阅读说明：本技术 一种用于成像和治疗的过氧化氢响应性化合物及其合成方法 (Hydrogen peroxide responsive compound for imaging and therapy and synthesis method thereof ) 是由 张建祥 闫新豪 郭嘉伟 吴鹏 李兰兰 窦寅 于 2020-05-07 设计创作，主要内容包括：本发明公开了一种用于成像和治疗的过氧化氢响应性化合物及其合成方法。该响应性化合物的化学结构如下图所示。本发明还提供了该类过氧化氢响应性化合物的合成方法：将单羟基化合物和草酰氯在含有缚酸剂的有机溶剂中反应,即得到过氧化氢响应性化合物。该类响应性化合物的合成方法简单,易于规模化合成；且可通过不同方法制备成纳米粒,其辅助化学发光特性具有过氧化氢响应性；当单羟基化合物为小分子药物时,可得到过氧化氢响应性前体药物,用于制备炎症和氧化应激损伤相关疾病及过氧化氢高表达肿瘤防治的纳米药物；该类过氧化氢响应性化合物具有良好的体内安全性。<Image he="390" wi="397" file="DDA0002480953080000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The invention discloses a hydrogen peroxide responsive compound for imaging and therapy and a synthesis method thereof. The chemical structure of the responsive compound is shown in the following figure. The invention also provides a synthesis method of the hydrogen peroxide responsive compound, which comprises the following steps: reacting a monohydroxy compound with oxalyl chloride in an organic solvent containing an acid-binding agent to obtain the hydrogen peroxide responsive compound. The synthesis method of the responsive compound is simple and easy for large-scale synthesis; the nano particles can be prepared by different methods, and the auxiliary chemiluminescence property of the nano particles has hydrogen peroxide responsiveness; when the monohydroxy compound is a micromolecular drug, a hydrogen peroxide responsive prodrug can be obtained and used for preparing nano-drugs for treating inflammation, oxidative stress injury related diseases and hydrogen peroxide high-expression tumor prevention and treatment; the hydrogen peroxide responsive compound has good in-vivo safety.)

1. A hydrogen peroxide responsive compound for use in imaging and therapy, characterized by: the chemical structural general formula is as follows

Wherein:

2. a method of synthesizing a hydrogen peroxide responsive compound for imaging and therapy comprising the steps of: mixing a single hydroxyl compound and an acid-binding agent in an organic solvent, slowly dropwise adding oxalyl chloride into the solution under the ice bath condition under the protection of nitrogen, removing the ice bath after dropwise adding, reacting for 2-12h, adding methanol or ethanol after the raw materials completely react, and quenching the reaction to obtain a crude product of the hydrogen peroxide responsive compound.

3. A method of synthesizing a hydrogen peroxide responsive compound for imaging and therapy according to claim 2, wherein: extracting the crude product with dichloromethane/water system for 3 times, collecting dichloromethane solution, extracting with saturated sodium chloride water solution for 1 time, drying the organic phase with anhydrous magnesium sulfate to remove excessive water, vacuum filtering, evaporating to remove dichloromethane, concentrating to obtain crude product, and separating and purifying with silica gel chromatographic column to obtain final product.

4. A method of synthesizing a hydrogen peroxide responsive compound for imaging and therapy according to claim 2 or 3, characterized in that: the monohydroxy compound is selected from tocopherol, idebenone, camptothecin, zidovudine, 4-hydroxymethylphenylboronic acid pinacol ester, 4-hydroxy-2, 2,6, 6-tetramethylpiperidine nitroxide, 3-methoxy-4-hydroxybenzaldehyde, 4-methoxymethylphenol, 4-ethoxymethylphenol, amyl salicylate, (E) -3, 5-dimethoxy-4' -hydroxystilbene or 2, 4-dinitrophenol.

5. A method of synthesizing a hydrogen peroxide responsive compound for imaging and therapy according to claim 2 or 3, characterized in that: the acid-binding agent is selected from N, N-diisopropylethylamine or triethylamine.

6. A method of synthesizing a hydrogen peroxide responsive compound for imaging and therapy according to claim 2 or 3, characterized in that: the organic solvent is dichloromethane.

7. A method of synthesizing a hydrogen peroxide responsive compound for imaging and therapy according to claim 2 or 3, characterized in that: the concentration of the monohydroxy compound in the organic solvent is between 0.1mmol/ml and 0.4 mmol/ml.

8. A method of synthesizing a hydrogen peroxide responsive compound for imaging and therapy according to claim 2 or 3, characterized in that: the molar ratio of the monohydroxy compound to the oxalyl chloride is between 1:0.5 and 1:1, and the molar ratio of the monohydroxy compound to the acid-binding agent is between 1:2 and 1: 5.

9. Use of the hydrogen peroxide-responsive compound of claim 1 for the preparation of a medicament and/or an imaging probe for the prevention and treatment of diseases associated with inflammation and/or oxidative stress injury.

10. Use according to claim 9, characterized in that: the inflammation includes allergic inflammation, non-specific inflammation, and infectious inflammation; the oxidative stress injury comprises acute heart injury, acute lung injury, acute/chronic liver injury, acute/chronic kidney injury, and acute/chronic intestinal tract injury.

Technical Field

The invention relates to the field of hydrogen peroxide responsive materials, in particular to a hydrogen peroxide responsive compound with auxiliary chemiluminescence imaging and treatment functions and a synthesis method thereof.

Background

When stimulated by growth factors or immune stimulation, cells produce endogenous hydrogen peroxide¹. The hydrogen peroxide has lipophilicity, is easy to pass through cell membranes to realize intercellular diffusion, is used as a second chemical messenger in cells, and plays an important role in regulating physiological reactions such as cell proliferation, differentiation, migration and the like^2-3. In addition, hydrogen peroxide is also involved in cellular redox signaling⁴Regulating the normal physiological functions of the body, and is closely related to the occurrence and development of various diseases, including angiogenesis, oxidative stress, aging, tumor and the like⁵. During the course of the disease, the expression and activity of hydrogen peroxide affect the pathophysiological changes of the disease, and therefore, studies on the diagnosis and treatment of diseases by means of hydrogen peroxide-responsive materials are receiving increasing attention^6-11。

A peroxyoxalate ester chemiluminescence system is a chemiluminescence system mainly composed of diaryl oxalate, hydrogen peroxide, fluorescent agent and solvent¹². The light-emitting mechanism is that chemical energy is transferred to fluorophore to transfer to excited state, and the excited state is transferred to ground state to emit light of certain wavelength¹³. The luminescent system is mainly applied in two aspects: detection application in analytical chemistry and development of chemical cold light source¹⁴. The peroxyoxalate ester compound is a small molecular substance sensitive to hydrogen peroxide, when the peroxyoxalate ester compound reacts with the hydrogen peroxide, an oxalate ester bond can be broken to generate intermediate and raw material molecules, and the intermediate can excite a dye to emit light for imaging¹⁵The responsiveness can be used for preparing a hydrogen peroxide responsive disease imaging diagnosis material or a hydrogen peroxide responsive drug release material^16-18Thereby being applied to the treatment of inflammation, oxidative stress injury and tumor related diseases^19-22. However, the in vivo safety of the products of hydrolysis of currently available peroxyoxalate compounds severely limits their use in disease imaging and therapy; meanwhile, the existing peroxy oxalate compounds can only be used for auxiliary chemiluminescence imaging and have no disease treatment function.

Disclosure of Invention

In view of the above-mentioned deficiencies of the prior art, it is a first object of the present invention to provide a hydrogen peroxide-responsive compound having a function of assisting in chemiluminescent imaging and therapy. Another object of the present invention is to provide a method for synthesizing the hydrogen peroxide-responsive compound.

In order to achieve the first object, the invention adopts the technical scheme that: a hydrogen peroxide responsive compound, the chemical structure general formula of which is as follows

Wherein:

in order to achieve the second object, the invention adopts the technical scheme that: the invention provides a synthesis method of a hydrogen peroxide responsive compound, which comprises the following steps: mixing a single hydroxyl compound and an acid-binding agent in an organic solvent, slowly dropwise adding oxalyl chloride into the solution under the ice bath condition under the protection of nitrogen, removing the ice bath after dropwise adding, reacting for 2-12h, adding methanol or ethanol after the raw materials completely react, and quenching the reaction to obtain a crude product of the hydrogen peroxide responsive compound.

In order to further purify the crude product to obtain a final product, the synthesis method of the invention further comprises the following steps: extracting the crude product with dichloromethane/water system for 3 times, collecting dichloromethane solution, extracting with saturated sodium chloride water solution for 1 time, drying the organic phase with anhydrous magnesium sulfate to remove excessive water, vacuum filtering, evaporating to remove dichloromethane, concentrating to obtain crude product, and separating and purifying with silica gel chromatographic column to obtain final product.

Further, the monohydroxy compound is selected from tocopherol, idebenone, camptothecin, zidovudine, 4-hydroxymethylphenylboronic acid pinacol ester, 4-hydroxy-2, 2,6, 6-tetramethylpiperidine nitroxide, 3-methoxy-4-hydroxybenzaldehyde, 4-methoxymethylphenol, 4-ethoxymethylphenol, amyl salicylate, (E) -3, 5-dimethoxy-4' -hydroxystilbene or 2, 4-dinitrophenol.

Further, the acid-binding agent is selected from N, N-diisopropylethylamine or triethylamine.

Further, the organic solvent is dichloromethane.

Further, the concentration of the monohydroxy compound in the organic solvent is between 0.1mmol/ml and 0.4 mmol/ml.

Further, the molar ratio of the monohydroxy compound to the oxalyl chloride is between 1:0.5 and 1:1, and the molar ratio of the monohydroxy compound to the acid-binding agent is between 1:2 and 1: 5.

The technical scheme of the invention also comprises the application of the hydrogen peroxide responsive compound in preparing a medicament and/or an imaging probe for preventing and treating diseases related to inflammation and/or oxidative stress injury.

The inflammation includes allergic inflammation, non-specific inflammation, and infectious inflammation; the nonspecific inflammation comprises red swelling and pain caused by trauma or operation; infectious inflammation includes inflammation caused by bacteria, bacterial products or viruses; allergic inflammation includes inflammatory diseases such as peritonitis, inflammatory bowel disease, arthritis, asthma, atherosclerosis, and non-alcoholic fatty liver disease.

The oxidative stress injury comprises acute heart injury, acute lung injury, acute/chronic liver injury, acute/chronic kidney injury, and acute/chronic intestinal tract injury.

Wherein the hydrogen peroxide-responsive compound is administered by a route selected from the group consisting of oral, intravenous, subcutaneous, intramuscular, and any combination thereof.

By combining the technical scheme, the invention has the beneficial technical effects that:

(1) the responsive compound is simple and convenient in synthesis method, when the selected compound is the monohydroxy active compound, the reaction site is single, namely one equivalent of oxalyl chloride is combined with two equivalents of monohydroxy active compound, the target compound is obtained by room temperature reaction, the condition is mild, and the scale synthesis is easy.

(2) The responsive compounds have significant and sensitive hydrogen peroxide dependent hydrolysis characteristics.

(3) The responsive compounds can be hydrolyzed in the presence of physiological levels of reactive oxygen species to the corresponding starting compounds.

(4) The responsive compound can be prepared into hydrogen peroxide responsive nanoparticles by different methods, and can effectively load fluorescent dye, so that the luminescent imaging nanoprobes with different self-luminescent wavelengths can be conveniently obtained.

(5) The nanoparticles prepared from the responsive compound can effectively load hydrophobic nano-drugs and realize the responsive release of hydrogen peroxide loaded with the drugs.

(6) When the responsive compound is synthesized by selecting the hydroxyl-containing drug, the hydrogen peroxide responsive prodrug can be conveniently prepared, and hydrogen peroxide responsive prodrug nanoparticles are prepared by adopting different methods, so that hydrogen peroxide responsive activation and release of the corresponding drug are realized.

(7) The nanoparticles prepared from the responsive compound have good in-vivo safety.

(8) When the obtained responsive compound is a solid, recrystallization can be used for removing impurities in the purification process, so that the silica gel chromatographic column is not used for separation and purification, and the method is easy to realize industrial production.

Drawings

FIG. 1 is a reaction of tocopherol with hydrogen peroxide responsive tocopherol compounds in deuterated chloroform, obtained by reacting tocopherol with oxalyl chloride¹And H NMR spectrum, and the structural correctness is confirmed according to the spectrum.

FIG. 2 is mass spectral data of a compound having a hydrogen peroxide-responsive tocopherol obtained by reacting tocopherol with oxalyl chloride.

FIG. 3 is a graph of light intensity measurements of 0.5mM hydrogen peroxide-responsive tocopherol compound and 0.5mM sulforhodamine 101 under stimulation with different concentrations of hydrogen peroxide; as can be seen, the luminous intensity increases as the hydrogen peroxide concentration increases.

FIG. 4 is a graph showing the luminous intensity test of 0.5mM hydrogen peroxide-responsive tocopherol compound and 0.5mM 1-chloro-9, 10-diphenylethynylanthracene under different concentrations of hydrogen peroxide, and it can be seen that the luminous intensity increases with increasing hydrogen peroxide concentration, and has concentration dependence.

Fig. 5 is a photograph of a sample, a particle size distribution diagram, and a transmission electron microscope photograph of nanoparticles prepared from the hydrogen peroxide-responsive tocopherol compound, the average particle size of which is 188 nm.

Fig. 6 is a graph showing that the sulforhodamine 101 nanoparticle encapsulated by the hydrogen peroxide-responsive tocopherol compound stimulates the light-responsive measurement to different types of active oxygen, and the nanoparticle has selective response to hydrogen peroxide.

Fig. 7 is an imaging diagram of a pore plate with the luminescent responsiveness of hydrogen peroxide-responsive tocopherol compound-loaded 1-chloro-9, 10-diphenylethynyl anthracene nanoparticles to hydrogen peroxide with different concentrations, and it can be seen from the graph that the luminescent intensity of the nanoparticles increases with the increase of the hydrogen peroxide concentration, and the nanoparticles have concentration dependence.

Fig. 8 is an imaging diagram of a pore plate of a hydrogen peroxide-responsive tocopherol compound-loaded 1-chloro-9, 10-diphenylethynyl anthracene nanoparticle in mouse peritoneal neutrophils, wherein the luminous intensity of the nanoparticle is gradually enhanced along with the increase of the number of the neutrophils, and the imaging of the nanoparticle has neutrophil responsiveness.

FIG. 9 shows imaging functional verification of hydrogen peroxide-responsive tocopherol compound-loaded 1-chloro-9, 10-diphenylethynyl anthracene nanoparticles in thioglycollate-induced peritonitis in mice.

Fig. 10 shows imaging functional verification of hydrogen peroxide-responsive tocopherol compound-loaded 1-chloro-9, 10-diphenylethynyl anthracene nanoparticles in alcohol-induced liver injury in mice.

Fig. 11 is an imaging functional test of hydrogen peroxide-responsive tocopherol compound-loaded 1-chloro-9, 10-diphenylethynyl anthracene nanoparticles in subcutaneous tumors.

Detailed Description

The present invention will be described in further detail with reference to specific embodiments. It is to be understood that the embodiments of the present invention are merely for illustrating the present invention and not for limiting the present invention, and that various substitutions and alterations made according to the common knowledge and conventional means in the art without departing from the technical idea of the present invention are included in the scope of the present invention.