CN114716420B - Fluorescent compound containing 7-cyano coumarin structural group - Google Patents
Fluorescent compound containing 7-cyano coumarin structural group Download PDFInfo
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Classifications
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- 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
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- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- 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"
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- 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
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Abstract
The invention discloses a fluorescent compound containing a 7-cyano coumarin structural group, which is a 7-cyano coumarin fluorescent compound formed by connecting a 7-cyano coumarin report group with a 2- (6, 7-dimethoxy-3, 4-dihydro-1H-isoquinoline-2-yl) phenethyl binding group through 2-formamide-4, 5-dimethoxy benzamide, and is used as a P-glycoprotein specific fluorescent probe. The invention develops a 7-cyano coumarin fluorescent compound as a P-glycoprotein specific fluorescent probe, and the fluorescent probes can be reliably used for positioning and tracing the P-glycoprotein and have the advantages of high specificity, high sensitivity and good safety; provides a new view angle and selection for the problems of high expression region positioning of P-glycoprotein tissue, intracellular P-glycoprotein positioning, visibility of research and the like of diseases such as tumor, epilepsy and the like.
Description
Technical Field
The invention belongs to the technical field of fluorescent compounds, and particularly relates to a fluorescent compound containing a 7-cyano coumarin structural group.
Background
P-glycoprotein (P-gp) is a transmembrane glycoprotein with a molecular weight of 170KD, and has an energy-dependent 'drug pumping' function, so that intracellular drugs are pumped out of cells, the concentration of the intracellular drugs is reduced, and the cells generate drug resistance. The presence of P-gp affects the absorption profile of a number of therapeutic agents, including anticancer, antiviral, antihistamine, antiepileptic, and analgesic agents.
Currently, P-gp is imaged primarily by positron emission tomography (positron emissiontomography, PET) techniques. Wherein the PET tracer used for studying P-gp function is isotopically labelledP-gp substrates, e.g. [ 11 C]Verapamil [ sic ] 11 C]Lomonamide [ sic ] 11 C]Colchicine [ A.C. ] and 11 C]carvedilol [ sic ] 64 Cu]Complexes [ 68 Ga]Complexes [ 99m Tc]Complexes, etc., which are used only to monitor decreases in P-gp in Parkinson and Alzheimer's patients, cannot be used to observe increases in P-gp in patients due to their low baseline brain absorption. Small amounts of isotopically labelled P-gp inhibitors, e.g. [ 11 C]elacridar、[ 11 C]laniquidar [ 11 C]tarquidar was also studied for P-gp expression imaging, but its practical effect remains to be further investigated.
In addition, the cost of PET technical inspection is relatively high, and the security of a detected person, the influence of medicines used simultaneously on PET and the like have difficulty in actual operation and result credibility.
Disclosure of Invention
Aiming at the defects and the problems in the prior art, the invention aims to provide a fluorescent compound containing a 7-cyano coumarin structural group, develops a visual P-glycoprotein tracer agent, and provides a new view angle and selection for the problems of P-glycoprotein tissue high-expression region positioning, intracellular P-glycoprotein positioning and the visibility of research thereof and the like of diseases such as tumors, epilepsy and the like. The excellent P-glycoprotein co-localization capability and safety of the compounds are verified by adopting methods such as computer-aided drug design, fluorescence property measurement, cytotoxicity measurement, cell and tissue imaging evaluation and the like.
The invention is realized by the following technical scheme:
the invention provides a fluorescent compound containing a 7-cyano coumarin structural group, which is a 7-cyano coumarin fluorescent compound formed by connecting a 7-cyano coumarin reporter group with a 2- (6, 7-dimethoxy-3, 4-dihydro-1H-isoquinoline-2-yl) phenethyl binding group through 2-formamide-4, 5-dimethoxy benzamide, and the structural formula of the fluorescent compound is shown in the following formula (1):
the 7-cyano coumarin fluorescent compound is used for a P-glycoprotein specific fluorescent probe.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention develops a 7-cyano coumarin fluorescent compound as a P-glycoprotein specific fluorescent probe, and the fluorescent probes can be reliably used for positioning and tracing the P-glycoprotein and have the advantages of high specificity, high sensitivity and good safety; provides a new view angle and selection for the problems of high expression region positioning of P-glycoprotein tissue, intracellular P-glycoprotein positioning, visibility of research and the like of diseases such as tumor, epilepsy and the like.
2. Compared with the currently used PET tracer, the fluorescent imaging method has the advantages of no radioactive injury, high safety, simple and visual result analysis, and only needs the auxiliary operation of a fluorescent imager.
Drawings
FIG. 1 is a schematic diagram of a fluorescent compound of the present invention;
FIG. 2 is a synthetic route diagram of fluorescent compounds S1-M04 of the present invention;
FIG. 3 is a graph showing the ultraviolet absorption of fluorescent compounds S1-M04 of the present invention;
FIG. 4 is a graph showing fluorescence intensity of fluorescent compounds S1 to M04 according to the present invention;
FIG. 5 is a graph showing cell survival of fluorescent compounds S1-M04 of the present invention;
FIG. 6 is a co-localized imaging of fluorescent compounds S1-M04 of the invention with P-glycoprotein;
FIG. 7 is a staining of liver sections with fluorescent compounds S1-M04 of the invention.
Detailed Description
The invention will be further described with reference to the accompanying drawings.
Example 1 design and Synthesis of fluorescent Compounds
As shown in FIG. 1, S1-M04 is 7-cyano coumarin fluorescent compound S1-M04 formed by connecting 7-cyano coumarin reporter group with 2- (6, 7-dimethoxy-3, 4-dihydro-1H-isoquinoline-2-yl) phenethyl binding group through 2-formamide-4, 5-dimethoxy benzamide.
To determine whether a designed small molecule can bind to P-gp and its potential in practical applications, we performed physicochemical properties and molecular docking calculations using the chemical calculation and molecular modeling software MOE developed by canadian chemical computing group company.
The results show that S1-M04 has good predicted binding capacity and proper physicochemical properties, the specific results are shown in the following table 1, the docking score of the fluorescent compound S1-M04 is-17.1115 kcal/mol, the fluorescent compound S1-M04 shows higher P-gp binding capacity and has good physicochemical properties, the log P (o/w) of the fluorescent compound S1-M04 is 4.82, and the molecular weight of the fluorescent compound S1-M04 is 688.74, so that the fluorescent compound S1-M04 is suitable for animal application.
TABLE 1 physicochemical Properties and Butt score of S1-M04
| Compound | Log P(o/w) | Mol.weight | S(kcal/mol) |
| S1-M04 | 4.82 | 688.74 | -17.1115 |
The 7-cyano coumarin fluorescent compound is prepared from p-nitrophenyl ethyl bromide, hydrochloric acid-1, 2,3, 4-tetrahydro-6, 7-dimethoxy isoquinoline, 4, 5-dimethoxy-2-nitro-benzoic acid, 2-hydroxy-4-methyl-benzaldehyde, 2-dimethyl-1, 3-dioxane-4, 6-dione and the like serving as raw materials through multi-step organic reaction. The specific synthetic route is shown in FIG. 2.
Example 2 determination of relevant optical Properties of fluorescent Compounds S1-M04
Fluorescence property measurement after chemical Synthesis of S1-M04
Measurement of (one) ultraviolet absorbance
The standard substance Coumarin-153 (Coumarin-153, C-153) was dissolved in absolute ethanol, and the compounds S1-M04 were dissolved in DMSO at a concentration of 30. Mu.M. The absorbance was measured by an ultraviolet-visible spectrophotometer UV-2450 manufactured by Shimadzu corporation, japan, and as a result, as shown in FIG. 3, the maximum absorption wavelength of S1-M04 was 424-447nm, and the absorbance was 0.170.
(II) measurement of fluorescence Spectrum
The maximum absorption wavelength for measuring ultraviolet absorbance is used as a fixed emission wavelength, an excitation curve is scanned to obtain a maximum excitation wavelength, and then the maximum excitation wavelength is used as the fixed excitation wavelength, the emission curve is scanned to obtain the maximum emission wavelength. The excitation curve and emission curve of the compound were measured by a fluorescence spectrometer F-7000 FL Spectrophotometer manufactured by HITACHI Co., japan. EX Slit 10.0nm or 5.0nm,EM Slit:10.0nm or 5.0nm,PMT Voltage:400V was adjusted, and as a result, as shown in FIG. 4, the maximum excitation wavelength of S1-M04 was 425nm and the maximum emission wavelength was 498nm.
Example 3 cytotoxicity detection of fluorescent Compounds
Compound cytotoxicity assays were performed by thiazole blue (MTT) assay.
1. Taking cells in logarithmic growth phase, centrifugally collecting, re-suspending with complete culture medium to prepare single cell suspension, regulating the cell concentration to 1.5X10-4/ml, and adding 200ul of cell suspension into each well of a 96-well plate.
2. The experimental groups were each dosed 24h after plating with a concentration gradient of 100nM,200nM, 1. Mu.M, 2. Mu.M, 10. Mu.M. 37 ℃,5% CO 2 Culturing under the condition for 2-3 days.
3. The culture broth was aspirated, 5mg/ml MTT 10ul was added, the culture was continued for 4 hours, MTT was aspirated, DMSO 100ul was added, and absorbance was measured at 492 nm. At the same time, zeroing holes (culture medium, MTT, dimethyl sulfoxide) were set.
4. As shown in FIG. 5, the cell viability in the S1-M04 medium solution containing 100nM,200nM, 1. Mu.M, 2. Mu.M, and 10. Mu.M concentrations was 75% or more, and the safety was high.
EXAMPLE 4 use of fluorescent Compounds S1-M04
In a specific implementation, fluorescent compounds S1-M04 act as P-glycoprotein tracers.
Co-localization of S1-M04 with P-glycoprotein in LLC-PK1-MDR1-Apple cells is shown in FIG. 6.
1. LLC-PK1-MDR1-Apple cells are transfected MDR1-Apple (Apple is red fluorescent marker). LLC-PK1-MDR1-Apple cells are planted on a climbing plate of a 24-pore plate two days before the experiment;
2. cells were washed 1 time with PBS prior to dosing, and 150 μl of compound (5 μM) was added to each well;
3. after culturing in incubator for 1h, washing the cells with PBS for 2 times, taking out the slide, covering the slide with the anti-fluorescence quenching sealing tablet, fixing with nail polish, exciting under 488nm (green) and 568nm (red) channel, and imaging under 60-fold oil immersion objective lens under laser scanning confocal microscope A1R HD25 produced by NICON corporation.
4. The results show that S1-M04 coincides with the P-glycoprotein fluorescence site and that S1-M04 has the ability to localize the P-glycoprotein.
(II) detection of the binding of the fluorescent compound S1-M04 to P-glycoprotein in ex vivo liver sections, as shown in FIG. 7.
1. Freezing the liver coronal plane, wherein the slice thickness is 15 mu m, and the slice is adhered on a glass slide;
2. circling around the tissue with a organizing pen, and respectively dripping the compound (5 mu M) into an experimental group and a control group;
3. after 30min incubation, the cells were washed twice with TBS, and 5 slice fields were photographed under a fluorescence inverted microscope Ti (Nikon, japan) with a 10-fold objective (FIG. 7A is only one representative field), and the filter used was ECFP.
4. The ratio of the combined area of fluorescent compounds to the total area is calculated by using an Image Pro Plus 6.0 pair of photographed fluorescent pictures, the combined area ratio of the fluorescent compounds to liver tissue sections of mice in a control group and an MDR1 group is counted by using a Graphpad prism6.01, the data are analyzed by a Multiple t test-one per row method, and the result shows that after the two groups are subjected to statistical analysis, P <0.05, the staining fluorescence of S1-M04 in a human P-glycoprotein high expression group (MDR 1++ group) is considered to be obviously higher than that of the control group, namely S1-M04 can specifically target P-glycoprotein in the liver.
In conclusion, the compound S1-M04 has a butt joint score of-17.1115 kcal/mol, shows higher P-glycoprotein binding capacity and better physicochemical property, has a Log P (o/w) of 4.82 and a molecular weight of 688.74, and is suitable for application to living animals. After chemical synthesis of S1-M04, fluorescence property measurement and cytotoxicity detection were performed. In LLC-PK1-MDR1-Apple cells, the co-localization effect of the compound and the P-glycoprotein is good, and the accumulation amount of the compound in the high-expression P-glycoprotein group is larger than that of a control group on the tissue level, which indicates that the compound specifically targets the P-glycoprotein, and further verifies the localization capability of the fluorescent probe on the P-glycoprotein.
The foregoing description of the preferred embodiments of the present invention has been presented only in terms of those specific and detailed descriptions, and is not, therefore, to be construed as limiting the scope of the invention. It should be noted that modifications, improvements and substitutions can be made by those skilled in the art without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (2)
1. A fluorescent compound comprising a 7-cyanocoumarin structural group, characterized in that: the fluorescent compound is a 7-cyano coumarin fluorescent compound formed by connecting a 7-cyano coumarin reporter group with a 2- (6, 7-dimethoxy-3, 4-dihydro-1H-isoquinoline-2-yl) phenethyl binding group through 2-formamide-4, 5-dimethoxy benzamide, and the structural formula of the 7-cyano coumarin fluorescent compound is shown in the following formula (1):
，
formula (1).
2. A fluorescent compound comprising a 7-cyanocoumarin structural group as claimed in claim 1, wherein: the fluorescent compound is used in a P-glycoprotein specific fluorescent probe, and the fluorescent probe is used for positioning and tracing of the P-glycoprotein.
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| WO2008098789A2 (en) * | 2007-02-16 | 2008-08-21 | Ktb Tumorforschungsgesellschaft Mbh | Dual acting prodrugs |
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| WO2008098789A2 (en) * | 2007-02-16 | 2008-08-21 | Ktb Tumorforschungsgesellschaft Mbh | Dual acting prodrugs |
| CN104910069A (en) * | 2014-12-15 | 2015-09-16 | 北京科技大学 | Anthranilic acid derivative, preparation method and application thereof in medicine |
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| In Vivo Imaging of Multidrug Resistance Using a Third Generation MDR1 Inhibitor;Melissa M. Sprachman et al;Bioconjugate Chem.;第25卷;第1137-1142页 * |
| Mariagrazia Rullo et al.1,2,3,4-Tetrahydroisoquinoline/2H-chromen-2-one conjugates as nanomolar P-glycoprotein inhibitors: Molecular determinants for affinity and selectivity over multidrug resistance associated protein 1.European Journal of Medicinal Chemistry.2018,第161卷第433-444页. * |
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