Organic diphosphate ligand as well as preparation method and application thereof

Abstract

The invention discloses an organic diphosphate ligand and a preparation method and application thereof, the structural formula of the ligand is shown in the specification, and the preparation method comprises the following steps: dissolving tetraethyl methylene diphosphate in an organic solvent, adding a catalyst and ethyl bromoacetate, and reacting to obtain an ethyl diphosphate intermediate; and reacting the diphosphate ethyl ester intermediate with acid to obtain the organic diphosphate ligand. The organic diphosphate ligand can efficiently chelate nuclide ions, can be used for removing nuclides contaminated on the body surface and can also promote discharge of nuclides in the body.

Description

Technical Field

[0001] The present invention relates to the field of chemical synthesis, and in particular to an organic bisphosphate ligand and its preparation method and application Background Art

[0002] With the rapid development of the nuclear energy industry, nuclear testing, nuclear accidents, and spent fuel disposal processes can all potentially release radionuclides into the environment. These radionuclides can then be introduced into the body through contamination of body surfaces and wounds, as well as through the digestive and respiratory tracts. The skin, being the body's largest organ, is susceptible to radionuclides. Once contaminated with radionuclides or wounds, they can penetrate the epidermis, enter the dermis, and then reach the subcutaneous tissues and enter the bloodstream. This rapid process requires prompt decontamination measures to prevent radionuclides from entering the body. Once these radionuclides enter the bloodstream, they can rapidly deposit in organs, causing internal radiation exposure. Furthermore, the chemical toxicity of heavy metals can cause renal failure, hepatic and renal dysfunction, lung cancer, osteosarcoma, and other conditions, which can be fatal in severe cases. Therefore, developing highly effective and low-toxic radionuclide decontamination solutions and excretion-promoting agents is a top priority for radiation protection.

[0003] To address the issue of surface contamination with radionuclides, the International Atomic Energy Agency's Emergency Health Organization (WHO-IAEA 2002) recommends potassium permanganate (KMnO4), ZnNa3-DTPA, and H2O2 solutions as decontamination agents for radionuclides. my country's GBZ / T216-2009, "Specifications for the Treatment of Radionuclides on Human Surfaces," recommends the use of 1% DTPA or 10% EDTA solutions for the decontamination of plutonium and transplutonium elements, while 1.4% bicarbonate solutions can be used for uranium contamination. Currently, internationally, water, weakly acidic soapy water, or decontamination solutions containing a certain amount of DTPA are primarily used to remove radionuclides from the skin surface. Water and soapy water are the most common methods, primarily due to the potential for skin irritation caused by the addition of chemicals, such as bleach, potassium permanganate, and hydrogen peroxide, which are highly oxidizing and can cause damage to the skin. However, the decontamination effect of simply using water or soapy water is very limited, especially for injured skin. Adding a highly effective chelating agent to the decontamination solution can significantly improve decontamination efficiency. In recent years, researchers at home and abroad have attempted to enhance the decontamination efficiency of decontamination solutions by incorporating actinide chelating agents. Regarding radionuclide decontamination agents, the geometric configuration of DTPA does not meet the coordination requirements for uranium, limiting its application in the decontamination of uranium surface contamination. To further enhance radionuclide decontamination capabilities, studies have shown that 1-hydroxyethane-1,10-diphosphoric acid (HEDP) solution is more effective than DTPA for decontamination of Pu(IV) and U(VI). Research has also shown that a calixarene nanoemulsion can effectively decontaminate uranium. For uranium-contaminated pig ears, the calixarene nanoemulsion effectively adsorbs uranium and prevents its penetration into the dermis. However, this type of decontamination agent is highly hepatotoxic and hematotoxic, and once absorbed through the skin and into the bloodstream, it poses a threat to the human body.

[0004] To address the problem of radionuclide contamination in the body, radionuclide chelators or excretion promoters can be used to accelerate the excretion of radionuclides in the body. The current clinical excretion promoter for actinides is diethylenetriaminepentaacetic acid (DTPA) salt. DTPA has a good excretion-promoting effect on transuranium elements plutonium and americium, but its excretion-promoting effect on uranium, the main raw material of the nuclear industry, is very limited. The reason is that the geometric configuration of DTPA does not conform to the coordination mode of uranium, resulting in weak chelation ability for uranium. In a complex physiological environment, it cannot compete for chelation of uranium and promote uranium excretion. Based on the limitations of the clinical excretion promoter DTPA salt, domestic and foreign researchers have developed a series of new actinide excretion promoters, including carboxylic acid ligands, siderophore ligands (o-phenol, hydroxypyridone, amine oxime) and phosphate ligands. Among these, hydroxypyridone, a siderophore ligand, has a strong affinity for actinides. Studies have shown that the octadentate ligand 3,4,3-LI (1,2-HOPO) and the tetradentate ligand 5-LIO (Me-3,2-HOPO) have excellent excretion-promoting effects on actinides such as plutonium, americium, neptunium, and uranium. However, their only drawback is that these two ligands have little effect on uranium deposited in bone. To enhance the excretion-promoting effect on uranium in bone, researchers have obtained the tetradentate ligand 5LIO-1-Cm-3,20HOPO and the hexadentate ligand TAM-2LI-MAM2 by weakening intramolecular hydrogen bonds and matching the uranium coordination environment, significantly enhancing the uranium excretion-promoting effect on bone. Secondly, phosphate ligands generally have good bone targeting properties. 1-Hydroxyethane-1,10-diphosphate (HEDP) is a therapeutic compound widely used to treat hypercalcemia and bone disease. Its metabolism and toxicity have been well studied, and it has been shown to be effective against internal uranium contamination, making it a promising actinide chelator. Both oral and subcutaneous injections of HEDP can effectively improve renal function in animals exposed to lethal doses of uranium. However, to date, most research on phosphate-based excretion enhancers has only focused on in vitro chelation ability, and no detailed studies have been reported on the toxicity, selectivity, and in vivo excretion-promoting effects of the ligands.

[0005] Based on the above research progress, there is still an urgent need to develop new chelating agents for the treatment of surface radionuclide contamination and in-body radionuclide contamination. Phosphate ligands have strong uranium chelating ability, but their performance in surface radionuclide decontamination and in-body radionuclide excretion promotion has not been fully verified. Therefore, designing new phosphate chelating agents that conform to the radionuclide coordination pattern to achieve efficient surface radionuclide decontamination and in-body radionuclide excretion promotion is of great significance for radiation protection and ensuring the safety of public life. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an organic bisphosphate ligand (DPC) and its preparation method and application. DPC can not only remove radionuclides contaminated on the body surface but also promote the excretion of radionuclides in the body. At the same time, it has low biological toxicity and little harm, and can be widely used in surface radionuclide decontamination and in vivo radionuclide excretion promotion.

[0007] In order to solve the above technical problems, the first aspect of the present invention provides an organic bisphosphate ligand, the structural formula of the ligand is:

[0008]

[0009] The second aspect of the present invention provides a method for preparing the organic bisphosphate ligand described in the first aspect, comprising the following steps:

[0010] S1. Dissolve tetraethyl methylene diphosphate of formula (1) in an organic solvent, add a catalyst and ethyl bromoacetate, and react to obtain an ethyl bisphosphate intermediate of formula (2). The reaction route is:

[0011]

[0012] S2. The ethyl bisphosphate intermediate of formula (2) reacts with an acid to obtain the organic bisphosphate ligand.

[0013] Furthermore, the organic solvent is tetrahydrofuran (THF) and / or N,N-dimethylformamide (DMF).

[0014] Furthermore, the catalyst is one or more of sodium bis(trimethylsilyl)amide, sodium methoxide, sodium ethoxide, and quaternary ammonium base.

[0015] Furthermore, the molar ratio of tetraethyl methylene diphosphate to ethyl bromoacetate is 1:(2-10), preferably 1:(2-4).

[0016] Furthermore, the molar ratio of the tetraethylmethylene diphosphate to the catalyst is 1:(1.2-2.0).

[0017] Furthermore, the reaction of S1 was monitored by thin layer chromatography (EA / MeOH=10 / 1), and the reaction time was 20-30 h.

[0018] Furthermore, after the reaction of S1 is completed, the solvent is distilled off under reduced pressure, dichloromethane (DCM) and / or chloroform are added to dissolve, the insoluble matter is removed by filtration, the filtrate is collected and concentrated, and the product is purified by column chromatography (gradually increasing the polarity from pure EA to EA / MeOH = 10 / 1).

[0019] Furthermore, the acid is hydrochloric acid or glacial acetic acid.

[0020] Furthermore, the reaction condition in S1 is an ice water bath, and the reaction condition in S2 is a boiling water bath.

[0021] Furthermore, step S1 is performed under an argon and / or nitrogen atmosphere.

[0022] The third aspect of the present invention provides the use of the organic bisphosphate ligand described in the first aspect in the surface washout or in vivo excretion promotion of radionuclides.

[0023] Furthermore, the radioactive nuclides are one or more of actinide elements such as uranium, thorium, neptunium, plutonium, americium, etc., lanthanide elements such as europium, cerium, lanthanum, etc., and heavy metals such as mercury, chromium, and lead.

[0024] Beneficial effects of the present invention:

[0025] The organic bisphosphate ligand of the present invention can not only remove radioactive nuclides contaminated on the body surface but also promote the excretion of nuclides in the body. In terms of nuclide decontamination, the organic bisphosphate ligand DPC has a strong chelating ability for actinides and can efficiently remove radioactive nuclides contaminated on the body surface, especially in terms of delayed decontamination. In addition, the ligand has excellent water solubility and does not require the introduction of organic solvents for pre-dissolution, which can reduce irritation and damage to the skin. In terms of nuclide excretion promotion, the organic bisphosphate ligand has low toxicity and can efficiently promote the excretion of actinide uranium in the kidneys and bones, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the H NMR spectrum of the ethyl bisphosphate intermediate;

[0027] Figure 2 is the H NMR spectrum of the organic bisphosphate ligand;

[0028] Figure 3 is the crystal structure of compound 1 in the b direction;

[0029] Figure 4 is the connection mode and coordination pattern of compound 1 on the c-axis;

[0030] Figure 5 To characterize the comprehensive cellular toxicity of uranyl and ligands;

[0031] Figure 6 This is a diagram representing the effect of DPC on promoting uranium excretion in the kidney and bones;

[0032] Figure 7 To characterize the decontamination effect of DPC and DTPA on uranium. DETAILED DESCRIPTION

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0034] Example 1

[0035] This embodiment relates to a method for preparing an organic bisphosphate ligand.

[0036] A 25 mL three-necked flask was evacuated and then flushed with nitrogen. 490 mg (1.70 mmol, 1 eq) of tetraethylmethylene diphosphate and 10 mL of anhydrous THF were added. Under an ice-water bath, 1.10 g (40% in THF, 2.75 mmol, 1.6 eq) of sodium bis(trimethylsilyl)amide was slowly added by injection, causing the solution to turn light brown. 710 mg of ethyl bromoacetate (4.25 mmol, 2.5 eq) was then added by injection, causing the solution to turn red. The reaction was stirred at approximately 0°C. The reaction was monitored by thin-layer chromatography (EA / MeOH = 10 / 1) during the reaction. The reaction was complete after stirring for approximately 22 hours. The solvent was removed by distillation under reduced pressure to yield a yellow viscous product. DCM was added for dissolution, and the insoluble material was removed by filtration. The filtrate was collected and concentrated. Column chromatography (gradually increasing the polarity of EA from pure EA to EA / MeOH = 10 / 1) afforded 460 mg of the ethyl bisphosphate intermediate as a pale yellow oil in approximately 73% yield. The results of H NMR spectrum test are shown in Figure 1 , specifically: 1 H NMR (400MHz, CDCl3): δ4.18 (m, 10H), 3.11 (tt, J = 16Hz, 4.4Hz, 1H), 6.72 (td, J = 10.4Hz, 4Hz, 2H), 1.33 (td, J = 4.4Hz, 1.6Hz, 12H), 1.28 (t, J = 4.8Hz, 3H).

[0037] 500 mg (1.34 mmol) of ethyl bisphosphate intermediate was added to a round-bottom flask, and 30 mL of 1M hydrochloric acid solution was added. The mixture was refluxed for 24 h and the solvent was removed by distillation under reduced pressure to obtain a colorless transparent liquid. After vacuum drying, 300 mg of a white solid organic bisphosphate ligand was obtained. The yield was approximately 96%. The results of the H NMR test are shown in Figure 2 , specifically: 1 H NMR (400 MHz, D2O): δ 2.81 (m, 3H); according to LC-MS [MH] - The calculated m / z was 232.96, and the experimental result was 232.93.

[0038] Example 2

[0039] This example relates to the study of the coordination mode between an organic bisphosphate ligand and uranyl.

[0040] (H3O) 4.5 K 3.5(Synthesis of (UO2)4[CH2CH(COOH)(PO3)2]4 (Compound 1): UO2(NO3)2·6H2O (0.1004 g, 0.25 mmol), DPC (0.0936 g, 0.25 mmol), KNO3 (0.0505 g, 0.075 mmol), and CH3OH (2 mL) were loaded into a 20 mL internal volume polytetrafluoroethylene-lined autoclave. The reaction vessel was sealed and heated to 150 °C in a box furnace. After reacting for three days, it was slowly cooled to 50 °C with a cooling period of 12 h. The reaction product was washed multiple times with distilled water and ethanol, and then dried to obtain yellow plate-like crystals, yielding Compound 1 (with a uranium-based yield of 50.1%).

[0041] Single crystal X-ray diffraction (SC-XRD): A single crystal of Compound 1 was selected using a polarized light microscope, then mounted on a low-temperature sample loop, and optically aligned on a Bruker D8 Venture single crystal X-ray diffractometer. A turbo X-ray source (Mo Kα radiation, ) was used to provide X-rays, and a CMOS detector was used at room temperature. Data frames were collected using the APEX II program and processed using SAINT in the program. For Compound 1, within the range of 2.20 < 2θ < 24.62, -15 < h < 15, -16 < k < 16, -17 < l < 17, a total of 9054 diffractions were collected, and 6688 diffraction points were used for data fitting. All structures were solved by the direct method and refined by full-matrix least squares on F2 using the SHELXTL-2014 / 2017 program. Residual electron cloud density was removed by SQUEEZE operation in the PLATON program. Table 1 summarizes the crystallographic data of Compound 1. Tables 1 and 2 list the selected bond lengths and bond valence sums (BVS) values of Compound 1.

[0042] Table 1: Crystallographic data of Compound 1

[0043]

[0044]

[0045] Table 2: Uranium-oxygen bond lengths and BVS values of Compound 1

[0046] [[ID=​​​​Cation. All four uranyl ions present a seven-coordinate pentagonal bipyramidal configuration, with the five equatorial oxygen atoms coming from the four oxygen atoms of two DPC ligands and one oxygen atom of one DPC ligand. The uranyl ions are connected to the adjacent uranyl ions along the c direction through the DPC ligands. In this structure, the carboxyl functional group on the ligand DPC never participates in the coordination. The crystal structure of compound 1 in the b direction is shown in Figure 3 The black, red, blue, and yellow balls represent carbon, oxygen, phosphorus, and uranium atoms, respectively. The connection mode of compound 1 on the c-axis is shown in Figure 4 (a), the coordination mode of compound 1 is shown in Figure 4 (b).

[0048] Example 3

[0049] This example relates to a study on the effect of organic bisphosphate ligands on uranium excretion.

[0050] Evaluation of biological toxicity of materials at the cellular level:

[0051] The combined cytotoxicity of DPC and ZnNa3-DTPA with uranyl was evaluated at the cellular level. Because actinide uranium is primarily deposited in renal tubular epithelial cells, rat proximal tubular epithelial cells (NRK-52E) were used in this study. Cell survival was measured using the CCK-8 assay in response to varying concentrations of chelating agents and uranium.

[0052] NRK-52E cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum, 1% penicillin, and streptomycin at 37°C in a cell culture incubator with 5% CO2. Cells in the logarithmic growth phase were collected and broken up to ensure uniform distribution. The cells were then seeded into a sterile 96-well culture dish at a seeding rate of 3×10 cells per well. 3 The cells were then placed in a cell culture incubator at 37°C with 5% CO2 for 24 hours. The experiment was divided into a control group and an experimental group. After 24 hours, the culture medium was removed after the cells adhered to the wall. 100 μL of 12.4 μM uranium and different concentrations of ligand (3.125 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL) were added to the experimental group. Each experimental group contained 6 parallel samples. The control group was given blank culture medium, and the two groups were placed in a cell culture incubator and incubated for 48 hours. After 48 hours, the old culture medium was removed, and 100 μL of DMEM / F12 (1:10) culture medium containing 10 μL of CCK-8 was added to each well. After incubation for another 1-2 hours, the absorbance (OD) of each well at 450 nm was measured using a microplate reader.

[0053] Cell survival rate = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0054] refer to Figure 5 As the drug concentration increased, cell survival rates in the ZnNa3-DTPA and DPC groups did not show a significant decrease. In particular, when the drug concentration reached 100 μg / mL, the overall cell survival rate remained around 80%. The cell survival rate in the DPC group was slightly lower than that in the ZnNa3-DTPA group at different concentrations, indicating that DPC has low cytotoxicity and meets the basic requirements of a low-toxic ovulation-promoting agent.

[0055] Evaluation of DPC's ability to promote uranium excretion at the mouse individual level:

[0056] This part uses Kunming mice to study the effect of the ligand DPC on the excretion of internally contaminated uranium. The effect of DPC on the excretion of uranium is studied 72 hours after pre-administration. The specific experiments are as follows:

[0057] Preparation of stock solution: U(VI) toxicity dose is 0.5mg / kg, ZnNa3-DTPA dosage is 100mg / kg, DPC dosage is 49.16mg / kg. Preparation of toxicity and drug storage solution: 1.6mg 238 UO2(NO3)2·6H2O was dissolved in 10 mL of 0.14 M NaCl solution (pH 4-5). At the same time, 10.7 mg of DPC was dissolved in 1.4 mL of 0.14 M NaCl solution, and 21.77 mg of ZnNa3-DTPA was dissolved in 3.0 mL of 0.14 M NaCl solution. The pH was adjusted to 7-8 with 0.1 M NaOH.

[0058] The mice used in this experiment were all female Kunming mice, about 8 weeks old, weighing about 31.10±0.74g. Fifteen mice were randomly divided into three groups of five. The three groups of mice were injected with 0.2mL of normal saline, ZnNa3-DTPA, and ligand DPC through the tail vein, respectively. One hour later, the three groups of mice were injected with 0.2mL of uranium solution (0.5mg 238 U / kg). Rats were fed normally for 72 hours before being sacrificed. The kidneys and femurs, the primary organs for uranium deposition, were harvested. The tissues were weighed, digested using a microwave digester, and then brought to a constant volume. The tissues were then filtered through a 220 nm filter and uranium content was measured using ICP-MS and converted to μg / g tissue.

[0059] The radionuclide content per gram of tissue = radionuclide concentration in the test solution × solution volume / tissue weight.

[0060] like Figure 6As shown, 72 hours after administration, the uranium content in the kidney and femur of the control mice was 8.2±1.73μg / g and 3.09±0.35μg / g, respectively. In the DPC group, the uranium content in the kidney and femur of mice was effectively reduced to 0.65±0.1μg / g and 1.7±0.14μg / g, respectively. Compared with the control group, uranium removal was achieved by 92.07% in the kidney and 44.98% in the femur, significantly enhancing the excretion effect. In the ZnNa3-DTPA group, uranium content in the kidney and femur was 6.52±2.4μg / g and 4.0±0.52μg / g, respectively. Compared with the control group, the excretion rate of uranium in the kidney was 20.49%, while there was almost no excretion effect in the femur. These results indicate that DPC has excellent excretion-promoting properties for uranium under internal contamination conditions, effectively removing not only uranium deposited in the kidney but also uranium deposited in the bones, suggesting promising application prospects.

[0061] Example 4

[0062] This example relates to a study of the effect of organic bisphosphate ligands on uranium decontamination.

[0063] First, prepare 8g / L uranium solution as the simulated poison solution, 2% DTPA and 2% DPC solution as the decontamination solution. Cut the pig skin into 1.2×1.2cm 2 Use a marker to mark a 1×1cm rectangle. 2 The infected area was large enough to be cleaned with tweezers, and the remaining area was large enough for the skin to be grasped with tweezers. According to the different disinfection materials, the patients were divided into deionized water, 2% DTPA and 2% DPC groups.

[0064] Use a pipette to apply 40 μL of an 8 g / L uranium solution to a designated area of ​​pig skin, evenly covering the affected area. Before and after exposure, weigh the pig skin on an electronic balance to determine the exact amount of exposure. After 3 minutes of exposure, allow the surface solution to dry. Use a pipette to apply 5 mL of 2% DTPA and 2% DPC detergent, respectively, for decontamination. Rinse with 5 mL of deionized water. Finally, use ICP-AES to determine the uranium concentration in each sample. Calculate the decontamination rate using the following formula: Decontamination rate (%) = (uranium concentration in the test solution × total mass of the test solution) / (concentration of the uranium solution × mass of the uranium solution) × 100%.

[0065] The cleaning effect is as follows Figure 7 As shown, the decontamination efficacy of 2% DPC was 88.77%, while that of DTPA was 59.47%. The immediate decontamination efficacy of 2% DPC was significantly higher than that of 2% DTPA. Combined with DPC's low cytotoxicity, it is expected to be rapidly converted into a radionuclide decontamination product for nuclear emergency treatment.

[0066] In summary, the organic bisphosphate ligand DPC has low cytotoxicity. At the same time, it can not only remove radionuclide contamination on the body surface but also promote the excretion of radionuclide in the body. Its excretion-promoting and decontamination effects are significantly better than DTPA. It is expected to be rapidly transformed and applied in radionuclide decontamination.

[0067] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. An organic bisphosphate ligand, characterized in that The structural formula of the ligand is:

2. A method for preparing the organic bisphosphate ligand according to claim 1, characterized in that: The steps include: S1. Dissolve tetraethyl methylene diphosphate of formula (1) in an organic solvent, add a catalyst and ethyl bromoacetate, and react to obtain an ethyl bisphosphate intermediate of formula (2). The reaction route is: S2. The ethyl bisphosphate intermediate of formula (2) reacts with an acid to obtain the organic bisphosphate ligand.

3. The method for preparing an organic bisphosphate ligand according to claim 2, wherein: The catalyst is one or more of sodium bis(trimethylsilyl)amide, sodium methoxide, sodium ethoxide and quaternary ammonium base.

4. The method for preparing an organic bisphosphate ligand according to claim 2, wherein: The molar ratio of tetraethyl methylene diphosphate to ethyl bromoacetate is 1:(2-10).

5. The method for preparing an organic bisphosphate ligand according to claim 2, wherein: The molar ratio of the tetraethylmethylene diphosphate to the catalyst is 1:(1.2-2.0).

6. The method for preparing an organic bisphosphate ligand according to claim 2, wherein: The acid is hydrochloric acid or glacial acetic acid.

7. The method for preparing an organic bisphosphate ligand according to claim 2, wherein: The reaction conditions in S1 were an ice water bath, and the reaction conditions in S2 were a boiling water bath.

8. The method for preparing an organic bisphosphate ligand according to claim 2, wherein: Step S1 is performed under an argon and / or nitrogen atmosphere.

9. Use of the organic bisphosphate ligand according to claim 1 in surface decontamination or in vivo excretion promotion of radionuclides.

10. The use according to claim 9, wherein the radioactive nuclide is one or more of the actinide elements uranium, thorium, neptunium, plutonium, americium, the lanthanide elements europium, cerium, lanthanum, and the heavy metals mercury, chromium, and lead.

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