CN114751896A - Fluorescent compound containing 7-methylcoumarin structural group - Google Patents
Fluorescent compound containing 7-methylcoumarin structural group Download PDFInfo
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- CN114751896A CN114751896A CN202210194958.3A CN202210194958A CN114751896A CN 114751896 A CN114751896 A CN 114751896A CN 202210194958 A CN202210194958 A CN 202210194958A CN 114751896 A CN114751896 A CN 114751896A
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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
-
- 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
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- 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
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- 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
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- 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"
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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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
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
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- 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
Abstract
The invention discloses a fluorescent compound containing a 7-methylcoumarin structural group, which is a 7-methylcoumarin fluorescent compound formed by connecting a 7-methylcoumarin 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 fluorescent compound containing a 7-methylcoumarin structural group as a P-glycoprotein tracer to be used in a P-glycoprotein specific fluorescent probe, and the fluorescent probe can be reliably used for the positioning tracing of P-glycoprotein and has the advantages of high specificity, high sensitivity and good safety in vivo; provides a new visual angle and selection for the problems of positioning of P-glycoprotein tissue high expression regions, positioning of P-glycoprotein in cells, visualization of research thereof 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-methylcoumarin structure group.
Background
P-glycoprotein (P-glycoprotein, P-gp) is a transmembrane glycoprotein with a molecular weight of 170KD and has a drug pump function, so that intracellular drugs can be pumped out of cells, the drug concentration in the cells can be reduced, and the cells can generate drug resistance. The presence of P-gp affects the absorption profile of a number of therapeutic drugs, including anticancer, antiviral, antihistamine, antiepileptic, and analgesic drugs.
Currently, imaging of P-gp is primarily by Positron Emission Tomography (PET) technology. Wherein the PET tracer for studying P-gp function is an isotopically labeled P-gp substrate, e.g., [ 2 ]11C]Verapamil [ alpha ], [ beta ], [ alpha ], [ beta ] and a11C]Rocarbamide [ alpha ], [ beta ], [ alpha ], [ beta ], [ alpha ], [ beta ]11C]Colchicine (colchicine, and colchicine11C]Carvedilol [ alpha ], [ beta ] -a64Cu]Composition [ 2 ]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]laniquard and [ 2 ]11C]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 practical operation and the result credibility are difficult due to the safety guarantee of the examinee, the influence of the concurrent use of the medicine 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-methylcoumarin structural group, develops a visible P-glycoprotein tracer, and has important significance for disease prediction, lesion tissue location, intracellular P-glycoprotein location and expression quantity detection, further verification of P-glycoprotein structure and function and the like. According to the invention, a P-glycoprotein specific fluorescent probe is designed by adding a 7-methylcoumarin fluorescent group on a 2- (6, 7-dimethoxy-3, 4-dihydro-1H-isoquinoline-2-yl) phenethyl binding group; the excellent P-glycoprotein 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-methylcoumarin structural group, which is a 7-methylcoumarin fluorescent compound formed by connecting a 7-methylcoumarin fluorescent group with 2- (6, 7-dimethoxy-3, 4-dihydro-1H-isoquinoline-2-yl) phenethyl through 2-formamide-4, 5-dimethoxybenzamide, and the structural formula of the fluorescent compound is shown as the following formula (1):
the 7-methylcoumarin 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-methylcoumarin 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 in vivo; provides a new visual angle and selection for the problems of positioning of P-glycoprotein tissue high expression regions, positioning of P-glycoprotein in cells, visualization of research thereof and the like of diseases such as tumors, epilepsy and the like.
2. The invention adopts the P-glycoprotein specificity tracer for fluorescence imaging, has no radioactive damage to human bodies 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 the fluorescent compound of the present invention.
FIG. 2 is a scheme showing the synthesis of fluorescent compound S1-M01 according to the present invention.
FIG. 3 is a graph showing the UV absorption curves of fluorescent compounds S1-M01 according to the present invention.
FIG. 4 is a fluorescence spectrum of fluorescent compound S1-M01 according to the present invention.
FIG. 5 is a graph showing the cell survival of fluorescent compound S1-M01 of the present invention.
FIG. 6 is a graph showing the staining of the fluorescent compound S1-M01 in LLC-PK1 and LLC-PK1-MDR1 cells.
FIG. 7 shows the co-localization imaging of fluorescent compound S1-M01 with P-glycoprotein according to the present invention.
FIG. 8 shows the staining of a liver section with the fluorescent compound S1-M01 of the present 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, 2- (6, 7-dimethoxy-3, 4-dihydro-1H-isoquinoline-2-yl) phenethyl is taken as a binding group to position P-gp, 7-methylcoumarin is taken as a fluorescent group for imaging, and 2-formamide-4, 5-dimethoxybenzamide is connected with the two to synthesize a 7-methylcoumarin fluorescent compound S1-M01 capable of carrying out P-gp specific positioning imaging.
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-M01 has good predicted binding capacity and appropriate physicochemical properties, and the specific results are shown in the following table 1, the docking score of the fluorescent compound S1-M01 is-16.2267 kcal/mol, the fluorescent compound shows higher calculated P-gp binding capacity, has good physicochemical properties, has the log P (o/w) of 5.46 and the molecular weight of 677.75, and is suitable for animal and human body application.
TABLE 1 physicochemical Properties and docking scores of S1-M01
| Compounds | Log P(o/w) | Mol.weight | S(kcal/mol) |
| S1-M01 | 5.46 | 677.75 | -16.2267 |
The 7-methylcoumarin 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 a 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-M01
The fluorescence properties were determined after chemical synthesis of S1-M01.
(ii) measurement of ultraviolet absorbance
The standard substance Coumarin-153 (Coumarin-153, C-153) was dissolved in absolute ethanol, compound S1-M01 was dissolved in DMSO, and the sample concentration was 30. mu.M. The absorbance was measured by an ultraviolet-visible spectrophotometer UV-2450 manufactured by SHIMADZU, Japan, and the result is shown in FIG. 3.
(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 the emission curve of the compound were measured by a fluorescence spectrometer F-7000 FL Spectrophotometer manufactured by HITACHI corporation, Japan. Adjusting EX Slit: 10.0nm or 5.0nm, EM Slit: 10.0nm or 5.0nm, PMT Voltage: 400V, the results are shown in FIG. 4.
(III) measurement of Absolute Quantum yield
The absolute quantum yield of the compound is detected by using a steady-state-transient fluorescence spectrometer FluoroMax-4-TCSPC produced by HORIBA company of Japan, and an appropriate excitation wavelength range and an emission wavelength range are selected to fit a quantum yield curve to obtain the absolute quantum yield, and the quantum yield of the compound relative to C-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-M01
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 solution was aspirated, 5mg/ml MTT was added thereto in an amount of 10ul, the culture was further continued for 4 hours, MTT was aspirated, DMSO was added in an amount of 100ul, and the absorbance was measured at a wavelength of 492 nm. And setting a zero setting hole (culture medium, MTT and dimethyl sulfoxide).
4. As shown in FIG. 5, the cell viability of S1-M01 was more than 75% at 100nM, 200nM, 1. mu.M, 2. mu.M, 10. mu.M, and the safety was high.
EXAMPLE 4 use of fluorescent Compound S1-M01
In a specific implementation, the fluorescent compound S1-M01 acts as a P-glycoprotein tracer. It has good effect of positioning P-glycoprotein in cells and liver tissues.
(one) binding of S1-M01 to P-glycoprotein in LLC-PK1 and LLC-PK1-MDR1 cells, as shown in FIG. 6.
1. LLC-PK1-MDR1 is a cell transfected with human MDR1 (gene encoding P-glycoprotein). The cells (LLC-PK1 cells and LLC-PK1-MDR1 cells) are respectively planted on the slide of a 24-pore plate two days before the experiment;
2. cells were washed 1 time with PBS before dosing, and 150. mu.l of compound (2.5. mu.M, 5. mu.M or 10. mu.M) was added to each well;
3. culturing in incubator for 1 hr, washing with PBS for 2 times, taking out the slide, covering the slide on the glass slide with the fluorescence quenching resisting tablet, fixing with nail polish, and air drying. The exposure time was adjusted and imaged on a 20-fold objective lens under a Nikon inverted fluorescence microscope ECFP filter.
4. The results show that S1-M01 showed good specific binding capacity to P-glycoprotein at three concentrations, wherein LLC-PK1 cells are indicated as LLC cells in the figure, and LLC-PK1-MDR1 cells are indicated as LLC-MDR1 cells in the figure.
(II) the co-localization of S1-M01 with P-glycoprotein in LLC-PK1-MDR1-Apple cells, as shown in FIG. 7.
1. LLC-PK1-MDR1-Apple cells are cells after being transfected with MDR1-Apple (Apple is red fluorescence marker). LLC-PK1-MDR1-Apple cells are planted on a slide of a 24-pore plate two days before the experiment;
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 1h, washing cells for 2 times by PBS, taking out a slide, covering the slide on a glass slide on which an anti-fluorescence quenching sealing tablet is dripped, fixing by nail polish, observing red fluorescence expression of the P-glycoprotein of the section and the combination condition of each part and a compound under a Nikon fluorescence inverted microscope with an objective lens of 20 times, wherein the optical filters used are TRITC and ECFP.
4. The results show that S1-M01 is consistent with the fluorescent position of P-glycoprotein, and S1-M01 has the capability of positioning P-glycoprotein.
(III) detecting the binding of the fluorescent compound S1-M01 to P-glycoprotein in ex vivo liver slices, as shown in FIG. 8.
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. incubating for 30min, washing with TBS twice, observing the binding condition of each part of the section and the compound under a fluorescence inverted microscope Ti (Nikon Japan) 10 times objective lens, wherein the used optical filter is ECFP;
4. the results showed that S1-M01 stained significantly more fluorescence in the highly expressed group of human P-glycoprotein (MDR1+ + group) than in the control group, indicating that S1-M01 specifically targets P-glycoprotein in liver.
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-methylcoumarin structural group is characterized in that: the fluorescent compound is a 7-methylcoumarin fluorescent compound formed by connecting a 7-methylcoumarin reporter group with a 2- (6, 7-dimethoxy-3, 4-dihydro-1H-isoquinolin-2-yl) phenethyl binding group through 2-formamide-4, 5-dimethoxybenzamide, wherein the structural formula of the 7-methylcoumarin fluorescent compound is shown as the following formula (1):
2. the fluorescent compound containing a 7-methylcoumarin structural group according to claim 1, wherein: the fluorescent compound is used in a P-glycoprotein specific probe.
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| Title |
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| 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》, vol. 161, pages 434 * |
| MELISSA M.SPRACHMAN ET AL: ""In Vivo Imaging of Multidrug Resistance Using a Third Generation MDR1 Inhibitor"", 《BIOCONJUGATE CHEM》, vol. 25, pages 1138 * |
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