CN114736189A - Fluorescent compound containing naphthalimide structural group - Google Patents
Fluorescent compound containing naphthalimide structural group Download PDFInfo
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- CN114736189A CN114736189A CN202210190983.4A CN202210190983A CN114736189A CN 114736189 A CN114736189 A CN 114736189A CN 202210190983 A CN202210190983 A CN 202210190983A CN 114736189 A CN114736189 A CN 114736189A
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- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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- 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
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Abstract
The invention discloses a fluorescent compound containing a naphthalimide structural group, which is a naphthalimide fluorescent compound formed by connecting a 6-aminonaphthalimide 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; the naphthalimide fluorescent compound is used for a P-glycoprotein specific fluorescent probe. The invention designs a P-glycoprotein tracer containing naphthalimide structural groups as a P-glycoprotein specific fluorescent probe, can be reliably used for positioning and 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 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 naphthalimide 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-glycoprotein 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 [ 2 ], [11C]Rocarbamide [ alpha ], [ beta ], [ alpha ], [ beta ], [ alpha ], [ beta ]11C]Colchicine (colchicine, and colchicine11C]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 Parkinson's and Alzheimer's patients and cannot be used to observe the 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 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 naphthalimide structural group, develops a visible P-glycoprotein tracer, has important significance for disease prediction, lesion excision, intracellular P-glycoprotein positioning and expression quantity detection, further verification of P-glycoprotein structure and function and the like, and designs a P-glycoprotein specific fluorescent probe by adding a 6-aminonaphthalimide 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:
a fluorescent compound containing naphthalimide structural groups is a naphthalimide fluorescent compound formed by connecting 6-aminonaphthalimide fluorescent groups with 2- (6, 7-dimethoxy-3, 4-dihydro-1H-isoquinoline-2-yl) phenethyl through 2-formamide-4, 5-dimethoxy benzamide, and the structural formula of the naphthalimide fluorescent compound is shown as the following formula (I):
the naphthalimide fluorescent compound is used as a p-glycoprotein specific fluorescent probe.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention designs a fluorescent compound containing a naphthalimide structural group, which is used as a P-glycoprotein tracer for a P-glycoprotein specific fluorescent probe, and the fluorescent probes can be reliably used for 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 intracellular P-glycoprotein and research visibility and the like of diseases such as tumors, epilepsy and the like.
2. Compared with the currently used PET tracer, the fluorescent imaging of the P-glycoprotein specificity tracer has no radioactive damage to a human body, high safety, simple and visual result analysis and only needs the auxiliary operation of a fluorescence imaging instrument. .
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 S3-M01 according to the present invention.
FIG. 3 is a graph showing the UV absorption curves of fluorescent compounds S3-M01 according to the present invention.
FIG. 4 is a fluorescence spectrum of fluorescent compound S3-M01 according to the present invention.
FIG. 5 is a graph showing the cell survival of fluorescent compound S3-M01 of the present invention.
FIG. 6 is a graph showing the staining of the fluorescent compound S3-M01 in LLC-PK1 and LLC-PK1-MDR1 cells.
FIG. 7 shows the co-localization imaging of fluorescent compound S3-M01 with P-glycoprotein according to the present invention.
FIG. 8 shows the staining of a liver section with the fluorescent compound S3-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, 6-aminonaphthalimide is taken as a fluorescent group for developing, and the two are connected by 2-formamide-4, 5-dimethoxybenzamide to synthesize a naphthalimide fluorescent compound S3-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 S3-M01 has good predicted binding capacity and appropriate physicochemical properties, and the specific results are shown in the following table 1. The compound S3-M01 has a docking score of-15.9827 kcal/mol, shows higher calculated P-gp binding capacity, has better physicochemical properties, has a log P (o/w) of 4.65 and a molecular weight of 743.82, and is suitable for animal and human body application.
TABLE 1 physicochemical Properties and docking scores of S3-M01
| Compounds | lopP(o/w) | Mol.weight | S(kcal/mol) |
| S3-M01 | 4.65 | 743.82 | -15.9827 |
The naphthalimide fluorescent compound is prepared by taking p-nitrophenylethyl bromide, hydrochloric acid-1, 2, 3, 4-tetrahydro-6, 7-dimethoxy isoquinoline, 4, 5-dimethoxy-2-nitro-benzoic acid 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 S3-M01
After chemical synthesis of S3-M01, fluorescence properties were measured.
(ii) measurement of ultraviolet absorbance
The standard substance Coumarin-153 (Coumarin-153, C-153) was dissolved in absolute ethanol, compound S3-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 emission curve of the compound were measured by a fluorescence spectrometer F-7000 FL Spectrophotometer manufactured by HITACHI, Japan. Adjusting EX Slit: 10.0nm or 5.0nm, EM Slit: 10.0nm or 5.0nm, PMT Voltage: 400V, results are shown in FIG. 4
(III) measurement of Absolute Quantum yield
Absolute Quantum yield of the Compound Steady-transient fluorescence Spectroscopy manufactured by HORIBA, Japan
And (3) carrying out FluoroMax-4-TCSPC detection, selecting a proper excitation wavelength range and an emission wavelength range to fit a quantum yield curve to obtain an absolute quantum yield, and calculating the quantum yield of the compound relative to C-153 (coumarin 153) 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 S3-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 S3-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 S3-M01
In a specific implementation, the fluorescent compound S3-M01 acts as a P-glycoprotein tracer. It has good effect of positioning P-glycoprotein in cells and liver tissues.
The binding of the compound (I) to P-glycoprotein in LLC-PK1 and LLC-PK1-MDR1 cells is 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 climbing film 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 fluorescence inverted microscope ECFP filter.
4. The results show that S3-M01 shows good specific binding capacity to P-glycoprotein at three concentrations. Wherein LLC-PK1 cells are indicated in the figure as LLC cells, and LLC-PK1-MDR1 cells are indicated in the figure as LLC-MDR1 cells.
The binding of the compound (II) to P-glycoprotein in LLC-PK1-MDR1-Apple cells is shown in FIG. 7.
1. LLC-PK1-MDR1-Apple cells are cells after being transfected with MDR1-Apple (Apple is red fluorescence marker). LLC-PK 1MDR1-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 blocking tablet is dripped, fixing by nail polish, observing red fluorescence expression of the P-glycoprotein of the slide and the combination condition of each part and a compound under a fluorescence inverted microscope Ti (Nikon Japan) 20 times objective lens, wherein the optical filters used are TRITC and ECFP.
4. The results show that S3-M01 is consistent with the fluorescent position of P-glycoprotein, and S3-M01 has the capability of positioning P-glycoprotein.
(III) test the binding of compounds to p-glycoprotein in ex vivo liver sections, 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. the compound (5. mu.M) was added dropwise to each of the experimental and control groups by circling the tissue using a pen.
3. After incubation for 30min, the cells were washed twice with PBS, and the binding of the compound to each part of the sections was observed under a fluorescence inverted microscope Ti (Nikon, Japan) 10-fold objective lens using ECFP as a filter.
4. The results showed that S3-M01 stained significantly more fluorescence in the highly expressed group of human P-glycoprotein (MDR1+ + group) than in the control group, indicating that S3-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 naphthalimide structural group is characterized in that: the fluorescent compound is a naphthalimide fluorescent compound formed by connecting a 6-aminonaphthalimide fluorescent 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 naphthalimide fluorescent compound is shown as the following formula (I):
2. the fluorescent compound containing a naphthalimide structural group according to claim 1, wherein: the fluorescent compound is used in a P-glycoprotein specific fluorescent probe.
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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 433 - 444, XP085529120, DOI: 10.1016/j.ejmech.2018.10.043 * |
| MELISSA M. SPRACHMAN ET AL: "In Vivo Imaging of Multidrug Resistance Using a Third Generation MDR1 Inhibitor", BIOCONJUGATE CHEM., vol. 25, pages 1137 - 1142, XP055860372, DOI: 10.1021/bc500154c * |
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