CN114736192B - 一种含7-羟基香豆素结构基团的荧光化合物 - Google Patents
一种含7-羟基香豆素结构基团的荧光化合物 Download PDFInfo
- Publication number
- CN114736192B CN114736192B CN202210190982.XA CN202210190982A CN114736192B CN 114736192 B CN114736192 B CN 114736192B CN 202210190982 A CN202210190982 A CN 202210190982A CN 114736192 B CN114736192 B CN 114736192B
- Authority
- CN
- China
- Prior art keywords
- glycoprotein
- hydroxycoumarin
- fluorescent
- fluorescent compound
- group
- 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.)
- Active
Links
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 30
- CJIJXIFQYOPWTF-UHFFFAOYSA-N 7-hydroxycoumarin Natural products O1C(=O)C=CC2=CC(O)=CC=C21 CJIJXIFQYOPWTF-UHFFFAOYSA-N 0.000 title claims abstract description 22
- ORHBXUUXSCNDEV-UHFFFAOYSA-N umbelliferone Chemical compound C1=CC(=O)OC2=CC(O)=CC=C21 ORHBXUUXSCNDEV-UHFFFAOYSA-N 0.000 title claims abstract description 22
- 102100033350 ATP-dependent translocase ABCB1 Human genes 0.000 claims abstract description 45
- 108010047230 Member 1 Subfamily B ATP Binding Cassette Transporter Proteins 0.000 claims abstract description 33
- 230000027455 binding Effects 0.000 claims abstract description 9
- -1 2- (6, 7-dimethoxy-3, 4-dihydro-1H-isoquinoline-2-yl) phenethyl Chemical group 0.000 claims abstract description 5
- 230000003834 intracellular effect Effects 0.000 abstract description 5
- 239000000700 radioactive tracer Substances 0.000 abstract description 4
- 201000010099 disease Diseases 0.000 abstract description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 3
- 206010028980 Neoplasm Diseases 0.000 abstract description 2
- 239000003795 chemical substances by application Substances 0.000 abstract description 2
- 206010015037 epilepsy Diseases 0.000 abstract description 2
- 238000001727 in vivo Methods 0.000 abstract description 2
- 238000011160 research Methods 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 150000001875 compounds Chemical class 0.000 description 26
- 101001017818 Homo sapiens ATP-dependent translocase ABCB1 Proteins 0.000 description 13
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 11
- 238000006862 quantum yield reaction Methods 0.000 description 7
- 230000005284 excitation Effects 0.000 description 6
- 239000003814 drug Substances 0.000 description 5
- 238000003384 imaging method Methods 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 210000004185 liver Anatomy 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 108091005942 ECFP Proteins 0.000 description 3
- 230000009194 climbing Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- IAKHMKGGTNLKSZ-INIZCTEOSA-N (S)-colchicine Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(OC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC IAKHMKGGTNLKSZ-INIZCTEOSA-N 0.000 description 2
- IUNJCFABHJZSKB-UHFFFAOYSA-N 2,4-dihydroxybenzaldehyde Chemical compound OC1=CC=C(C=O)C(O)=C1 IUNJCFABHJZSKB-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000008045 co-localization Effects 0.000 description 2
- VSSSHNJONFTXHS-UHFFFAOYSA-N coumarin 153 Chemical compound C12=C3CCCN2CCCC1=CC1=C3OC(=O)C=C1C(F)(F)F VSSSHNJONFTXHS-UHFFFAOYSA-N 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002189 fluorescence spectrum Methods 0.000 description 2
- 238000012632 fluorescent imaging Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000002600 positron emission tomography Methods 0.000 description 2
- 238000010791 quenching Methods 0.000 description 2
- 230000000171 quenching effect Effects 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- NTURQZFFJDCTMZ-UHFFFAOYSA-N 1-(2-bromoethyl)-4-nitrobenzene Chemical compound [O-][N+](=O)C1=CC=C(CCBr)C=C1 NTURQZFFJDCTMZ-UHFFFAOYSA-N 0.000 description 1
- SGTNSNPWRIOYBX-KTXUZGJCSA-N 2-(3,4-dimethoxyphenyl)-5-[2-(3,4-dimethoxyphenyl)ethyl-methylamino]-2-propan-2-ylpentanenitrile Chemical compound C1=C(OC)C(OC)=CC=C1CCN([11CH3])CCCC(C#N)(C(C)C)C1=CC=C(OC)C(OC)=C1 SGTNSNPWRIOYBX-KTXUZGJCSA-N 0.000 description 1
- WWCMFGBGMJAJRX-UHFFFAOYSA-N 4,5-dimethoxy-2-nitrobenzoic acid Chemical compound COC1=CC(C(O)=O)=C([N+]([O-])=O)C=C1OC WWCMFGBGMJAJRX-UHFFFAOYSA-N 0.000 description 1
- SHOWAGCIRTUYNA-UHFFFAOYSA-N 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline;hydron;chloride Chemical compound [Cl-].C1C[NH2+]CC2=C1C=C(OC)C(OC)=C2 SHOWAGCIRTUYNA-UHFFFAOYSA-N 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- GUGOEEXESWIERI-UHFFFAOYSA-N Terfenadine Chemical compound C1=CC(C(C)(C)C)=CC=C1C(O)CCCN1CCC(C(O)(C=2C=CC=CC=2)C=2C=CC=CC=2)CC1 GUGOEEXESWIERI-UHFFFAOYSA-N 0.000 description 1
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical compound C1=CSC=N1 FZWLAAWBMGSTSO-UHFFFAOYSA-N 0.000 description 1
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000000730 antalgic agent Substances 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000003556 anti-epileptic effect Effects 0.000 description 1
- 230000001387 anti-histamine Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 239000001961 anticonvulsive agent Substances 0.000 description 1
- 239000000739 antihistaminic agent Substances 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- NPAKNKYSJIDKMW-UHFFFAOYSA-N carvedilol Chemical compound COC1=CC=CC=C1OCCNCC(O)COC1=CC=CC2=NC3=CC=C[CH]C3=C12 NPAKNKYSJIDKMW-UHFFFAOYSA-N 0.000 description 1
- 229960004195 carvedilol Drugs 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 101150074451 clpP gene Proteins 0.000 description 1
- 229960001338 colchicine Drugs 0.000 description 1
- 238000002784 cytotoxicity assay Methods 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 102000053576 human ABCB1 Human genes 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 210000001503 joint Anatomy 0.000 description 1
- 210000005228 liver tissue Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- TULGGJGJQXESOO-BJUDXGSMSA-N methyl 11-[1-[2-[4-(quinolin-2-ylmethoxy)phenyl]ethyl]piperidin-4-ylidene]-5,6-dihydroimidazo[2,1-b][3]benzazepine-3-carboxylate Chemical compound C12=CC=CC=C2CCN2C(C(=O)O[11CH3])=CN=C2C1=C1CCN(CCC=2C=CC(OCC=3N=C4C=CC=CC4=CC=3)=CC=2)CC1 TULGGJGJQXESOO-BJUDXGSMSA-N 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000003032 molecular docking Methods 0.000 description 1
- OSFCMRGOZNQUSW-BJUDXGSMSA-N n-[4-[2-(6,7-dimethoxy-3,4-dihydro-1h-isoquinolin-2-yl)ethyl]phenyl]-5-methoxy-9-oxo-10h-acridine-4-carboxamide Chemical compound N1C2=C(O[11CH3])C=CC=C2C(=O)C2=C1C(C(=O)NC1=CC=C(C=C1)CCN1CCC=3C=C(C(=CC=3C1)OC)OC)=CC=C2 OSFCMRGOZNQUSW-BJUDXGSMSA-N 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000006053 organic reaction Methods 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- JGVWCANSWKRBCS-UHFFFAOYSA-N tetramethylrhodamine thiocyanate Chemical compound [Cl-].C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=C(SC#N)C=C1C(O)=O JGVWCANSWKRBCS-UHFFFAOYSA-N 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 238000001161 time-correlated single photon counting Methods 0.000 description 1
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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
- C07D405/12—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 chain containing hetero atoms as chain links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- 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
- A61K49/0039—Coumarin dyes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0052—Small organic molecules
-
- 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
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1007—Non-condensed systems
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Biomedical Technology (AREA)
- Optics & Photonics (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Materials Engineering (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明公开了一种含7‑羟基香豆素结构基团的荧光化合物，其为7‑羟基香豆素报告基团通过2‑甲酰胺‑4，5‑二甲氧基苯甲酰胺连接2‑(6，7‑二甲氧基‑3，4‑二氢‑1H‑异喹啉‑2‑基)苯乙基结合基团形成的7‑羟基香豆素类荧光化合物，用于P‑糖蛋白特异性荧光探针。本发明开发了一种含7‑羟基香豆素结构基团的P‑糖蛋白示踪剂作为P‑糖蛋白特异性荧光探针，此荧光探针能够可靠地用于P‑糖蛋白的定位示踪，在体内具有特异性高、灵敏度高、安全性好的优点；对肿瘤、癫痫等疾病的P‑糖蛋白组织高表达区定位、细胞内P‑糖蛋白定位及其研究的可视性等问题提供了新的视角和选择。
Description
技术领域
本发明属于荧光化合物技术领域，具体涉及一种含7-羟基香豆素结构基团的荧光化合物。
背景技术
P-糖蛋白(P-glycoprotein，P-gp)是一种分子量170KD的跨膜糖蛋白，它具有能量依赖性″药泵″功能，使细胞内药物泵出细胞外，减低了细胞内的药物浓度，使细胞产生耐药性。P-gp的存在影响了大量治疗药物的吸收分布，包括抗癌药物、抗病毒药物、抗组胺药、抗癫痫药和镇痛药。
目前，对P-gp的成像主要通过正电子发射计算机断层扫描(positronemissiontomography，PET)技术。其中用于研究P-gp功能的PET示踪剂是用同位素标记的P-gp底物，如[11C]维拉帕米，[11C]洛培酰胺，[11C]秋水仙碱，[11C]卡维地洛，[64Cu]配合物，[68Ga]配合物和[99mTc]配合物等，由于其低基线脑吸收，它们仅被用于监测帕金森和阿尔兹海默病患者P-gp的下降，不能用于观察患者P-gp的增加。少量的同位素标记的P-gp抑制剂，如[11C]elacridar、[11C]laniquidar和[11C]tarquidar也被研究用于P-gp表达成像，但其实际效果仍待进一步考察。
此外，PET技术检查费用相对较高，并且受检者安全保障、同期使用药物对PET的影响等对实际操作与结果可信性带来困难。
发明内容
针对现有技术中的不足与难题，本发明旨在提供一种含7-羟基香豆素结构基团的荧光化合物，开发可视性P-糖蛋白示踪剂，对疾病预测、病灶组织定位、细胞内P-糖蛋白定位和表达量检测、P-糖蛋白结构及功能的进一步验证等具有重要意义。本发明通过在2-(6，7-二甲氧基-3，4-二氢-1H-异喹啉-2-基)苯乙基结合基团上添加7-羟基香豆素荧光基团，设计了P-糖蛋白特异性荧光探针。采用计算机辅助药物设计、荧光量子产率测定、细胞毒性实验、细胞和组织成像评价等方法，证实了化合物出色的P-糖蛋白共定位能力和安全性。
本发明通过以下技术方案予以实现：
一种含7-羟基香豆素结构基团的荧光化合物，其为7-羟基香豆素荧光基团通过2-甲酰胺-4，5-二甲氧基苯甲酰胺连接2-(6，7-二甲氧基-3，4-二氢-1H-异喹啉-2-基)苯乙基形成的7-羟基香豆素荧光化合物，其结构式如下式(I)所示：
所述7-羟基香豆素荧光化合物用于P-糖蛋白特异性荧光探针中。
与现有技术相比，本发明有益效果包括：
1、本发明开发了一种含7-羟基香豆素结构基团的荧光化合物用于P-糖蛋白特异性荧光探针，这些荧光探针能够可靠地用于P-糖蛋白的定位示踪，在体内具有特异性高、灵敏度高、安全性好的优点；对肿瘤、癫痫等疾病的P-糖蛋白组织高表达区定位、细胞内P-糖蛋白定位及其研究的可视性等问题提供了新的视角和选择。
2、本发明采用P-糖蛋白特异性示踪剂荧光显像，与目前使用的PET示踪剂相比，对人体无放射性伤害，安全性高，仅需荧光成像仪辅助操作，且结果分析简便直观。
附图说明
图1为本发明荧光化合物的设计图。
图2为本发明荧光化合物S1-M02合成路线图。
图3为本发明荧光化合物S1-M02紫外吸收曲线图。
图4为本发明荧光化合物S1-M02的荧光光谱图。
图5为本发明荧光化合物S1-M02细胞存活曲线图。
图6为本发明荧光化合物S1-M02在LLC-PK1和LLC-PK1-MDR1细胞中的染色图。
图7为本发明荧光化合物S1-M02与P-糖蛋白共定位成像。
图8为本发明荧光化合物S1-M02肝脏切片染色。
具体实施方式
下面结合附图，对本发明作进一步地说明。
实施例1荧光化合物的设计与合成
如图1所示，2-(6，7-二甲氧基-3，4-二氢-1H-异喹啉-2-基)苯乙基为结合基团对P-gp进行定位，7-羟基香豆素作为荧光基团显像，通过2-甲酰胺-4，5-二甲氧基苯甲酰胺连接两者合成可进行P-gp特异性定位成像的7-羟基香豆素荧光化合物S1-M02。
为确定设计的小分子是否能与P-gp结合及其在实际应用中的可能性，我们采用加拿大化学计算集团公司开发的化学计算及分子模拟软件MOE进行了理化性质和分子对接计算。
结果显示，S1-M02具有良好的预测结合能力及适宜的理化性质，具体如表1所示，化合物S1-M02对接评分为-15.0457kcal/mol，表现出计算出较高的p-糖蛋白结合能力，且具有较好的理化性质，其log P(o/w)为4.86，分子量为679.73，适合动物和人体应用。
表1 S1-M02的理化性质及对接得分
| Compounds | lopP(o/w) | Mol.weight | S(kcal/mol) |
| S1-M02 | 4.86 | 679.73 | -15.0457 |
7-羟基香豆素荧光化合物S1-M02以对硝基苯乙基溴、盐酸--1，2，3，4-四氢-6，7-二甲氧基异喹啉、4，5-二甲氧基-2-硝基-苯甲酸、2，4-二羟基苯甲醛、、2，2-二甲基-1，3-二恶烷-4，6-二酮等为原料，经多步有机反应生成。具体合成路线如图2所示。
实施例2荧光化合物S1-M02的相关光学性质测定
化学合成S1-M02后，进行了荧光性质测定。
(一)紫外吸光度的测量
标准物质香豆素-153(Coumarin-153，C-153)溶于无水乙醇中，化合物S1-M02，溶于DMSO中，样品浓度均为30μM。检测吸光度用日本SHIMADZU公司生产的紫外-可见光分光光度计UV-2450检测，结果如图3所示。
(二)荧光光谱的测量
首先用上述测量紫外吸光度的最大吸收波长作为固定的发射波长，扫描出激发曲线，得到最大激发波长，再用最大激发波长作为固定激发波长，扫描出发射曲线，得到最大发射波长。化合物的激发曲线和发射曲线用日本HITACHI公司生产的荧光光谱仪F-7000 FLSpectrophotometer检测。调整EX Slit：10.0nm or 5.0nm，EM Slit：10.0nm or 5.0nm，PMTVoltage：400V，上样量200μl。结果如图4所示
(三)绝对量子产率的测量
化合物的绝对量子产率用日本HORIBA公司生产的稳态-瞬态荧光光谱仪FluoroMax-4-TCSPC检测，选择合适的激发波长范围和发射波长范围拟合量子产率曲线，得到绝对量子产率，由绝对量子产率可以计算出化合物相对于C-153的量子产率。其中对照品C-153用无水乙醇校正，荧光化合物用DMSO校正，结果如表2所示。
表2 S1-M02的量子产率
实施例3荧光化合物的细胞毒性检测
通过噻唑蓝(MTT)实验进行了化合物细胞毒性检测。
1、取对数生长期细胞，离心收集后，用完全培养基重悬，制成单细胞悬液，调整细胞浓度至1.5×10^4个/ml，96孔板每孔加入200ul细胞悬液。
2、实验组分别于铺板后24h分别加入化合物，浓度梯度为100nM，200nM，1μM，2μM，10μM。37℃，5％CO2条件下继续培养2-3天。
3、吸除培养液，加5mg/ml MTT 10ul，再继续培养4小时，吸除MTT，加入DMSO100ul，于492nm波长下检测吸光度。同时设置调零孔(培养基、MTT、二甲基亚砜)。
4、结果如图5所示，S1-M02在100nM，200nM，1μM，2μM，10μM浓度下细胞存活率皆在75％以上，安全性较高。
实施例4荧光化合物S1-M02的应用
在具体实施中，荧光化合物S1-M02作为P-糖蛋白示踪剂。其在细胞与肝脏组织中定位P-糖蛋白效果良好。
(一)S1-M02在LLC-PK1和LLC-PK1-MDR1细胞内与P-糖蛋白的结合情况，如图6所示。
1、LLC-PK1-MDR1为转染人源MDR1(编码P-糖蛋白的基因)后的细胞。实验前两天分别将细胞(LLC-PK1细胞和LLC-PK1-MDR1细胞)种植于24孔板的爬片上；
2、给药前用PBS将细胞洗1遍，每孔加入150μl的化合物(2.5μM、5μM or10μM)；
3、放入孵箱内培养1h后，用PBS将细胞洗2遍，取出爬片，将爬片覆盖在滴有抗荧光淬灭封片剂的载玻片上，指甲油固定，晾干。调节曝光时间，在Nikon荧光倒置显微镜ECFP滤光片下20倍镜成像。
4、结果显示，S1-M02在5μM浓度下对P-糖蛋白显示良好的特异性结合能力，其中LLC-PK1细胞在图中表示为LLC细胞，LLC-PK1-MDR1细胞在图中表示为LLC-MDR1细胞。
(二)S1-M02在LLC-PK1-MDR1-Apple细胞内与P-糖蛋白的共定位情况，如图7所示。
1、LLC-PK1-MDR1-Apple细胞为转染MDR1-Apple(Apple为红色荧光标记)后的细胞。实验前两天将LLC--PK1-MDR1-Apple细胞种植于24孔板的爬片上；
2、给药前用PBS将细胞洗1遍，每孔加入150μl的化合物(5μM)；
3、放入孵箱内培养1h后，用PBS将细胞洗2遍，取出爬片，将爬片覆盖在滴有抗荧光淬灭封片剂的载玻片上，指甲油固定，在荧光倒置显微镜Ti(日本尼康)20倍物镜下观察切片P-糖蛋白红色荧光表达及各部位与化合物结合情况，使用的滤光片为TRITC和ECFP。
4、结果显示，S1-M02与P-糖蛋白荧光位置一致，S1-M02具有定位P-糖蛋白的能力。
(三)检测化合物在离体肝脏切片中与P-糖蛋白的结合情况，如图8所示。
1、将肝脏冠状面进行冰冻切片，切片厚度15μm，切片粘附于载玻片上；
2、用组化笔在组织周围画圈，实验组和对照组分别滴加化合物(5μM)；
3、孵育30min后用TBS洗两遍，在荧光倒置显微镜Ti(日本尼康)20倍物镜下观察切片各部位与化合物结合情况，使用的滤光片为ECFP；
4、结果显示，S1-M02在人源P-糖蛋白高表达组(MDR1++组)中染色荧光明显多于对照组，表示S1-M02在肝脏中可特异性靶向P-糖蛋白。
以上所述仅表达了本发明的优选实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形、改进及替代，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
Claims (2)
1.一种含7-羟基香豆素结构基团的荧光化合物，其特征在于：所述荧光化合物为7-羟基香豆素荧光基团通过2-甲酰胺-4,5-二甲氧基苯甲酰胺连接2-（6,7-二甲氧基-3,4-二氢-1H-异喹啉-2-基）苯乙基结合基团形成的7-羟基香豆素荧光化合物，所述7-羟基香豆素荧光化合物的结构式如下式（1）所示：
，
式（Ⅰ）。
2.根据权利要求1所述的一种含7-羟基香豆素结构基团的荧光化合物，其特征在于：所述荧光化合物用于P-糖蛋白特异性荧光探针中，所述荧光探针用于P-糖蛋白的定位示踪。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210190982.XA CN114736192B (zh) | 2022-03-01 | 2022-03-01 | 一种含7-羟基香豆素结构基团的荧光化合物 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210190982.XA CN114736192B (zh) | 2022-03-01 | 2022-03-01 | 一种含7-羟基香豆素结构基团的荧光化合物 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114736192A CN114736192A (zh) | 2022-07-12 |
| CN114736192B true CN114736192B (zh) | 2023-10-31 |
Family
ID=82274868
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210190982.XA Active CN114736192B (zh) | 2022-03-01 | 2022-03-01 | 一种含7-羟基香豆素结构基团的荧光化合物 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114736192B (zh) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5696157A (en) * | 1996-11-15 | 1997-12-09 | Molecular Probes, Inc. | Sulfonated derivatives of 7-aminocoumarin |
| US5990336A (en) * | 1997-01-13 | 1999-11-23 | The University Of Tennessee Research Corporation | Synthesis and purification of zosteric acid |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6327565B2 (ja) * | 2012-10-03 | 2018-05-23 | 国立大学法人弘前大学 | クマリン誘導体が結合した蛍光標識糖誘導体を用いた細胞イメージング方法及びイメージング剤 |
| US10399964B2 (en) * | 2017-03-30 | 2019-09-03 | The University Of Kansas | Coumarin-linked taxanes for detection and circumvention of cellular efflux |
-
2022
- 2022-03-01 CN CN202210190982.XA patent/CN114736192B/zh active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5696157A (en) * | 1996-11-15 | 1997-12-09 | Molecular Probes, Inc. | Sulfonated derivatives of 7-aminocoumarin |
| US5990336A (en) * | 1997-01-13 | 1999-11-23 | The University Of Tennessee Research Corporation | Synthesis and purification of zosteric acid |
Non-Patent Citations (5)
| Title |
|---|
| "A coumarin derivative as a fluorogenic glycoproteomic probe for biological imaging";Lei Rong,et.;《Chem. Commun.》;第50卷;第667--669页 * |
| "Functionalized Coumarine Fragment to Obtain Fluorescent and Selective P-Glycoprotein Ligands";Elena Capparelli，et.;《Arch. Pharm. Chem.》;第349卷;第161–167页 * |
| "In Vivo Imaging of Multidrug Resistance Using a Third Generation MDR1 Inhibitor";Sprachman,et.;《Bioconjugate Chem.》;第25卷;第1137-1142页 * |
| "白芷香豆素类化合物与P-糖蛋白体外亲和作用研究";董伟等;《中草药》;第47卷(第16期);第2893-2896页 * |
| "香豆素类新基质在基质辅助激光解吸／电离飞行时间质谱 测定多糖和糖蛋白中的应用";张珍英等;《分析化学研究简报》;第36卷(第10期);第1419-1422页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114736192A (zh) | 2022-07-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| He et al. | Near-infrared fluorescent macromolecular reporters for real-time imaging and urinalysis of cancer immunotherapy | |
| Wani et al. | A potential anticancer dihydropyrimidine derivative and its protein binding mechanism by multispectroscopic, molecular docking and molecular dynamic simulation along with its in-silico toxicity and metabolic profile | |
| Yuan et al. | Near‑infrared fluorescence imaging of prostate cancer using heptamethine carbocyanine dyes | |
| Chen et al. | BODIPY-substituted hydrazine as a fluorescent probe for rapid and sensitive detection of formaldehyde in aqueous solutions and in live cells | |
| Yang et al. | A ratiometric two-photon fluorescent probe for selective detection of endogenous formaldehyde via Aza-Cope reaction | |
| CN110129037A (zh) | 一种检测粘度的荧光探针及其制备方法和应用 | |
| Liu et al. | Two-photon fluorescent probe for detection of nitroreductase and hypoxia-specific microenvironment of cancer stem cell | |
| US9958434B2 (en) | Fluorescent probe sensing tyrosine kinase and use thereof | |
| Guan et al. | Synthesis and biological evaluation of genistein-IR783 conjugate: cancer cell targeted delivery in MCF-7 for superior anti-cancer therapy | |
| Zheng et al. | A small, steady, rapid and selective TICT based fluorescent HSA sensor for pre-clinical diagnosis | |
| Sang et al. | Peroxynitrite/lipid droplet sequence-activated dual-lock fluorescent probes enable precise intraoperative imaging of atherosclerotic plaques | |
| Shieh et al. | Characterization of MB-102, a new fluorescent tracer agent for point-of-care renal function monitoring | |
| Kabir et al. | Intrinsically fluorescent anti-cancer drugs | |
| Jia et al. | Displacement Induced Off–On Fluorescent Biosensor Targeting IDO1 Activity in Live Cells | |
| Ma et al. | A polyamine-based dinitro-naphthalimide conjugate as substrates for polyamine transporters preferentially accumulates in cancer cells and minimizes side effects in vitro and in vivo | |
| Wu et al. | Novel near-infrared frequency up-conversion luminescence probe for monitoring biothiols in vitro and in vivo | |
| Stalinska et al. | Anti-glioblastoma effects of phenolic variants of benzoylphenoxyacetamide (BPA) with high potential for blood brain barrier penetration | |
| CN114736192B (zh) | 一种含7-羟基香豆素结构基团的荧光化合物 | |
| CN114751896B (zh) | 一种含7-甲基香豆素结构基团的荧光化合物 | |
| CN114736189B (zh) | 一种含萘酰亚胺结构基团的荧光化合物 | |
| Ugwu et al. | Organic cation rhodamines for screening organic cation transporters in early stages of drug development | |
| Wang et al. | In‐vivo two‐photon visualization and quantitative detection of redox state of cancer | |
| Luikham et al. | Structural insight into groove binding of yohimbine with calf thymus DNA: A spectroscopic, calorimetric, and computational approach | |
| CN114716420B (zh) | 一种含7-氰基香豆素结构基团的荧光化合物 | |
| CN114716419B (zh) | 一种含7-氨基香豆素结构基团的荧光化合物 |
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 |