CN111138415B - Application of bifunctional chelating agent in uranium excretion promotion and radiation protection - Google Patents

Abstract

The invention discloses a novel bifunctional chelating agent and its application in promoting the excretion of nuclide uranium and radiation protection. The present invention utilizes the uranium chelating effect of cyclophanamine and the free radical scavenging and radiation damage protection effect of Tempol nitroxide free radicals, and couples cyclophanamine and Tempol to obtain a novel bifunctional chelating agent, which simultaneously achieves the promotion of uranium contamination and the protection against radiation damage. The purpose of internal radiation damage protection plays the role of double-effect drug therapy. It has the advantages of simple synthesis process, low production cost, strong uranium chelation promotion and damage prevention double effects, and can be prepared into dosage forms such as tablets, capsules, solutions, injections and dripping pills, and is suitable for nuclear weapons, nuclear weapons, etc. Injury treatment of people exposed to nuclide uranium in accidents and special environments.

Description

Application of bifunctional chelating agent in uranium excretion promotion and radiation protection

technical field

The invention relates to a dual-functional chelating agent and its application in promoting the emission of nuclide uranium and radiation protection, which can be used for injury treatment of people exposed to nuclide uranium in nuclear weapons, nuclear accidents and special environments, and belongs to the technical field of medicine.

Background technique

With the rapid development of nuclear energy in my country, the demand for uranium is increasing day by day. However, a large number of radioactive and non-radioactive pollutants will be produced during the mining and production process of uranium, which poses a serious threat to the entire ecological environment and human health. Uranium is not only a heavy metal element but also a very harmful radioactive element. Once it is transferred into the human body through food, water or other means, it is extremely difficult to excrete it out of the body. These uranium elements will form a lifetime radioactive internal exposure in the human body, resulting in radiation damage and Chemical damage causes body disease and causes great harm to health.

Generally, uranium compounds are most common with tetravalent and hexavalent uranium, and the uranium that enters the body fluid usually exists in the form of uranyl ions (UO ₂ ²⁺ ). After UO ₂ ^{2 +} enters the blood circulation, it can quickly invade various tissues and organs, kidneys, bones, etc. , lungs are vulnerable to uranium damage organs. At the same time, uranium will produce alpha rays and a small amount of beta rays and gamma rays during the decay process. The α-rays radiated by uranium will form a large number of free radicals, resulting in an imbalance in the proportion of endogenous free radicals, degeneration or apoptosis of nearby cells, causing material metabolism and energy metabolism disorders, and inducing pathological changes in the body. If uranium accumulates in the body for a long time, it will bring long-term harm to the human body. "Gulf War Syndrome" and "Balkan Syndrome" are typical cases caused by uranium poisoning.

Drug treatment measures have always been an important guarantee for the protection of radiation damage in nuclear emergencies. The damage of uranium should not only consider the acute chemical damage of uranium, but also the radiation damage of uranium. Therefore, the treatment of uranium contamination should consider two aspects at the same time , that is, uranium excretion stimulation and radiation protection therapy.

Contents of the invention

The object of the present invention is to provide a chelating agent (I) for uranium contamination with double functions of excretion promotion and radiation protection and its synthesis method.

The realization process of the present invention is as follows:

The compound shown in general structural formula (I),

。

.

The preparation method of the compound shown in general structural formula (I), comprises the following steps:

(1) Using 1,2-diphenylethylenediamine as a substrate, reacting with acid chloride represented by structural formula (II) to obtain amide product a;

(2) hydrolyzing the ester group in the amide product a to make an acid chloride and reacting with substituted 1,2-diphenylethylenediamine to obtain the amide product, and further reducing to obtain compound b;

(3) compound b is oxidized to obtain compound c;

(4) Compound c is further reacted with 4-OH-Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine oxide) to synthesize target compound (I);

。

.

In step (1) of the above preparation method, the molar ratio of 1,2-diphenylethylenediamine, acid chloride represented by structural formula (II) to NaOH is 1:(2~5):(2~5), and the reaction temperature is 0-100°C.

In step (2) of the above preparation method, the molar ratio of amide product a, oxalyl chloride and 1,2-diphenylethylenediamine is 1:(1~3):(0.5~1), and the reaction temperature is -20- 100°C.

In step (4) of the above preparation method, the molar ratio of compound c, DCC, DMAP and 4-OH-Tempol is 1:(4~8):(4~8):(4~12), and the reaction temperature is 0- 100°C.

Chelation and pharmacological test results prove that compound I-0 shown in formula (I) of the present invention, I-1, I-2, I-3 and I-4 (corresponding to n=0,1,2, 3,4) It can form a 1:1 type chelate with uranium, through the uranium chelation of cyclophanamine and the free radical scavenging and radiation damage protection of Tempol nitroxide free radicals, it shows good effect on uranium-infected animals It can reduce the accumulation of uranium in the kidney and bone, increase the urinary excretion of uranium in the body, and is obviously better than the positive drug DTPA-Ca. Moreover, administration of I-0, I-1, I-2, I-3 and I-4 can significantly reduce the generation of cellular ROS and alleviate cell damage caused by radiation. In the whole animal uranyl exposure experiment, I-0, I-1, I-2, I-3 and I-4 can significantly improve the toxicity of the poisoned animals through their strong chelation and excretion promotion and radiation damage protection. The survival rate has a good therapeutic effect on uranium-infected animals.

The compound of formula (I) of the present invention can be used as a pharmaceutical composition formed with active ingredients and suitable pharmaceutical carriers or excipients. After the compound is mixed with one or more physiologically acceptable excipients or carriers (such as physiological saline, water for injection, starch, dextrin, etc.), it can be formulated into injection, oral, oral, sublingual, implanted , a pharmaceutical composition for local administration or rectal administration, prepared into various pharmaceutical dosage forms. The carrier includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field, and sweeteners can also be added if necessary. Flavoring agents, flavoring agents, etc. The present invention is preferably administered by injection.

The present invention utilizes the uranium chelating effect of cyclophanamine and the free radical scavenging and radiation damage protection effect of Tempol nitroxide free radicals, and couples cyclophanamine and Tempol to obtain a novel bifunctional chelating agent (I), simultaneously achieving the effect of uranium contamination. For the purpose of promoting discharge and protecting against internal radiation damage, it plays the role of double-effect drug therapy, and effectively solves the problem of injury treatment of people exposed to nuclide uranium in nuclear weapons, nuclear accidents, and special environments.

Detailed ways

The present invention will be further described below by way of examples, but the scope of the present invention is not limited to the following examples.

Embodiment 1: the synthesis of chelating agent I-2 in structural general formula (I)

Dissolve 1,2 diphenylethylenediamine (2.12g, 10 mmol), NaOH (880mg, 22 mmol) in ³⁰ mL of dichloromethane solution, add acetyl chloride cinnamate (984mg, 60 mmol) stirred overnight. TLC was used to detect the reaction process. After the reaction was completed, add 2 M HCl to adjust the pH to 1, extract with ethyl acetate (30 mL x 3), collect the organic phase, wash with saturated sodium chloride, dry with anhydrous sodium sulfate, spin dry, 200-300 By silica gel column chromatography, 3.97 g of the target compound 1 was obtained, with a yield of 82%.

To a 100 mL flask containing compound 1 (3.97g, 8.2 mmol) was added tetrahydrofuran: methanol: water = 4:1:1 (volume ratio) solvent 30 mL, then NaOH (1.28g, 32 mmol) was added at 70 ^o C under reflux for 3h. After the reaction was detected by TLC, the pH was adjusted to 1 with 2M HCl, extracted with ethyl acetate (30 mL x 3), the organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and spin-dried to obtain the target compound 2 (3.47g, 8.1 mmol). It was directly put into the next reaction without purification.

Take a dry 50 mL flask, weigh compound 2 (1.7 g, 4 mmol), add 20 mL of dichloromethane, 2 mL of oxalyl chloride, a drop of N, N dimethylformamide, a large number of bubbles are generated, and stir at room temperature for 1 h Finally, spin dry and set aside. Take another dry 100 mL flask, add 20 mL of dichloromethane, 3 mL of triethylamine, 1,2 diphenylethane-1,2 diamine (2.12 g, 10 mmol), and use 10 mL of spin-dried acid chloride Dichloromethane was dissolved, and it was slowly added to the reaction solution with a syringe, and a large amount of white smoke was generated. After reacting at room temperature for 3 h, TLC detected that the raw material had reacted completely. Adjust the pH to 1 with 2M HCl, extract the reaction solution with dichloromethane (30 mL×3), combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, and rotary evaporate under reduced pressure to obtain a light yellow oily liquid, 200-300 mesh Silica gel column chromatography (PE:EA=10:1~5:1) yielded 2.0 g of target compound 3 with a yield of 85%.

Take a 250 mL flask, add compound 3 (2.0 g, 3.4 mmol), THF 30 mL, and place it in a low-temperature reactor at -78 ^o C. Slowly add 6 mL (15 mmol, 2.5M) of lithium aluminum hydride in n-hexane. After the dropwise addition, the reaction solution was placed at 0 ^o C and continued to stir for 6 h. After the completion of the reaction detected by TLC, slowly add 50 mL of saturated ammonium chloride solution to quench the reaction, extract with ethyl acetate (30 mL x 3), collect the organic phase, wash with saturated sodium chloride, dry over anhydrous sodium sulfate, spin dry, 200 ~300 mesh silica gel column chromatography (PE:EA=10:1~5:1) yielded 1.62 g of target compound 4 with a yield of 90%.

Take a 100 mL flask, under CO gas protection, add compound 4 (1.62g, 3.0 mmol), add tin tetrachloride (3.12g, 12 mmol), silver oxide (277.2mg, 1.2 mmol), 1,1- Dichloromethyl ether 30 mL. Stir at normal pressure for 10 hours. After the completion of the reaction as detected by TLC, the reaction solution was concentrated to dryness. Adjust pH = 13 with 2M NaOH solution. The aqueous phase was extracted with ether to remove impurities, and the pH was adjusted to 1 with concentrated hydrochloric acid. (30 mL x 3), collected the organic phase, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, spin-dried, 200~300 mesh silica gel column chromatography (PE:EA=10:1~5:1), and 1.66 g target compound 5, yield 78%. ¹ H NMR (500 MHz, CDCl ₃ ) δ 7.97-7.91 (m, 2H), 7.41 - 7.35 (m, 2H), 4.39- 4.30 (m, 1H), 2.74 (dtd, J = 14.5, 6.5, 4.0 Hz , 1H), 2.63 (dtd, J =14.5, 6.5, 4.0 Hz, 1H), 2.09 (dt, J = 7.0, 4.0 Hz, 1H), 1.71 – 1.53 (m, 2H). ¹³ C NMR (125 MHz, CDCl ₃ ) δ 168.2, 143.9, 130.0, 129.6, 127.4, 67.3, 46.3, 26.1. MS: m/z: 708 (32%), 648 (23%), 540 (100%), 480 (14%).

Take a dry 50 mL flask, weigh compound 5 (1.41 g, 2 mmol), add dichloromethane 20 mL, dicyclohexylcarbodiimide (454 mg, 2.2 mmol), dimethylaminopyridine (0.12 g, 1 mmol), 4-hydroxy-2,2,6,6-tetramethylpiperidine oxide (344 mg, 2 mmol). After reacting at room temperature for 3 h, TLC detected that the raw material had reacted completely. Adjust the pH to 1 with 2M HCl, extract the reaction solution with dichloromethane (30 mL×3), combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, and rotary evaporate under reduced pressure to obtain a light yellow oily liquid, 200-300 mesh Silica gel column chromatography (PE:EA=10:1~5:1) yielded 2.0 g of the target compound 3 as light yellow oily compound, with a yield of 85%. Structure by. IR = 2977, 1726, 1592, 1585, 1469, 881, 748cm-1. MS: m/z: 1325(21), 1269 (13), 1205 (5), 1041 (99), 1001 (27), 937 (100), 877 (91), 857 (25).

Example 2 Chelation evaluation experiment of compounds represented by formula (I)

Accurately weigh an appropriate amount of uranyl acetate, add deionized water to make a solution with a metal ion concentration of 0.2 mmol/L. Accurately weigh an appropriate amount of bifunctional chelating agent (n=0, 1, 2, 3, 4, respectively named as I-0, I-1, I-2, I-3 and I-4) shown in (I), Add deionized water to make a solution with a concentration of 0.2 mmol/L. Different volumes of each metal ion solution and the compound solution represented by formula (I) are mixed separately to form a series of solutions to be tested. After forming a stable complex, use a UV-Vis spectrophotometer to scan a series of solutions in the range of 200-500 nm to find the maximum absorption peak of the complex, and record the absorbance value at this point. Take the mole fraction χ (the ratio of the mass concentration of the metal ion to the total mass concentration) as the abscissa, and the absorbance as the ordinate, and determine the maximum absorbance A _T when the metal and the chelating agent form a complex approximately completely by linear fitting , according to the results to find the composition of the complex. At the same time, the maximum absorbance A _E at coordination equilibrium was obtained according to the ultraviolet measurement results. When the mole fraction χ is 0.5, the metal and the chelating agent form a 1:1 complex, and the formula for calculating the K value is Equation (1); when the mole fraction χ is 0.4, the metal and the chelating agent form a 2:3 complex, The formula for calculating the K value is Equation (3), and the logarithm of the K value is the stability constant (lg K ).

c is the concentration when the complex does not dissociate completely, which is equal to the initial concentration of metal ions. c` is the concentration of the complex at coordination equilibrium, χ is the molar fraction of the formal metal ion (0.4), and k is the total mass concentration.

Experimental results: The research results of the interaction system between the compound represented by formula (I) and uranyl ions show that uranyl ions can form 1:1 chelate with I-0, I-1, I-2, I-3 and I-4 The chelate stability constants (lgK) are 15.78, 16.32, 22.21, 17.52 and 10.43, respectively, and I-2 has the strongest chelating ability.

Example 3 Evaluation experiment of the uranium excretion promoting effect of the compound represented by formula (I)

SD rats, weighing 200±20 g, 12 in each group. Randomly divided into ① blank control group; ② exposure model group; ③ uranium exposure + 100 mg/kg DTPA-Ca positive drug group; ④ uranium exposure + 100 mg/kg bifunctional chelating agent shown in formula (I) ( n=0,1,2,3,4, named as I-0, I-1, I-2, I-3 and I-4) groups. Rats were intraperitoneally injected with 500 μg/kg uranyl acetate and immediately intramuscularly injected with DTPA-Ca and a bifunctional chelator group. Rats in the poisoning control group were intraperitoneally injected with uranyl acetate and immediately intramuscularly injected with equal volume of normal saline. Rats in the blank control group were intraperitoneally injected with normal saline and immediately intramuscularly injected with the same volume of normal saline, and the result was used as the background value.

After the injection, each rat was individually placed in a metabolic cage with free access to water. Three rats in each group were killed by cervical dislocation after ether anesthesia 24 hours after exposure, and the urine of the rats at the corresponding time points was collected, and bilateral kidneys and femurs were dissected. Put the collected urine, kidney and femur into a heat-resistant beaker, add a mixed acid composed of perchloric acid and concentrated nitric acid (V/V=1:4), and place it on a flat electric stove in a fume hood for digestion to white residual orange, then dissolved and diluted to 30mL with 2% HNO ₃ , and the uranium content in the three samples was determined by ICP-MS. Subtract the background value from the measured value and multiply by 30 to calculate the uranium content in urine, kidney and femur, and divide it by the exposure dose, which is the percentage of urinary uranium excretion or the percentage of uranium accumulation in kidney and femur.

Experimental results: After intraperitoneal injection of uranyl acetate, rats were immediately intramuscularly injected with DTPA-Ca and bifunctional chelating agents I-0, I-1, I-2, I-3 and I-4, and the 24 hours after exposure were determined respectively. h Percentage of uranium excreted in urine and accumulated in kidney and femur. The results showed that, compared with the poisoned model group, DTPA-Ca and five bifunctional chelating agents could reduce the accumulation of uranium in the kidney and bone to varying degrees, and increase the excretion of uranium in urine. Administration of the same dose of DTPA-Ca and bifunctional chelating agents I-0, I-1, I-2, I-3 and I-4, group I-2 reduced renal and bone uranium accumulation and increased urinary uranium excretion within 24 hours The best, obviously better than the positive drug DTPA-Ca, which is consistent with the result that I-2 has the strongest chelating ability with uranium.

Example 4 Evaluation experiment of the radiation protection effect of the compound represented by formula (I)

HK-2 cells in the logarithmic growth phase were seeded in 6-well plates according to the number of cells 6×10 ³ /well. 25 μg/mL uranyl nitrate containing uranium and drugs acted together for 48 h on HK-2 cells, or uranyl nitrate pre-infected for 24 h and then added drugs for 24 h, the positive drug Tempol, chelating agent formula (I) The concentration of the indicated compound is 50 μM/mL. After drug treatment, DCFH-DA was added to make the final concentration 10 μmol/L, and incubated in a cell incubator for 20 min. After washing the cells with serum-free medium for 3 times, the flow cytometer (FC-500, Becton Dickinson Company, USA) detected the fluorescence intensity at the excitation wavelength of 488nm and the emission wavelength of 525nm, and calculated the amount of intracellular ROS generated. Intracellular ROS amount (% of the blank control group) = (fluorescence intensity of each experimental group/fluorescence intensity of the blank control group) × 100%.

Experimental results: After administration of Tempol and I-0, I-1, I-2, I-3 and I-4, the radiation-induced cell damage was significantly reduced, and the generation of ROS was significantly reduced. Compared with Tempol (55% reduction), I-0, I-1, I-2, I-3 and I-4 produced 65%, 67%, 72%, 69% and 63% less ROS, respectively.

Example 5 In vivo therapeutic effect evaluation test of compounds represented by formula (I)

Kunming mice, 20 male and 20 each, acclimatized to the environment for 7 days before the experiment. The room temperature was controlled at 22°C ± 3°C, the relative humidity was 60% ± 10%, free access to water, 10 mice per cage. Fasting 12 hours before the start of the experiment, the animals were randomly divided into 4 groups, 10 in each group, half male and half male. One group is the poisoning control group. After intraperitoneal injection of 200 mg/kg of uranyl acetate, the same volume of normal saline is given intragastrically immediately. Afterwards, the DTPA-Ca-positive drug and the compound represented by formula (I) were immediately administered intramuscularly at 300 mg/kg respectively. There were 5 experimental mice per cage, free access to water, and the 7-day survival of the mice was recorded. GraphPad Prism 6 software was used to draw survival curves, and SPSS17.0 statistical software was used to compare multiple groups (one-way analysis of variance) and two groups (t test). P < 0.05 indicated a statistical difference.

Experimental results: After Kunming mice were exposed to uranyl acetate, all the mice in the control group died within 4 days. When the DTPA-Ca-positive drug and the compound represented by formula (I) were given respectively, 3 animals survived the DTPA-Ca-positive drug, 5 animals survived respectively from I-0, I-1, I-2, I-3 and I-4, 6, 9, 7 and 5, the compounds represented by the formula (I) all have good activity, among which I-2 has the best therapeutic effect on uranium exposure, which is due to its strong chelating effect and radiation damage related to protection.

Claims (9)

1. A compound shown in a structural general formula (I),

2. a compound according to claim 1, characterized in that: n is 2.

3. A process for the preparation of a compound according to claim 1, characterized in that it comprises the following steps:

(1) Using 1,2-diphenylethylenediamine as a substrate, and reacting with acyl chloride shown as a structural formula (II) to obtain an amide product a;

(2) Hydrolyzing ester groups in the amide product a to prepare acyl chloride, reacting the acyl chloride with substituted 1,2-diphenylethylenediamine to obtain an amide product, and reducing to obtain a compound b;

(3) Oxidizing the compound b to obtain a compound c;

(4) The compound c is reacted with 4-OH-Tempol (4-hydroxy-2,2,6,6-tetramethyl piperidine oxide) to synthesize a target compound (I);

4. a process for the preparation of a compound according to claim 3, characterized in that: in the step (1), 1,2-diphenylethylenediamine, acyl chloride shown in a structural formula (II) and NaOH are in a molar ratio of 1 (2-5) to 2-5, and the reaction temperature is 0-100 ℃.

5. A process for the preparation of a compound according to claim 3, characterized in that: in the step (2), the molar ratio of the amide product a, the oxalyl chloride and the 1,2-diphenylethylenediamine is 1 (1-3) to 0.5-1, and the reaction temperature is-20-100 ℃.

6. A process for the preparation of a compound according to claim 3, characterized in that: in the step (4), the molar ratio of the compound c, DCC, DMAP and 4-OH-Tempol is 1 (4-8) to (4-12), and the reaction temperature is 0-100 ℃.

7. The use of a compound according to claim 1 for the preparation of a medicament for the prevention and treatment of uranium staining and radiation damage.

8. Use according to claim 7, characterized in that: the medicament is a pharmaceutical composition comprising an effective amount of a compound of claim 1 and a pharmaceutically acceptable carrier or excipient.

9. Use according to claim 7, characterized in that: the medicine is injection, tablet, capsule, powder, pill, granule or emulsion.