阅读说明：本技术 诊疗联用分子先导化合物合成方法及在线粒体成像中应用 (Diagnosis and treatment combined molecular lead compound synthesis method and application in mitochondrial imaging ) 是由 高杰 袁泽利 麦宇涵 于 2020-05-14 设计创作，主要内容包括：诊疗联用分子先导化合物合成方法及在线粒体成像中应用,该先导化合物由吲哚花菁母核和苯丁酸氮芥构成,在同一分子结构中集线粒体靶向、荧光成像诊断、光动力治疗与化疗于一体。本发明还公开了在细胞线粒体成像的应用。本发明中的先导化合物结构简单、分子量小,具有确定的化学结构,易于制备、纯化和进一步修饰。通过细胞成像实验证明,借助近红外荧光成像可发现先导化合物结构中的正电性和线粒体负电性作用可以显著靶向于细胞的线粒体。因此,本发明涉及的线粒体靶向诊疗联用单分子多功能先导化合物在细胞器成像、肿瘤治疗方面具有良好的应用前景。(The lead compound is composed of indocyanine mother nucleus and chlorambucil, and integrates mitochondrial targeting, fluorescence imaging diagnosis, photodynamic therapy and chemotherapy in the same molecular structure. The invention also discloses application of the method in cell mitochondrial imaging. The lead compound has the advantages of simple structure, small molecular weight, determined chemical structure and easy preparation, purification and further modification. Cell imaging experiments prove that the electropositive and mitochondrial electronegative effects in the structures of the lead compounds can be found by means of near-infrared fluorescence imaging and can be obviously targeted to the mitochondria of cells. Therefore, the mitochondrion targeted diagnosis and treatment combined single-molecule multifunctional lead compound has good application prospect in the aspects of organelle imaging and tumor treatment.)

1. A method for synthesizing a molecular lead compound for diagnosis and treatment comprises the following steps: (i) taking IR780(0.334g, 0.5mmol) and dissolving in 10mL of dehydrated DMF under the condition of keeping out of the light, adding a dehydrated DMF solution dissolved with piperazine (0.173g, 2mmol) under the protection of nitrogen and heating to 80 ℃ for reaction for 4 h; gradually changing the reaction solution from green to blue, monitoring the reaction completion degree by TLC, cooling the reaction system to room temperature after the reaction is completed, and concentrating under reduced pressure to obtain a crude product; purification was performed by silica gel column chromatography using methanol: dichloromethane (v: v) ═ 1:30, yielding the intermediate as a red solid (75% yield); see the following reaction scheme:

(ii) under the condition of keeping out of the sun, taking 30mL of dehydrated dichloromethane, adding the intermediate (5mg, 6.6 mu mol) and chlorambucil (2mg, 6.6 mu mol) with stirring, then adding BOP reagent (3mg, 6.6 mu mol), adding several drops of triethylamine, and reacting for 1-2h with stirring at room temperature under the protection of nitrogen; monitoring the reaction completion degree by TLC, cooling the reaction system to room temperature after the reaction is completed, and concentrating under reduced pressure to obtain a crude product; purification was performed by silica gel column chromatography using methanol: dichloromethane (v: v) ═ 0:100 to 1:20, giving the red solid desired product, see the following reaction scheme:

2. the method for synthesizing the diagnosis and treatment combined molecular lead compound according to claim 1, wherein the method comprises the following steps: the lead compound is an organic small molecule, has the functions of mitochondrion targeting, near-infrared fluorescence imaging diagnosis and near-infrared photodynamic therapy synergistic chemotherapy in the same molecular structure, and the molecular structure of the lead compound is composed of indocyanine parent nucleus and chlorambucil. The electropositivity of the parent nucleus of the indocyanine in the molecular structure of the indocyanine target mitochondrial group; the indocyanine mother nucleus is a photodynamic group; chlorambucil is a chemotherapy group, and the functional structural formula of the chlorambucil is shown as a formula I,

3. the method for synthesizing the diagnosis and treatment combined molecular lead compound according to claim 1, wherein the method comprises the following steps: the lead compounds are useful in mitochondrial imaging.

Technical Field

The invention relates to a synthesis method of a molecular lead compound for diagnosis and treatment and application of the molecular lead compound in mitochondrial imaging, and belongs to the field of biomedicine.

Background

The real-time and original taste screening and diagnosis of related diseases such as cell surface receptor, signal channel, cell metabolism change, gene abnormal mutation and the like at the cellular and molecular level is always the direction of the molecular imaging technology and is one of the limited receptions for realizing accurate medical treatment, and the rapid development is realized in scientific research and clinical application. Currently, the main molecular imaging diagnostic techniques include optical imaging, nuclear species imaging, magnetic resonance imaging, and computed tomography. Wherein, the optical molecular imaging has low energy, no radiation and high sensitivity to signal detection, and has real-time monitoring labeled in vivo cell activity and gene behavior, and is becoming a research hotspot in molecular imaging [ Chen C, Tian R, Zeng Y, et al, bioconjugate Chemistry,2020,31:276-]. The fluorescence imaging technology in the optical imaging can dynamically mark the space-time distribution of target molecules in cells under specific tissues and pathological states on a three-dimensional scale, realize the 'targeting' tracing in living cells and tissues, carry out the visual detection of the target molecules in living bodies on the cell and tissue/organ level, track the physiological process of the target molecules in living organisms and present the structure-activity relationship of the target molecules and the organisms in real time and original taste. Meanwhile, the fluorescence imaging technology has the characteristics of non-invasiveness, safety and high space-time resolution, so that the fluorescence imaging technology is widely applied to various fields such as tumor diagnosis, drug distribution and metabolism, endogenous biomolecule detection and the like, and becomes an important tool in the current biomedical field^[6-12]。

In optical imaging, the fluorescent dye used and its intrinsic absorption and fluorescence wavelengths become the key to the diagnostic technique, mainly because the relationship between wavelength and tissue penetration can image its diagnostic condition in vivo. Near infrared light has the characteristics of strong tissue penetration and significantly reduced interference from autofluorescence of endogenous molecules as compared with light of other wavelengths, and therefore, a fluorescent photosensitizer in the near infrared spectral region has the combined advantages of good tissue penetration and low background, and is readily transmitted through biological tissues for in vivo fluorescence imaging [ Kobayashi H, Ogawa M, Alford R, et al. chemical reviews,2010,110: 2620-. In the biological imaging research, the cyanine dye has the advantages of small background interference, high sensitivity, strong tissue penetrating power, suitability for tissue fluorescence and the like, and becomes a hot-spot mother-nucleus structure of the near-infrared fluorescent dye. Meanwhile, the biocompatibility of the phthalocyanine dye and the low toxicity of the biological sample enable the phthalocyanine dye to be suitable for the living body real-time and original-taste near-infrared fluorescence imaging diagnosis of the biological sample. In addition, the near-infrared indocyanine dye not only has excellent near-infrared fluorescence imaging diagnosis performance, but also shows light-dependent cytotoxicity, and can be used as a photosensitizer with photodynamic therapy performance [ Yang Q, Jin H, Gao Y, et al. ACS Applied Materials & Interfaces,2019,11: 15417-.

Disclosure of Invention

The invention aims to solve the technical problems of single imaging and treatment mode, poor diagnosis and treatment effect, tumor insensitivity of a monotherapy and the like in the field of malignant tumor diagnosis and treatment in the prior art aiming at the current clinical requirements of targeted drugs and diagnosis and treatment combined drugs on future personalized medical treatment, and the invention realizes near-infrared fluorescence imaging diagnosis in the early stage of malignant tumor, in-situ photodynamic therapy and chemotherapy cooperative therapy and treatment of the malignant tumor and the curative effect evaluation of the treatment process.

In order to solve the technical problems, the invention adopts the following technical scheme:

a method for synthesizing a molecular lead compound for diagnosis and treatment comprises the following steps: (i) taking IR780(0.334g, 0.5mmol) and dissolving in 10mL of dehydrated DMF under the condition of keeping out of the light, adding a dehydrated DMF solution dissolved with piperazine (0.173g, 2mmol) under the protection of nitrogen and heating to 80 ℃ for reaction for 4 h; gradually changing the reaction solution from green to blue, monitoring the reaction completion degree by TLC, cooling the reaction system to room temperature after the reaction is completed, and concentrating under reduced pressure to obtain a crude product; purification was performed by silica gel column chromatography using methanol: dichloromethane (v: v) ═ 1:30, yielding the intermediate as a red solid (75% yield); see the following reaction scheme:

(ii) under the condition of keeping out of the sun, taking 30mL of dehydrated dichloromethane, adding the intermediate (5mg, 6.6 mu mol) and chlorambucil (2mg, 6.6 mu mol) with stirring, then adding BOP reagent (3mg, 6.6 mu mol), adding several drops of triethylamine, and reacting for 1-2h with stirring at room temperature under the protection of nitrogen; monitoring the reaction completion degree by TLC, cooling the reaction system to room temperature after the reaction is completed, and concentrating under reduced pressure to obtain a crude product; purification was performed by silica gel column chromatography using methanol: dichloromethane (v: v) ═ 0:100 to 1:20, giving the red solid desired product, see the following reaction scheme:

the lead compound is an organic small molecule, has the functions of mitochondrion targeting, near-infrared fluorescence imaging diagnosis and near-infrared dynamic therapy synergistic chemotherapy in the same molecular structure, and the molecular structure of the lead compound is composed of indocyanine parent nucleus and chlorambucil. The electropositivity of the parent nucleus of the indocyanine in the molecular structure of the indocyanine target mitochondrial group; the indocyanine mother nucleus is a photodynamic group; chlorambucil is a chemotherapy group, and the functional structural formula of the chlorambucil is shown as a formula I,

the lead compounds are useful in mitochondrial imaging.

The beneficial effect of adopting above-mentioned technical scheme is:

the monomolecular multifunctional diagnosis and treatment combined lead compound can perform near-infrared positioning on tumors through fluorescence imaging, can monitor targeted tumor enrichment of the first-come compound through living body fluorescence imaging, and guides accurate photodynamic to generate a large amount of ROS at tumor focuses, so that photodynamic therapy and chemotherapy synergistic therapy are achieved, and the curative effect is effectively improved.

Drawings

FIG. 1 shows 1H NMR of a lead compound for single-molecule multifunctional diagnosis and treatment according to the present invention.

FIG. 2 shows 13C NMR of the lead compound for single-molecule multifunctional diagnosis and treatment according to the present invention.

FIG. 3 is a high-resolution mass spectrum of the lead compound for single-molecule multifunctional diagnosis and treatment.

FIG. 4 is a diagram of the ultraviolet-visible absorption spectrum of the lead compound for single-molecule multifunctional diagnosis and treatment in accordance with the present invention.

Fig. 5 is a fluorescence excitation spectrum and an emission spectrum of the single-molecule multifunctional diagnosis and treatment combined lead compound.

Fig. 6 is a diagram illustrating mitochondrion targeting imaging of the single molecule multifunctional diagnosis and treatment combined lead compound of the present invention. HepG2 cells were co-stained with Mito-Tracker green and lead compounds. (a) A lead compound channel; (b) the Mito-Trackergreen channel; (c) a superimposed image of image (a) and image (b); (d) corresponding differential interference contrast images; (e) intensity correlation plots of Mito-Tracker green and lead compounds (Pearson coefficient 0.95 and overlap coefficient 0.94); (f) Mito-Tracker green in Linear ROI 1 and intensity curves of the lead compound.

Detailed Description

The present invention will be described in detail with reference to specific embodiments. It should be noted that the following description is illustrative of the invention and is not to be construed as limiting.

1. The invention relates to a synthesis method of a diagnosis and treatment combined molecular lead compound;

the method comprises the following steps: (i) taking IR780(0.334g, 0.5mmol) and dissolving in 10mL of dehydrated DMF under the condition of keeping out of the light, adding a dehydrated DMF solution dissolved with piperazine (0.173g, 2mmol) under the protection of nitrogen and heating to 80 ℃ for reaction for 4 h; gradually changing the reaction solution from green to blue, monitoring the reaction completion degree by TLC, cooling the reaction system to room temperature after the reaction is completed, and concentrating under reduced pressure to obtain a crude product; purification was performed by silica gel column chromatography using methanol: dichloromethane (v: v) ═ 1:30, yielding the intermediate as a red solid (75% yield); see the following reaction scheme:

(ii) under the condition of keeping out of the sun, taking 30mL of dehydrated dichloromethane, adding the intermediate (5mg, 6.6 mu mol) and chlorambucil (2mg, 6.6 mu mol) with stirring, then adding BOP reagent (3mg, 6.6 mu mol), adding several drops of triethylamine, and reacting for 1-2h with stirring at room temperature under the protection of nitrogen; monitoring the reaction completion degree by TLC, cooling the reaction system to room temperature after the reaction is completed, and concentrating under reduced pressure to obtain a crude product; purification was performed by silica gel column chromatography using methanol: dichloromethane (v: v) ═ 0:100 to 1:20, giving the red solid desired product, see the following reaction scheme:

2. the molecular lead compound for diagnosis and treatment provided by the invention has the maximum absorption wavelength of 775nm, the maximum excitation wavelength of 798nm and the maximum fluorescence emission spectrum of 806nm after being modified, and is a near-infrared absorption region.

The diagnosis and treatment combined molecular lead compound provided by the invention is formed by indocyanine and chlorambucil, and realizes that the same molecule has multiple functions of mitochondrion targeting, near-infrared fluorescence imaging diagnosis, near-infrared light dynamic therapy and chemotherapy synergy.

The diagnosis and treatment combined molecular lead compound is dissolved by ethanol to prepare 5.0 mu mol.L^-1The absorbance value is measured by an ultraviolet-visible spectrophotometer, and the fluorescence excitation spectrum and the fluorescence emission spectrum are measured by a Vary Eclipse fluorescence spectrophotometer, the results are shown in figures 1-1 and 1-2, the maximum absorption wavelength is 775nm, the maximum excitation wavelength is 798nm, the maximum fluorescence emission spectrum length is 806nm, and the optical characteristics of the near infrared fluorescent dye accord with 650-900 nm.

3. The diagnosis and treatment combined molecular lead compound has mitochondrion targeting property.

The invention uses HepG2 cell as 5 × 10⁴Each well was inoculated in a petri dishAnd incubating for 24 hours, then changing into a culture solution containing 1 mu M of single-molecule multifunctional diagnosis and treatment combined lead compound, incubating for 8 hours, then changing into a culture solution containing 150nM mitochondrion targeting agent Mito-tracker green, incubating for half an hour again, then washing the culture dish for 3 times by using PBS, adding 1mL of PBS, placing the culture dish in an inverted fluorescence microscope for fluorescence imaging shooting, shooting the fluorescence of the lead compound under a green fluorescence channel, exciting the fluorescence of the Mito-tracker green by using a blue channel, and processing a fluorescence picture by using Image J software.

While some embodiments of the present invention have been shown, it will be understood by those skilled in the art that changes may be made to the embodiments herein without departing from the spirit of the invention. The above examples are merely illustrative and should not be taken as limiting the scope of the invention.