CN114349794B

CN114349794B - Platinum bisphosphonate complex and synthesis method and application thereof

Info

Publication number: CN114349794B
Application number: CN202111457415.8A
Authority: CN
Inventors: 张振琴; 谢安琦; 居一春; 马露
Original assignee: Nanjing Medical University
Current assignee: Nanjing Medical University
Priority date: 2021-10-31
Filing date: 2021-12-02
Publication date: 2023-11-24
Anticipated expiration: 2041-12-02
Also published as: CN114349794A

Abstract

The invention discloses a biphosphonate platinum complex and a preparation method thereof, wherein a non-leaving group of the complex is composed of ammonia and biphosphonate bipyridine ligand, and the biphosphonate bipyridine ligand and a platinum center form an eight-membered ring. The synthesis method of the complex comprises the synthesis of 2-chloromethylpyridine, [1, 3-di (2-pyridyl) propane-2, 2-diyl ] diethyl ester and the synthesis of the biphosphonate platinum complex, and the synthesis method has the advantages of high synthesis yield, high implementation feasibility and stable product. The complex is difficult to chelate with glutathione, can greatly reduce side effects caused by combination of the platinum drugs and the glutathione, and can be used for overcoming drug resistance of the platinum drugs; the complex has a certain inhibition effect on osteosarcoma cells; the complex may also interact with human serum albumin by means of static quenching. The research provides a new strategy for solving the problem of the drug resistance of the bisphosphonate platinum, and achieves good technical progress.

Description

Platinum bisphosphonate complex and synthesis method and application thereof

Technical Field

The invention belongs to the field of chemical biology, and particularly relates to synthesis and biological application of a platinum bisphosphonate complex.

Background

Cisplatin, also known as cis-diamminedichloroplatinum, is a clinically useful platinum-based anticancer drug. The platinum anticancer drugs such as cisplatin and the like can effectively improve prognosis of osteosarcoma, and the survival rate of osteosarcoma patients in 5 years is improved to 60% -80%. The main mechanism of action of cisplatin is to inhibit proliferation of rapidly dividing cells by damaging DNA, but the acquired drug resistance and serious toxic and side effects of cisplatin greatly limit the wide use of platinum drugs.

Glutathione (GSH) is a soluble tripeptide formed by combining glutamic acid, cysteine and glycine, is a non-protein tripeptide with rich content in cells, has the functions of antioxidation and integration detoxification, and has the intracellular concentration of 0.5-10 mM. GSH as a nucleophile can be chelated with platinum (II) to form Pt (GS) ₂ Chelate, reduce DNA damage caused by bivalent platinum, and reduce the curative effect of platinum drugs. Thus, the interaction between GSH and Pt (II) complexes is believed to play a key role in the mechanisms associated with inactivation of platinum-based drugs, development of drug resistance, and toxic side effects.

Bisphosphonates (BPs) have long been used to treat bone diseases such as osteoporosis, bone metastasis, and multiple myeloma, and are the most commonly used bone targeting molecules in platinum complex design. Pt-BP complexes were found to selectively inhibit Osteosarcoma (OS) cell line U2OS, exhibiting lower acute toxicity, and some of these complexes were found to be dual targeting complexes.

Lipophilicity refers to the ability of a substance to dissolve in fats, oils, lipids, or nonpolar solvents. The lipophilicity parameter (log P) is a key factor affecting the transport of a substance across a membrane, from which the substance's ability to cross a membrane can be determined initially.

DNA is the main target of platinum anti-tumor drugs. To gain a deeper understanding of the anti-tumor activity of DBPP, this patent investigated the effect of DBPP on the conformation of calf thymus DNA (CT-DNA) and the ability to unwind supercoiled pUC19 DNA.

Human Serum Albumin (HSA) plays a vital role in drug transport due to its remarkable binding properties and abundance in plasma.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a bipyridine platinum bisphosphonate complex, a synthesis method and biological application thereof.

In order to achieve the purpose, the technical scheme of the invention is to design a bipyridine platinum bisphosphonate complex, which is called DBPP for short. The non-leaving group of the bipyridyl platinum bisphosphonate complex consists of ammonia and a bipyridyl bisphosphonate ligand, and the bipyridyl bisphosphonate ligand and the platinum center form an eight-membered ring; the structural formula is as follows:

the platinum bisphosphonate complex is in CDCl ₃ In (a) and (b) ¹ H NMR is shown in FIG. 1.

In addition, DBPP is in CDCl ₃ In (a) and (b) ³¹ P NMR is shown in FIG. 2, which shows two signal peaks at 22.88ppm and 20.55ppm, indicating that the two P atoms in DBPP are magnetically unequal.

DBPP in CDCl ₃ In (a) and (b) ¹³ C NMR is shown in FIG. 3, which shows a C signal peak at nine positions, consistent with the number of carbon atom species in DBPP.

DBPP (DBPP) ¹⁹⁵ Pt nuclear magnetic resonance spectrum is shown in figure 4, which shows DBPP ¹⁹⁵ Pt NMR showed a single peak at-2287.651 ppm.

Electrospray mass spectrometry (ESI-MS) of DBPP is shown in FIG. 5, which shows that DBPP has a peak at m/z 698.33, which is consistent with the calculated value (m/z 699.19) in FIG. 5.

In order to facilitate the synthesis and application of the above-mentioned platinum bisphosphonate complex, a synthesis method of the platinum bisphosphonate complex is now proposed, comprising the following steps:

s1: synthesizing 2-chloromethylpyridine, dissolving a certain amount of 2-chloromethylpyridine hydrochloride in a certain amount of distilled water, and adding a certain amount of sodium hydroxide into a certain amount of distilled water to prepare an alkaline solution. Mixing the two solutions, extracting, and purifying the dried filter residues to obtain 2-chloromethylpyridine for later use;

s2: synthesis of [1, 3-bis (2-pyridyl) propane-2, 2-diyl ] bis (phosphonic acid) tetraethyl ester, and adding a certain proportion of 2- [ 2-pyridylethyl ] bis (phosphonic acid) tetraethyl ester and anhydrous DMSO to a mixture of NaH and DMSO in a certain proportion under the protection of nitrogen. The reaction mixture was stirred, 2-chloromethylpyridine was added dropwise, and quenched after completion of the reaction. The mixture was extracted, and the extracts were combined and concentrated. The mixture was dissolved and washed, and after drying, the yellow product was concentrated to give [1, 3-di (2-pyridyl) propane-2, 2-diyl ] di (phosphonic acid) tetraethyl ester.

S3: synthesis of a platinum bisphosphonate complex, cisplatin and AgNO ₃ Stirring in anhydrous DMF in dark, filtering to remove precipitate, and adding [1, 3-di (2-pyridyl) propane-2, 2-diyl into the filtrate]Tetra ethyl di (phosphonate). After stirring again, silver nitrate was added, and after continuing the reaction, the precipitate was removed and CH was added to the reaction mixture ₂ Cl ₂ And stirring was continued, the white precipitate was removed by filtration, and the solvent was removed by concentration. Dissolving the product in CHCl ₃ Diethyl ether is added, and the yellow precipitate is the platinum bisphosphonate complex DBPP.

As a preferred embodiment, in the step S1, 2-chloromethylpyridine hydrochloride (5.292 g,32 mmol) is dissolved in distilled water (7 mL), and sodium hydroxide (1.34 g,32 mmol) is added to distilled water (14 mL) to prepare an alkaline solution. Chloroform extraction, the organic layer was dried over anhydrous magnesium sulfate and the solvent was removed in vacuo. The filter residue is distilled and purified under reduced pressure to obtain light red 2-chloromethylpyridine.

As a preferable solution, in the step S2, the specific operations are as follows: the method for synthesizing the [1, 3-di (2-pyridyl) propane-2, 2-diyl ] di (phosphonic acid) tetraethyl ester according to claim 2, wherein in the step S2, the specific operations are as follows: 273K of tetraethyl 2- [ 2-pyridylethyl ] bis (phosphonic acid) (9.31 g,24.56 mmol) in 20mL dry DMSO was added to a mixture of 60% NaH (1.08 g,29.47 mmol) in 20mL DMSO under nitrogen. The reaction mixture was stirred at 273K for 30min, then at room temperature for 2h, 2-chloromethylpyridine (2.82 g,20 mmol) was added dropwise to the stirred reaction mixture. The reaction solution was stirred at room temperature for another 96 hours, and then quenched by addition of a saturated aqueous ammonium chloride solution. The crude reaction product was extracted with dichloromethane, the organic layers were combined and concentrated under reduced pressure to remove a portion of the solvent. Toluene was added to the crude product, which was washed with water and saturated brine, dried over anhydrous sodium sulfate and then distilled off. Separating by isopropanol/dichloromethane mixed liquid column chromatography to obtain yellow product [1, 3-di (2-pyridyl) propane-2, 2-diyl ] di (phosphonic acid) tetraethyl ester.

As a preferable solution, in the step S3, the specific operations are as follows: cisplatin (75 mg,0.25 mmol) and silver nitrate (42.5 mg,0.25 mmol) were placed in anhydrous DMF (4.0 mL) and stirred at 325K under dark conditions for 24h, the resulting AgCl precipitate was removed by filtration, and tetraethyl [1, 3-bis (2-pyridinyl) propane-2, 2-diyl ] bis (phosphonic acid) (94.27 mg,0.2 mmol) was added to the filtrate. After stirring for 24h at 325K, silver nitrate (34.0 mg,0.20 mmol) was added to the reaction solution, stirring was continued for 24h at 325K, and after removal of the AgCl precipitate by centrifugation, dichloromethane (100 mL) was added to the reaction mixture, stirring was continued for 15min, the white precipitate was removed by filtration, and the solvent was removed under reduced pressure. After the resulting oil was dissolved in chloroform (4 mL), diethyl ether (100 mL) was added and a yellow precipitate was formed as the platinum bisphosphonate complex DBPP.

In order to facilitate the application and implementation of the above-mentioned bisphosphonate platinum complex, several applications of the bisphosphonate platinum complex are proposed, and the bisphosphonate platinum complex can be used for preparing antitumor drugs. The biphosphonate platinum complex is difficult to chelate with glutathione, can overcome the toxic and side effects caused by combination with glutathione when the platinum drug is used for treating osteosarcoma, and can relieve drug resistance of the platinum drug. The bisphosphonate platinum complex has a certain inhibition effect on osteosarcoma cells. The cytostatic effect of the platinum bisphosphonate complex is related to the conformational transition of B-DNA to A-DNA and the unwinding of pUC19 DNA. The complex can also interact with human serum albumin by means of static quenching.

Compared with the prior art, the invention has the advantages that:

the invention provides an octa-ring biphosphonate platinum complex with a novel structure, which adopts bipyridine biphosphonate as a ligand. The complex has high synthesis yield, stable product and difficult chelation with glutathione, can greatly reduce side effects caused by the glutathione, and can be used for relieving the drug resistance of platinum drugs. The complex has a certain inhibition effect on osteosarcoma cells. The complex may also interact with human serum albumin by means of static quenching. The invention provides a new thought for relieving the drug resistance of the biphosphonate platinum complex and has important application value.

Drawings

FIG. 1 is a chart showing the results of DBPP according to example 1 using Bruker DRX-500 test ¹ H-NMR(500MHz,CDCl ₃ ) The data show that DBPP contains amino, bipyridine bisphosphonates.

FIG. 2 is a chart showing the DBPP test result from example 1 using Bruker DRX-500 ³¹ P-NMR(202MHz,CDCl ₃ ) The data show that DBPP contains two magnetically unequal P atoms.

FIG. 3 is a chart showing the DBPP test result from example 1 using Bruker DRX-500 ¹³ C-NMR(125MHz,CDCl ₃ ) The data show that DBPP contains nine different carbon atoms.

FIG. 4 is a chart showing the DBPP test result from example 1 using Bruker DRX-500 ¹⁹⁵ Pt-NMR(107MHz,CDCl ₃ ) The data shows that Pt peaks at-2287.651 ppm in DBPP.

FIG. 5 is an electrospray ionization mass spectrometry (ESI-MS) plot of DBPP from example 1 in methanol using an LCQ jet ESI-MS spectrometer (Thermo Scientific) and using the ISOPRO 3.0 procedure to simulate the isotopic distribution pattern of the complex.

FIG. 6 is a graph of GSH at 310K (2.64×10) in example 2 ^-2 DBPP (1.12X10) in the presence of M ^-2 M) nuclear magnetic resonance spectra obtained at different time intervals using Bruker DRX-500; (a) Is that ³¹ P-NMR chart, (b) is ¹ H-NMR chart.

FIG. 7 is a graph showing the ultraviolet absorption spectrum obtained by using Shimadzu UV 3600 (UV-vis-near-IR) test of DBPP at various concentrations in phosphate buffer and 1-octanol in example 3: before (a) shaking and after (b) shaking. The inset is the maximum absorbance plot of DBPP at 267 nm.

FIG. 8 is a graph showing uptake of DBPP by U2OS obtained by ELAN9000 ICP-MS test after 24, 48, 72 hours of incubation in example 4.

FIG. 9 is a graph showing changes in proliferation of U2OS cells obtained by measuring Optical Density (OD) by Thermo Scientific Varioskan Flash after incubation with DBPP for 24 hours in example 5.

FIG. 10 is a graph showing the change in cell proliferation obtained by measuring Optical Density (OD) using Thermo Scientific Varioskan Flash after treating cells with DBPP for 24, 48 and 72 hours in example 5.

FIG. 11 shows the difference [ DBPP ] after incubation of (a) CT-DNA (0.1 mM) in a buffer (5.0 mM Tris-HCl,50mM NaCl,pH 7.4) for 24 hours at 310K in example 6]/[DNA]Testing the obtained CD spectrogram by using a Jasco J-810 spectrometer in the presence of DBPP with a molar ratio; (b) pUC19 plasmid DNA was co-precipitated with DBPP at 310K for 24 hours and agarose gel electrophoresis images were obtained using a Bio-Rad gel Doc XR imaging system and quantitatively analyzed using quality One software. Lanes 1 are DNA controls, lanes 2-7 r _i 0.30,0.60,0.90,1.20,1.50,1.60 respectively.

FIG. 12 is an electron absorption spectrum of HSA (3.0. Mu.M) in the presence of DBPP (0.0,3.0,6.0, 12.0,18.0,24.0,30.0, 48.0. Mu.M) at various concentrations obtained in example 7 using Shimadzu UV 3600 (UV-vis-near-IR). Arrows show the direction of change in absorbance as the DBPP concentration increases.

Detailed Description

The following describes the embodiments of the present invention further with reference to the drawings and examples. The following examples are only for more clearly illustrating the technical aspects of the present invention, and are not intended to limit the scope of the present invention.

Example 1

The synthesis method of the biphosphonate platinum complex comprises the following steps:

s1: synthesis of 2-chloromethylpyridine:

2-chloromethylpyridine hydrochloride (5.292 g,32 mmol) was dissolved in distilled water (7 mL), sodium hydroxide (1.34 g,32 mmol) was added to distilled water (14 mL) to prepare an alkaline solution, the above two solutions were mixed, extracted with chloroform, and the extracted chloroform layer was dried over anhydrous magnesium sulfate and the solvent was removed in vacuo. The filter residue is distilled and purified under reduced pressure to obtain light red 2-chloromethylpyridine.

S2: synthesis of [1, 3-bis (2-pyridyl) propane-2, 2-diyl ] bis (phosphonic acid) tetraethyl ester:

273K of nitrogen was added to a 20mL DMSO mixture of 60% NaH (1.08 g,29.47 mmol) in 20mL dry DMSO with 2- [ 2-pyridylethyl ] tetra-ethyl bis (phosphonate) (9.31 g,24.56 mmol). The reaction mixture was stirred at 273K for 30min, then at room temperature for 2h, 2-chloromethylpyridine (2.82 g,20 mmol) was added dropwise to the stirred reaction mixture. The reaction solution was stirred at room temperature for another 96 hours, and then quenched by addition of a saturated aqueous ammonium chloride solution. The crude reaction product was extracted with dichloromethane, the organic layers were combined and concentrated under reduced pressure to remove a portion of the solvent. Toluene was added to the crude product, which was washed with water and saturated brine, and dried over anhydrous sodium sulfate, followed by rotary evaporation to remove the solvent. Separating by isopropanol/dichloromethane mixed liquid column chromatography to obtain yellow product [1, 3-di (2-pyridyl) propane-2, 2-diyl ] di (phosphonic acid) tetraethyl ester.

S3: synthesis of a bipyridine bisphosphonate platinum complex DBPP:

cisplatin (75 mg,0.25 mmol) and silver nitrate (42.5 mg,0.25 mmol) were placed in anhydrous DMF (4.0 mL) and stirred at 325K in the absence of light for 24h, the resulting AgCl precipitate was removed by filtration, and tetraethyl [1, 3-bis (2-pyridinyl) propane-2, 2-diyl ] bis (phosphonic acid) (94.27 mg,0.20 mmol) was added to the filtrate. After stirring for 24h at 325K, silver nitrate (34.0 mg,0.20 mmol) was added to the reaction solution, stirring was continued for 24h at 325K, and after removal of the AgCl precipitate by centrifugation, dichloromethane (100 mL) was added to the reaction mixture, stirring was continued for 15min, the white precipitate was removed by filtration, and the solvent was removed under reduced pressure. After the resulting oil was dissolved in chloroform (4 mL), diethyl ether (100 mL) was added and a yellow precipitate was formed as a platinum bisphosphonate complex (DBPP). The characterization of the complexes is shown in FIGS. 1-5.

Example 2

The reactivity of the platinum bisphosphonate complex DBPP prepared in example 1 with GSH was investigated as follows:

platinum bisphosphonate complex DBPP (0.0056 mmol) was dissolved in deuterium water and phosphate buffer solution (PBS 7.87mM, pH 7.4, D ₂ O0.5 mL), and the above-mentioned liquid was put into a nuclear magnetic resonance tube. GSH (2.45 mg,0.0132 mmol) is added into the same nuclear magnetic resonance tube, the nuclear magnetic resonance tube is placed into 310K water bath for hatching, and the GSH is adopted at different time intervals ³¹ P NMR ¹ The H NMR was performed on the solution in the tube, and the detection results are shown in FIG. 6.

The data in FIG. 6a shows that the peaks of DBPP are located at 24.88 and 20.09ppm, respectively, no significant change in the data is observed after 168h of incubation, indicating that DBPP is somewhat inert to glutathione; similar results can be observed for the data of fig. 6 b. The result shows that DBPP is difficult to chelate with glutathione, can overcome toxic and side effects caused by glutathione, and achieves obvious technical progress.

Example 3

The lipophilicity of the platinum bisphosphonate complex DBPP prepared in example 1 was investigated as follows:

and (3) determining the lipophilicity coefficient by adopting a n-octanol shake flask method. To 10mM phosphate buffer, pH 7.4, (50,100,150,200. Mu.M) DBPP solution was added, 2.0mL of the above solution was mixed with an equal volume of 1-octanol, and the mixture was placed in an air bath orbital shaker at constant temperature (25.0.+ -. 0.1 ℃ C.) and centrifuged at 200rpm for 4 hours. After centrifugation at 2500rpm for 15min, the sample separated into two phases; by means of an ultraviolet-visible spectrophotometer (lambda) _max =267 nm), the DBPP concentration in the aqueous solution was measured, the DBPP concentration in the n-octanol phase and the logarithmic P value of the DBPP concentration were calculated to obtain the lipid water distribution coefficient of DBPP, the measurement results were shown in fig. 7, and the calculation results were shown in table 1.

The data in fig. 7 shows that DBPP is regularly distributed between n-octanol and water. The data in Table 1 show that the calculated log P is 1.14.+ -. 0.13, indicating good DBPP lipid solubility.

TABLE 1

。

Example 4

The platinum bisphosphonate complex DBPP prepared in example 1 was investigated for its cellular uptake, as follows:

u2OS cells (15×10) ⁴ ) Inoculated in 6-well plate, cultured for 18h, and then incubated with (50.0. Mu.M, 100.0. Mu.M) DBPP for 24h, 48h and 72h, respectively. At the end of incubation, the culture medium was removed, each well was washed three times with Phosphate Buffered Saline (PBS), pancreatin digested, living cells were counted, and heated with 0.5mL concentrated nitric acid approximately 90℃for 2h. ICP-MS determines the total platinum uptake per well of cells, the results of the analysis are shown in FIG. 8, and the platinum content data are shown in Table 2.

The data in FIG. 8 and Table 2 show that (50.0 and 100.0. Mu.M) DBPP treated U2OS cells for 24h had intracellular platinum concentrations of 0.58.+ -. 0.46 and 1.38.+ -. 0.54. Mu.g/10, respectively ⁶ And (3) cells. After 48 and 72h of DBPP (50.0 and 100.0. Mu.M) treatment, the cells were isolatedThe inner platinum concentration increased to 0.78+ -0.92 and 3.70+ -0.51,1.34 + -0.86 and 4.97+ -0.33 μg/10, respectively ⁶ And (3) cells.

TABLE 2

。

Example 5

The antitumor activity of the platinum bisphosphonate complex prepared in example 1 was investigated, comprising the following steps:

u2OS, MG-63, MCF-7 and HeLa cells were cultured in different media in 96-well plates. U2OS in 10% foetal calf serum DMEM culture. MG-63, MCF-7 and HeLa cells were cultured in 1640 with 10% fetal bovine serum. Subsequently, the above culture broth was added with different concentrations of the platinum bisphosphonate complex DBPP. After incubation for 48h MTT (20. Mu.L, 5mg mL in PBS) was added to each well ^-1 ). After incubation for 4h, the solution in each well was removed, DMSO (200 μl) was added to each well, and absorbance of the dissolved formazan (formazan) was measured at 570nm using an enzyme-linked immunosorbent assay (ELISA) plate-mounted instrument, and the experimental results are shown in fig. 9, fig. 10, and table 3. Inhibition rate IC ₅₀ Is the average of three independent results.

As the data in FIG. 9 show, there was no significant death of MG-63, MCF-7 and HeLa cells after incubation for 24 hours using DBPP concentrations of 50 and 100. Mu.M. As shown in fig. 10, DBPP showed a strong inhibition on U2OS cells under the same conditions. The data in Table 3 show that after 24h incubation at DBPP concentrations of 50 and 100. Mu.M, the relative cell viability was 85.76.+ -. 1.65% and 55.72.+ -. 3.02%, respectively; as incubation time was prolonged from 24h to 48h, relative cell viability decreased to 65.62±3.77% and 48.15 ±0.88%, respectively; when the culture time was prolonged to 72 hours, the relative cell viability was reduced to 58.32.+ -. 4.83% and 30.75.+ -. 1.56%, respectively. The half-inhibition of U2OS by DBPP was about 60. Mu.M, higher than CDDP, consistent with the uptake of DBPP and CDDP by U2OS cells.

TABLE 3 Table 3

。

Example 6

The interaction of the platinum bisphosphonate complex DBPP prepared in example 1 with DNA was investigated, comprising the following steps:

w1: DBPP was bound to CT-DNA at various concentrations in Tris-HCl/NaCl buffer (5 mM Tris-HCl,50mM NaCl,pH 7.4). After incubation of CT-DNA with DBPP in different proportions at 310K for 24 hours in the absence of light, CD spectra were recorded in the wavelength range 220-320 nm. After background subtraction, each sample was assayed three times and averaged. The experimental results are shown in fig. 11.

FIG. 11a shows a CD spectrum of CT-DNA in the presence of DBPP at different concentrations, wherein the negative band at 245nm and the positive band at 275nm represent characteristics of B-DNA. As the [ DBPP ]/[ DNA ] ratio increases from 0 to 0.8, the negative band absorption intensity decreases and the positive band absorption intensity increases. Furthermore, the maximum wavelength of the negative band has a tendency to red shift. This trend indicates a conformational transition from B-DNA to A-DNA.

W2: cleavage of plasmid DNA by DBPP was detected by agarose gel electrophoresis. pUC19 plasmid DNA (200 ng) was treated with DBPP at various concentrations for 24h at 310K. Finally, all samples were added to the mixture containing EB (0.5. Mu.g mL) ^-1 ) In a 1% agarose gel of (2). Electrophoresis was performed in TAE buffer (50 mM acetate and 1mM EDTA) at 100V for 2h. Imaging was performed using a Bio-Rad Gel Doc XR imaging system. The experimental results are shown in FIG. 11b.

FIG. 11b is a DBPP de-swirling pUC19 DNA agarose gel electrophoresis. After co-incubation with DBPP, a blocking of supercoiled DNA was observed, indicating that DBPP bound to pUC19 DNA to unwind pUC19 DNA. Furthermore, the separation between supercoiled DNA and relaxed DNA decreases with increasing molar ratio (ri). The coalescing point ri (c) for complete removal of supercoiled DNA was 1.8, high Yu Shunbo (ri (c) =0.076). The results indicate that DBPP does not interfere as effectively with the tertiary structure of supercoiled DNA as cisplatin, consistent with their cytotoxic activity.

Example 7

The interaction of the platinum bisphosphonate complex prepared in example 1 with HSA was investigated, comprising the following steps:

DBPP was reacted with HSA in PBS (10 mM, pH 7.4) and the reaction was detected by ultraviolet visible spectroscopy (UV-Vis). The absorbance spectra were obtained after continuous reaction of 310K in PBS with HSA (3.0. Mu.M) and DBPP at different concentrations (0.0,3.0,6.0,12.0,18.0,24.0,30.0,48.0. Mu.M) in the absence of light for 24 hours, see FIG. 12.

The data in FIG. 12 shows that the absorption intensity of HSA is enhanced upon addition of DBPP, with a pronounced blue shift of about 12nm (from 278 to 266 nm), indicating a change in the microenvironment of tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe) residues. The results indicate that there is static quenching between DBPP and HSA.

The foregoing is merely a preferred embodiment of the present invention, and it should be noted that it will be apparent to those skilled in the art that several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the scope of the invention.

Claims

1. A platinum bisphosphonate complex, characterized in that the non-leaving group of the complex consists of ammonia and a bipyridine bisphosphonate ligand, the bipyridine bisphosphonate ligand forming an eight membered ring with the platinum centre; the structural formula is as follows:

2. a method of synthesizing the platinum bisphosphonate complex of claim 1, comprising the steps of:

s1: synthesis of 2-chloromethylpyridine: dissolving a certain amount of 2-chloromethylpyridine hydrochloride in a proper amount of distilled water, adding sodium hydroxide solution, extracting a reaction liquid by using chloroform after reaction, and drying an extract liquid to obtain 2-chloromethylpyridine for later use;

s2: synthesis of [1, 3-bis (2-pyridyl) propane-2, 2-diyl ] bis (phosphonic acid) tetraethyl ester: under the protection of nitrogen, adding a certain proportion of 2- [ 2-pyridylethyl ] bis (phosphonic acid) tetraethyl ester and dry DMSO into a mixture of NaH and DMSO in a certain proportion, stirring the reaction mixture, dropwise adding 2-chloromethylpyridine, continuing to react for a plurality of hours after the dropwise adding is finished, and quenching; extracting the mixture, combining the extracts, removing the solvent, dissolving the mixture with toluene, washing, drying, concentrating and separating a yellow product to obtain [1, 3-di (2-pyridyl) propane-2, 2-diyl ] diethyl ester of phosphonic acid;

s3: synthesizing a biphosphinate platinum complex, putting cisplatin and silver nitrate into anhydrous DMF, stirring for reaction in a dark place, centrifuging to remove precipitate, and adding [1, 3-di (2-pyridyl) propane-2, 2-diyl into filtrate]Tetra ethyl di (phosphonate); after stirring, adding a proper amount of silver nitrate into the reaction solution, centrifuging to remove precipitate after the reaction is completed, and adding CH into the reaction mixture ₂ Cl ₂ Stirring continuously, centrifuging to remove white precipitate, spin-evaporating centrifugate to remove solvent, and dissolving the product in CHCl ₃ Diethyl ether is added to obtain yellow precipitate, namely the bipyridine platinum bisphosphonate complex.

3. The method for synthesizing a platinum bisphosphonate complex according to claim 2, wherein in the step S1, 5.292g of 2-chloromethylpyridine hydrochloride is dissolved in 7mL of distilled water, a sodium hydroxide solution is added, the reaction solution is extracted with chloroform after the reaction, the extract is dried with anhydrous magnesium sulfate, and the 2-chloromethylpyridine is obtained by distillation under reduced pressure for standby.

4. The method for synthesizing a platinum bisphosphonate complex according to claim 2, wherein in the step S2, the specific operations are as follows: in a three-necked flask, 20mL of anhydrous DMSO solution containing 9.31g of 2- [ 2-pyridylethyl ] tetra-ethyl bis (phosphonate) was added to 20mL of DMSO mixture containing 1.08g of NaH under 273K nitrogen protection, and the reaction mixture was stirred at 273K for 30min and then at room temperature for 2h; 2.82g of 2-chloromethylpyridine is dripped into the reaction system, the reaction is continuously stirred for 96 hours at room temperature, and then saturated ammonium chloride aqueous solution is added for quenching; extracting with dichloromethane, combining organic layers, concentrating, dissolving the mixture in excess toluene, washing with distilled water and brine sequentially, drying over anhydrous sodium sulfate and concentrating under vacuum; the yellow product was separated by isopropanol/dichloromethane column chromatography to give tetraethyl [1, 3-di (2-pyridyl) propane-2, 2-diyl ] di (phosphonate).

5. The method for synthesizing a platinum bisphosphonate complex according to claim 2, wherein in the step S3, the specific operations are as follows: 75mg of cisplatin and 42.5mg of silver nitrate were put into 4.0mL of anhydrous DMF, stirred for 24 hours under dark conditions at 325K, the resulting AgCl precipitate was removed by filtration, and 94.27mg,0.20mmol of tetraethyl [1, 3-bis (2-pyridyl) propane-2, 2-diyl ] bis (phosphonic acid) was added to the filtrate;

stirring for 24 hours under 325K in dark, continuously adding 34.0mg of silver nitrate into the reaction solution, continuously stirring for 24 hours under 325K, centrifuging to remove AgCl precipitate, adding 100mL of dichloromethane into the reaction mixture, stirring for 15min, filtering to remove white precipitate, and removing the solvent; after the generated oil is dissolved in 4mL of chloroform, 100mL of diethyl ether is added, and the yellow precipitate formed is the platinum bisphosphonate complex DBPP.

6. Use of a platinum bisphosphonate complex of claim 1 in the preparation of a medicament for the treatment of osteosarcoma.

7. The use according to claim 6, wherein the bisphosphonate platinum complex is difficult to chelate with glutathione, which can overcome the toxic and side effects caused by the combination with glutathione when the platinum-based drug is used for treating osteosarcoma, and reduce the drug resistance of the platinum-based drug.

8. The use according to claim 6, wherein the platinum bisphosphonate complex interacts with human serum albumin by means of static quenching to treat osteosarcoma.