Method for improving catalytic click reaction activity of copper ions through ionizing radiation and application

Abstract

The invention relates to the technical field of modern medicine, in particular to a method for improving the catalytic click reaction activity of copper ions through ionizing radiation and application. The copper ion catalyst ligand is used for inducing or enhancing the activation of the radiation catalysis medicine, the problem that high dose is needed by ionizing radiation and the problems of limitation of transition metal reduction catalysis and the like can be solved by combining radiation chemistry and catalytic chemistry, medicine release can be controlled under the condition of low radiation dose, and the application range is wide. The controllable activation in space and time is realized, so that the cytotoxicity of the whole body is greatly reduced, and the potential side effects of the medicine are reduced.

Description

Technical Field

[0001] The invention relates to the field of modern medical technology, and in particular to a method and application of improving the activity of copper ion-catalyzed click reaction by ionizing radiation. Background Art

[0002] Malignant tumors have become a global public health problem that poses a serious threat to human health. According to statistics from 2020, nearly 10 million deaths (accounting for nearly one-sixth of the total global deaths) were caused by cancer. What is more serious is that the incidence and mortality rates of malignant tumors have shown a rising trend in the past decade, and the prevention and control situation has become increasingly severe. It is particularly noteworthy that in 2020, the number of new cancer cases and deaths in China ranked first in the world. Cancer has become the most challenging threat to human health in China and around the world.

[0003] At present, the clinical treatment methods for malignant tumors mainly include surgical treatment, chemotherapy and radiotherapy. According to the report of the World Health Organization, about 67% of all patients with malignant tumors can be cured, of which 30% of the cured cases are attributed to radiotherapy. Radiotherapy uses high doses of radiation to damage the DNA of tumor cells, causing tumor cells with damaged and unrepaired DNA to stop dividing or die. However, while radiotherapy destroys tumor cells, it may also cause damage to surrounding healthy tissues, and the severity of its toxic effects varies significantly between individuals. When the DNA chain breaks caused by radiotherapy are not complete, the cells can recover to become normal cells through repair mechanisms. Therefore, most radiotherapy requires repeated irradiation to enhance the killing effect on tumor cells, but this repeated irradiation will also cause collateral damage to adjacent normal tissues.

[0004] Due to the complexity, diversity and heterogeneity of tumors, a single treatment method is often difficult to achieve ideal therapeutic effects in the treatment of highly recurrent and easily metastatic malignant tumors. As a treatment model formed after more than ten years of clinical exploration, radiotherapy and chemotherapy can significantly improve the cure rate of patients with advanced tumors. Commonly used first-line chemotherapy drugs can inhibit cancer cell proliferation by interfering with nucleic acid anabolism, inhibiting mitosis, inhibiting protein synthesis and interfering with DNA replication. However, traditional chemotherapy drugs have many shortcomings such as rapid clearance in the body, non-specific distribution, and drug resistance, which seriously reduce the anti-tumor effect and are prone to toxic side effects on normal tissues. In addition, the pathophysiological characteristics of the same tumor in different physiological states and locations may be different. Therefore, tumor treatment requires a multimodal combined treatment strategy to achieve personalized medicine.

[0005] Ionizing radiation-induced activation of chemotherapy drugs is a strategy with broad application prospects. It can be used for radiotherapy-induced precision targeted therapy to achieve precise activation of drugs. The importance of this strategy is reflected in the following aspects: 1. Spatial synergy: radiotherapy and chemotherapy act on different spatial locations of the body respectively. Radiotherapy acts on local and regional lesions, while chemotherapy uses the permeability and retention effect of nanoparticles to accumulate drug molecules in the tumor site. The two have a synergistic effect; 2. Act on different cell cycles: When the two treatments are carried out simultaneously, the cell cycle is most sensitive to radioactivity when it is in the G2 / M phase, and most resistant to radioactivity when it is in the S phase. Some chemotherapy drugs have specific cytotoxicity to S phase cells, so the combination of the two has a complementary killing effect on tumor cells. Clinical data also show that synchronous treatment has better efficacy for a variety of tumors; 3. Enhance the sensitivity of cancer cells to chemotherapy drugs: This strategy can not only reduce the toxicity of chemotherapy drugs to a certain extent, but also reduce the occurrence of tumor cell resistance, thereby improving the anti-cancer effect. Therefore, the radiotherapy-induced drug activation strategy can regulate the biological effects of drugs in time and space, so that precision radiotherapy and chemotherapy have broad application prospects in tumor treatment.

[0006] In order to achieve precise activation of drug molecules in biological environment systems, click chemistry has become a new and powerful tool for studying the structure and function of biological systems in recent years. Click reactions can be carried out selectively and effectively under the corresponding physiological environment, and have the characteristics of fast reaction kinetics and good tolerance to water environment. + The cycloaddition reaction between azides and alkynes (CuAAC) is one of the most popular metal-catalyzed reactions in the past few decades. The local synthesis of drugs at the corresponding drug targets through the CuAAC reaction can maximize the efficacy and reduce side effects. The team of researcher Xiaogang Qu of the Chinese Academy of Sciences reported an adaptive bioorthogonal catalytic system that encapsulates prodrugs and sodium ascorbate in adenosine triphosphate (ATP) aptamer-functionalized metal-organic framework (MOF) nanoparticles, releases sodium ascorbate and prodrugs under high ATP concentration conditions, and reduces Cu 2+ Realize local synthesis of drugs in cells. In recent years, the team has used MOF-Cu, DNA enzymes and other catalyst materials to achieve local synthesis of resveratrol analogs in tumor cells. Professor Bu Wenbo's team at Fudan University designed and synthesized copper cysteine ​​nanoparticles. 2+ It undergoes unsaturated coordination with cysteine ​​and is subsequently reduced by reduced glutathione (GSH) in tumor cells to generate Cu + , catalyzing the Fenton-like reaction to produce the highly oxidizing OH. The team of Professor Bai Yugang from Hunan University reported a membrane-embedded macromolecular catalyst based on the cationic dense shell nanoparticle skeleton to deliver Cu+ , and can still show substrate selectivity and excellent catalytic effect when working in a living cell system. Professor Xing Hang's team at Hunan University has established a membrane-anchored catalyst with precise spatial control, which can be used to bind Cu + The complex is delivered to the membrane, thereby greatly reducing the amount of catalyst required for effective intracellular catalysis. Professor Bradley's team at Edinburgh designed a heterogeneous copper catalyst with a size of about 150 μm, which can catalyze the synthesis of drugs from the two components of anticancer drugs in the extracellular matrix. The development of CuAAC reaction catalysts can not only increase the reaction rate, but also further reduce the amount of copper ions used, greatly reducing toxicity to cells or organisms.

[0007] The copper-catalyzed click chemistry reaction accelerated by catalyst ligands is a powerful and highly adaptable bio-coupling tool. Its further improvement is of great significance for the discovery of more versatile catalytic systems. The research group of Professor Chen Peng of Peking University used the CuAAC reaction to specifically label proteins in the cytoplasm of Escherichia coli. By systematically comparing a series of click chemistry reactions catalyzed by copper complexes, they found a copper ion ligand (BTTP) suitable for labeling proteins in the bacterial cytoplasm. By combining the click chemistry reaction catalyzed by BTTP-Cu (I) with the non-natural amino acid insertion technology, they successfully specifically labeled the acidic chaperone proteins in the bacterial cytoplasm. The teams of Professor Finn and Professor Wu Peng in the United States have respectively developed a series of water-soluble ligands for bioorthogonal reactions. These ligands avoid Cu 2+ Oxidative damage caused by "reactive oxygen" species (ROS) is produced in cells, and its biotoxicity is reduced by copper ion complexes. The click chemistry reaction without the participation of a catalyst takes 1 hour, while the new ligand-accelerated copper-catalyzed click chemistry reaction can be effectively completed in just 3 to 5 minutes. In summary, although terminal alkynes and azides can be used to obtain the desired compounds or drugs through a simple click reaction, the in situ synthesis of drugs through CuAAC reactions in living cells is still in its infancy. This "bottom-up" drug synthesis method is suitable for the synthesis of a variety of desired drugs and avoids complex pre-synthesis and purification steps. However, due to the biocompatibility and functionality of the catalyst, the problem of targeted and precise synthesis of drugs at the lesion site remains unsolved. For example, the CuAAC reaction requires the use of Cu + As a catalyst, copper catalyst has certain toxicity. In addition, since copper catalyst needs to maintain catalytic activity in an oxygen-free environment, it is necessary to inhibit Cu + Oxidation requires the addition of reducing agents such as ascorbic acid, and the resulting byproducts are also cytotoxic. These problems limit the application of CuAAC in vivo.

[0008] Compared with traditional small molecule induction methods, photoinduced reactions are not only easy to obtain, but also can be precisely controlled in time and space. Researchers have long been pursuing the use of light to specifically induce the release of bioactive molecules. For example, the team of researcher Zhu Guangyu from the City University of Hong Kong, China, reported a small molecule Pt prodrug activated by near-infrared light that directly oxidizes intracellular biomolecules in an oxygen-independent manner. The team of researcher Qu Xiaogang from the Chinese Academy of Sciences used near-infrared light to promote the CuAAC reaction of biocompatible heterogeneous copper nanocatalysts through photodynamic and photothermal effects, and promoted Cu through photodynamics. + The photoinduced reduction of copper ions is also widely used in the synthesis of small molecules, polymers and other materials with spatial and temporal selectivity. Professor Bowman's team at the University of Colorado reported the light-induced CuAAC reaction. 2+ The complex improves the photoinitiation efficiency with Cu + In summary, photoreactions have problems such as only acting on molecules that selectively absorb specific wavelengths, low light energy utilization, and insufficient penetration depth, which greatly limit the application and development of photoresponsive reactions.

[0009] High-energy ionizing radiation has the advantages of high temporal and spatial controllability, deep tissue penetration, strong clinical applicability, and chemotherapy can improve the sensitivity of radiotherapy to tumor cells. So far, the use of medical radiotherapy rays to induce chemical bond breakage to achieve controllable drug delivery has attracted widespread attention from researchers. Based on strategies such as traditional polymers, new responsive polymers, inorganic composite materials and organic small molecule compounds, researchers have achieved a certain dose of radiotherapy induced drug delivery and activation. Professor Li Jinghong's team at Tsinghua University designed a radiotherapy-triggered PROTAC prodrug activation strategy, which precisely and spatiotemporally controls protein degradation through X-ray radiation. X-ray-induced benzene azide is combined with PROTAC to achieve effective activation under X-rays. This strategy shows anti-tumor efficacy synergistic with radiotherapy in tumor cells and can effectively reduce the adverse systemic toxicity caused by PROTACs. Professor Xu Huaping's team at Tsinghua University has developed a series of selenium-containing nanodrugs for application in combined therapy research. The drug delivery carrier was prepared by encapsulating the chemotherapy drug doxorubicin. Under the 5 Gy low-dose gamma ray irradiation commonly used in clinical radiotherapy, the diselenide bond and the triple hydrogen bond were broken to release the encapsulated drug, and organic selenious acid was generated, thereby realizing the combination of chemotherapy, radiotherapy and immunotherapy. Professor Liu Zhibo's team at Peking University reported a new prodrug activation strategy using radiotherapy (X-rays). A series of nitrogen oxides were designed and synthesized, and this type of prodrug can be reduced to the original drug by X-rays. It was proved in living cells and living bodies that this strategy can accurately activate chemotherapy prodrugs and realize radiotherapy-driven radiotherapy and chemotherapy combined treatment. Professor Lin Wenbin's team at the University of Chicago reported a nanometal organic framework material. The heavy metal MOF containing covalently bound drugs can be triggered by X-rays to release drugs by enhancing ROS generation and ROS-induced prodrug cleavage. Professor Bradley's team at the University of Edinburgh reported a strategy for releasing prodrugs containing sulfonyl azide and phenyl azide groups with clinical doses of radiation, realizing "real-time" drug activation at the site of ionizing radiation irradiation. Professor Miller's team in the United States reported a drug conjugate strategy to release active therapeutic drugs during ionizing radiation, and 50% of the drug can be released under 8 Gy of local X-ray irradiation. Kazuhito Tanabe's research group in Japan proposed a copper-catalyzed azide-alkyne cycloaddition reaction triggered by hypoxic X-ray irradiation. The radiolysis and reduction of Cu(II) generates monovalent copper ions (Cu(I)). The generated metal ions accelerate the coupling between azide and acetylene molecules. The reaction conditions are non-biocompatible solvents and hypoxic conditions.

[0010] In summary, the radiation response dose of most materials is still much higher than the 2 Gy daily dose that chemotherapy patients can usually accept, and this type of strategy is mainly limited by the radiochemical yield of aqueous solutions, which fundamentally limits the sensitivity of radiation response reactions.

[0011] However, the existing technology still has the following technical problems: (1) Problems with phototherapy include: limited penetration depth, good treatment effect for epidermal tumors, but unable to treat deep parts; skin phototoxicity, free drugs in non-tumor parts will damage the skin. (2) Problems with copper catalysts: The efficiency of Cu(I)-catalyzed azide-alkyne click chemistry (CuAAC) depends largely on the presence of copper ions Cu(I). The level of free copper in the human body is low, most of the copper is bound to proteins or other molecules, and Cu(I) ions are easily oxidized and disproportionated, and are also cytotoxic to living cells. The method of using chemical reducing agents has poor spatiotemporal controllability, resulting in off-target effects of drugs. The targeted activation of Cu(I) ions has greatly limited the use of CuAAC in vivo. (3) Low-dose radiation has limited therapeutic effect on tumors, and high-dose radiation is difficult for the human body to withstand, so there is a certain contradiction in tumor treatment. (4) Previous strategies usually focused on active species such as free radicals, hydrated electrons and H2O2 produced by water radiolysis, which participate in or induce secondary reactions. The main limiting factor is that the active species produced by radiation in water are usually low in yield, which fundamentally restricts the sensitivity of its radiation response.

[0012] In view of this, the present invention is proposed. Summary of the invention

[0013] In order to solve the above technical problems, the present invention provides a method and application of improving the activity of copper ion-catalyzed click reaction by ionizing radiation.

[0014] Specifically, the technical solution of the present invention is as follows: In the first aspect, the present invention provides a method for improving the catalytic activity of copper ion click reaction, comprising: using a catalyst ligand to coordinate with Cu(II) ions in an aqueous solution, and obtaining a Cu(I) complex by ionizing radiation; wherein the water-soluble catalyst ligand is a nitrogen-containing water-soluble molecule; in the Cu(I) complex, the molar ratio of Cu(I) ions to the catalyst ligand is 1:2-10.

[0015] Preferably, in the present invention, the concentration of Cu(II) ions in the aqueous solution is 50-300 μM; more preferably 100-150 μM.

[0016] Preferably, in the present invention, the concentration of the catalyst ligand in the aqueous solution is 100-800 μM; more preferably 300-600 μM.

[0017] Preferably, in the Cu(I) complex of the present invention, the molar ratio of Cu(I) ions to catalyst ligands is 1:4-6; more preferably 1:5.

[0018] Preferably, in the present invention, the irradiation dose of the ionizing radiation is 0.1-100 Gy.

[0019] In a second aspect, the present invention provides the application of the method for improving the catalytic activity of copper ion click reaction in catalyzing the cycloaddition reaction between azide and alkyne.

[0020] In a third aspect, the present invention provides application of the method for improving the catalytic activity of copper ion click reaction in living cell imaging.

[0021] In a fourth aspect, the present invention provides application of the method for improving the catalytic activity of copper ion click reaction in in situ synthesis of drugs.

[0022] Preferably, in the present invention, the above-mentioned applications are not for the purpose of disease diagnosis and treatment.

[0023] In a fifth aspect, the present invention provides a method for in situ synthesis of drugs by radiation click chemistry, characterized in that it includes: using the method for improving the catalytic activity of copper ion click reaction to catalyze a cycloaddition reaction between azide and alkyne in living cells.

[0024] Beneficial effects: The present invention provides a method and application for improving the catalytic activity of copper ion click reaction, using copper ion catalyst ligands to induce or enhance the activation of radiation catalytic drugs. By combining radiation chemistry and catalytic chemistry, the problem of high doses of ionizing radiation and the limitations of transition metal reduction catalysis can be solved. Drug release can be controlled at lower radiation doses to achieve controllable activation in space and time, thereby greatly reducing systemic cytotoxicity and reducing potential side effects of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0026] Figure 1 In Example 1 of the present invention, benzyl azide (compound 1) and phenylacetylene (compound 2) are catalyzed by Cu(I) to generate azide-alkyne (triazole 3).

[0027] Figure 2 is 1-benzyl-4-phenyl-1H-[1,2,3]triazole in Example 1 of the present invention 1 H NMR spectrum (H2O δ=3.3; DMSO-d6 δ=2.5).

[0028] Figure 3 is the HPLC spectra of compound 1, compound 2 and triazole 3 in Example 1 of the present invention ( Figure 3A) and a standard curve of triazole 3 concentration ( Figure 3 (Figure B).

[0029] Figure 4 The effect of a series of catalyst ligands on the model reaction yield in Example 1 of the present invention is shown.

[0030] Figure 5 The effect of a series of copper salts on the model reaction yield in Example 1 of the present invention is shown.

[0031] Figure 6 The radiation catalytic model reaction before and after the embodiment 1 of the present invention 1 Comparison of changes in H NMR spectra.

[0032] Figure 7 The figure shows the influence of Cu(II) ion concentration and BTTP catalyst ligand concentration on the model reaction yield in Example 1 of the present invention.

[0033] Figure 8 The model reaction solution under different irradiation dose conditions in Example 1 of the present invention is 1 Changes in the H NMR spectrum ( Figure 8 Figure A) and the relationship between the yield of triazole 3 generated by the model reaction and the irradiation dose ( Figure 8 (Figure B).

[0034] Fig. 9 This is the Cu 2P spectrum of the copper ion aqueous solution (0.1 mM, Cu:BTTP=1:5) before irradiation in Example 2 of the present invention ( Fig. 9 Figure A), Cu 2P spectrum after irradiation of copper ion aqueous solution (0.1mM, Cu:BTTP=1:5) ( Fig. 9 (B in the middle). Cu 2P spectrum of copper ion aqueous solution (1mM) after 100 Gy gamma ray irradiation ( Fig. 9 C) and Cu LMM spectrum ( Fig. 9 (D in the middle).

[0035] Fig.10 is the standard curve of Cu(II) ion in Example 2 of the present invention ( Fig.10 Figure A), the amount of Cu(Ⅱ) ion conversion in aqueous solution after irradiation with different doses of gamma rays ( Fig.10 2,9-Dimethyl-1,10-phenanthroline reacts with cuprous copper to form an orange-yellow Cu(I) complex ( Fig.10 Figure C in the middle), UV absorption curves of aqueous solution after irradiation with different doses of gamma rays ( Fig.10 (D in the middle).

[0036] Fig.11The yield of triazole 3 generated by benzyl azide and phenylacetylene changes with time after the aqueous solution of the Cu(II) complex is irradiated with 100 Gy gamma rays in Example 2 of the present invention.

[0037] Fig.12 : The radiochemical yield of triazole 3 generated by benzyl azide and phenylacetylene at different absorbed doses in Example 2 of the present invention.

[0038] Fig.13 In Example 3 of the present invention, alkynyl coumarin and 3,5-dihydroxybenzeneazide undergo CuAAC reaction in HeLa cells at an irradiation dose of 30 Gy. DETAILED DESCRIPTION

[0039] The invention provides a method for improving the catalytic activity of a copper ion click reaction and application thereof.

[0040] First, the present invention realizes Cu by using catalyst ligand under ionizing radiation conditions. 2+ To Cu + Controllable and efficient reduction of ionizing radiation in aqueous solution. The radiochemical yield of active species produced by ionizing radiation in aqueous solution is low. The radiochemical yield (G value) is the number of molecules, ions, free radicals or electrons in the substance that causes chemical changes when a substance absorbs 100 eV of energy, reflecting the generation of various radiolysis products during the radiolysis process. Taking the radiochemical reaction of water as an example, the yield of hydroxyl radicals is 2.72, the yield of hydrated electrons is 2.63, and the yield of hydrogen radicals is only 0.55. Theoretical calculations show that the concentration of hydrated electrons in aqueous solution is about 5.4 μM under 20 Gy gamma ray irradiation. The low concentration of active species greatly restricts the reduction efficiency of metal ions in aqueous solution. At the same time, the radiolysis products react with each other and with Cu(Ⅱ) ions to generate a series of secondary products, which then participate in various reactions to form a complex set of reaction equations, including 9 reactions related to Cu ions. Therefore, the controllable and efficient reduction of copper ions is crucial for irradiation-catalyzed click reactions.

[0041] The key to the controllable and efficient reduction of copper ions is to enhance the stability of Cu(I) ions in aqueous solution. In this study, the catalyst ligand can improve the radiation reduction efficiency of Cu(II) and improve the stability of Cu(I) complexes. Specifically, the catalyst ligand is used as a carrier to coordinate and load transition metal ions, and the radiation reduction of transition metals and the activation of catalytic ability are achieved under the action of ionizing radiation. High-energy ray irradiation can produce active species such as hydrated electrons, hydroxyl radicals, hydrogen radicals, and hydrogen peroxide in water. Among them, hydrated electrons or hydrogen radicals have a lower reduction potential and can be reduced to produce catalytically active Cu(I) complexes, which can catalyze the cycloaddition reaction of azide and alkyne (CuAAC). The bioorthogonality of acetylene and azide groups prevents them from interfering with biological systems, thus providing powerful innovation in the field of chemical biology. Moreover, the click reaction activates drug release only at the tumor site, improving the therapeutic effect at the tumor site and reducing damage to normal tissues. The water-soluble catalyst ligand enables the irradiated CuAAC to be carried out on the cell surface or in the cell by a small amount of Cu catalyst loading, and does not show cytotoxicity. The present invention uses a copper complex with biocompatibility and bioorthogonality as a catalyst for the radiation click reaction, thereby reducing the minimum radiation dose required for radiation, and provides a new material for synchronous chemoradiotherapy.

[0042] In the present invention, the carrier can be a nitrogen-containing small molecule, for example, a small molecule without aromatic rings and aromatic heterocycles: ammonia, methylamine, ethylamine, propylamine, dimethylamine, trimethylamine, acetyleneamine, urea, pentamethylenediamine, acridine, thiazole, acridinedione, imidazole, pyridone, acridinol, thiazoline, acridinone, imidazolone, pyridazinol, acridinone, pentanoneamine, acridinyl sulfonamide, pyridazinol, acridinol, pentamethylenediamine ketone, imidazolinone, pentanolamine, thiazoloneamine, propylamine ketone, etc.; nitrogen-containing aromatic and heteroaromatic groups: aminophenyl, methyleneaniline, dimethylaminophenyl, pyridyl, pyridazinyl, imidazolyl, acridinyl, pyranyl, pyrrolyl, quinolyl, pyrimidinyl, acridinyl, pyridazinyl, pyrrolyl The ligands include dioxadione, carbazole, quinolinone, acridone, pyridazinone, pyridinemethanol, pyridazinesulfonyl, acridone, imidazolinone, acridone methyl ester, pyridazinyl methyl ester, imidazolinedione, pyridone, thienyl, acrandione, pyrone, cyanurea, isocyanuric acid, triazinol, diethylene glycol triazine, triazinone, triazineamine, triazine ester, triazine thioether, triazinemethanol, etc., and some nitrogen-containing multidentate organic ligands, the structural formula is shown below; it can also be some polymers, such as metal organic frameworks (MOFs), covalent organic frameworks (COFs), molecular sieves, mesoporous materials, porous polymers, etc., which are not limited here.

[0043] The structural formula of the nitrogen-containing multidentate organic ligand is as follows: Among them, 1a: R=benzyl (TBTA); 1b: R=tert-butyl (TTTA); 1c: R=CH2CH2CH2OH (THPTA); 1d: R=CH2CH2CO2H; 1e: R=CH2C8H4CO2H; Among them, 4a: R = OH; 4b: R = OBn.

[0044] Preferably, the carrier needs to be pretreated by adsorbing copper ion exchange resin, and the catalyst ligands are pretreated to remove copper.

[0045] In the present invention, the radiolytically reduced Cu(I) ions and the catalyst ligands generate stable Cu(I) complexes, and therefore, the ratio of the Cu(I) ions to the catalyst ligands will affect the formation rate of the complexes and the click reaction activity. Therefore, the present invention uses water-soluble catalyst ligands to stabilize the Cu(I) ions and improve the click reaction catalytic activity of the Cu(I) ions.

[0046] Preferably, the molar ratio of the Cu(I) ions to the catalyst ligands is 1:5.

[0047] Furthermore, the present invention provides a method for in-situ synthesis of drugs by radiation click chemistry, comprising: The copper ion complex is irradiated with a radiation source to induce reduction of the procatalyst into Cu(I) ions, and then the Cu(I) ion complex performs an in-situ click reaction on the prodrug molecule to synthesize the drug molecule.

[0048] In the present invention, the radiation catalytic drug activation method is not only applicable to the cycloaddition reaction of azide and alkyne, but also can be used for click elimination reaction.

[0049] Preferably, the method for in situ synthesis of drugs by radiation click chemistry comprises: incubating cancer cells in a culture medium, adding the procatalyst, water and the prodrug molecule to the culture medium for continued cultivation, and then subjecting the culture medium to ionizing radiation; More preferably, the method for activating the radiation-catalyzed drug is performed in the following order: S1: incubating cancer cells in a culture medium, adding the procatalyst and water to the culture medium for further culturing, and then subjecting the culture medium to ionizing radiation; S2: adding the prodrug molecule into the culture medium for reaction.

[0050] The present invention has found that by following the above-mentioned sequence of operations, that is, adding the prodrug molecule after ionizing radiation, a Cu(I) complex with higher activity can be obtained, which can more effectively catalytically activate the prodrug molecule.

[0051] Preferably, the radiation source includes alpha rays, beta rays or gamma rays produced by decay of radioactive nuclides; or X-rays, gamma rays, high-energy electrons, protons, heavy ions or alpha particles produced by boron neutron capture therapy generated by external radiation sources, and other possible exogenous or endogenous radiation.

[0052] More preferably, the irradiation dose of the radiation source is 0.1~100Gy; further preferably, the irradiation dose of the radiation source is 1~30Gy.

[0053] Preferably, the method for activating a radiation-catalyzed drug further comprises: adding isopropanol or DMSO to the culture medium.

[0054] Preferably, the catalytic activation time is 0 to 72 h; more preferably 20 to 30 h.

[0055] The above method provided by the present invention can be applied to living cell imaging by utilizing radiation-catalyzed click reactions; or, it can be applied to the in-situ synthesis of anti-tumor active drugs. The present invention proposes a new concept of radiation-driven catalysis by combining radiation chemistry, catalytic chemistry and coordination chemistry: ionizing radiation activates the precatalytic center, catalyzing the activation of prodrug compounds or the release of drugs, thereby achieving the production of active drugs and reducing or avoiding the restriction of primary active species produced by radiation. Using the copper ion catalyst ligand and method of the present invention, low-dose irradiation can produce significant cytotoxicity.

[0056] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0057] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0058] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementations", or "some specific implementations" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0059] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0060] Example 1 1.1 Radiation catalytic reaction.

[0061] Benzyl azide (compound 1) and phenylacetylene (compound 2) were reacted by Cu(I)-catalyzed CuAAC reaction to generate 1-benzyl-4-phenyl-1H-[1,2,3]triazole (triazole 3) ( Figure 1 ).

[0062] like Figure 2 As shown, the chemical shift of H (a) on the methylene group in compound triazole 3 is at δ = 5.65 ppm, and the chemical shift of H (b) on the triazole ring in compound triazole 3 is at δ = 8.65 ppm. Therefore, the NMR signals of H at δ = 5.65 ppm and δ = 8.65 ppm can be used to monitor whether the model reaction occurs. A high performance liquid chromatography (HPLC) detection method for benzyl azide, phenylacetylene and triazole 3 was established. Figure 3 As shown, the retention times of benzyl azide, phenylacetylene and triazole 3 are 24.3min, 24.9min and 26.1min respectively. The retention time of the raw material and triazole 3 are different. At the same time, the standard curve of triazole 3 concentration and peak area is y=6186.5x+268.5, R 2 =0.999, indicating that the UV absorption peak area and concentration of triazole 3 in the range of 0.2-2 mM have a good linear relationship. Subsequent experiments can quantitatively monitor the generation of triazole 3 by HPLC.

[0063] Then, the effects of a series of catalyst ligands on the irradiation catalytic model reaction were studied. The catalysts were all ligand molecules that have been reported to have CuAAC reaction activity ( Figure 4), the aqueous solution of Cu(Ⅱ) complex was irradiated with 100 Gy of gamma ray to obtain an activated complex solution, and 1 mM benzyl azide and 1 mM phenylacetylene were added to react for 24 hours, and the formation of triazole 3 was quantitatively monitored by HPLC. The results showed that TBTA, THPTA, TEOTA, and BTTP had catalytic effects, among which BTTP had the best catalytic effect.

[0064] Secondly, the effects of a series of copper salts on the catalyst ligand in the irradiation catalytic model reaction were studied. The aqueous solution of Cu(Ⅱ) complexes of different copper salts was irradiated with a gamma ray dose of 100 Gy to obtain an activated complex solution. After adding 1 mM benzyl azide and 1 mM phenylacetylene, the formation of triazole 3 was quantitatively monitored by HPLC after 24 hours of reaction. The results showed that CuCl2 had a better catalytic effect ( Figure 5 ).

[0065] Subsequently, the aqueous solution of the Cu(Ⅱ) / BTTP complex prepared by CuCl2 was irradiated with a gamma ray dose of 100 Gy to obtain an activated complex solution. 1 mM benzyl azide and 1 mM phenylacetylene were added and reacted for 24 h. The NMR spectrum of the mixture was observed ( Figure 6 ), the NMR signal of H (a) on the methylene group of triazole 3 was observed at δ = 5.65ppm, and the chemical shift of H on the benzene ring was also observed to change at δ = 7.8ppm. It was proved that under the conditions of gamma-ray irradiation activated Cu(Ⅱ) / BTTP complex aqueous solution, benzyl azide and phenylacetylene generated triazole 3. The results showed that gamma-ray irradiation of Cu(Ⅱ) / BTTP complex aqueous solution can induce click reaction.

[0066] 1.2 Optimization of radiation catalytic reaction conditions.

[0067] The relationship between the radiation catalytic yield and the concentration of Cu(Ⅱ) ions and catalyst ligand (BTTP) was studied. Figure 7As shown in the figure, the concentration range of Cu(Ⅱ) ions is 50 ~ 300 µM, and the concentration range of the catalyst ligand BTTP is 100 ~ 800 µM. In the figure, dark blue indicates a lower yield, and red indicates a higher yield. When the copper ion concentration is between 100 ~ 150 µM and the BTTP concentration is within the concentration range of 300 ~ 600 µM, the radiation catalytic yield can reach a high level. When the copper salt concentration is less than 100 µM or greater than 150 µM, and the BTTP concentration is less than 200 µM or greater than 700 µM, the radiation catalytic yield is low. When the BTTP concentration is fixed, the yield first increases and then decreases with the increase of the copper salt concentration. Similarly, when the copper salt concentration is fixed, the yield first increases and then decreases with the increase of the BTTP concentration. In summary, the radiation catalytic reaction is sensitive to the concentrations of copper ions and BTTP. Too high or too low concentrations will lead to a decrease in yield. When Cu: BTTP = 1:5, the radiation catalytic click reaction has the best catalytic effect with a yield of 53%.

[0068] 1.3 Effect of irradiation dose on radiation catalytic reaction.

[0069] like Figure 8 As shown in the figure, when the radiation dose gradually increases, the yield of triazole 3 in the radiation-catalyzed click reaction increases with the increase of radiation dose. The model reaction solution was irradiated with 2 Gy, 10 Gy, 60 Gy and 100 Gy doses, 1 The HNMR spectrum showed that the signal at δ = 5.5 ppm of the methylene hydrogen of triazole 3 gradually increased, and the HPLC results showed that the yield of triazole 3 gradually increased when the model reaction solution was irradiated with a dose of 10 Gy, 30 Gy, and 60 Gy, and the yields were 5.3%, 19.8%, and 41.5%, respectively ( Figure 8 Figure B). NMR and HPLC results show that the yield of CuAAC reaction increases with the increase of irradiation dose. It is speculated that increasing the irradiation dose will increase the reduction yield of Cu(Ⅱ) ions, and the increase of Cu(Ⅰ) ion concentration will accelerate the CuAAC reaction.

[0070] Example 2 Mechanistic study of gamma-ray catalyzed click reactions.

[0071] 2.1 Controllable reduction of copper ions under radiation conditions.

[0072] In order to explore the reduction reaction of Cu(Ⅱ) ion complexes in aqueous solution under gamma-ray irradiation conditions, this example studies the valence distribution information of copper element in copper coordination compounds by measuring X-ray photoelectric spectroscopy (XPS). Fig. 9). As shown in the figure, when the Cu(Ⅱ) ion concentration is 100 µM and the Cu(Ⅱ) ion concentration: BTTP concentration is 1:5, the change of copper ion valence state under 100 Gy irradiation dose is studied by X-ray photoelectric spectroscopy (XPS) ( Fig. 9 Figure A and Figure B). As shown in the figure, Figure A is the Cu 2P spectrum of copper ion aqueous solution (0.1 mM, Cu:BTTP=1:5) before irradiation. A group of satellite characteristic peaks of Cu(Ⅱ) can be clearly observed at 943 eV. The binding energy of Cu(Ⅱ) Cu 2p 3 / 2 is 934.5 eV, and the binding energy with copper oxide Cu 2p 3 / 2 Figure B is the Cu 2P spectrum after irradiation of copper ion aqueous solution (0.1 mM, Cu:BTTP=1:5). 932.8 eV and 952.5 eV are the Cu 2p of Cu(I), respectively. 3 / 2 and Cu 2p 1 / 2 The characteristic peaks at 934.5 eV are the characteristic peaks of Cu(Ⅱ). The XPS spectrum shows that the Cu(Ⅱ) aqueous solution is a mixed system of Cu(Ⅱ) and Cu(I) after irradiation. The peak fitting shows that the content of Cu(Ⅱ) is 85.5% and the content of Cu(I) is 14.5%. In the Cu 2p spectrum of 1mM Cu(Ⅱ) ion aqueous solution irradiated with a dose of 100 Gy, 932.8 eV and 952.5 eV are the Cu 2p peaks of Cu(I), respectively. 3 / 2 and Cu 2p 1 / 2 The characteristic peak of Cu LMM spectrum is 569.9 eV, which is the signal of Cu(I). Fig. 9 In Figures C and D), no signal absorption of Cu(0) was detected in the Cu LMM spectrum. This is because the hydrated electrons generated by irradiation reduce Cu(Ⅱ) ions to Cu(I) ions, and the Cu(I)\BTTP complex generated by the BTTP ligand and Cu(I) ions improves the stability of Cu(I). In summary, the hydrated electrons generated by irradiation can reduce Cu(Ⅱ) ions, and the generated Cu(I) complex can exist stably.

[0073] The change of copper ion concentration in aqueous solution was detected by sodium diethyldithiocarbamate spectrophotometry. Copper reacted with sodium diethyldithiocarbamate to form a yellow-brown complex. The absorbance was measured at a wavelength of 440 nm, and a standard curve of Cu(Ⅱ) ion concentration and absorbance was drawn ( Fig.10 Then, the changes of Cu(Ⅱ) ion concentration in aqueous solution after irradiation under different conditions were detected, such as Fig.10As shown in Figure B, under the condition of no ligand added, the amount of Cu(Ⅱ) ions after irradiation decreased by 9.3 μM. With the increase of ligand equivalent, the amount of Cu(Ⅱ) ion conversion after irradiation decreased from 13.7, 6.5 to 1.3 μM. This is due to the strong competitive effect between the ligand and the probe. When hydroxyl radical quenchers tert-butanol and ethanol were added, the amount of Cu(Ⅱ) ion conversion was 12.3 and 10.6 μM, respectively, indicating that hydroxyl radical quenchers can increase the amount of Cu(Ⅱ) ion conversion. When potassium nitrate, a hydrated electron quencher, was added, the amount of Cu(Ⅱ) ion conversion decreased to 3.9 μM, indicating that hydrated electron quenchers can reduce the amount of Cu(Ⅱ) ion conversion. An orange-yellow Cu(I) complex ( Fig.10 Figure C in the middle shows the UV absorption curves of aqueous solutions after being irradiated with different doses of gamma rays ( Fig.10 D in the middle). After irradiation with gamma rays at doses of 10 Gy, 20 Gy, 30 Gy, 60 Gy and 100 Gy, the absorption peak of the aqueous solution at 457 nm gradually increased. This is because the generated cuprous ions reacted with 2,9-dimethyl-1,10-phenanthroline to form a colored cationic complex with strong ultraviolet absorption at 457 nm. The results show that the hydrated electrons generated by irradiation can reduce Cu(Ⅱ) ions to Cu(I) ions. With the increase of irradiation dose, the concentration of Cu(Ⅱ) ions in the aqueous solution gradually decreased, while the concentration of Cu(I) ions gradually increased.

[0074] 2.2 Chemical kinetics of radiation-catalyzed click reactions.

[0075] After gamma ray irradiation of the aqueous solution of Cu(Ⅱ) complex, the substrates benzyl azide and phenylacetylene were added, and the yield of the product triazole 3 was detected as a function of reaction time ( Fig.11 After irradiation, the yield of triazole 3 increased with time, and the yield had a good linear relationship with time, indicating that the Cu(I)\BTTP complex generated by radiation reduction avoided the oxidation of Cu(I) ions by free radicals and other oxides, and could exist stably for a long time when exposed to air, and had good catalytic activity within 72 hours.

[0076] 2.3 Relationship between gamma ray dose and irradiation chemical yield.

[0077] The radiation chemical yield (G value) is defined as the number of molecules, ions, free radicals or electrons in the substance that cause chemical changes when the substance absorbs 100 eV of energy. It reflects the generation of various radiolysis products during the radiolysis process and is related to the linear energy transfer (LET) value of the ray and temperature. The G value of γ-ray irradiation at 25 °C is shown in Table 1.

[0078] Table 1 Radiolysis products of water irradiated by γ-rays at 25°C G Value (100 eV -1 ) Note: Considering the material balance, G (•H) Through the material balance equation G (•H)= G (•OH)+2 G (H2O2)- G (e aq - )-2 G (H2)+3 G (HO2•) is obtained.

[0079] The radiolysis products react with each other and with Cu(Ⅱ) ions to generate a series of secondary products, which then participate in various reactions to form a complex set of reaction equations, including 9 reactions related to Cu ions (Table 2).

[0080] Table 2 Partial reaction equations in the radiolysis calculation model of aqueous solution containing copper ions To investigate the effect of radiation on the click reaction, benzyl azide and phenylacetylene were used to generate triazole 3 ( Figure 1 ) to study the efficiency of click reactions under gamma irradiation conditions. Fig.12 As shown in the figure, at an absorbed dose of 10 Gy, the G value of triazole 3 was 47.3 and the yield was 4.9 μM\Gy. At an absorbed dose of 30 Gy, the G value of triazole 3 was 63.6 and the yield was 6.6 μM\Gy. At an absorbed dose of 100 Gy, the G value of triazole 3 was 40 and the yield was 4.14 μM\Gy. Compared with the G value of hydrated electrons of 2.63, the G value of the reaction can be greatly increased by using gamma irradiation to induce click reactions. At the same time, the yield of irradiation reduction of Cu(Ⅱ) ions is 0.2 μM\Gy, and the yield of triazole 3 in the click reaction is greatly improved. In theory, the radiation yield of highly active species is certain, and each mole of highly active substances participating in subsequent reactions can only produce an equivalent amount of activated molecules. By using radiation-induced click reactions, the limitation of equivalent activation of molecules such as prodrugs can be broken through, and the yield of radiation-induced click reactions can be greatly increased.

[0081] Example 3 Gamma-ray catalyzed click reactions for live cell imaging.

[0082] In order to demonstrate the application of radiation-catalyzed click reaction in cells, HeLa cells were selected as a model system to further study the radiation-catalytic effect of this method in cells ( Fig.13). The mixture of 3,5-dihydroxybenzeneazide (4) and alkynyl coumarin (5) has no fluorescence. After the click reaction, a fluorescent molecule (Dye 6) is generated. The fluorescent molecule Dye 6 emits cyan-blue fluorescence. The lysosomal green fluorescent probe (Lyso-Tracker Green) is used for specific fluorescent staining of lysosomes in living cells. As shown in the figure, under the conditions of 3,5-dihydroxybenzeneazide (4) and alkynyl coumarin (5), there is no fluorescence in the cell. In the presence of the catalyst ligand BTTP, a weak blue fluorescence is generated in HeLa cells. After adding sodium ascorbate, the Cu(Ⅱ)\BTTP complex is chemically reduced to the Cu(I)\BTTP complex. The Cu(I)\BTTP complex can catalyze the CuAAC reaction of 3,5-dihydroxybenzeneazide (4) and alkynyl coumarin (5) to generate cyan-blue fluorescent Dye 6, and the fluorescence in the cell is enhanced. In the presence of the catalyst ligand BTTP, after irradiation of 30 Gy, a strong blue fluorescence is generated in the cell. Fluorescence imaging showed that gamma rays can reduce intracellular Cu(Ⅱ)\BTTP complexes to Cu(I)\BTTP complexes, catalyzing the CuAAC reaction to generate the cyan fluorescent molecule Dye 6. Under the conditions of catalyst ligands, gamma rays can catalyze click reactions in living cells.

[0083] Example 4 Application of gamma-ray catalyzed click reaction in in situ synthesis of antitumor active drugs.

[0084] This example explores whether gamma-ray catalysis can catalyze prodrugs 4 and 5 to generate resveratrol (5-(4-(4-hydroxyphenyl)-1H-1,2,3-triazol-1-yl)benzene-1,3-diol) analog 6 in tumor cells. According to literature reports, prodrug 6 can inhibit tumor growth by inhibiting cyclooxygenase activity. The experimental results of CCK-8 showed that prodrugs 4 and 5 were less toxic in Hela cells, and no obvious cytotoxicity was observed at a high concentration of 200 μM. The Cu(Ⅱ)\BTTP complex had a small amount of cytotoxicity at a high concentration (200 μM), with a survival rate of 85%. After treatment with prodrugs 4, 5 and Cu(Ⅱ)\BTTP complexes, the effect on cell viability was relatively small in the entire concentration range, and the cell viability was always maintained at a high level. At high concentration (200 μM), there was a small amount of cytotoxicity, and the survival rate was 84.4%. Subsequently, after treatment with irradiation conditions at a dose of 30 Gy, the cell viability decreased slightly in the entire concentration range. When the prodrug 4 / 5 was at a high concentration of 100 μM, the cell survival rate was 86.7%, and when the prodrug 4 / 5 was at a high concentration of 200 μM, the cell survival rate was 71.2%. These results indicate that gamma rays can effectively catalyze the in situ synthesis of 6.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for improving the catalytic activity of copper ion click reaction, characterized in that: include: A catalyst ligand is used to coordinate with Cu(II) ions in an aqueous solution, and a Cu(I) complex is obtained by ionizing radiation; wherein the water-soluble catalyst ligand is a nitrogen-containing water-soluble molecule; in the Cu(I) complex, the molar ratio of Cu(I) ions to the catalyst ligand is 1:2-10.

2. The method for improving the catalytic activity of copper ion click reaction according to claim 1, characterized in that: The concentration of Cu(II) ions in the aqueous solution is 50-300 μM.

3. The method for improving the catalytic activity of copper ion click reaction according to claim 1 or 2, characterized in that: The concentration of the catalyst ligand in the aqueous solution is 100-800 μM.

4. The method for improving the catalytic activity of copper ion click reaction according to any one of claims 1 to 3, characterized in that: In the Cu(I) complex, the molar ratio of Cu(I) ions to catalyst ligands is 1:4-6.

5. The method for improving the catalytic activity of copper ion click reaction according to any one of claims 1 to 4, characterized in that: The irradiation dose of the ionizing radiation is 0.1-100 Gy.

6. Use of the method for improving the catalytic activity of copper ion click reaction according to any one of claims 1 to 5 in catalyzing the cycloaddition reaction between azide and alkyne.

7. Use of the method for improving the catalytic activity of copper ion click reaction according to any one of claims 1 to 5 in living cell imaging.

8. Use of the method for improving the catalytic activity of copper ion click reaction according to any one of claims 1 to 5 in in-situ synthesis of drugs.

9. The use according to any one of claims 6 to 8, characterized in that: The application is not intended for disease diagnosis and treatment.

10. A method for in-situ synthesis of drugs by radiation click chemistry, characterized in that: include: The method for improving the catalytic activity of copper ion click reaction according to any one of claims 1 to 5 is used to catalyze a cycloaddition reaction between azide and alkyne in living cells.

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