CN113683645B - A dinuclear Pt(II) complex with anti-tumor activity and its preparation method and application - Google Patents

Abstract

The invention belongs to the technical field of development of novel platinum complexes, and particularly relates to a binuclear Pt (II) complex with anti-tumor activity, and a preparation method and application thereof. The invention takes 2, 6-dibromo methyl pyridine as an initial raw material and carries out 1:2 substitution reaction with dimethyl pyridine amine to generate intermediate BPA-TPA, BPA-TPA and K ₂ PtCl ₄ Formation of the target complex Pt of the invention through coordination reaction ₂ -BPA-TPA. The invention designs and synthesizes symmetrical dual-core single-function platinum (II) complex by taking a polypyridine compound BPA-TPA containing 5 pyridine rings as a ligand for the first time, and the complex has good anti-tumor activity and potential medicinal value, and is expected to become one of alternatives of novel non-classical platinum anti-cancer drugs, thereby overcoming the problems of more outstanding drug resistance and more obvious toxic and side effects of the existing platinum drugs. The binuclear Pt (II) complex with anti-tumor activity has the following structural formula:

Description

A kind of dinuclear Pt (Ⅱ) complex with anti-tumor activity and its preparation method and application

Technical field

The invention belongs to the technical field of new platinum complex development, and specifically relates to a dinuclear Pt(II) complex with anti-tumor activity and its preparation method and application.

Background technique

According to data released by the World Health Organization (WHO) in December 2020, 19.3 million people around the world were diagnosed with cancer in 2020, and 10 million people died from cancer. Cancer has become the second leading cause of death in the world. The current clinical treatments for various tumors mainly include surgery, chemotherapy and radiotherapy. Among a wide variety of chemotherapy drugs, platinum complexes are undoubtedly one of the classic anti-tumor drugs, such as cisplatin, carboplatin, oxaliplatin, etc. Statistics show that about 40%-80% of patients with various tumors have received or are receiving chemotherapy containing platinum drugs (alone or in combination), and it has become the standard or first-line regimen for the treatment of many cancers.

Classic platinum drugs have been very successful in the field of cancer treatment, but so far only cisplatin, carboplatin and oxaliplatin have been approved for use globally, while nedaplatin, idaplatin and loplatin have been approved in Japan (1995). , South Korea (1999) and China (2010) were listed separately. There are two main reasons for this: (1) The toxic and side effects are obvious, including nephrotoxicity, neurotoxicity, bone marrow suppression, nausea, vomiting, hair loss and fatigue; (2) The drug resistance is prominent, including endogenous drug resistance. and acquired resistance. The existence of toxic side effects and drug resistance limits the wider application of current platinum-based drugs, and also prompts people to continue to research and develop new platinum-based drugs with high anti-cancer activity, weak side effects, and no cross-resistance.

Contents of the invention

In view of the technical problems existing in the prior art, the present invention provides a binuclear Pt(II) complex with anti-tumor activity and its preparation method and application. The toxicity is very small.

The technical solutions of the present invention to solve the above technical problems are as follows:

A dinuclear Pt(II) complex with anti-tumor activity, its structural formula is as follows:

On the basis of the above technical solution, the present invention can also make the following improvements.

Another object of the present invention is to provide a method for preparing the above-mentioned dinuclear Pt(II) complex.

The specific technical solutions are as follows:

A method for preparing a binuclear Pt(II) complex with anti-tumor activity, including the following steps:

1) Dissolve 2,6-dibromomethylpyridine, lutidineamine and potassium carbonate in acetonitrile, stir and react at 30°C until the solution changes from the initial yellow color to orange-red, then filter to remove the potassium carbonate, The filtrate is concentrated to dryness under reduced pressure to obtain a yellow-brown oily liquid, which is the crude product of BPA-TPA;

2) Add petroleum ether with a boiling range of 60-90°C to the yellow-brown oily liquid obtained in step 1), stir vigorously at 80°C, take out the clear petroleum ether solution, let it stand and cool, and white flocculent crystals will precipitate, which is BPA. -TPA ligand pure product, collected after vacuum drying;

3) Dissolve the pure BPA-TPA ligand obtained in step 2) in methanol, and dissolve K ₂ PtCl ₄ in water. After mixing the two, stir and react for 48 hours at room temperature and in the dark to obtain a mixture;

4) Centrifuge the mixture obtained in step 3), discard the supernatant, and wash the solid product with methylene chloride, ethanol, acetone and anhydrous ether in sequence until the washing tower is clear. After vacuum drying, a white powder is obtained, which is Pt ₂ -BPA-TPA.

Further, in step 1), the ratio of the amounts of 2,6-dibromomethylpyridine, lutidineamine and potassium carbonate is 1:2:2.

Further, in step 1), the stirring reaction time is 2 h.

Further, in step 2), stir vigorously at 80°C, repeatedly add petroleum ether with a boiling range of 60-90°C to the yellow-brown oily liquid obtained in step 1) several times for extraction, and purify the resulting BPA-TPA ligand. The products were combined and collected after vacuum drying.

Further, in step 2), the ratio of the amounts of BPA-TP and K ₂ PtCl ₄ is 1:2.

The final purpose of the present invention is to provide the application of the above-mentioned binuclear Pt(II) complex.

The specific technical solutions are as follows:

Application of a dinuclear Pt(II) complex in the preparation of drugs for preventing and treating liver cancer, lung cancer, cervical cancer and breast cancer.

The beneficial effects of the present invention are:

1) For the first time, the present invention uses the polypyridine compound BPA-TPA containing 5 pyridine rings as a ligand to creatively design and synthesize a binuclear platinum (II) complex. This complex is a new compound and is well known in this field and existing literature. There are no reports yet.

2) The present invention determines the structure of the above-mentioned new binuclear platinum (II) complex through characterization means such as ¹ H-NMR and HRMS, and proves that the stated method can successfully prepare the target binuclear platinum (II) complex.

3) Through in vitro anti-tumor experiments and measurement of the expression levels of related proteins such as p21 and casepase-3, the present invention confirms that the target dinuclear platinum (II) complex has excellent anti-tumor activity and is less toxic to normal cells. ; Its anti-tumor activity makes it have good potential medicinal value, and it is expected to become one of the candidates for new non-classical platinum anti-cancer drugs, especially for the preparation of anti-non-small cell lung cancer drugs, thereby overcoming the The existing problems of prominent drug resistance and obvious side effects of platinum-based drugs provide some useful exploration and valuable reference for the future design and synthesis of new platinum-based complex drugs.

Description of the drawings

Figure 1 is the ¹ H-NMR spectrum of BPA-TPA in Example 1 of the present invention;

Figure 2 is the ¹³ C-NMR spectrum of BPA-TPA in Example 1 of the present invention;

Figure 3 is the ¹ H-NMR spectrum of Pt ₂ -BPA-TPA in Example 1 of the present invention;

Figure 4 is the HRMS spectrum of Pt ₂ -BPA-TPA in Example 1 of the present invention;

Figure 5 is a diagram showing the inhibition of HepG2 cells by Pt ₂ -BPA-TPA in Example 1 of the present invention;

Figure 6 is a diagram showing the inhibition of A549 cells by Pt ₂ -BPA-TPA in Example 1 of the present invention;

Figure 7 is a diagram showing the effect of Pt ₂ -BPA-TPA on the expression of p21 and casepase-3 in HepG2 cells in Example 1 of the present invention;

Figure 8 is a diagram showing the effect of Pt ₂ -BPA-TPA on the expression of p21 and casepase-3 in A549 cells in Example 1 of the present invention;

Figure 9 is a diagram showing the effect of Pt ₂ -BPA-TPA on the expression of p21 and casepase-3 in H9c2 cells in Example 1 of the present invention.

Detailed ways

The principles and features of the present invention are described below. The examples cited are only used to explain the present invention and are not intended to limit the scope of the present invention.

Unless otherwise stated, the raw materials and equipment used in the embodiments of the present invention are conventional raw materials and equipment in this technical field (conventionally commercially available products), and can be purchased in the market.

The raw material 2,6-dibromomethylpyridine mentioned in the embodiments of the present invention is prepared with reference to the existing literature (Zhang X. WO Pat, 97/13763, 1997.).

Example 1

Preparation of dinuclear Pt(Ⅱ) complex with the following structural formula:

In a 100 ml round-bottomed flask, add 8 ml of acetonitrile solution dissolved in 2,6-dibromomethylpyridine (1.2g, 4.5mmol), and then add 16 ml of lutidine amine (1.8g, 9.0mmol) dissolved in it. acetonitrile solution, and then add potassium carbonate powder (1.3g, 9.4mmol). The reaction was stirred at 30°C for 2 h, and the solution changed from the initial yellow color to orange-red. Filter and spin the filtrate to dryness to obtain a yellowish-brown oily liquid. Then stir vigorously at 80°C and extract multiple times with petroleum ether with a boiling range of 60-90°C. Combine the petroleum ether extracts and let stand to cool, and white flocculent crystals precipitate. After suction filtration, 1.3g of product BPA-TPA was obtained, yield: 56%.

As shown in Figure 1 and Figure 2, BPA-TPA: ¹ H-NMR (400MHz, CDCl ₃ ) ppm δ8.51 (d, 4H), 7.60 (m, 9H), 7.43 (d, 2H), 7.12 (m, 4H),3.91(s,8H),3.89(s,4H). ¹³ C-NMR (100MHz, CDCl ₃ ) δ 159.32, 158.61, 149.05, 136.87, 136.45, 123.00, 122.00, 121.17, 60.20.

In another 100 ml round-bottomed flask, add BPA-TPA (248 mg, 0.5 mmol) and dissolve it in 10 ml of methanol. Weigh K ₂ PtCl ₄ (410 mg, 1.0 mmol), dissolve it in 10 ml of pure water, and add it to the round-bottomed flask. The reaction was stirred at room temperature and in the dark for 48 h. Centrifuge, discard the supernatant, and wash the solid product with dichloromethane, ethanol, acetone and anhydrous ether in sequence until the washing tower is clear. After vacuum drying, 108 mg of white powder solid was obtained, which was the target dinuclear Pt(II) complex Pt ₂ -BPA-TPA. The yield was 21%.

As shown in Figure 3 and Figure 4, Pt ₂ -BPA-TPA: ¹ H-NMR (400MHz, DMSO-d ₆ )ppmδ8.60(d,4H),8.18(t,4H),7.74(d,4H) ,7.59(s,3H),7.55(t,4H),7.12(m,4H),3.91(s,8H),3.89(s,4H). HRMS (m/z): [Pt ₂ Cl ₂ -BPA-TPA+6H ₂ O] ^{2 +} theoretical value 534.0958, measured value 534.0923; [Pt ₂ Cl ₂ -BPA-TPA] ^{2 +} theoretical value 480.0629, measured value 480.0631 ; [Pt ₂ Cl ₂ -BPA-TPA+Cl] ⁺ theoretical value 995.0981, measured value 995.0963.

In this example, 2,6-dibromomethylpyridine is used as the starting material, and a 1:2 substitution reaction occurs with lutidine amine to generate the intermediate BPA-TPA. BPA-TPA is coordinated with K ₂ PtCl ₄ The reaction produces the target complex Pt ₂ -BPA-TPA of this embodiment.

Example 2

An in vitro anti-tumor activity experiment was conducted on the Pt ₂ -BPA-TPA prepared in Example 1.

2.1 Cell lines and cell culture

Three cell lines were selected: human liver cancer cell HepG2, human non-small cell lung cancer cell A549, and rat normal cardiomyocyte H9c2.

All cell lines were cultured in RPMI-1640 culture medium containing 100 U/ml penicillin, 10 wt% fetal bovine serum, and 100 U/ml streptomycin, and cultured in a 37°C incubator containing 5% CO ₂ volume concentration.

2.2 Solution preparation of the complex to be tested

The complex was first dissolved in DMSO to prepare a 10mM stock solution. Then use physiological buffer to dilute to final solutions of 12.5, 25, 50, and 100 μM (final concentration of DMSO ≤ 1%), and test the inhibitory degree of the complex on selected tumor cells and normal cells at various concentrations.

2.3 Cell growth inhibition experiment (CCK-8 method)

HepG2, A549 and H9c2 cells in the logarithmic growth phase were seeded into a 96-well plate at 4 × 10 ³ cells/well, and cultured at 37°C and 5% CO ₂ until the cells were 90% confluent, and then cultured with serum-free Incubate for 2 hours to synchronize the cells. Subsequently, the supernatant was discarded, and DMEM medium containing each complex (0 μM, 12.5 μM, 25 μM, 50 μM, 100 μM) was added and incubated for 48 h. 3 h before the end of the incubation, 10 μL of CCK-8 solution was added to each well. After the incubation, the supernatant of each well was discarded, 150 μL DMSO was added to each well, and the cell shaker was shaken for 10 min. After the crystals were fully dissolved, a microplate reader was used to measure OD ₄₅₀ at a wavelength of 450 nm.

2.4 Analysis of measurement results

It can be seen from Figure 5 and Figure 6:

1) The binuclear Pt(II) complex synthesized in Example 1 of the present invention shows a certain inhibitory effect on both HepG2 and A549 cells.

2) Regardless of HepG2 or A549, the inhibitory activity of the binuclear Pt(Ⅱ) complex against them is better than that of the control group cisplatin; and for two different types of cancer cells, the binuclear Pt(Ⅱ) complex has an inhibitory effect on human non-small cell lung cancer. The inhibitory activity of cell A549 is significantly stronger than that of human liver cancer cell HepG2; its IC ₅₀ value against A549 cells is below 24 μM.

Example 3

The Pt ₂ -BPA-TPA prepared in Example 1 was subjected to a measurement experiment to determine the impact on the expression levels of p21 and casepase-3.

3.1 Western blot experiment

HepG2, A549 or H9c2 was lysed and homogenized with cell lysis buffer (#9803, CST, USA), and then centrifuged at 4°C and 12000 rpm for 15 min to extract the total protein. The concentration of extracted total protein was determined using BCA protein kit (A53225, Thermo Fisher Scientific, USA). Western blot analysis of the above proteins was performed using α-tublin (GB11200, ServiceBio, Wuhan, China), activated caspase 3 (9661S, CST, USA) and p21 (A1483, ABclonal, Wuhan, China) as labeled antibodies. For detailed operations and analysis methods, please refer to the literature (Meng X, He C, Chen X, et al. J. Ethnopharmacol., 2021, 268, 113579).

3.2 Analysis of experimental results

It can be seen from Figure 7, Figure 8 and Figure 9:

1) In human liver cancer cells HepG2 and human non-small cell lung cancer cells A549, the dinuclear platinum (II) complex prepared in Example 1 of the present invention significantly up-regulated both activated caspase 3 and p21, indicating that the dinuclear platinum (II) complex of the present invention (II) The complex has good anticancer activity.

2) In HepG2, the binuclear platinum (II) complex prepared in Example 1 of the present invention makes the expression levels of activated caspase 3 and p21 5 times and 4 times that of the control group CON, respectively.

3) In A549, the binuclear platinum (II) complex prepared in Example 1 of the present invention caused the expression levels of activated caspase 3 and p21 to be 4 times and 6 times that of the control group CON, respectively.

4) In rat normal cardiomyocytes H9c2, the binuclear platinum (II) complex prepared in Example 1 of the present invention caused the expression of activated caspase 3 and p21 to be twice that of the control group CON, indicating that the present invention Binuclear platinum (II) complexes are less toxic to normal cells.

In summary, the dinuclear platinum (II) complex Pt ₂ -BPA-TPA according to the present invention exhibits excellent anti-tumor activity in vitro and has weak toxicity to normal cells. The anti-tumor activity exhibited by the new binuclear platinum (II) complex Pt ₂ -BPA-TPA synthesized by the present invention makes it have good potential medicinal value, and is expected to become one of the candidates for new non-classical platinum anti-cancer drugs. , is especially expected to be used in the preparation of anti-non-small cell lung cancer drugs, thereby overcoming the problems of prominent drug resistance and obvious toxic and side effects of existing platinum drugs.

The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

Claims (7)

1. The binuclear Pt (II) complex with anti-tumor activity is characterized by having the following structural formula:

2. the method for preparing a binuclear Pt (ii) complex having antitumor activity as defined in claim 1, comprising the steps of:

1) Dissolving 2, 6-dibromomethylpyridine, dimethylpyridine amine and potassium carbonate in acetonitrile, stirring at 30 ℃ to react until the solution turns from the initial yellow to orange red, filtering to remove the potassium carbonate, and concentrating the filtrate under reduced pressure to dryness to obtain yellow brown oily liquid which is a crude product of BPA-TPA;

2) Adding petroleum ether with the boiling range of 60-90 ℃ into the yellow brown oily liquid obtained in the step 1), vigorously stirring at the temperature of 80 ℃, taking out clarified petroleum ether solution, standing and cooling, separating out white flocculent crystals, namely a pure BPA-TPA ligand product, and collecting after vacuum drying;

3) Dissolving the BPA-TPA ligand pure product obtained in the step 2) in methanol, K ₂ PtCl ₄ Dissolving in water, mixing the two, stirring at room temperature under dark condition, and reacting for 48 hr to obtain a mixture;

4) Centrifuging the mixture obtained in the step 3), removing the supernatant, washing the solid product with dichloromethane, ethanol, acetone and anhydrous diethyl ether in sequence until the washing tower is clear, and drying in vacuum to obtain white powder, namely the binuclear Pt (II) complex.

3. The method according to claim 2, wherein in step 1), the ratio of the amounts of 2, 6-dibromomethylpyridine, dimethylpyridine amine, and potassium carbonate is 1:2:2.

4. The process according to claim 2, wherein in step 1), the stirring reaction time is 2 hours.

5. The process according to claim 2, wherein in step 2), stirring is vigorously carried out at 80 ℃, petroleum ether having a boiling range of 60 to 90 ℃ is repeatedly added to the tan oily liquid obtained in step 1) to extract, and the resulting purified BPA-TPA ligand product is combined, dried in vacuo and collected.

6. The process of claim 2, wherein in step 2), BPA-TPA is combined with K ₂ PtCl ₄ The ratio of the amounts of the substances is 1:2.

7. The use of the binuclear Pt (ii) complex as defined in claim 1 for the preparation of a medicament for the prevention and treatment of liver cancer and lung cancer.