WO2023029638A1 - N,N-DIPHENYLAMINO-MODIFIED β-CARBOLINE INDOLIUM SALT, PREPARATION METHOD AND USE - Google Patents

N,N-DIPHENYLAMINO-MODIFIED β-CARBOLINE INDOLIUM SALT, PREPARATION METHOD AND USE Download PDF

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WO2023029638A1
WO2023029638A1 PCT/CN2022/097845 CN2022097845W WO2023029638A1 WO 2023029638 A1 WO2023029638 A1 WO 2023029638A1 CN 2022097845 W CN2022097845 W CN 2022097845W WO 2023029638 A1 WO2023029638 A1 WO 2023029638A1
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carboline
diphenylamino
compound
modified
anion
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Chinese (zh)
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凌勇
杨宇民
李鹏
钱建强
孟迟
张延安
许中原
丁倩
缪道鑫
张雨婷
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南通大学
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    • C07—ORGANIC CHEMISTRY
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    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
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    • A61K49/00—Preparations for testing in vivo
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    • A61K49/0013—Luminescence
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    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
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    • C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1092—Heterocyclic compounds characterised by ligands containing sulfur as the only heteroatom

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  • the invention relates to the field of biomedicine, and relates to a class of N,N-diphenylamino-modified ⁇ -carboline indolium salts, a preparation method and application thereof, in particular to a class of pH-sensitive N,N-diphenylamino-modified ⁇ -carboline indolium and its preparation method and application.
  • Cancer is one of the deadliest diseases faced by human beings. According to the statistics of the World Health Organization in 2018, there were 18.1 million new cancer cases and 9.6 million cancer deaths worldwide. However, if it were possible to image tumors early in their development, this would greatly improve cancer mortality.
  • Tumor fluorescence imaging diagnosis technology a key is to design fluorescent probes that can respond quickly and accurately to tumor markers.
  • Tumor cells mainly rely on aerobic glycolysis to provide energy, and the lactic acid produced during this process will be effluxed out of the cell, thereby creating an acidic tumor microenvironment.
  • pH-responsive probes are often based on acid-sensitive bonds to achieve "switching" effects, such as controlling fluorescence by controlling the breakage of acid-sensitive hydrazone bonds, imine bonds, or acetals.
  • this type of pH probe has obvious defects. First, the process of covalent bond breaking takes a certain amount of time, and it cannot perform rapid and real-time diagnosis of tumors in spray mode; second, it does not have fluorescence reversibility, so it cannot It can dynamically observe tumor and normal tissues with different pH.
  • NIR fluorescent probes Compared with ultraviolet-visible fluorescence, near-infrared (NIR) fluorescent probes have the advantages of less damage to organisms, better tissue penetration, and less interference from tissue autofluorescence, so they are more suitable for in vivo imaging. Therefore, in order to enable real-time, accurate and rapid imaging diagnosis of tumors, the present invention modifies ⁇ -carboline.
  • NIR near-infrared
  • ⁇ -carboline compounds are a large class of naturally occurring indole alkaloids, which have a planar tricyclic structure similar to carbazole-like pyrido[3,4-b]indole, and have low toxicity.
  • the present invention introduces an electron-donating group NN-diphenylamino group at the 6-position of ⁇ -carboline, which can quickly generate stable pH-sensitive fluorescence, and connects different substituted indolium salts or different combinations by introducing a vinyl group at the 3-position
  • the benzoindolium salt fragment in the form, the NN-diphenylamino group of the 6-position electron-donating effect of ⁇ -carboline can enhance the electron-donating ability to obtain a fluorescent compound with a longer fluorescence wavelength and a larger Stokes shift value, Introduce water-soluble side chains containing morpholinyl and piperazinyl at the N9 position of ⁇ -carboline to improve the lipid-water distribution coefficient of the entire molecule and obtain NIR
  • this fluorescent probe can quickly achieve pH-sensitive fluorescence through intramolecular charge transfer (ICT), and has acidic pH-regulated fluorescence. reversibility.
  • ICT intramolecular charge transfer
  • the compound of the present invention has pH-sensitive near-infrared fluorescence characteristics, and the fluorescence is reversible with pH changes, and can be prepared as a spray, which can be sprayed or locally injected on the surface of tumor cells or tissues for rapid, real-time and selective fluorescence imaging.
  • the compound of the present invention will be a good supplement and expansion of the clinical application field.
  • a class of pH-sensitive N,N-diphenylamino-modified ⁇ -carboline indolium salts having the structure shown in general formula I:
  • R 1 is selected from H, C1-C6 alkyl, C1-C6 linear alkyl morpholine;
  • R 2 is selected from one of H, F, Cl, Br, I;
  • n is 0 or 1.
  • a class of pH-sensitive N,N-diphenylamino modified ⁇ -carboline indolium salts in the structure of formula I, R 1 , R 2 , Y and n are selected from the following combinations:
  • Another object of the present invention is to provide the following preparation method of the compound described in general formula I of the present invention:
  • the preparation method route is:
  • R 1 is selected from H, C1-C6 alkyl, C1-C6 linear alkyl morpholine;
  • R 2 is selected from one of H, F, Cl, Br, I;
  • n is 0 or 1.
  • the preparation method specifically comprises the following steps:
  • the present invention also provides the application of the above-mentioned N,N-diphenylamino-modified ⁇ -carboline indolium salt in preparing a pH-responsive fluorescent developer.
  • the fluorescent contrast agent is a selective fluorescence imaging contrast agent for tumor tissue or tumor cells in vivo and in vitro.
  • the application is specifically dissolving the N,N-diphenylamino-modified ⁇ -carboline indolium salt with a cosolvent/surfactant/solvent system to obtain a sprayable N,N-diphenyl Amino-modified ⁇ -carboline indolium salt solution.
  • the content of the co-solvent is 1-30%, and the content of the surfactant is 1-30%; the co-solvent 1,2-propanediol, DMSO or ethanol; the solvent is water; the surfactant is Tween 20, Tween 40 or Tween 80.
  • the tumor is one of liver cancer, colon cancer, breast cancer, lung cancer and cervical cancer.
  • the present invention Compared with the prior art, the present invention has the application effect: the compound of the present invention utilizes the principle of ICT to activate in the acidic microenvironment of the tumor tissue, and selectively rapidly generate pH-sensitive near-infrared fluorescence at the tumor site.
  • the specific implementation method is to Spray or locally inject the compound solution of the present invention on the tumor lesion site and the surrounding tissue before or during the operation, and use a fluorescent endoscope or an in vivo imager to perform rapid and selective fluorescence imaging and tracing of the tumor lesion tissue, which has a high
  • the selectivity and low background fluorescence interference of tumor tissue fluorescence imaging enable accurate diagnosis of tumors to guide surgery and/or drug treatment.
  • Fig. 1 is the ultraviolet absorption spectrum of compound of the present invention, fluorescence spectrum, and wherein A figure is the ultraviolet absorption spectrum of I 1 , and B figure is the fluorescence spectrum of I 1 , and C figure is the ultraviolet absorption spectrum of I 4 , and D figure is I 4 's Fluorescence spectrum;
  • Fig. 2 is the selective fluorescence imaging of liver cancer cells by compound I 1 of the present invention
  • Figure 3 shows the fluorescence imaging of lung cancer cells by Compound I 2 of the present invention
  • Fig. 4 is the selective fluorescence imaging of isolated breast cancer tumor by compound I 1 of the present invention.
  • Fig. 5 is the selective fluorescence imaging of clinical colon cancer tumor by compound I 1 of the present invention.
  • Fig. 6 is the selective fluorescence imaging of clinical colon cancer tumors by Compound I 4 of the present invention.
  • compound 1 (500mg, 1.57mmol) was dissolved in anhydrous DMF, NaH (301mg, 12.56mmol) was added under ice-bath condition and stirred for 0.5h, then CH 3 I (1ml, 15.7mmol) was added and stirred at room temperature for 1h, After the reaction was monitored by TLC, the reaction solution was poured into ice water, adjusted to pH 7 with 0.1 mol hydrochloric acid solution, the filtrate was suction filtered, and the filter cake was dried to obtain compound 2 with a yield of 85%.
  • First compound 2 (480mg, 1.45mmol), diphenylamine (1.47g, 8.7mmol), Pd(dBa) 3 (83.375g, 0.145mmol), sodium tert-butoxide (430mg, 5.8mmol), tri-tert-butylphosphine
  • Add 0.1ml into a sealed tube add 4ml of toluene, protect with N2 , and react at 110°C for 12h. After the reaction is completed and monitored by TLC, the reaction solution is concentrated, sanded, and passed through the column to obtain compound 3 with a yield of 76 %.
  • compound 3 (530mg, 1.3mmol) was dissolved in anhydrous THF (10ml). After ice bathing for 10min, LiAlH 4 (74.1mg, 1.95mmol) was added in batches. After 3 hours, after the reaction was monitored by TLC, 10 ml of methanol was added until no bubbles were generated, then 1 ml of water was added, suction filtered, the filter cake was washed with methanol, and spin-dried to obtain compound 4 with a yield of 80%.
  • compound 3 (200mg, 0.508mmol), I 2 (280mg, 1.103mmol), K 3 PO 4 (186mg, 0.876mmol) were added into a sealed tube, dissolved in 2ml of acetonitrile, protected by N 2 , and reacted at 110°C for 18h. After the reaction, about 1.2 ml of triethylamine was added to continue the reaction at 150° C. for 4 h, concentrated under reduced pressure, and separated by column chromatography to obtain compound 6 with a yield of 65%.
  • the fluorescent compound of the present invention is dissolved in an aqueous solution containing 1% DMSO to prepare a 5-20 ⁇ M detection solution.
  • the ultraviolet absorption spectrum data is tested by an ultraviolet-visible spectrophotometer, and the result shows that the ultraviolet maximum absorption wavelength of the fluorescent compound of the present invention is within the range of 520-650nm.
  • compounds I 1 and I 4 have maximum ultraviolet absorption wavelengths around 592 and 583 nm respectively (Fig. 1A and Fig. 1C); Note: Fig. 1A is the ultraviolet absorption spectrum of I 1 , Fig. 1B is the fluorescence spectrum of I 1 , Fig. 1C It is the ultraviolet absorption spectrum of I 4 , and Fig. 1D is the fluorescence spectrum of I 4 .
  • Embodiment 6 The pH-sensitive fluorescence test of the compound of the present invention
  • the fluorescent compound of the present invention is dissolved in an aqueous solution containing 1% DMSO to prepare a 5-20 ⁇ M detection solution.
  • the results show that the maximum emission wavelength of the fluorescent compound of the present invention is in the range of 680-750nm.
  • the maximum emission wavelengths of compounds I1 and I4 of the present invention between pH 3.5 and 6.5 are respectively about 740 and 692nm, and the fluorescence wavelength reaches the near-infrared region, but there is almost no fluorescence at neutral pH.
  • the Stokes of the compounds of the present invention The displacement value reaches 150-200nm, and has good fluorescence characteristics;
  • Example 7 The compounds of the present invention are tested for selective fluorescence imaging of tumor cells
  • Human liver cancer cells HepG2, normal liver cells LO2 and human lung cancer cells A549 were cultured in confocal culture dishes at a density of 1 ⁇ 10 5 cells at 37°C for 24 h with 1 mL of culture medium. Then, the culture medium of HepG2 and LO2 cells was replaced with fresh medium containing 1-100 ⁇ M I1 , and the medium of A549 cells was replaced with fresh medium containing 1-100 ⁇ M I2 , and incubated at 37 °C for 10 ⁇ 30min, and then wash the cells 3 times with PBS.
  • the imaging results shown in Figure 2 and Figure 3 show that the compound I 1 (10 ⁇ M) of the present invention can clearly perform fluorescence imaging on the liver tumor cell HepG2 after 4 hours, while the LO2 fluorescence in normal cells is very weak.
  • HepG2 The fluorescence intensity is 6.5 times that of LO2 cells, indicating that the compound of the present invention can selectively perform fluorescence imaging on liver tumor cells; at the same time, the compound I 2 (50 ⁇ M) of the present invention can clearly perform fluorescence imaging on A549 cells after 4 hours.
  • Example 8 Fluorescence imaging test of compound of the present invention in spray mode on isolated tumor tissue
  • Embodiment 9 Compounds of the present invention are tested on clinical tumor tissue fluorescence imaging

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Abstract

The present invention relates to the field of biological medicines, and relates to an N,N-diphenylamino-modified β-carboline indolium salt, a preparation method and the use. The β-carboline indolium salt has a structure as represented by general formula (I), can be activated in the acidic microenvironment of tumor tissue by the ICT principle, and selectively and rapidly generates pH-sensitive near-infrared fluorescence at a tumor site. The specific implementation method comprises: spraying or locally injecting a solution of the β-carboline indoliumm salt of the present invention onto/into a tumor lesion site and the surrounding tissue, and performing rapid and selective fluorescence imaging and tracing on the tumor lesion tissue by means of using a fluorescence endoscope or an in vivo imager. The method has a relatively high tumor tissue fluorescence imaging selectivity and a relatively low background fluorescence interference, and can be used for accurately detecting tumors.

Description

N,N二苯基氨基修饰的β-咔啉吲哚鎓盐、制备方法与应用N, N diphenylamino modified β-carboline indolium salt, preparation method and application 技术领域technical field
本发明涉及生物医药领域，涉及一类N,N二苯基氨基修饰的β-咔啉吲哚鎓盐、制备方法与应用，具体涉及一类具有pH敏感的N,N-二苯基氨基修饰的β-咔啉吲哚鎓及其制备方法与应用。The invention relates to the field of biomedicine, and relates to a class of N,N-diphenylamino-modified β-carboline indolium salts, a preparation method and application thereof, in particular to a class of pH-sensitive N,N-diphenylamino-modified β-carboline indolium and its preparation method and application.
背景技术Background technique
癌症是人类面临的最致命的疾病之一，根据世界卫生组织2018年的统计，全球新增癌症病例1810万例，癌症死亡960万例。但是，如果能够对在肿瘤发展的早期对肿瘤进行成像诊断，这将大大改善癌症的死亡率。Cancer is one of the deadliest diseases faced by human beings. According to the statistics of the World Health Organization in 2018, there were 18.1 million new cancer cases and 9.6 million cancer deaths worldwide. However, if it were possible to image tumors early in their development, this would greatly improve cancer mortality.
临床上常见的成像技术如CT、MRI和PET等因其空间分辨率有限而难以对肿瘤进行精确的诊断指导。相比之下，荧光成像技术因其高灵敏度、高空间分辨率和能够实时成像诊断成等优势而受到越来越多的关注。在肿瘤荧光成像诊断技术中，一个关键就是设计出能够对肿瘤标志物快速准确响应的荧光探针。肿瘤细胞主要依赖于有氧糖酵解提供能量，在此过程中产生的乳酸会外排到细胞外，进而造成肿瘤酸性微环境，已有一些文献报道利用较低的pH来设计低pH响应的肿瘤诊断剂。以往报道的pH响应探针常常基于酸性敏感键来达到“开关”效果，如通过控制酸敏感的腙键、亚胺键或缩醛等断裂来控制荧光。但是该类pH探针具有明显的缺陷，第一，共价键断键过程需要一定的时间，不能够对肿瘤进行喷洒模式的快速、实时诊断；第二，其不具备荧光可逆性，所以不能够动态观察不同pH的肿瘤和正常组织。Common clinical imaging techniques such as CT, MRI, and PET are difficult to provide accurate diagnostic guidance for tumors due to their limited spatial resolution. In contrast, fluorescence imaging technology has attracted more and more attention due to its advantages of high sensitivity, high spatial resolution, and the ability to perform real-time imaging and diagnosis. In tumor fluorescence imaging diagnosis technology, a key is to design fluorescent probes that can respond quickly and accurately to tumor markers. Tumor cells mainly rely on aerobic glycolysis to provide energy, and the lactic acid produced during this process will be effluxed out of the cell, thereby creating an acidic tumor microenvironment. There have been some reports in the literature that use lower pH to design low-pH response Tumor diagnostic agent. Previously reported pH-responsive probes are often based on acid-sensitive bonds to achieve "switching" effects, such as controlling fluorescence by controlling the breakage of acid-sensitive hydrazone bonds, imine bonds, or acetals. However, this type of pH probe has obvious defects. First, the process of covalent bond breaking takes a certain amount of time, and it cannot perform rapid and real-time diagnosis of tumors in spray mode; second, it does not have fluorescence reversibility, so it cannot It can dynamically observe tumor and normal tissues with different pH.
与紫外-可见荧光相比，近红外(NIR)荧光探针具有对生物体损伤小、对组织穿透性好、组织自身荧光干扰少等优点而更适合体内成像。因此为了使其能够实时、精确、快速地对肿瘤进行成像诊断，本发明对β-咔啉进行修饰改造。Compared with ultraviolet-visible fluorescence, near-infrared (NIR) fluorescent probes have the advantages of less damage to organisms, better tissue penetration, and less interference from tissue autofluorescence, so they are more suitable for in vivo imaging. Therefore, in order to enable real-time, accurate and rapid imaging diagnosis of tumors, the present invention modifies β-carboline.
发明内容Contents of the invention
β-咔啉化合物是一大类天然存在的吲哚生物碱，具有类似于咔唑的吡啶[3,4-b]并吲哚的平面三环结构骨架，同时具有较低的毒性。本发明在β-咔啉的6位引入供电子基团NN-二苯基氨基，可快速产生稳定的pH敏感型荧光，通过在3位引入乙烯基连接不同取代的吲哚鎓盐或不同拼合形式的苯并吲哚鎓盐片段，β-咔啉6位给电子效应的NN-二苯基氨基可以增强给电子能力，以获得更长的荧光波长和更大的Stokes位移值的荧光化合物，在β-咔啉的N9位引入含有吗啉基、哌嗪基等水溶性侧链，以改善整个分子的脂水分布系数，获得基于“供体-受体”型大Stokes位移的NIR荧光分子。与传统的pH荧光探针通过酸性敏感的腙键或缩醛基团激活的方式不同，该荧光探针可以通过分子内电荷转移(ICT)快速实现pH敏感性荧光，且具有酸性pH调控的荧光可逆性。β-carboline compounds are a large class of naturally occurring indole alkaloids, which have a planar tricyclic structure similar to carbazole-like pyrido[3,4-b]indole, and have low toxicity. The present invention introduces an electron-donating group NN-diphenylamino group at the 6-position of β-carboline, which can quickly generate stable pH-sensitive fluorescence, and connects different substituted indolium salts or different combinations by introducing a vinyl group at the 3-position The benzoindolium salt fragment in the form, the NN-diphenylamino group of the 6-position electron-donating effect of β-carboline can enhance the electron-donating ability to obtain a fluorescent compound with a longer fluorescence wavelength and a larger Stokes shift value, Introduce water-soluble side chains containing morpholinyl and piperazinyl at the N9 position of β-carboline to improve the lipid-water distribution coefficient of the entire molecule and obtain NIR fluorescent molecules based on the "donor-acceptor" large Stokes shift . Unlike traditional pH fluorescent probes that are activated by acid-sensitive hydrazone bonds or acetal groups, this fluorescent probe can quickly achieve pH-sensitive fluorescence through intramolecular charge transfer (ICT), and has acidic pH-regulated fluorescence. reversibility.
本发明化合物具有pH敏感的近红外荧光特性，且荧光随pH变化具有可逆性，能够制备成喷剂，可喷洒或局部注射在肿瘤细胞或组织表面进行快速、实时、选择性的荧光成像。目前临床上尚未有任何荧光显影剂采用喷洒方式对肿瘤组织进行快速、实时、选择性的 荧光成像，本发明化合物将为临床这一应用领域进行很好的补充和扩展。The compound of the present invention has pH-sensitive near-infrared fluorescence characteristics, and the fluorescence is reversible with pH changes, and can be prepared as a spray, which can be sprayed or locally injected on the surface of tumor cells or tissues for rapid, real-time and selective fluorescence imaging. At present, there is no fluorescent contrast agent in the clinic to perform rapid, real-time and selective fluorescence imaging on tumor tissue by spraying, and the compound of the present invention will be a good supplement and expansion of the clinical application field.
本发明具体技术方案如下：Concrete technical scheme of the present invention is as follows:
一类具有pH敏感的N,N-二苯基氨基修饰的β-咔啉吲哚鎓盐，具有通式Ⅰ所示结构：A class of pH-sensitive N,N-diphenylamino-modified β-carboline indolium salts, having the structure shown in general formula I:
Figure PCTCN2022097845-appb-000001
Figure PCTCN2022097845-appb-000001
其中，R 1选自H，C1-C6烷基，C1-C6直链烷基吗啉；R 2选自H、F、Cl、Br、I中的一种；Y -代表卤负离子、六氟磷酸根负离子、对甲苯磺酸负离子、或者甲磺酸负离子；n为0或1。 Wherein, R 1 is selected from H, C1-C6 alkyl, C1-C6 linear alkyl morpholine; R 2 is selected from one of H, F, Cl, Br, I; Y -represents halide anion, hexafluoro Phosphate anion, p-toluenesulfonate anion, or methanesulfonate anion; n is 0 or 1.
进一步的，一类具有pH敏感的N,N-二苯基氨基修饰的β-咔啉吲哚鎓盐，所述式I的结构中，R 1、R 2、Y及n选自如下组合： Further, a class of pH-sensitive N,N-diphenylamino modified β-carboline indolium salts, in the structure of formula I, R 1 , R 2 , Y and n are selected from the following combinations:
R 1＝CH 3，R 2＝I，Y＝I，n＝0，
Figure PCTCN2022097845-appb-000002
R 1 =CH 3 , R 2 =I, Y=I, n=0,
Figure PCTCN2022097845-appb-000002
或者
Figure PCTCN2022097845-appb-000003
R 2＝H，Y＝CH 3SO 3，n＝0，
Figure PCTCN2022097845-appb-000004
or
Figure PCTCN2022097845-appb-000003
R 2 =H, Y=CH 3 SO 3 , n=0,
Figure PCTCN2022097845-appb-000004
或者R 1＝CH 3CH 2，R 2＝H，Y＝Br，n＝1，
Figure PCTCN2022097845-appb-000005
or R 1 =CH 3 CH 2 , R 2 =H, Y=Br, n=1,
Figure PCTCN2022097845-appb-000005
或者R 1＝CH 3，R 2＝I，Y＝PF 6，n＝1，
Figure PCTCN2022097845-appb-000006
or R 1 =CH 3 , R 2 =I, Y=PF 6 , n=1,
Figure PCTCN2022097845-appb-000006
上述通式结构化合物优选结构如表1所示：The preferred structure of the above-mentioned general formula structure compound is as shown in table 1:
表1通式Ⅰ部分化合物代号及其对应的结构Table 1 Part of the compound code of general formula Ⅰ and its corresponding structure
Figure PCTCN2022097845-appb-000007
Figure PCTCN2022097845-appb-000007
本发明的另一目的在于提供本发明通式Ⅰ所述化合物的如下制备方法：Another object of the present invention is to provide the following preparation method of the compound described in general formula I of the present invention:
制备方法路线为：The preparation method route is:
Figure PCTCN2022097845-appb-000008
Figure PCTCN2022097845-appb-000008
其中，R 1选自H，C1-C6烷基，C1-C6直链烷基吗啉；R 2选自H、F、Cl、Br、I中的一种；Y -代表卤负离子、六氟磷酸根负离子、对甲苯磺酸负离子、或者甲磺酸负离子；n为0或1。 Wherein, R 1 is selected from H, C1-C6 alkyl, C1-C6 linear alkyl morpholine; R 2 is selected from one of H, F, Cl, Br, I; Y -represents halide anion, hexafluoro Phosphate anion, p-toluenesulfonate anion, or methanesulfonate anion; n is 0 or 1.
制备方法具体包括以下步骤：The preparation method specifically comprises the following steps:
S1.制备中间体5：6-溴-β-咔啉1与卤代烃R 1Br或R 1I经NaH作用反应生成化合物2； S1. Preparation of intermediate 5: 6-bromo-β-carboline 1 reacts with halogenated hydrocarbon R 1 Br or R 1 I through the action of NaH to generate compound 2;
S2.化合物2通过与二苯胺在叔丁醇钠条件下经Pd(dBa) 3、P(t-Bu) 3催化反应得到胺化产物3；S3.经LiAlH 4还原胺化产物3的羧基变成醇中间体4；醇中间体4经DMP氧化醇获得醛中间体5； S2. Compound 2 reacted with diphenylamine under the condition of sodium tert-butoxide through Pd(dBa) 3 , P(t-Bu) 3 to obtain amination product 3; Alcohol intermediate 4 is formed; alcohol intermediate 4 is oxidized by DMP to obtain aldehyde intermediate 5;
S4.胺化产物3在K 3PO 4和I 2催化下脱羧碘代获得化合物6，再在K 2CO 3、Pd(PPh) 4催化下与甲酰基噻吩硼酸酯进行Suzuki偶联得到中间体7； S4. Decarboxylation and iodolation of the aminated product 3 under the catalysis of K 3 PO 4 and I 2 to obtain compound 6, and then Suzuki coupling with formylthiophene borate under the catalysis of K 2 CO 3 and Pd(PPh) 4 to obtain the intermediate Body 7;
S5.醛中间体5或中间体7与吲哚鎓盐或苯并吲哚鎓盐在催化量的哌啶中加热回流，通过Knoevenagel反应获得化合物I，具有pH敏感的N,N-二苯基氨基修饰的β-咔啉吲哚鎓盐。S5. Aldehyde intermediate 5 or intermediate 7 and indolium salt or benzindolium salt are heated to reflux in a catalytic amount of piperidine, and compound I is obtained by Knoevenagel reaction, with pH-sensitive N,N-diphenyl Amino-modified β-carboline indolium salts.
本发明还提供了上述N,N-二苯基氨基修饰的β-咔啉吲哚鎓盐在制备pH响应的荧光显影剂中的应用。The present invention also provides the application of the above-mentioned N,N-diphenylamino-modified β-carboline indolium salt in preparing a pH-responsive fluorescent developer.
进一步的，所述荧光显影剂为用于体内外肿瘤组织或肿瘤细胞的选择性荧光成像显影剂。Further, the fluorescent contrast agent is a selective fluorescence imaging contrast agent for tumor tissue or tumor cells in vivo and in vitro.
进一步的，所述应用具体为以助溶剂/表面活性剂/溶剂体系溶解所述N,N-二苯基氨基修饰的β-咔啉吲哚鎓盐，得到可喷洒的N,N-二苯基氨基修饰的β-咔啉吲哚鎓盐溶液。Further, the application is specifically dissolving the N,N-diphenylamino-modified β-carboline indolium salt with a cosolvent/surfactant/solvent system to obtain a sprayable N,N-diphenyl Amino-modified β-carboline indolium salt solution.
进一步的，所述助溶剂/表面活性剂/溶剂体系中，按体积百分比计，所述助溶剂的含量为1～30％，所述表面活性剂的含量为1～30％；所述助溶剂为1,2-丙二醇、DMSO或乙醇；所述溶剂为水；所述表面活性剂为吐温20、吐温40或吐温80。Further, in the co-solvent/surfactant/solvent system, by volume percentage, the content of the co-solvent is 1-30%, and the content of the surfactant is 1-30%; the co-solvent 1,2-propanediol, DMSO or ethanol; the solvent is water; the surfactant is Tween 20, Tween 40 or Tween 80.
进一步的，所述肿瘤为肝癌、结肠癌、乳腺癌、肺癌和宫颈癌肿瘤中的一种。Further, the tumor is one of liver cancer, colon cancer, breast cancer, lung cancer and cervical cancer.
与现有技术相比，本发明具有的应用效果：本发明化合物利用ICT原理，在肿瘤组织酸性微环境下激活，选择性地在肿瘤部位快速产生pH敏感的近红外荧光，具体实施方法是将喷洒或局部注射本发明化合物溶液于术前或术中肿瘤病灶部位及周围的组织上，利用荧光腔镜或活体成像仪对肿瘤病灶组织进行快速、选择性荧光成像和示踪，具有较高的肿瘤组织荧光成像选择性和较低的背景荧光干扰，能够对肿瘤进行精确诊断，以指导手术和/或药物治疗。Compared with the prior art, the present invention has the application effect: the compound of the present invention utilizes the principle of ICT to activate in the acidic microenvironment of the tumor tissue, and selectively rapidly generate pH-sensitive near-infrared fluorescence at the tumor site. The specific implementation method is to Spray or locally inject the compound solution of the present invention on the tumor lesion site and the surrounding tissue before or during the operation, and use a fluorescent endoscope or an in vivo imager to perform rapid and selective fluorescence imaging and tracing of the tumor lesion tissue, which has a high The selectivity and low background fluorescence interference of tumor tissue fluorescence imaging enable accurate diagnosis of tumors to guide surgery and/or drug treatment.
附图说明Description of drawings
图1为本发明化合物紫外、荧光光谱，其中A图为I 1的紫外吸收谱图，B图为I 1的荧光谱图，C图为I 4的紫外吸收谱图，D图为I 4的荧光谱图； Fig. 1 is the ultraviolet absorption spectrum of compound of the present invention, fluorescence spectrum, and wherein A figure is the ultraviolet absorption spectrum of I 1 , and B figure is the fluorescence spectrum of I 1 , and C figure is the ultraviolet absorption spectrum of I 4 , and D figure is I 4 's Fluorescence spectrum;
图2为本发明化合物I 1对肝癌细胞选择性荧光成像； Fig. 2 is the selective fluorescence imaging of liver cancer cells by compound I 1 of the present invention;
图3位本发明化合物I 2对肺癌细胞荧光成像； Figure 3 shows the fluorescence imaging of lung cancer cells by Compound I 2 of the present invention;
图4为本发明化合物I 1对离体乳腺癌肿瘤选择性荧光成像； Fig. 4 is the selective fluorescence imaging of isolated breast cancer tumor by compound I 1 of the present invention;
图5为本发明化合物I 1对临床结肠癌肿瘤选择性荧光成像； Fig. 5 is the selective fluorescence imaging of clinical colon cancer tumor by compound I 1 of the present invention;
图6为本发明化合物I 4对临床结肠癌肿瘤选择性荧光成像。 Fig. 6 is the selective fluorescence imaging of clinical colon cancer tumors by Compound I 4 of the present invention.
具体实施方式Detailed ways
为了进一步阐明本发明，下面给出一系列实施例，这些实施例完全是例证性的，它们仅用来对本发明具体描述，不应当理解为对本发明的限制。In order to further clarify the present invention, a series of examples are given below, these examples are completely illustrative, they are only used to specifically describe the present invention, and should not be construed as limiting the present invention.
实施例1：(E)-2-(2-(6-(二苯基氨基)-1,9-二甲基-9H-吡啶并[3,4-b]吲哚-3-基)乙烯基)-7-碘-1,1,3-三甲基-1H-苯并[e]吲哚-3-碘盐(化合物I 1) Example 1: (E)-2-(2-(6-(diphenylamino)-1,9-dimethyl-9H-pyrido[3,4-b]indol-3-yl)ethylene base)-7-iodo-1,1,3-trimethyl-1H-benzo[e]indole-3-iodide salt (compound I 1 )
(1)6-溴-9-甲基-9H-吡啶甲基[3,4-b]吲哚-3-羧酸酯(化合物2)的制备(1) Preparation of 6-bromo-9-methyl-9H-pyridylmethyl[3,4-b]indole-3-carboxylate (compound 2)
首先将化合物1(500mg,1.57mmol)溶于无水DMF，冰浴条件下加入NaH(301mg,12.56mmol)搅拌0.5h后，加入CH 3I(1ml,15.7mmol)，继续室温搅拌1h后，经TLC监测反应结束后，将反应液倒入冰水中，用0.1mol盐酸溶液调至pH为7，将滤液抽滤，滤饼干燥，得到化合物2，产率85％。 1H NMR(400MHz,DMSO)δ12.21(s,1H,NH),8.84(s,1H,ArH),8.66(d,J＝1.7Hz,1H,ArH),7.70(dd,J＝8.7,1.9Hz,1H,ArH),7.61(d,J＝8.7Hz,1H,ArH),3.90(s,3H,OCH 3),2.81(s,3H,CH 3). Firstly, compound 1 (500mg, 1.57mmol) was dissolved in anhydrous DMF, NaH (301mg, 12.56mmol) was added under ice-bath condition and stirred for 0.5h, then CH 3 I (1ml, 15.7mmol) was added and stirred at room temperature for 1h, After the reaction was monitored by TLC, the reaction solution was poured into ice water, adjusted to pH 7 with 0.1 mol hydrochloric acid solution, the filtrate was suction filtered, and the filter cake was dried to obtain compound 2 with a yield of 85%. 1 H NMR (400MHz, DMSO) δ 12.21 (s, 1H, NH), 8.84 (s, 1H, ArH), 8.66 (d, J = 1.7Hz, 1H, ArH), 7.70 (dd, J = 8.7, 1.9Hz, 1H, ArH), 7.61(d, J=8.7Hz, 1H, ArH), 3.90(s, 3H, OCH 3 ), 2.81(s, 3H, CH 3 ).
(2)6-(二苯氨基)-1,9-二甲基-9H-吡啶并[3,4-b]吲哚-3-羧酸(化合物3)的制备(2) Preparation of 6-(diphenylamino)-1,9-dimethyl-9H-pyrido[3,4-b]indole-3-carboxylic acid (compound 3)
首先将化合物2(480mg,1.45mmol)，二苯胺(1.47g,8.7mmol)，Pd(dBa) 3(83.375g,0.145mmol)，叔丁醇钠(430mg,5.8mmol)，三叔丁基膦0.1ml加入密封管中，加入4ml甲苯，N 2保护，110℃反应12h，待反应结束后，经TLC监测反应结束后，将反应液用浓缩，制砂，过柱得化合物3，产率76％。 1H NMR(400MHz,DMSO)δ10.8(s,1H,COOH),8.70(s,1H,ArH),8.19(s,1H,ArH),7.79(d,J＝8.9Hz,1H,ArH),7.42(dd,J＝8.8,1.9Hz,1H,ArH),7.27(t,J＝7.9Hz,4H,4ArH),6.98(dd,J＝10.7,7.7Hz,6H,2CH＝C,4ArH),4.21(s,3H,CH 3),3.08(s,3H,CH 3). First compound 2 (480mg, 1.45mmol), diphenylamine (1.47g, 8.7mmol), Pd(dBa) 3 (83.375g, 0.145mmol), sodium tert-butoxide (430mg, 5.8mmol), tri-tert-butylphosphine Add 0.1ml into a sealed tube, add 4ml of toluene, protect with N2 , and react at 110°C for 12h. After the reaction is completed and monitored by TLC, the reaction solution is concentrated, sanded, and passed through the column to obtain compound 3 with a yield of 76 %. 1 H NMR(400MHz,DMSO)δ10.8(s,1H,COOH),8.70(s,1H,ArH),8.19(s,1H,ArH),7.79(d,J＝8.9Hz,1H,ArH) ,7.42(dd,J=8.8,1.9Hz,1H,ArH),7.27(t,J=7.9Hz,4H,4ArH),6.98(dd,J=10.7,7.7Hz,6H,2CH=C,4ArH) ,4.21(s,3H,CH 3 ),3.08(s,3H,CH 3 ).
(3)6-(二苯氨基)-1,9-二甲基-9H-吡啶并[3,4-b]吲哚-3-甲醇(化合物4)(3) 6-(diphenylamino)-1,9-dimethyl-9H-pyrido[3,4-b]indole-3-carbinol (compound 4)
首先将化合物3(530mg,1.3mmol)用无水THF(10ml)溶解，冰浴10min后，分批加入LiAlH 4(74.1mg,1.95mmol)，加完后搅拌30min后，恢复至室温继续反应约3h，经TLC监测反应结束后，加入10ml甲醇至无气泡产生后加入1ml水，抽滤，用甲醇清洗滤饼，旋干得化合物4，产率80％。 1H NMR(400MHz,DMSO)δ8.70(s,1H,ArH),8.19(s,1H,ArH),7.79(d,J＝8.9Hz,1H,ArH),7.42(dd,J＝8.8,1.9Hz,1H,ArH),7.27(t,J＝7.9Hz,4H,4ArH),6.98(dd,J＝10.7,7.7Hz,6H,2CH＝C,4ArH),5.21(m,2H,CH 2),4.21(s,3H,CH 3),3.89(s,1H,OH),3.08(s,3H,CH 3). First, compound 3 (530mg, 1.3mmol) was dissolved in anhydrous THF (10ml). After ice bathing for 10min, LiAlH 4 (74.1mg, 1.95mmol) was added in batches. After 3 hours, after the reaction was monitored by TLC, 10 ml of methanol was added until no bubbles were generated, then 1 ml of water was added, suction filtered, the filter cake was washed with methanol, and spin-dried to obtain compound 4 with a yield of 80%. 1 H NMR (400MHz, DMSO) δ8.70(s, 1H, ArH), 8.19(s, 1H, ArH), 7.79(d, J=8.9Hz, 1H, ArH), 7.42(dd, J=8.8, 1.9Hz, 1H, ArH), 7.27(t, J=7.9Hz, 4H, 4ArH), 6.98(dd, J=10.7, 7.7Hz, 6H, 2CH=C, 4ArH), 5.21(m, 2H, CH 2 ),4.21(s,3H,CH 3 ),3.89(s,1H,OH),3.08(s,3H,CH 3 ).
(4)6-(二苯氨基)-1,9-二甲基-9H-吡啶并[3,4-b]吲哚-3-甲醛(化合物5)的制备(4) Preparation of 6-(diphenylamino)-1,9-dimethyl-9H-pyrido[3,4-b]indole-3-carbaldehyde (compound 5)
首先将化合物4(500mg,1.27mmol)用无水DCM(10ml)溶解，加入DMP(1.076g,2.54mmol)，大约反应2h，待反应完毕后，将反应液用滴管加入50ml碳酸氢钠饱和溶液中，抽滤获得产物5，产率67％。 1H NMR(400MHz,DMSO)δ9.75(s,1H,CHO),8.70(s,1H,ArH),8.19(s,1H,ArH),7.79(d,J＝8.9Hz,1H,ArH),7.42(dd,J＝8.8,1.9Hz,1H,ArH),7.27(t,J＝7.9Hz,4H,4ArH),6.98(dd,J＝10.7,7.7Hz,6H,2CH＝C,4ArH),4.21(s,3H,CH 3),3.08(s,3H,CH 3). First, compound 4 (500mg, 1.27mmol) was dissolved in anhydrous DCM (10ml), and DMP (1.076g, 2.54mmol) was added, and reacted for about 2 hours. In the solution, product 5 was obtained by suction filtration with a yield of 67%. 1 H NMR(400MHz,DMSO)δ9.75(s,1H,CHO),8.70(s,1H,ArH),8.19(s,1H,ArH),7.79(d,J＝8.9Hz,1H,ArH) ,7.42(dd,J=8.8,1.9Hz,1H,ArH),7.27(t,J=7.9Hz,4H,4ArH),6.98(dd,J=10.7,7.7Hz,6H,2CH=C,4ArH) ,4.21(s,3H,CH 3 ),3.08(s,3H,CH 3 ).
(5)(E)-2-(2-(6-(二苯基氨基)-1,9-二甲基-9H-吡啶并[3,4-b]吲哚-3-基)乙烯基)-7-碘-1,1,3-三甲基-1H-苯并[e]吲哚-3-碘盐(化合物I 1)的制备 (5) (E)-2-(2-(6-(diphenylamino)-1,9-dimethyl-9H-pyrido[3,4-b]indol-3-yl)ethenyl Preparation of )-7-iodo-1,1,3-trimethyl-1H-benzo[e]indole-3-iodide salt (compound I 1 )
首先将化合物5(480mg,1.22mmol)用无水乙醇(约2ml)溶解，加入7-碘-1,1,2,3-四甲基-1H-苯并[e]吲哚-3-碘盐(379mg,1.22mmol)，大约反应0.5h，待反应完毕后，抽滤获得产物I 1，产率85％。 1HNMR(400MHz,CDCl 3)δ8.13(d,J＝8.5Hz,1H,ArH),8.08(d,J＝8.9Hz,2H,2ArH),7.99(d,J＝8.1Hz,2H,2ArH),7.84(m,1H,CH＝C),7.66(s,2H,2ArH),7.59(d,J＝7.9Hz,2H,2ArH),7.43(dd,J＝10.1,8.4Hz,3H,3ArH),7.04(d,J＝7.7Hz,5H,5ArH),6.95(t,J＝7.4Hz,3H,2ArH,CH＝C),4.45(s,3H,CH 3),4.18(s,3H,CH 3),2.06(s,6H,2CH 3),1.92(s,3H,CH 3). First, compound 5 (480mg, 1.22mmol) was dissolved in absolute ethanol (about 2ml), and 7-iodo-1,1,2,3-tetramethyl-1H-benzo[e]indole-3-iodo was added Salt (379mg, 1.22mmol), reacted for about 0.5h. After the reaction was completed, the product I 1 was obtained by suction filtration with a yield of 85%. 1 HNMR (400MHz, CDCl 3 ) δ8.13 (d, J = 8.5Hz, 1H, ArH), 8.08 (d, J = 8.9Hz, 2H, 2ArH), 7.99 (d, J = 8.1Hz, 2H, 2ArH ), 7.84(m, 1H, CH=C), 7.66(s, 2H, 2ArH), 7.59(d, J=7.9Hz, 2H, 2ArH), 7.43(dd, J=10.1, 8.4Hz, 3H, 3ArH ), 7.04(d, J=7.7Hz, 5H, 5ArH), 6.95(t, J=7.4Hz, 3H, 2ArH, CH=C), 4.45(s, 3H, CH 3 ), 4.18(s, 3H, CH 3 ), 2.06(s,6H,2CH 3 ),1.92(s,3H,CH 3 ).
实施例2(E)-2-(2-(6-(6-(二苯氨基)-1-甲基-9-(2-吗啉代乙基)-9H-吡啶并[3,4-b]吲哚-3-基)乙烯基)-1,1,3-三甲基-1H-苯并[e]吲哚-3-磺酸盐(化合物I 2) Example 2 (E)-2-(2-(6-(6-(diphenylamino)-1-methyl-9-(2-morpholinoethyl)-9H-pyrido[3,4- b] indol-3-yl)vinyl)-1,1,3-trimethyl-1H-benzo[e]indole-3-sulfonate (compound I 2 )
参考实施例1中2的制备方法，将化合物碘甲烷替代方法中的溴乙基吗啉，最后得到深红色产物I 2，产率76％。 1H NMR(400MHz,DMSO)δ8.67(s,1H,ArH),8.23(d,J＝2.1Hz,1H,ArH),8.23(m,2H,2ArH),8.06(m,2H,2ArH),7.82(d,J＝8.9Hz,1H,ArH),7.54(m,3H,3ArH)7.44(d,J＝2.1Hz,1H,ArH),7.30(m,4H,4ArH),6.99(t,J＝8.2Hz,6H,6ArH),4.78(dt,2H,CH 2),3.54(m,4H,2CH 2),3.11(s,3H,CH 3),2.75(m,2H,CH 2),2.47(m,4H,2CH 2),2.06(s,6H,2CH 3),1.9(s,3H,CH 3). Referring to the preparation method of 2 in Example 1, the compound iodomethane was substituted for bromoethylmorpholine in the method, and finally a dark red product I 2 was obtained with a yield of 76%. 1 H NMR(400MHz,DMSO)δ8.67(s,1H,ArH),8.23(d,J=2.1Hz,1H,ArH),8.23(m,2H,2ArH),8.06(m,2H,2ArH) ,7.82(d,J=8.9Hz,1H,ArH),7.54(m,3H,3ArH)7.44(d,J=2.1Hz,1H,ArH),7.30(m,4H,4ArH),6.99(t, J＝8.2Hz, 6H, 6ArH), 4.78(dt, 2H, CH 2 ), 3.54(m, 4H, 2CH 2 ), 3.11(s, 3H, CH 3 ), 2.75(m, 2H, CH 2 ), 2.47(m,4H,2CH 2 ),2.06(s,6H,2CH 3 ),1.9(s,3H,CH 3 ).
实施例3(E)-2-(2-(5-(6-(二苯氨基)-1,9-二甲基-9H-吡啶并[3,4-b]吲哚-3-基)噻吩-2-基)乙烯基)-1,3,3-三甲基-3H-吲哚-1-溴盐(化合物I 3) Example 3 (E)-2-(2-(5-(6-(diphenylamino)-1,9-dimethyl-9H-pyrido[3,4-b]indol-3-yl) Thiophen-2-yl)vinyl)-1,3,3-trimethyl-3H-indole-1-bromide salt (compound I 3 )
(1)3-碘-1-甲基-N,N-二苯基-9H-吡啶并[3,4-b]吲哚-6-胺(化合物6)的制备(1) Preparation of 3-iodo-1-methyl-N,N-diphenyl-9H-pyrido[3,4-b]indol-6-amine (Compound 6)
首先将化合物3(200mg,0.508mmol)、I 2(280mg,1.103mmol)、K 3PO 4(186mg,0.876mmol)加入密封管中，用乙腈2ml溶解，N 2保护，110℃反应18h，待反应结束后，加入三乙胺约1.2ml继续150℃反应4h，减压浓缩，柱层析分离获得化合物6，产率65％。 1HNMR(400MHz,DMSO)δ8.70(s,1H,ArH),8.19(s,1H,ArH),7.79(d,J＝8.9Hz,1H,ArH),7.42(dd,J ＝8.8,1.9Hz,1H,ArH),7.27(t,J＝7.9Hz,4H,4ArH),6.98(dd,J＝10.7,7.7Hz,6H,2CH＝C,4ArH),4.21(s,3H,CH 3),3.08(s,3H,CH 3). First, compound 3 (200mg, 0.508mmol), I 2 (280mg, 1.103mmol), K 3 PO 4 (186mg, 0.876mmol) were added into a sealed tube, dissolved in 2ml of acetonitrile, protected by N 2 , and reacted at 110°C for 18h. After the reaction, about 1.2 ml of triethylamine was added to continue the reaction at 150° C. for 4 h, concentrated under reduced pressure, and separated by column chromatography to obtain compound 6 with a yield of 65%. 1 HNMR (400MHz, DMSO) δ8.70 (s, 1H, ArH), 8.19 (s, 1H, ArH), 7.79 (d, J = 8.9Hz, 1H, ArH), 7.42 (dd, J = 8.8, 1.9 Hz, 1H, ArH), 7.27(t, J=7.9Hz, 4H, 4ArH), 6.98(dd, J=10.7, 7.7Hz, 6H, 2CH=C, 4ArH), 4.21(s, 3H, CH 3 ) ,3.08(s,3H,CH 3 ).
(2)5-(6-(二苯氨基)-1,9-二甲基-9H-吡啶并[3,4-b]吲哚-3-基)噻吩-2-甲醛(化合物7)的制备(2) 5-(6-(diphenylamino)-1,9-dimethyl-9H-pyrido[3,4-b]indol-3-yl)thiophene-2-carbaldehyde (compound 7) preparation
首先将化合物6(170mg,0.730mmol)，碳酸钾(202mg,1.46mmol)，Pd(PPh 3) 4(69.44mg,0.073mmol)，噻吩硼酸酯醛(163mg,1.46mmol)加入密封管中，N 2保护，用4ml甲苯溶解，110℃反应3h。待反应结束后，可以点板看见黄色荧光，然后用EA:PE＝1:2过柱，然后约500ml溶剂可以过下来，获得黄色固体化合物7，产率82％。 1H NMR(400MHz,DMSO)δ9.84(s,1H,CHO),8.13(s,1H,ArH),8.00(d,1H,CH＝C),7.37-7.20(m,6H,6ArH),6.81(m,2H,ArH),6.73-6.63(m,4H,4ArH),6.12(m,1H,ArH),3.82(s,3H,CH 3),2.89(s,3H,CH 3). First, compound 6 (170mg, 0.730mmol), potassium carbonate (202mg, 1.46mmol), Pd(PPh 3 ) 4 (69.44mg, 0.073mmol), and thiophene borate aldehyde (163mg, 1.46mmol) were added to the sealed tube, N 2 protected, dissolved in 4ml of toluene, and reacted at 110°C for 3h. After the reaction is finished, yellow fluorescence can be seen by pointing the plate, and then EA:PE=1:2 is used to pass through the column, and then about 500ml of solvent can be passed down to obtain yellow solid compound 7 with a yield of 82%. 1 H NMR (400MHz, DMSO) δ9.84 (s, 1H, CHO), 8.13 (s, 1H, ArH), 8.00 (d, 1H, CH=C), 7.37-7.20 (m, 6H, 6ArH), 6.81(m,2H,ArH),6.73-6.63(m,4H,4ArH),6.12(m,1H,ArH),3.82(s,3H, CH3 ),2.89(s,3H, CH3 ).
(3)(E)-2-(2-(5-(6-(二苯氨基)-1,9-二甲基-9H-吡啶并[3,4-b]吲哚-3-基)噻吩-2-基)乙烯基)-1,3,3-三甲基-3H-吲哚-1-溴盐(化合物I 3)的制备首先将化合物7(121mg,0.256mmol)用无水乙醇(约2ml)溶解，加入1,2,3,3-四甲基-3H-吲哚溴盐(58.3mg,0.256mmol)，大约反应0.5h，待反应完毕后，将反应液抽滤获得产物I 3，产率78％。 1H NMR(400MHz,DMSO)δ8.92(d,1H,ArH),8.13(s,1H,ArH),8.02(m,1H,CH＝C),7.73(d,1H,ArH),7.37-7.20(m,9H,8ArH,CH＝C),6.81(m,2H,2ArH),6.65(m,5H,4ArH,CH＝C),6.20(m,1H,ArH),5.67(m,1H,ArH),3.82(s,3H,CH 3),2.89(s,3H,CH 3),2.06(s,6H,2CH 3),1.92(s,3H,CH 3). (3)(E)-2-(2-(5-(6-(Diphenylamino)-1,9-dimethyl-9H-pyrido[3,4-b]indol-3-yl) Preparation of thiophen-2-yl)vinyl)-1,3,3-trimethyl-3H-indole-1-bromide salt (compound I 3 ) First, compound 7 (121mg, 0.256mmol) was dissolved in absolute ethanol (about 2ml) was dissolved, and 1,2,3,3-tetramethyl-3H-indole bromide salt (58.3mg, 0.256mmol) was added, reacted for about 0.5h, after the reaction was completed, the reaction solution was suction filtered to obtain the product I 3 , yield 78%. 1 H NMR (400MHz, DMSO) δ8.92 (d, 1H, ArH), 8.13 (s, 1H, ArH), 8.02 (m, 1H, CH=C), 7.73 (d, 1H, ArH), 7.37- 7.20(m, 9H, 8ArH, CH=C), 6.81(m, 2H, 2ArH), 6.65(m, 5H, 4ArH, CH=C), 6.20(m, 1H, ArH), 5.67(m, 1H, ArH), 3.82(s,3H,CH 3 ), 2.89(s,3H,CH 3 ), 2.06(s,6H,2CH 3 ), 1.92(s,3H,CH 3 ).
实施例4(E)-2-(2-(5-(6-(二苯氨基)-1,9-二甲基-9H-吡啶并[3,4-b]吲哚-3-基)噻吩-2-基)乙烯基)-7-碘-1,1,3-三甲基-1H-苯并[e]吲哚-3-六氟磷酸盐(化合物I 4)的制备 Example 4 (E)-2-(2-(5-(6-(diphenylamino)-1,9-dimethyl-9H-pyrido[3,4-b]indol-3-yl) Preparation of thiophen-2-yl)vinyl)-7-iodo-1,1,3-trimethyl-1H-benzo[e]indole-3-hexafluorophosphate (compound I 4 )
参考实施例3中I 3的制备方法，将化合物7-碘-1,1,2,3-四甲基-1H-苯并[e]吲哚-3-六氟磷酸盐代替方法中的1,2,3,3-四甲基-3H-吲哚-1-溴盐，最后得到深红色产物I 4，产率78％。 1H NMR(400MHz,DMSO)δ8.92(d,1H,ArH),8.13(s,1H,ArH),8.02(m,3H,3ArH),7.73(d,1H,ArH),7.54(d,2H,2ArH)7.37-7.20(m,9H,9ArH),6.81(m,2H,2ArH),6.65(m,5H,4ArH,CH＝C),6.20(m,1H,ArH),5.67(m,1H,ArH),3.82(s,3H,CH 3),2.89(s,3H,CH 3),2.06(s,6H,2CH 3),1.92(s,3H,CH 3). With reference to the preparation method of I3 in Example 3, the compound 7-iodo-1,1,2,3-tetramethyl-1H-benzo[e]indole-3-hexafluorophosphate replaces 1 in the method , 2,3,3-Tetramethyl-3H-indole-1-bromide salt, the dark red product I 4 was finally obtained with a yield of 78%. 1 H NMR (400MHz,DMSO)δ8.92(d,1H,ArH),8.13(s,1H,ArH),8.02(m,3H,3ArH),7.73(d,1H,ArH),7.54(d, 2H, 2ArH) 7.37-7.20(m, 9H, 9ArH), 6.81(m, 2H, 2ArH), 6.65(m, 5H, 4ArH, CH=C), 6.20(m, 1H, ArH), 5.67(m, 1H,ArH),3.82(s,3H,CH 3 ),2.89(s,3H,CH 3 ),2.06(s,6H,2CH 3 ),1.92(s,3H,CH 3 ).
实施例5本发明化合物的紫外吸收光谱测试The ultraviolet absorption spectrum test of embodiment 5 compounds of the present invention
将本发明荧光化合物溶于含1％DMSO的水溶液中，配制成5-20μM的检测液。采用紫外-可见分光光度计测试其紫外吸收光谱数据，结果显示本发明荧光化合物紫外最大吸收波长在520-650nm范围内。其中化合物I 1和I 4紫外最大吸收波长分别在592和583nm左右(图1A和图1C)；注：图1A为I 1的紫外吸收谱图，图1B为I 1的荧光谱图，图1C为I 4的紫外吸收谱图，图1D为I 4的荧光谱图。 The fluorescent compound of the present invention is dissolved in an aqueous solution containing 1% DMSO to prepare a 5-20 μM detection solution. The ultraviolet absorption spectrum data is tested by an ultraviolet-visible spectrophotometer, and the result shows that the ultraviolet maximum absorption wavelength of the fluorescent compound of the present invention is within the range of 520-650nm. Among them, compounds I 1 and I 4 have maximum ultraviolet absorption wavelengths around 592 and 583 nm respectively (Fig. 1A and Fig. 1C); Note: Fig. 1A is the ultraviolet absorption spectrum of I 1 , Fig. 1B is the fluorescence spectrum of I 1 , Fig. 1C It is the ultraviolet absorption spectrum of I 4 , and Fig. 1D is the fluorescence spectrum of I 4 .
实施例6本发明化合物的pH敏感荧光测试Embodiment 6 The pH-sensitive fluorescence test of the compound of the present invention
将本发明荧光化合物溶于含1％DMSO的水溶液中，配制成5-20μM的检测液。采用荧光光谱仪测试其在pH＝3～8的荧光光谱数据，结果如图1所示。结果显示本发明荧光化合物最大发射波长在680-750nm范围内。本发明化合物I 1和I 4在pH3.5～6.5之间的最大发射波长分别在740和692nm左右，荧光波长达到近红外区域，而在中性pH下几乎没有荧光，此外本发明化合物的Stokes位移值达到150～200nm，具有良好的荧光特性； The fluorescent compound of the present invention is dissolved in an aqueous solution containing 1% DMSO to prepare a 5-20 μM detection solution. Fluorescence spectrometer was used to test its fluorescence spectrum data at pH=3-8, and the results are shown in FIG. 1 . The results show that the maximum emission wavelength of the fluorescent compound of the present invention is in the range of 680-750nm. The maximum emission wavelengths of compounds I1 and I4 of the present invention between pH 3.5 and 6.5 are respectively about 740 and 692nm, and the fluorescence wavelength reaches the near-infrared region, but there is almost no fluorescence at neutral pH. In addition, the Stokes of the compounds of the present invention The displacement value reaches 150-200nm, and has good fluorescence characteristics;
此外，化合物I 1在740nm左右的荧光峰值随化合物I 1的pH减小而增加，相反其荧光峰值随pH增加而减小，其峰值相差8倍(图1B)。 In addition, the fluorescence peak of compound I1 around 740nm increased with the decrease of the pH of compound I1 , on the contrary its fluorescence peak decreased with the increase of pH, and the peak difference was 8 times (Fig. 1B).
实施例7本发明化合物对肿瘤细胞选择性荧光成像测试Example 7 The compounds of the present invention are tested for selective fluorescence imaging of tumor cells
通过使用40X物镜的共聚焦激光扫描显微镜(LeicaTCSSP8)进行细胞摄取和定位。将人肝癌细胞HepG2、正常肝细胞LO2和人肺癌细胞A549分别用1mL培养基以1×10 5细胞的密度在共聚焦培养皿中于37℃培养24h。然后，将HepG2和LO2细胞培养基替换为含有1～100μM的I 1的新鲜培养基，将A549细胞的培养基替换为含有1～100μM的I 2的新鲜培养基，并在37℃下孵育10～30min，然后用PBS洗涤细胞3次。最后，使用共聚焦激光扫描显微镜获得细胞荧光成像的图像，结果如图2(化合物I 1)和图3(化合物I 2)所示。图2和图3中左测图为细胞明场图，右测图为细胞荧光成像图。 Cell uptake and localization were performed by a confocal laser scanning microscope (Leica TCSSP8) using a 40X objective. Human liver cancer cells HepG2, normal liver cells LO2 and human lung cancer cells A549 were cultured in confocal culture dishes at a density of 1×10 5 cells at 37°C for 24 h with 1 mL of culture medium. Then, the culture medium of HepG2 and LO2 cells was replaced with fresh medium containing 1-100 μM I1 , and the medium of A549 cells was replaced with fresh medium containing 1-100 μM I2 , and incubated at 37 °C for 10 ~30min, and then wash the cells 3 times with PBS. Finally, the fluorescence imaging images of the cells were obtained using a confocal laser scanning microscope, and the results are shown in Figure 2 (Compound I 1 ) and Figure 3 (Compound I 2 ). In Figure 2 and Figure 3, the left side view is the bright field image of the cells, and the right side view is the cell fluorescence imaging picture.
图2和图3成像结果显示，本发明化合物I 1(10μM)在4h后能够清晰地对肝肿瘤细胞HepG2进行荧光成像，而在正常细胞LO2荧光很弱，根据细胞内荧光的量化，HepG2的荧光强度是LO2细胞的6.5倍，说明本发明化合物能够选择性地对肝肿瘤细胞荧光成像；同时，本发明化合物I 2(50μM)在4h后能够清晰地对A549细胞进行荧光成像。这些结果表明本发明化合物的对肿瘤细胞具有选择性荧光成像的能力。 The imaging results shown in Figure 2 and Figure 3 show that the compound I 1 (10 μM) of the present invention can clearly perform fluorescence imaging on the liver tumor cell HepG2 after 4 hours, while the LO2 fluorescence in normal cells is very weak. According to the quantification of the intracellular fluorescence, HepG2 The fluorescence intensity is 6.5 times that of LO2 cells, indicating that the compound of the present invention can selectively perform fluorescence imaging on liver tumor cells; at the same time, the compound I 2 (50 μM) of the present invention can clearly perform fluorescence imaging on A549 cells after 4 hours. These results demonstrate the ability of the compounds of the present invention to selectively fluorescently image tumor cells.
实施例8本发明化合物对离体肿瘤组织进行喷洒模式的荧光成像试验Example 8 Fluorescence imaging test of compound of the present invention in spray mode on isolated tumor tissue
取人乳腺癌细胞(MDA-MB-231)移植瘤模型裸鼠，将其处死，取出乳腺癌肿瘤及主要脏器进行喷洒成像分析。将配制好的本发明化合物I 1溶液(10～100μM)喷洒在组织上3～5次，用PBS清洗并用棉花吸干，采用活体成像仪进行组织荧光成像。结果如图4所示，乳腺癌组织的荧光强度值明显高于其他器官组织，而正常器官组织几乎看不到荧光。由此说明了本发明化合物可选择性、快速对肿瘤组织喷洒成像，以实现对临床肿瘤组织的快速检测。 Human breast cancer cell (MDA-MB-231) xenografted tumor model nude mice were taken and sacrificed, and the breast cancer tumor and major organs were taken out for spray imaging analysis. The prepared compound I 1 solution of the present invention (10-100 μM) was sprayed on the tissue for 3-5 times, washed with PBS and blotted dry with cotton, and the tissue fluorescence imaging was performed using an in vivo imager. The results are shown in Figure 4, the fluorescence intensity value of breast cancer tissue is significantly higher than that of other organ tissues, while almost no fluorescence can be seen in normal organ tissues. This shows that the compound of the present invention can selectively and rapidly spray and image tumor tissues, so as to realize rapid detection of clinical tumor tissues.
实施例9本发明化合物对临床肿瘤组织荧光成像试验Embodiment 9 Compounds of the present invention are tested on clinical tumor tissue fluorescence imaging
在此基础上，进一步研究本发明化合物对临床肿瘤组织的选择性成像能力。选取临床结肠癌组织及癌旁组织进行喷洒成像分析，将本发明化合物I 1和I 4溶液(10～100μM)均匀喷洒在结肠癌组织及癌旁组织1～3次，3～10min后再用适当生理盐水洗去表面多余的溶液，利用活体成像仪进行荧光成像，结果如图5(化合物I 1)和图6(化合物I 4)所示。 On this basis, the selective imaging ability of the compound of the present invention to clinical tumor tissue is further studied. Select clinical colon cancer tissue and paracancerous tissue for spray imaging analysis, evenly spray the compound I 1 and I 4 solutions (10-100 μM) of the present invention on the colon cancer tissue and para-cancerous tissue 1-3 times, and then use it after 3-10 minutes. Appropriate physiological saline was used to wash off excess solution on the surface, and the in vivo imager was used for fluorescence imaging. The results are shown in Figure 5 (Compound I 1 ) and Figure 6 (Compound I 4 ).
荧光成像结果表明，本发明化合物I 1和I 4能够在50μM下选择性、快速点亮临床结肠癌组织，而对周围正常组织不显色或显示较弱(图5-6)。由此进一步确认了本发明化合物对临床肝肿瘤组织的选择性成像能力。 The results of fluorescence imaging showed that compounds I 1 and I 4 of the present invention could selectively and rapidly light up clinical colon cancer tissues at 50 μM, while showing no or weaker coloration to surrounding normal tissues (Fig. 5-6). This further confirms the selective imaging ability of the compound of the present invention on clinical liver tumor tissue.
以上对本发明的实施例进行了详细说明，但所述内容仅为本发明的较佳实施例，不能被认为用于限定本发明的实施范围。凡依本发明申请范围所作的均等变化与改进等，均应仍归属于本发明的专利涵盖范围之内。The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention, and cannot be considered as limiting the implementation scope of the present invention. All equivalent changes and improvements made according to the application scope of the present invention shall still belong to the scope covered by the patent of the present invention.

Claims (9)

  1. 一类具有pH敏感的N,N-二苯基氨基修饰的β-咔啉吲哚鎓盐，其特征在于，具有通式Ⅰ所示结构：A class of pH-sensitive N,N-diphenylamino-modified β-carboline indolium salts, characterized in that it has the structure shown in general formula I:
    Figure PCTCN2022097845-appb-100001
    Figure PCTCN2022097845-appb-100001
    其中，R 1选自H、C1-C6烷基和C1-C6直链烷基吗啉中的一种；R 2选自H、F、Cl、Br和I中的一种；Y -代表卤负离子、六氟磷酸根负离子、对甲苯磺酸负离子和甲磺酸负离子中的一种；n为0或1。 Wherein, R 1 is selected from one of H, C1-C6 alkyl and C1-C6 linear alkyl morpholine; R 2 is selected from one of H, F, Cl, Br and I; Y - represents halogen One of anion, hexafluorophosphate anion, p-toluenesulfonate anion and methanesulfonate anion; n is 0 or 1.
  2. 根据权利要求1所述的一类具有pH敏感的N,N-二苯基氨基修饰的β-咔啉吲哚鎓盐，其特征在于：R 1选自H、甲基、乙基和乙基吗啉中的一种；R 2选自H或I；Y -代表卤负离子、六氟磷酸根负离子、对甲苯磺酸负离子或者甲磺酸负离子；n为0或1。 A class of pH-sensitive N, N-diphenylamino-modified β-carboline indolium salts according to claim 1, characterized in that: R is selected from H, methyl, ethyl and ethyl One of morpholines; R2 is selected from H or I; Y - represents halide anion, hexafluorophosphate anion, p-toluenesulfonate anion or methanesulfonate anion; n is 0 or 1.
  3. 根据权利要求1所述的一类具有pH敏感的N,N-二苯基氨基修饰的β-咔啉吲哚鎓盐，其特征在于，所述通式Ⅰ所示结构中，R 1、R 2、Y及n选自如下组合： A class of pH-sensitive N,N-diphenylamino-modified β-carboline indolium salts according to claim 1, characterized in that, in the structure shown in the general formula I, R 1 , R 2. Y and n are selected from the following combinations:
    R 1＝CH 3，R 2＝I，Y＝I，n＝0，
    Figure PCTCN2022097845-appb-100002
    R 1 =CH 3 , R 2 =I, Y=I, n=0,
    Figure PCTCN2022097845-appb-100002
    或者
    Figure PCTCN2022097845-appb-100003
    R 2＝H，Y＝CH 3SO 3，n＝0，
    Figure PCTCN2022097845-appb-100004
    or
    Figure PCTCN2022097845-appb-100003
    R 2 =H, Y=CH 3 SO 3 , n=0,
    Figure PCTCN2022097845-appb-100004
    或者R 1＝CH 3CH 2，R 2＝H，Y＝Br，n＝1，
    Figure PCTCN2022097845-appb-100005
    or R 1 =CH 3 CH 2 , R 2 =H, Y=Br, n=1,
    Figure PCTCN2022097845-appb-100005
    或者R 1＝CH 3，R 2＝I，Y＝PF 6，n＝1，
    Figure PCTCN2022097845-appb-100006
    or R 1 =CH 3 , R 2 =I, Y=PF 6 , n=1,
    Figure PCTCN2022097845-appb-100006
  4. 一类具有pH敏感的N,N-二苯基氨基修饰的β-咔啉吲哚鎓盐的制备方法，其特征在于，所述制备方法的路线如下式所示：A preparation method of a pH-sensitive N,N-diphenylamino-modified β-carboline indolium salt, characterized in that the route of the preparation method is shown in the following formula:
    Figure PCTCN2022097845-appb-100007
    Figure PCTCN2022097845-appb-100007
    Figure PCTCN2022097845-appb-100008
    Figure PCTCN2022097845-appb-100008
    其中，R 1选自H、C1-C6烷基和C1-C6直链烷基吗啉中的一种；R 2选自H、F、Cl、Br和I中的一种；Y -代表卤负离子、六氟磷酸根负离子、对甲苯磺酸负离子或者甲磺酸负离子；n为0或1； Wherein, R 1 is selected from one of H, C1-C6 alkyl and C1-C6 linear alkyl morpholine; R 2 is selected from one of H, F, Cl, Br and I; Y - represents halogen Anion, hexafluorophosphate anion, p-toluenesulfonate anion or methanesulfonate anion; n is 0 or 1;
    所述制备方法包括以下步骤：The preparation method comprises the following steps:
    S1.制备中间体5：6-溴-β-咔啉1与卤代烃R 1Br或R 1I经NaH作用反应生成化合物2； S1. Preparation of intermediate 5: 6-bromo-β-carboline 1 reacts with halogenated hydrocarbon R 1 Br or R 1 I through the action of NaH to generate compound 2;
    S2.化合物2通过与二苯胺在叔丁醇钠条件下经Pd(dBa) 3、P(t-Bu) 3催化反应得到胺化产物3；S3.经LiAlH 4还原胺化产物3的羧基变成醇中间体4；醇中间体4经DMP氧化醇获得醛中间体5； S2. Compound 2 reacted with diphenylamine under the condition of sodium tert-butoxide through Pd(dBa) 3 , P(t-Bu) 3 to obtain amination product 3; Alcohol intermediate 4 is formed; alcohol intermediate 4 is oxidized by DMP to obtain aldehyde intermediate 5;
    S4.胺化产物3在K 3PO 4和I 2催化下脱羧碘代获得化合物6，再在K 2CO 3、Pd(PPh) 4催化下与甲酰基噻吩硼酸酯进行Suzuki偶联得到中间体7； S4. Decarboxylation and iodolation of the aminated product 3 under the catalysis of K 3 PO 4 and I 2 to obtain compound 6, and then Suzuki coupling with formylthiophene borate under the catalysis of K 2 CO 3 and Pd(PPh) 4 to obtain the intermediate Body 7;
    S5.醛中间体5或中间体7与吲哚鎓盐或苯并吲哚鎓盐在催化量的哌啶中加热回流，通过Knoevenagel反应获得化合物I，化合物I为具有pH敏感的N,N-二苯基氨基修饰的β-咔啉吲哚鎓盐。S5. Aldehyde intermediate 5 or intermediate 7 and indolium salt or benzindolium salt are heated to reflux in a catalytic amount of piperidine, and compound I is obtained by Knoevenagel reaction. Compound I is a pH-sensitive N,N- Diphenylamino-modified β-carboline indolium salt.
  5. 权利要求1-3任一项所述N,N-二苯基氨基修饰的β-咔啉吲哚鎓盐或权利要求4制备得到的N,N-二苯基氨基修饰的β-咔啉吲哚鎓盐在制备pH响应的荧光显影剂中的应用。The N,N-diphenylamino-modified β-carboline indolium salt described in any one of claims 1-3 or the N,N-diphenylamino-modified β-carboline indolium salt prepared in claim 4 Application of Inylium Salts in the Preparation of pH-Responsive Fluorescent Developers.
  6. 根据权利要求5所述的应用，其特征在于，所述荧光显影剂为用于体内外肿瘤组织或肿瘤细胞的选择性荧光成像显影剂。The application according to claim 5, characterized in that the fluorescent contrast agent is a selective fluorescence imaging contrast agent for tumor tissue or tumor cells in vivo and in vitro.
  7. 根据权利要求5所述的应用，其特征在于，所述应用具体为以助溶剂/表面活性剂/溶剂体系溶解所述N,N-二苯基氨基修饰的β-咔啉吲哚鎓盐，得到可喷洒的N,N-二苯基氨基修饰的β-咔啉吲哚鎓盐溶液。The application according to claim 5, wherein the application is specifically dissolving the N,N-diphenylamino-modified β-carboline indolium salt with a cosolvent/surfactant/solvent system, A sprayable solution of N,N-diphenylamino-modified β-carboline indolium salt was obtained.
  8. 根据权利要求7所述的应用，其特征在于，所述助溶剂/表面活性剂/溶剂体系中，按体积百分比计，所述助溶剂的含量为1～30％，所述表面活性剂的含量为1～30％；所述助溶剂为1,2-丙二醇、DMSO或乙醇；所述溶剂为水；所述表面活性剂为吐温20、吐温40或吐温80。The application according to claim 7, characterized in that, in the cosolvent/surfactant/solvent system, by volume percentage, the content of the cosolvent is 1 to 30%, and the content of the surfactant 1-30%; the co-solvent is 1,2-propanediol, DMSO or ethanol; the solvent is water; the surfactant is Tween 20, Tween 40 or Tween 80.
  9. 根据权利要求6所述的应用，其特征在于，所述肿瘤为肝癌、结肠癌、乳腺癌、肺癌和宫颈癌肿瘤中的一种。The use according to claim 6, wherein the tumor is one of liver cancer, colon cancer, breast cancer, lung cancer and cervical cancer tumors.
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