CN114716419A - Fluorescent compound containing 7-aminocoumarin structural group - Google Patents
Fluorescent compound containing 7-aminocoumarin structural group Download PDFInfo
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- CN114716419A CN114716419A CN202210257719.8A CN202210257719A CN114716419A CN 114716419 A CN114716419 A CN 114716419A CN 202210257719 A CN202210257719 A CN 202210257719A CN 114716419 A CN114716419 A CN 114716419A
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- aminocoumarin
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- 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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Abstract
The invention discloses a fluorescent compound containing a 7-aminocoumarin structural group, which is a 7-aminocoumarin fluorescent compound formed by connecting a 7-aminocoumarin report group with a 2- (6, 7-dimethoxy-3, 4-dihydro-1H-isoquinoline-2-yl) phenethyl binding group through 2-formamide-4, 5-dimethoxybenzamide and is used as a P-glycoprotein specific fluorescent probe. The invention develops a 7-aminocoumarin fluorescent compound as a P-glycoprotein specific fluorescent probe, and the fluorescent probes can be reliably used for the positioning and tracing of P-glycoprotein and have the advantages of high specificity, high sensitivity and good safety; provides a new visual angle and selection for the problems of positioning of P-glycoprotein tissue high expression regions, positioning of intracellular P-glycoprotein and research visibility and the like of diseases such as tumors, 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-aminocoumarin structural group.
Background
P-glycoprotein (P-glycoprotein, P-gp) is a transmembrane glycoprotein with the molecular weight of 170KD, and has the function of an energy-dependent drug pump, 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 wide range of therapeutic drugs, including anticancer drugs, antiviral drugs, antihistamines, antiepileptics, and analgesics.
Currently, imaging of P-gp is primarily by Positron Emission Tomography (PET) technology. Wherein the PET tracer for studying P-gp function is a P-gp substrate labeled with an isotope, e.g., [ 2 ]11C]Verapamil [ alpha ], [ beta ], [ alpha ], [ beta ] and a11C]Rocarbamide [ alpha ], [ beta ], [ alpha ], [ beta ], [ alpha ], [ beta ]11C]Colchicine [ colchicine ], [ 2 ]11C]Carvedilol [ alpha ], [ beta ] -a64Cu]The composition [ alpha ], [ beta ]68Ga]The composition and99mTc]complexes, etc., which are used only to monitor the decline in P-gp in patients with Parkinson's and Alzheimer's disease, and cannot be used to observe an increase in P-gp in patients, due to their low baseline brain uptake. A small amount of an isotopically labeled P-gp inhibitor, e.g., [ 2 ]11C]elacridar、[11C]laniquida [ Lamiquidar ], [ alpha ], [ beta ], [ alpha ], [ beta ], and11C]tarquidar was also studied for imaging P-gp expression, but its actual effect remains to be examined further.
In addition, the inspection cost of the PET technology is relatively high, and the actual operation and the result credibility are difficult due to the safety guarantee of the examinees, the influence of the concurrent use of the drugs on the PET, and the like.
Disclosure of Invention
Aiming at the defects and problems in the prior art, the invention aims to provide a fluorescent compound containing a 7-aminocoumarin structural group, develop a visible P-gp tracer, and provide a new visual angle and selection for positioning a P-gp tissue high expression region of diseases such as tumors and epilepsy and positioning P-gp in cells and researching visibility and the like. The excellent P-gp co-localization ability and safety of the compound are verified by methods such as computer-aided drug design, fluorescence quantum yield determination, cytotoxicity experiments, 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-aminocoumarin structural group, which is a 7-aminocoumarin fluorescent compound formed by connecting a 7-aminocoumarin report group with a 2- (6, 7-dimethoxy-3, 4-dihydro-1H-isoquinoline-2-yl) phenethyl binding group through 2-formamide-4, 5-dimethoxybenzamide, and the structural formula of the fluorescent compound is shown as the following formula (1):
the 7-aminocoumarin fluorescent compound is used for a P-gp specific fluorescent probe.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention develops a 7-aminocoumarin fluorescent compound as a P-gp specific fluorescent probe, and the fluorescent probes can be reliably used for positioning and tracing P-gp and have the advantages of high specificity, high sensitivity and good safety; provides a new visual angle and selection for the problems of positioning of P-gp tissue high expression region, positioning of intracellular P-gp and the research visibility and the like of diseases such as tumor, epilepsy and the like.
2. The invention adopts the P-gp specific tracer for fluorescence imaging, has no radioactive damage and high safety compared with the currently used PET tracer, only needs the auxiliary operation of a fluorescence imaging instrument, and has simple, convenient and visual result analysis.
Drawings
FIG. 1 is a design drawing of a fluorescent compound of the present invention;
FIG. 2 is a scheme showing the synthesis of fluorescent compound S1-M03 according to the present invention;
FIG. 3 is a graph of the UV absorption of fluorescent compound S1-M03 according to the present invention;
FIG. 4 is a graph of the fluorescence intensity of fluorescent compound S1-M03 according to the invention;
FIG. 5 is a graph showing the cell survival curves of fluorescent compound S1-M03 according to the present invention;
FIG. 6 is a co-localization image of fluorescent compound S1-M03 with P-gp of the present invention;
FIG. 7 shows the staining of liver sections with fluorescent compound S1-M03 according to 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 figure 1, S1-M03 is a 7-aminocoumarin fluorescent compound formed by connecting a 7-aminocoumarin reporter group with a 2- (6, 7-dimethoxy-3, 4-dihydro-1H-isoquinolin-2-yl) phenethyl binding group through 2-formamide-4, 5-dimethoxybenzamide.
To determine whether the designed small molecule can bind to P-gp and its possibility in practical application, we performed physicochemical properties and molecular docking calculations using the chemical calculation and molecular simulation software MOE developed by the Canada chemical calculation group, Inc.
The results show that S1-M03 has good predicted binding capacity and appropriate physicochemical properties, and the specific results are shown in the following table 1, the fluorescent compound S1-M03 has a docking score of-16.2382 kcal/mol, shows high P-gp binding capacity and has good physicochemical properties, the log P (o/w) of the fluorescent compound is 4.49, the molecular weight is 678.74, and the fluorescent compound is suitable for animals.
TABLE 1 physicochemical Properties and docking scores of S1-M03
| Compound | LogP(o/w) | Mol.weight | S(kcal/mol) |
| S1-M03 | 4.49 | 678.74 | -16.2382 |
The 7-aminocoumarin fluorescent compound is prepared by taking p-nitrophenylethyl bromide, hydrochloric acid-1, 2,3, 4-tetrahydro-6, 7-dimethoxyisoquinoline, 4, 5-dimethoxy-2-nitro-benzoic acid, 2-hydroxy-4-methyl-benzaldehyde, 2-dimethyl-1, 3-dioxane-4, 6-diketone and the like as raw materials through multi-step organic reaction. The specific synthetic route is shown in figure 2.
EXAMPLE 2 determination of the relevant optical Properties of fluorescent Compound S1-M03
The fluorescence property was measured after chemical synthesis of S1-M03
(ii) measurement of ultraviolet absorbance
The standard substance Coumarin-153 (Coumarin-153, C-153) was dissolved in absolute ethanol, and the compound S1-M03 was dissolved in DMSO at a sample concentration of 30. mu.M. The absorbance was measured by an ultraviolet-visible spectrophotometer UV-2450 manufactured by SHIMADZU, Japan, and the results are shown in FIG. 3, in which the maximum absorption wavelength of S1-M03 was 410-.
(II) measurement of fluorescence Spectroscopy
Firstly, scanning an excitation curve by using the maximum absorption wavelength of the measured ultraviolet absorbance as a fixed emission wavelength to obtain the maximum excitation wavelength, and scanning the emission curve by using the maximum excitation wavelength as the fixed excitation wavelength to obtain the maximum emission wavelength. The excitation curve and emission curve of the compound were measured by a fluorescence spectrometer F-7000FL Spectrophotometer manufactured by HITACHI, Japan. As shown in FIG. 4, the maximum excitation wavelength of S1-M03 was 428nm and the maximum emission wavelength was 494nm, when EX Slit was adjusted to 10.0nm or 5.0nm, EM Slit was adjusted to 10.0nm or 5.0nm, and PMT Voltage was adjusted to 400V.
(III) measurement of Absolute Quantum yield
The absolute quantum yield of the compound is detected by a steady-state-transient fluorescence spectrometer FluoroMax-4-TCSPC produced by HORIBA company of Japan, a proper excitation wavelength range and an emission wavelength range are selected to fit a quantum yield curve, so that the absolute quantum yield is obtained, and the quantum yield of the compound relative to C-153 (coumarin 153) can be calculated according to the absolute quantum yield. Wherein control C-153 was calibrated with absolute ethanol and fluorescent compounds were calibrated with DMSO, the results are shown in Table 2.
TABLE 2 Quantum yields of S1-M03
Example 3 cytotoxicity assays of fluorescent Compounds
Compound cytotoxicity assays were performed by thiazole blue (MTT) assay.
1. Taking cells in logarithmic phase, centrifugally collecting, then using complete culture medium to carry out heavy suspension to prepare single cell suspension, adjusting the cell concentration to 1.5 multiplied by 10^ 4/ml, and adding 200ul cell suspension into each hole of a 96-hole plate.
2. The compounds were added to each experimental group at 24h after plating with a concentration gradient of 100nM, 200nM, 1. mu.M, 2. mu.M, 10. mu.M. 37 ℃ and 5% CO2Culturing for 2-3 days.
3. The culture medium was aspirated, 10ul of MTT (5 mg/ml) was added, the culture was continued for 4 hours, the MTT was aspirated, 100ul of DMSO was added, and the absorbance was measured at 492 nm. And setting a zero setting hole (culture medium, MTT and dimethyl sulfoxide).
4. As shown in FIG. 5, the cell viability was more than 75% and the safety was high in the S1-M03 medium solution containing 100nM, 200nM, 1. mu.M, 2. mu.M, and 10. mu.M concentrations.
EXAMPLE 4 use of fluorescent Compound S1-M03
In a specific implementation, the fluorescent compound S1-M03 acts as a P-glycoprotein tracer.
(one) co-localization of S1-M03 with P-glycoprotein in LLC-PK1-MDR1-Apple cells, as shown in FIG. 6.
1. LLC-PK1-MDR1-Apple cells are cells after being transfected with MDR1-Apple (Apple is red fluorescence marker). Two days before the experiment, LLC-PK1-MDR1-Apple cells are planted on a slide of a 24-pore plate;
2. cells were washed 1 time with PBS before dosing, and 150. mu.l of compound (5. mu.M) was added to each well;
3. after culturing in an incubator for 1 hour, the cells were washed 2 times with PBS, the slide was taken out, the slide was covered on a slide glass on which an anti-fluorescence quenching blocking agent was dropped, fixed with nail polish, excited at 488nm (green) and 568nm (red) channels, and imaged with a 60-fold oil-immersed objective lens under a scanning confocal laser microscope A1R HD25 manufactured by NICON, Japan.
4. The results show that S1-M03 is consistent with the fluorescent position of P-glycoprotein, and S1-M03 has the capability of positioning P-glycoprotein.
(II) detecting the binding of the fluorescent compound S1-M03 to P-glycoprotein in ex vivo liver slices, as shown in FIG. 7.
1. Carrying out frozen section on the coronal plane of the liver, wherein the section thickness is 15 mu m, and the section is adhered on a glass slide;
2. drawing circles around the tissues with a histochemical pen, and dripping the compound (5 mu M) into the experimental group and the control group respectively;
3. after 30min incubation, the sections were washed twice with TBS and photographed under a fluorescence inverted microscope Ti (Nikon Japan) 10-fold objective lens for 5 fields of view (FIG. 7A is only one representative field of view) using ECFP as a filter.
4. The ratio of the binding area of the fluorescent compound to the total area is calculated by using Image Pro Plus 6.0 to the taken fluorescence pictures, the binding area ratio of the compound to the liver tissue sections of mice in a control group and an MDR1 group is counted by Graphpad prism6.01, the data is analyzed by a Multiple test-one row method, and the mean + -SEM processing is performed, the result shows that after the two groups are analyzed in a statistical way, P is less than 0.0001, and S1-M03 is considered to stain the P-glycoprotein high expression group (MDR1+ + group) with obviously more fluorescence than the control group, namely S1-M03 can specifically target the P-glycoprotein in the liver.
In conclusion, the compound S1-M03 has a docking score of-16.2382 kcal/mol, shows higher P-glycoprotein binding capacity and has better physicochemical properties, the Log P (o/w) of the compound is 4.49, the molecular weight of the compound is 678.74, and the compound is suitable for being applied to animal living bodies. After chemical synthesis of S1-M03, fluorescence properties and cytotoxicity were measured. In LLC-PK1-MDR1-Apple cells, the co-localization effect of the compound and the P-glycoprotein is good, and in the tissue level, the accumulation amount of the compound in the high expression P-glycoprotein group is larger than that of the control group, which indicates that the compound specifically targets the P-glycoprotein, and further verifies the localization ability of the fluorescent probe to the P-glycoprotein.
The foregoing merely represents preferred embodiments of the invention, which are described in some detail and detail, and therefore should not be construed as limiting the scope of the invention. It should be noted that, for those skilled in the art, various changes, modifications and substitutions can be made without departing from the spirit of the present invention, and these are all within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (2)
1. A fluorescent compound containing a 7-aminocoumarin structural group is characterized in that: the fluorescent compound is a 7-aminocoumarin fluorescent compound formed by connecting a 7-aminocoumarin reporter group with a 2- (6, 7-dimethoxy-3, 4-dihydro-1H-isoquinoline-2-yl) phenethyl binding group through 2-formamide-4, 5-dimethoxybenzamide, wherein the structural formula of the 7-aminocoumarin fluorescent compound is shown as the following formula (1):
2. a fluorescent compound containing a 7-aminocoumarin structural group according to claim 1, characterized in that: the fluorescent compound is used in a P-glycoprotein specific probe.
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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 |
| US20190343827A1 (en) * | 2018-05-08 | 2019-11-14 | Izumi Technology, Llc | Deuterated analogs of tariquidar |
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