CN110655508A - 一种靶向活细胞线粒体的小分子荧光探针及其制备方法和应用 - Google Patents
一种靶向活细胞线粒体的小分子荧光探针及其制备方法和应用 Download PDFInfo
- Publication number
- CN110655508A CN110655508A CN201810720903.5A CN201810720903A CN110655508A CN 110655508 A CN110655508 A CN 110655508A CN 201810720903 A CN201810720903 A CN 201810720903A CN 110655508 A CN110655508 A CN 110655508A
- Authority
- CN
- China
- Prior art keywords
- unsubstituted
- substituted
- fluorescent probe
- alkyl
- small molecule
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 44
- 210000003470 mitochondria Anatomy 0.000 title claims abstract description 27
- 230000008685 targeting Effects 0.000 title claims abstract description 20
- 150000003384 small molecules Chemical class 0.000 title claims description 54
- 238000002360 preparation method Methods 0.000 title abstract description 13
- 230000002438 mitochondrial effect Effects 0.000 claims abstract description 16
- SMWDFEZZVXVKRB-UHFFFAOYSA-N Quinoline Chemical compound N1=CC=CC2=CC=CC=C21 SMWDFEZZVXVKRB-UHFFFAOYSA-N 0.000 claims abstract description 10
- 150000001875 compounds Chemical class 0.000 claims abstract description 10
- QXAMGWKESXGGNV-UHFFFAOYSA-N 7-(diethylamino)-1-benzopyran-2-one Chemical compound C1=CC(=O)OC2=CC(N(CC)CC)=CC=C21 QXAMGWKESXGGNV-UHFFFAOYSA-N 0.000 claims abstract description 5
- WSFSSNUMVMOOMR-UHFFFAOYSA-N formaldehyde Substances O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims abstract description 3
- 239000000126 substance Substances 0.000 claims abstract 2
- 210000004027 cell Anatomy 0.000 claims description 77
- 125000006297 carbonyl amino group Chemical group [H]N([*:2])C([*:1])=O 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- 229910052717 sulfur Inorganic materials 0.000 claims description 4
- 125000004178 (C1-C4) alkyl group Chemical group 0.000 claims description 2
- 125000004429 atom Chemical group 0.000 claims description 2
- 125000004432 carbon atom Chemical group C* 0.000 claims description 2
- 125000005842 heteroatom Chemical group 0.000 claims description 2
- 125000000623 heterocyclic group Chemical group 0.000 claims description 2
- 229910052739 hydrogen Inorganic materials 0.000 claims description 2
- 229910052760 oxygen Inorganic materials 0.000 claims description 2
- 238000001215 fluorescent labelling Methods 0.000 claims 1
- 239000008098 formaldehyde solution Substances 0.000 claims 1
- 239000000523 sample Substances 0.000 abstract description 24
- 229940079593 drug Drugs 0.000 abstract description 14
- 239000003814 drug Substances 0.000 abstract description 14
- 238000001514 detection method Methods 0.000 abstract description 8
- 230000004663 cell proliferation Effects 0.000 abstract description 2
- 238000012827 research and development Methods 0.000 abstract 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 20
- 239000006228 supernatant Substances 0.000 description 16
- 238000003384 imaging method Methods 0.000 description 15
- 238000004113 cell culture Methods 0.000 description 14
- 239000000975 dye Substances 0.000 description 14
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 12
- 230000004083 survival effect Effects 0.000 description 12
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 10
- NZCHHEFOTMKOJX-UHFFFAOYSA-K [6-[[3-carboxy-4-(3-oxido-6-oxoxanthen-9-yl)phenyl]carbamothioylamino]hexoxy-oxidophosphoryl] [5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound O1C(COP([O-])(=O)OP([O-])(=O)OCCCCCCNC(=S)NC=2C=C(C(=CC=2)C2=C3C=CC(=O)C=C3OC3=CC([O-])=CC=C32)C(O)=O)C(O)C(O)C1N1C=CC(=O)NC1=O NZCHHEFOTMKOJX-UHFFFAOYSA-K 0.000 description 10
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 10
- 231100000135 cytotoxicity Toxicity 0.000 description 10
- 230000003013 cytotoxicity Effects 0.000 description 10
- XCVSDCPWJRHMSZ-UHFFFAOYSA-M [6-(diethylamino)-9-[2-(4-methyl-2-oxochromen-7-yl)oxycarbonylphenyl]xanthen-3-ylidene]-diethylazanium;chloride Chemical compound [Cl-].C1=CC(=[N+](CC)CC)C=C2OC3=CC(N(CC)CC)=CC=C3C(C=3C(=CC=CC=3)C(=O)OC=3C=C4OC(=O)C=C(C)C4=CC=3)=C21 XCVSDCPWJRHMSZ-UHFFFAOYSA-M 0.000 description 9
- 239000003068 molecular probe Substances 0.000 description 9
- 239000007787 solid Substances 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 8
- 230000003698 anagen phase Effects 0.000 description 8
- 238000000799 fluorescence microscopy Methods 0.000 description 8
- 238000005406 washing Methods 0.000 description 8
- 210000003771 C cell Anatomy 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 6
- 229960000956 coumarin Drugs 0.000 description 5
- 235000001671 coumarin Nutrition 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 230000001988 toxicity Effects 0.000 description 5
- 231100000419 toxicity Toxicity 0.000 description 5
- 229930040373 Paraformaldehyde Natural products 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000010438 heat treatment Methods 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 229920002866 paraformaldehyde Polymers 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 231100000820 toxicity test Toxicity 0.000 description 4
- 210000004881 tumor cell Anatomy 0.000 description 4
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- OEDBHOAJDADPMY-UHFFFAOYSA-N 7-(diethylamino)-2-oxochromene-4-carbaldehyde Chemical compound O=CC1=CC(=O)OC2=CC(N(CC)CC)=CC=C21 OEDBHOAJDADPMY-UHFFFAOYSA-N 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000003501 co-culture Methods 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 230000031700 light absorption Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000003828 vacuum filtration Methods 0.000 description 3
- CYJWXRGJMXJKTO-UHFFFAOYSA-N 1,1,2,3-tetramethyltetrazol-1-ium Chemical compound CN1N([N+](C=N1)(C)C)C CYJWXRGJMXJKTO-UHFFFAOYSA-N 0.000 description 2
- 238000010923 batch production Methods 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- SMUQFGGVLNAIOZ-UHFFFAOYSA-N quinaldine Chemical compound C1=CC=CC2=NC(C)=CC=C21 SMUQFGGVLNAIOZ-UHFFFAOYSA-N 0.000 description 2
- GPIARMSVZOEZCV-UHFFFAOYSA-N 6-fluoro-2-methylquinoline Chemical compound C1=C(F)C=CC2=NC(C)=CC=C21 GPIARMSVZOEZCV-UHFFFAOYSA-N 0.000 description 1
- NAGJQQFMJKMXJQ-UHFFFAOYSA-N 6-methoxy-2-methylquinoline Chemical compound N1=C(C)C=CC2=CC(OC)=CC=C21 NAGJQQFMJKMXJQ-UHFFFAOYSA-N 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000010627 oxidative phosphorylation Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 125000003698 tetramethyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
- C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
- C07D405/06—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- 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
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- 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
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Life Sciences & Earth Sciences (AREA)
- Optics & Photonics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种靶向活细胞线粒体的小分子荧光探针及其制备方法和应用,该探针是由7‑(二乙胺基)香豆素‑3‑甲醛与喹啉类小分子化合物通过化学键连接而成。本发明的小分子荧光探针具有较好的生物安全性,对细胞增殖无影响;本发明的小分子荧光探针具有较强的线粒体靶向能力,为线粒体的检测、靶向线粒体药物的研发提供了新的方案。
Description
技术领域
本发明涉及荧光探针领域,具体涉及一种靶向活细胞线粒体的荧光探针及其制备方法和应用。
背景技术
线粒体作为细胞的能量工厂,参与细胞的氧化磷酸化和脂质氧化等众多新陈代谢过程。许多病理学过程均与它相关(Chaturvedi R K,Flint B M.Mitochondrial diseasesof the brain[J].Free Radical Biology&Medicine,2013,63(10):1-29.)。因此,线粒体成像对于亚细胞水平的诊断治疗具有重要意义。
香豆素及其衍生物因有独特的光物理性质和与不同离子高的结合性能而被广泛研究。香豆素及其衍生物是荧光探针设计中的优秀候选荧光团,具有Stokes位移大、荧光量子产率高、光稳定性好和明显的激发和发射波长等优点(罗先金等.新型香豆素荧光染料的合成及应用[J].中国科学,2001,31(6):542-547.)。
本发明以香豆素为7-(二乙胺基)香豆素-3-甲醛为发光基团,与喹啉类小分子化合物结合,制备具有线粒体靶向性的荧光探针。
发明内容
本发明的目的在于提供一种检测活细胞线粒体的小分子荧光探针及其制备和应用。本发明的小分子探针具有较好的线粒体靶向性,制备方式简单,可工业化批量生产,具有较好的生物相容性,对正常细胞和肿瘤细胞安全无毒,具有广阔的临床前和临床应用前景。
本发明提供了一种靶向活细胞线粒体的小分子荧光探针,该小分子探针结构式为:
其中,R1~R6相同或不同,并各自为H、D、F、Cl、Br、CN、CF3、OH、OD、取代或未取代的C1-C6烷基、取代或未取代的C1-C6烷基-O-、取代或未取代的C3-C10碳环-O-、取代或未取代的C1-C6烷基-CO-、取代或未取代的C3-C10碳环-CO-、取代或未取代的C1-C6烷基-COO-、取代或未取代的C3-C10碳环-COO-、取代或未取代的C3-C10碳环-S(O)n-、取代或未取代C1-C6烷基-S(O)n-、取代或未取代C1-C6烷基-S(O)n-取代或未取代C1-C6烷基、NO2、NH2、NH(C1-C4烷基)、NH(取代或未取代的C3-C10碳环)、-N(C1-C4烷基)2、-CONH2、-CONH(C1-C4烷基)、(C1-C4烷基)CONH-、(取代或未取代的C3-C10碳环)CONH-、-CONH(取代或未取代的C3-C10碳环)、-CON(C1-C4烷基)2、取代或未取代的C3-C10碳环、含碳原子及1-4个选自N、O、S及S(O)n的杂原子的3至10元杂环基等,但不限于举例范围。
本发明还提供了一种靶向活细胞线粒体的小分子荧光探针的制备方法,该方法步骤简单,可工业化大批量生产。
一种靶向活细胞线粒体的小分子荧光探针的制备方法,包括:将喹啉类小分子化合物溶于乙醇中,再加入哌啶,室温下搅拌后,加入化合物7-(二乙胺基)香豆素-3-甲醛的二氯甲烷溶液,升温回流,得到所述的靶向活细胞线粒体的小分子荧光探针。
作为优选,所述小分子荧光探针为(I-1)~(I-3)所示化合物:
本发明还提供了一种上述任一技术方案所述小分子探针的细胞安全性检测。取对数生长期细胞,接种于96孔培养板(5000个细胞/孔)。放入在37℃细胞培养箱中恒温培养24h后,加入小分子探针,取相同的7个浓度梯度(0、25、50、100、250、500、1000、2000nM),以不加药组作为空白对照组,每种药每个浓度4-6个重复值,加完药后将96孔细胞板放入细胞培养箱中继续培养24h,在96孔板的每孔内加入四甲基偶氮唑蓝(MTT)(20μL),放入细胞培养箱中继续培养4h,吸弃培养基,每孔加入二甲亚砜(DMSO)(150μL),用酶标仪检测吸光值(490nm)。计算细胞存活率,绘制出细胞存活曲线,得到药物对细胞生长的IC50(半数抑制浓度)。体外细胞毒性试验显示,与细胞共培养24h后,小分子探针对两种细胞(L02、HepG2)的存活率都无影响。
本发明还提供了一种靶向活细胞线粒体的小分子荧光探针的应用,具有靶向活细胞线粒体的功能,提供了一种用于检测活细胞线粒体的荧光小分子探针及其制备方法和应用。
取对数生长期细胞,接种于35mm玻底平皿中(5000个细胞/孔)。放入在37℃细胞培养箱中恒温培养24h后,加入MitoRed(一种商用线粒体染料,终浓度为300nmol/L)于37℃孵育20min。弃上清,PBS(磷酸缓冲液,pH7.4)清洗2次,加入小分子探针(100nmol/L)于37℃孵育20min。弃上清,PBS清洗2次,加入Hoechst(商用细胞核染料,1∶1000稀释)于37℃孵育20min。弃上清,PBS清洗2次,加入4%多聚甲醛室温固定20分钟,弃上清,PBS清洗2次,加PBS于激光共聚焦显微镜下观察细胞成像。本发明的小分子探针与商用染料MitoRed在细胞内的荧光成像基本重叠。该结果说明小分子探针可以定位到细胞的线粒体上,并实现线粒体成像。
本发明的小分子探针具有以下有益效果:
本发明中的小分子探针制备方式简单,适合工业化批量生产。
本发明中的小分子探针无明显的细胞毒性,安全性好,可用于细胞实验,具有广阔的临床前和临床应用前景。
本发明中的小分子探针可标记细胞中的线粒体,为线粒体标记提供新的方式,适合用于线粒体的荧光成像。
附图说明
图1为小分子荧光探针1的合成路线图;
图2为小分子荧光探针2的合成路线图;
图3为小分子荧光探针3的合成路线图;
图4为小分子荧光探针1对L02细胞的毒性检测图;
图5为小分子荧光探针1对HepG2细胞的毒性检测图;
图6为小分子荧光探针2对L02细胞的毒性检测图;
图7为小分子荧光探针2对HepG2细胞的毒性检测图;
图8为小分子荧光探针3对L02细胞的毒性检测图;
图9为小分子荧光探针3对HepG2细胞的毒性检测图;
图10为小分子荧光探针1对L02线粒体靶向性的激光共聚焦图;
图11为小分子荧光探针2对L02线粒体靶向性的激光共聚焦图;
图12为小分子荧光探针3对L02线粒体靶向性的激光共聚焦图;
图中,MitoRed为商用线粒体染料,Hoechst为商用细胞核染料
具体实施方式
下面结合具体实施方式和附图对本发明作进一步详细说明,但本发明并不受其限制。
实施例1 小分子荧光探针1的制备
合成路线如图1所示。
将2-甲基喹啉(200mg,1.40mmol)溶于乙醇(5mL)中,再加入哌啶(200μL),室温下搅拌30min,然后再加入化合物7-(二乙胺基)香豆素-3-甲醛(343mg,1.40mmol)的二氯甲烷溶液(2mL),升温回流2h,TLC点板监测。当反应完全时,停止加热,冷却到室温,有固体析出,将其真空抽滤,真空干燥,得产物小分子荧光探针1为橙黄色固体(70mg),产率13.5%。
1H NMR(400MHz,CDCl3)δ8.11-8.05(q,2H),7.86(s,1H),7.78-7.74(q,3H),7.71-7.63(m,2H),7.50-7.46(m,1H),7.33-7.30(d,J=12Hz,1H),6.62-6.59(dd,J=4Hz,1H),6.52-6.51(d,J=4Hz,1H),3.45-3.41(q,4H),1.24-1.21(t,6H).
13C NMR(101MHz,CHLOROFORM-D)δ161.44,156.30,156.09,150.98,148.36,140.42,136.39,129.77,129.73,129.43,129.25,129.02,127.62,127.46,126.10,120.36,116.92,109.33,109.15,97.18,45.04,12.59.
HRMS(ESI):理论值:C24H22N2O2[M+H]+371.1681,检测值:371.1760。
实施例2 小分子荧光探针2的制备
合成路线如图2所示。
将6-氟-2-甲基喹啉(200mg,1.24mmol)溶于乙醇(5mL)中,再加入哌啶(200μL),室温下搅拌30min,然后再加入化合物7-(二乙胺基)香豆素-3-甲醛(304mg,1.24mmol)的二氯甲烷溶液(2mL),升温回流2h,TLC点板监测。当反应完全时,停止加热,冷却到室温,有固体析出,将其真空抽滤,真空干燥,得产物小分子荧光探针2为橙黄色的固体(115mg),产率23.9%。
1H NMR(400MHz,CDCl3)δ8.04-8.00(t,2H),7.78(s,1H),7.69(s,2H),7.62-7.60(d,J=8Hz,1H),7.46-7.40(ddd,J=4Hz,1H),7.36-7.34(dd,J=8Hz,1H),6.58-6.55(dd,J=4Hz,1H),6.47(dd,1H),6.86-6.83(dd,J=4Hz,1H),3.43-3.38(q,4H),1.23-1.19(t,6H).
13C NMR(101MHz,CDCl3)δ161.23,155.96,155.61,150.91,145.36,140.41,135.58,131.59,131.50,129.33,129.31,128.94,121.03,119.80,119.55,116.71,110.71,110.49,109.02,97.04,44.93,12.49.
HRMS(ESI):理论值:C24H21FN2O2[M+H]+388.1587,检测值:388.1538。
实施例3 小分子荧光探针3的制备
合成路线如图3所示。
将6-甲氧基-2-甲基喹啉(200mg,1.15mmol)溶于乙醇(5mL)中,再加入哌啶(200μL),室温下搅拌30min,然后再加入化合物7-(二乙胺基)香豆素-3-甲醛(283mg,1.15mmol)的二氯甲烷溶液(2mL),升温回流2h,TLC点板监测。当反应完全时,停止加热,冷却到室温,有固体析出,将其真空抽滤,真空干燥,得产物小分子荧光探针3为橙黄色固体(105mg),总收率22.7%。
1H NMR(400MHz,CDCl3)δ7.94-7.87(q,2H),7.76(s,1H),7.62(s,2H),7.55-7.52(d,J=12Hz,1H),7.28-7.25(m,1H),7.22-7.19(d,J=12Hz,1H),6.98-6.97(d,J=4Hz,1H),6.54-6.51(dd,J=4Hz,1H),6.44(s,1H),3.86(s,3H),3.38-3.33(q,4H),1.15(s,6H).
13C NMR(101MHz,CHLOROFORM-D)δ161.50,157.64,156.01,154.00,150.87,139.91,135.18,130.66,129.34,128.40,127.83,122.37,120.60,117.16,109.29,105.32,97.20,55.65,45.02,29.81,22.80,14.24,12.59.
HRMS(ESI):理论值:C25H24N2O3[M+H]+401.1787,检测值:401.1865。
实施例4 小分子荧光探针1体外细胞毒性评价
考察实施例1中小分子探针1对细胞增殖的影响,具体方法如下:
取对数生长期细胞,接种于96孔培养板(5000个细胞/孔)。放入在37℃细胞培养箱中恒温培养24h后,加入小分子探针1,取相同的7个浓度梯度(0、25、50、100、250、500、1000、2000nM),以不加药组作为空白对照组,每种药每个浓度4-6个重复值,加完药后将96孔细胞板放入细胞培养箱中继续培养24h,在96孔板的每孔内加入20μL的四甲基偶氮唑蓝(MTT),放入细胞培养箱中继续培养4h,吸弃培养基,每孔加入150μL二甲亚砜(DMSO),用酶标仪检测490nm处的吸光值。计算细胞存活率,绘制出细胞存活曲线,得到药物对细胞生长的IC50(半数抑制浓度)。
小分子荧光探针1对L02、HepG2细胞的毒性检测曲线结果分别如图4和图5所示。结果显示,与L02和HepG2细胞共培养24h后,与空白对照组相比(0nM),小分子探针1对两种肿瘤细胞的存活率均无影响。
细胞毒性实验表明,小分子荧光探针1无明显细胞毒性,具有广阔的临床前和临床应用前景。
实施例5 小分子荧光探针2体外细胞毒性评价
考察实施例2中小分子探针2对细胞的增殖的影响,具体方法如下:
取对数生长期细胞,接种于96孔培养板(5000个细胞/孔)。放入在37℃细胞培养箱中恒温培养24h后,加入小分子探针2,取相同的7个浓度梯度(0、25、50、100、250、500、1000、2000nM),以不加药组作为空白对照组,每种药每个浓度4-6个重复值,加完药后将96孔细胞板放入细胞培养箱中继续培养24h,在96孔板的每孔内加入20μL的四甲基偶氮唑蓝(MTT),继续放入细胞培养箱中培养4h后,吸弃培养基,每孔加入150μL二甲亚砜(DMSO),用酶标仪检测490nm处的吸光值。计算细胞存活率,绘制出细胞存活曲线,得到药物对细胞生长的IC50(半数抑制浓度)。
小分子荧光探针2对L02、HepG2细胞的毒性检测曲线结果分别如图6和图7所示。结果显示,与L02和HepG2细胞共培养24h后,与空白对照组相比(0nM),小分子探针2对两种肿瘤细胞的存活率均无影响。
细胞毒性实验表明,小分子荧光探针2无明显细胞毒性,具有广阔的临床前和临床应用前景。
实施例6 小分子荧光探针3体外细胞毒性评价
考察实施例3中小分子探针3对细胞的增殖的影响,具体方法如下:
取对数生长期细胞,接种于96孔培养板(5000个细胞/孔)。放入在37℃细胞培养箱中恒温培养24h后,加入小分子探针3,取相同的7个浓度梯度(0、25、50、100、250、500、1000、2000nM),以不加药组作为空白对照组,每种药每个浓度4-6个重复值,加完药后将96孔细胞板放入细胞培养箱中继续培养24h,在96孔板的每孔内加入20μL的四甲基偶氮唑蓝(MTT),继续放入细胞培养箱中培养4h后,吸弃培养基,每孔加入150μL二甲亚砜(DMSO),用酶标仪检测490nm处的吸光值。计算细胞存活率,绘制出细胞存活曲线,得到药物对细胞生长的IC50(半数抑制浓度)。
小分子荧光探针3对L02、HepG2细胞的毒性检测曲线结果分别如图8和图9所示。结果显示,与L02和HepG2细胞共培养24h后,与空白对照组相比(0nM),小分子探针3对两种肿瘤细胞的存活率均无影响。
细胞毒性实验表明,小分子荧光探针3无明显细胞毒性,具有广阔的临床前和临床应用前景。
实施例7 小分子荧光探针1在L02细胞中的成像分析
本发明申请人应用小分子探针1对L02细胞进行了成像分析,具体实验方法如下:
取对数生长期细胞,接种于35mm玻底平皿中(5000个细胞/孔)。放入在37℃细胞培养箱中恒温培养24h后,加入MitoRed(一种商用线粒体染料,终浓度为300nmol/L)于37℃孵育20min。弃上清,PBS(磷酸缓冲液,pH7.4)清洗2次,加入小分子探针1(100nmol/L)于37℃孵育20min。弃上清,PBS清洗2次,加入Hoechst(商用细胞核染料,1∶1000稀释)于37℃孵育20min。弃上清,PBS清洗2次,加入4%多聚甲醛室温固定20min,弃上清,PBS清洗2次,加PBS于激光共聚焦显微镜下观察细胞成像。
荧光成像情况如图10所示,商用染料MitoRed与小分子探针1在细胞内的荧光成像基本重叠。该结果说明小分子探针1可以定位到细胞的线粒体上,并实现线粒体成像。
实施例8 小分子荧光探针2在L02细胞中的成像分析
本发明申请人应用小分子探针2对L02细胞进行了成像分析,具体实验方法如下:
取对数生长期细胞,接种于35mm玻底平皿中(5000个细胞/孔)。放入在37℃细胞培养箱中恒温培养24h后,加入MitoRed(一种商用线粒体染料,终浓度为300nmol/L)于37℃孵育20min。弃上清,PBS(磷酸缓冲液,pH7.4)清洗2次,加入小分子探针2(100nmol/L)于37℃孵育20min。弃上清,PBS清洗2次,加入Hoechst(商用细胞核染料,1∶1000稀释)于37℃孵育20min。弃上清,PBS清洗2次,加入4%多聚甲醛室温固定20min,弃上清,PBS清洗2次,加PBS于激光共聚焦显微镜下观察细胞成像。
荧光成像情况如图11所示,商用染料MitoRed与小分子探针2在细胞内的荧光成像基本重叠。该结果说明小分子探针2可以定位到细胞的线粒体上,并实现线粒体成像。
实施例9 小分子荧光探针3在L02细胞中的成像分析
本发明申请人应用小分子探针3对L02细胞进行了成像分析,具体实验方法如下:
取对数生长期细胞,接种于35mm玻底平皿中(5000个细胞/孔)。放入在37℃细胞培养箱中恒温培养24h后,加入MitoRed(一种商用线粒体染料,终浓度为300nmol/L)于37℃孵育20min。弃上清,PBS(磷酸缓冲液,pH7.4)清洗2次,加入小分子探针3(100nmol/L)于37℃孵育20min。弃上清,PBS清洗2次,加入Hoechst(商用细胞核染料,1∶1000稀释)于37℃孵育20min。弃上清,PBS清洗2次,加入4%多聚甲醛室温固定20min,弃上清,PBS清洗2次,加PBS于激光共聚焦显微镜下观察细胞成像。
荧光成像情况如图12所示,商用染料MitoRed与小分子探针1在细胞内的荧光成像基本重叠。该结果说明小分子探针3可以定位到细胞的线粒体上,并实现线粒体成像。
Claims (3)
1.一种靶向活细胞线粒体的小分子荧光探针,其特征在于,是由7-(二乙胺基)香豆素-3-甲醛通过化学键与喹啉类小分子化合物连接而成,结构式如(I)式:
其中,R1~R6相同或不同,并各自为H、D、F、Cl、Br、CN、CF3、OH、OD、取代或未取代的C1-C6烷基、取代或未取代的C1-C6烷基-O-、取代或未取代的C3-C10碳环-O-、取代或未取代的C1-C6烷基-CO-、取代或未取代的C3-C10碳环-CO-、取代或未取代的C1-C6烷基-COO-、取代或未取代的C3-C10碳环-COO-、取代或未取代的C3-C10碳环-S(O)n-、取代或未取代C1-C6烷基-S(O)n-、取代或未取代C1-C6烷基-S(O)n-取代或未取代C1-C6烷基、NO2、NH2、NH(C1-C4烷基)、NH(取代或未取代的C3-C10碳环)、-N(C1-C4烷基)2、-CONH2、-CONH(C1-C4烷基)、(C1-C4烷基)CONH-、(取代或未取代的C3-C10碳环)CONH-、-CONH(取代或未取代的C3-C10碳环)、-CON(C1-C4烷基)2、取代或未取代的C3-C10碳环、含碳原子及1-4个选自N、O、S及S(O)n的杂原子的3至10元杂环基等,但不限于举例范围。
2.一种权利要求1所述的靶向活细胞线粒体的小分子荧光探针的制备方法,其特征在于:将喹啉类小分子化合物溶液与7-(二乙胺基)香豆素-3-甲醛溶液反应,制备得到所述的选择性靶向活细胞线粒体的小分子荧光探针。
3.一种权利要求1所述靶向活细胞线粒体的小分子荧光探针在线粒体荧光标记中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810720903.5A CN110655508B (zh) | 2018-06-29 | 2018-06-29 | 一种靶向活细胞线粒体的小分子荧光探针及其制备方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810720903.5A CN110655508B (zh) | 2018-06-29 | 2018-06-29 | 一种靶向活细胞线粒体的小分子荧光探针及其制备方法和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN110655508A true CN110655508A (zh) | 2020-01-07 |
CN110655508B CN110655508B (zh) | 2022-06-03 |
Family
ID=69028474
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201810720903.5A Active CN110655508B (zh) | 2018-06-29 | 2018-06-29 | 一种靶向活细胞线粒体的小分子荧光探针及其制备方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110655508B (zh) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111217798A (zh) * | 2020-02-24 | 2020-06-02 | 山西大学 | 一种香豆素-喹啉衍生物及其合成方法与应用 |
CN111440143A (zh) * | 2020-02-25 | 2020-07-24 | 苏州大学 | 基于含氮杂环的中性线粒体荧光标记物及其制备方法与应用 |
CN113004257A (zh) * | 2021-02-26 | 2021-06-22 | 三峡大学 | 查尔酮结构的荧光探针及其制备方法和检测肼的应用 |
-
2018
- 2018-06-29 CN CN201810720903.5A patent/CN110655508B/zh active Active
Non-Patent Citations (1)
Title |
---|
SASA ZHU: "Development of a ratiometric fluorescent pH probe for cell imaging based on a coumarine-quinoline platform", 《DYES AND PIGMENTS》 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111217798A (zh) * | 2020-02-24 | 2020-06-02 | 山西大学 | 一种香豆素-喹啉衍生物及其合成方法与应用 |
CN111440143A (zh) * | 2020-02-25 | 2020-07-24 | 苏州大学 | 基于含氮杂环的中性线粒体荧光标记物及其制备方法与应用 |
CN111440143B (zh) * | 2020-02-25 | 2021-04-27 | 苏州大学 | 基于含氮杂环的中性线粒体荧光标记物及其制备方法与应用 |
CN113004257A (zh) * | 2021-02-26 | 2021-06-22 | 三峡大学 | 查尔酮结构的荧光探针及其制备方法和检测肼的应用 |
Also Published As
Publication number | Publication date |
---|---|
CN110655508B (zh) | 2022-06-03 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Liu et al. | A fluorescent probe for hydrazine based on a newly developed 1-indanone-fused coumarin scaffold | |
Zhang et al. | A ratiometric lysosomal pH probe based on the coumarin–rhodamine FRET system | |
CN110655508B (zh) | 一种靶向活细胞线粒体的小分子荧光探针及其制备方法和应用 | |
Arcau et al. | Luminescent alkynyl-gold (I) coumarin derivatives and their biological activity | |
Capodilupo et al. | Design and synthesis of fluorenone-based dyes: two-photon excited fluorescent probes for imaging of lysosomes and mitochondria in living cells | |
CN107407672B (zh) | 用于准确和灵敏检测s-期dna合成和细胞增殖的光稳定aie荧光团 | |
CN111303139B (zh) | 具有聚集诱导发光性能的化合物及其制备方法和应用 | |
Li et al. | A near-infrared fluorescent probe for Cu2+ in living cells based on coordination effect | |
CN112779001B (zh) | 一种近红外粘度荧光探针的制备及其应用 | |
Pålsson et al. | Two-photon absorption and photoluminescence of europium based emissive probes for bioactive systems | |
Yang et al. | BODIPY derivatives bearing borneol moieties: Enhancing cell membrane permeability for living cell imaging | |
Plater et al. | The synthesis and evaluation of o-phenylenediamine derivatives as fluorescent probes for nitric oxide detection | |
Hande et al. | Design and expeditious synthesis of quinoline‐pyrene‐based ratiometric fluorescent probes for targeting lysosomal pH | |
Yuan et al. | A novel highly selective near-infrared and naked-eye fluorescence probe for imaging peroxynitrite | |
CN114149441B (zh) | 一种氨基取代色烯并喹啉型荧光标记物及其制备和应用 | |
CN103382189B (zh) | 一类菁类化合物、其制备方法及应用 | |
KR102513508B1 (ko) | pH 검출용 염료 화합물, 이를 이용한 필름 및 키트 | |
Chen et al. | A mitochondria-specific fluorescent probe for rapidly assessing cell viability | |
CN105061515A (zh) | 一种磷光铱配合物的合成及其用于血吸虫尾蚴荧光标记 | |
KR101261791B1 (ko) | 생체 세포 내 알칼리 인산분해효소의 활성도 탐지를 위한 형광 프로브 | |
CN109503550B (zh) | 2-氮杂芳基-6-取代氨基喹唑啉酮化合物及其制备方法和应用 | |
CN113416196B (zh) | 一种苯并噻二唑-tb类化合物及其合成方法和应用 | |
CN114702447B (zh) | 一种萘酰亚胺衍生物及其制备方法与应用 | |
CN115557878A (zh) | 一种吲哚菁类衍生物及其制备和应用 | |
CN112479998B (zh) | 一种二氢喹啉类荧光探针及其制备方法与应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |