeled calreticulin (CRT) antibody at 4°C for 0.5 h. After washing with PBS three times, the exposure of CRT on the surface of tumor cells was tested by flow cytometry. The experimental results are shown in Fig. 9 As shown, Fig. 9 For trans-[PtCl 2 (Py-I)(NH 3 )] Flow cytometry quantification of the number of cells with surface exposed calreticulin after treatment with different drugs. The experimental results showed that trans-[PtCl 2 (Py-I)(NH 3 )] can cause significant CRT exposure and thus induce immunogenic death of tumor cells.

[0063] Embodiment 13

[0064] In vivo anti-tumor experiment: 4T1 cells were inoculated at a concentration of 1 million cells / mouse into the left axilla of mice 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 given intratumoral administration of cisplatin (saline, trans-[PtCl 2 (Py-I)(NH 3 )]), the dosage was Pt (3 mg / kg). The experiment was ended on the 14th day, the mice were killed, and the tumor tissues were removed. The experimental results are shown in Fig.10 As shown, Fig.10 For trans-[PtCl 2 (Py-I)(NH 3 )] in vivo tumor inhibition experiment results. At the end of the experiment, it was found that trans-[PtCl 2 (Py-I)(NH 3 )] can significantly inhibit the growth of proximal and distal tumors.

[0065] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A trans-platinum, characterized in that: It has the structure of formula I; Formula I; Wherein R1~R5 are independently selected from hydrogen, halogen, CHO, OH, NH2 or CH3.

2. The trans-platinum according to claim 1, characterized in that It has the structure of formula I-1; Formula I-1.

3. The trans-platinum according to claim 2, characterized in that The R3 is selected from H, CH3, Br or I.

4. The method for preparing trans-platinum according to any one of claims 1 to 3, characterized in that: The following steps are involved: React hydrochloric acid, cisplatin and the compound of the structure shown in formula a to obtain trans-platinum; Formula a.

5. The method for preparing trans-platinum according to claim 4, characterized in that: The specific steps include: S1) heat-treating cisplatin and the compound represented by the structure of formula a; S2) reacting hydrochloric acid with the reaction product obtained in step S1) to obtain trans-platinum.

6. The method for preparing trans-platinum according to claim 5, characterized in that: In step S1), the molar ratio of cisplatin to the compound of the structure represented by formula a is 1:(2-10).

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

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

9. The method for preparing trans-platinum according to claim 5, characterized in that: In step S2), the reaction temperature is 50°C to 100°C, and the reaction time is 2 h to 72 h.

10. Use of the trans-platinum according to any one of claims 1 to 3 or the trans-platinum obtained by the preparation method according to any one of claims 4 to 8 in the preparation of anti-tumor drugs.

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