CN117777114A - Large Stokes displacement near-infrared cell membrane targeting dye based on coumarin skeleton and preparation method and application thereof - Google Patents
Large Stokes displacement near-infrared cell membrane targeting dye based on coumarin skeleton and preparation method and application thereof Download PDFInfo
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- CN117777114A CN117777114A CN202311783724.3A CN202311783724A CN117777114A CN 117777114 A CN117777114 A CN 117777114A CN 202311783724 A CN202311783724 A CN 202311783724A CN 117777114 A CN117777114 A CN 117777114A
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- organic solvent
- reaction
- dye
- cell membrane
- dissolving
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- 230000008685 targeting Effects 0.000 title abstract description 17
- 125000000332 coumarinyl group Chemical group O1C(=O)C(=CC2=CC=CC=C12)* 0.000 title abstract description 13
- 238000006073 displacement reaction Methods 0.000 title abstract description 5
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- 150000001875 compounds Chemical class 0.000 claims abstract description 25
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 21
- 150000003839 salts Chemical class 0.000 claims abstract description 12
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- 239000003960 organic solvent Substances 0.000 claims description 49
- 238000006243 chemical reaction Methods 0.000 claims description 48
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical group CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 45
- FKNQCJSGGFJEIZ-UHFFFAOYSA-N 4-methylpyridine Chemical compound CC1=CC=NC=C1 FKNQCJSGGFJEIZ-UHFFFAOYSA-N 0.000 claims description 28
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical group CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 27
- 125000000217 alkyl group Chemical group 0.000 claims description 27
- 239000002904 solvent Substances 0.000 claims description 27
- NQRYJNQNLNOLGT-UHFFFAOYSA-N tetrahydropyridine hydrochloride Natural products C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 20
- 238000010438 heat treatment Methods 0.000 claims description 19
- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 claims description 18
- 238000010992 reflux Methods 0.000 claims description 17
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- 239000002585 base Substances 0.000 claims description 13
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- VMKOFRJSULQZRM-UHFFFAOYSA-N 1-bromooctane Chemical compound CCCCCCCCBr VMKOFRJSULQZRM-UHFFFAOYSA-N 0.000 claims description 6
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- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 6
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- LTMRRSWNXVJMBA-UHFFFAOYSA-L 2,2-diethylpropanedioate Chemical compound CCC(CC)(C([O-])=O)C([O-])=O LTMRRSWNXVJMBA-UHFFFAOYSA-L 0.000 claims description 5
- ZQOCDOFDUXPNGM-UHFFFAOYSA-N 3-(4-methylimidazol-1-yl)-5-(trifluoromethyl)benzoic acid Chemical compound C1=NC(C)=CN1C1=CC(C(O)=O)=CC(C(F)(F)F)=C1 ZQOCDOFDUXPNGM-UHFFFAOYSA-N 0.000 claims description 5
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- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N benzo-alpha-pyrone Natural products C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 5
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- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
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Landscapes
- Plural Heterocyclic Compounds (AREA)
Abstract
The invention provides a coumarin skeleton-based near-infrared cell membrane targeting dye with large Stokes displacement, and a preparation method and application thereof, and belongs to the technical field of biology. The dye is a compound shown in a formula I, a salt thereof, a stereoisomer thereof, a solvate thereof or a hydrate thereof. Compared with the prior art, the serial cell membrane targeting fluorescent dye developed by the invention has the advantages of simple and mild preparation process, high efficiency, near infrared emission, large Stokes shift, high fluorescence quantum yield, good biocompatibility and strong cell membrane targeting capability, and particularly has obviously better dyeing uniformity and retention than commercial dye and good application prospectThe scene.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a near-infrared cell membrane targeting dye with large Stokes displacement based on a coumarin skeleton, and a preparation method and application thereof.
Background
Fluorescent dyes refer to fluorescent substances that are capable of significantly changing their photophysical properties, which emit fluorescence or fluorescent signals upon various interactions with a detection object. The signal transmission mechanisms to be considered in dye design are Intramolecular Charge Transfer (ICT), excited Monomer-Excimer (FRET), fluorescence resonance energy transfer (PET), trans-bond energy transfer (TBET), bioluminescence Resonance Energy Transfer (BRET), aggregation-induced emission (AIE), excited state intramolecular proton transfer (esit), and the like. The dye luminescence characteristic is also related to the structure, and for pi conjugated system, the extension of the conjugated length can lead the emission wavelength of the molecule to be red-shifted, and the Stokes shift is enlarged; the higher the rigidity of the molecule is, the rotation or vibration relaxation of the excited state molecule can be restrained, so that the fluorescence quantum yield is enhanced; the electron-donating and electron-withdrawing groups introduced into the molecules can form an electron push-pull structure and can also lead the emission spectrum of the molecules to be red-shifted.
Small molecule organic fluorescent dyes have become powerful tools for understanding the characteristics of intracellular and extracellular microenvironments because of their advantages of easy functionalization, high environmental sensitivity, high biocompatibility and rapid and efficient uptake by cells. Reactive organelle dyes chemically react with target ions or molecules in the organelle and then produce photoactive products of different structures, the fluorescent properties of which change significantly compared to the dye itself, such as fluorescence enhancement or a significant shift in wavelength. Another design concept is to use different hydrophilia, lipophilicity and coordination capability of dye molecules to specifically target organelles, and cell membrane fluorescent dye can be designed and developed based on the design concept. The design principle is mainly based on two important properties of cell membranes: 1. the hydrophilic head of the phospholipid molecule is provided with a negatively charged phosphate group, and can be combined with an ammonium group through electrostatic action; 2. the tail of the phospholipid molecule is a long hydrophobic chain, and can be combined with a long alkyl chain of the dye through hydrophobic effect. Thus, cell membrane targeting dyes typically include a hydrophobic tail, a zwitterionic lipid or ammonium group, and a fluorescent parent nucleus, with the dye being bound to the phospholipid bilayer by electrostatic and hydrophobic interactions, targeting to the cell membrane.
The Shynkar team developed a cell membrane bicolor ratio fluorescent dye F2N12S with 4' -diethylamino-3-hydroxyflavone as a fluorescent parent nucleus, the structure of which is shown in a. The long alkyl chain in the dye molecule has hydrophobicity, and the quaternary ammonium salt and the sulfonic acid group have hydrophilicity, so that the dye can be stably combined with the phospholipid bilayer under the hydrophobic effect and the electrostatic effect. Based on esit, the change in cell membrane surface charge causes the dye to exhibit highly sensitive dual band emissions to the lipid component of the cell membrane. The dye can image the phenomenon of cell membrane asymmetry loss in early apoptosis, so that the dye can be used for apoptosis detection.
Team Feng Guojiang reports a polar sensitive cell membrane fluorescent dye MOM with tetrahydroquinoxaline coumarin amide as a fluorescent parent, the structure of which is shown as b. The fluorescent parent nucleus has hydrophobicity, and the introduced quaternary ammonium salt structure has hydrophilicity, so that the hydrophilicity and hydrophobicity of the fluorescent parent nucleus are relatively balanced. Polarity sensitive dyes can specifically target cancer cell membranes for imaging, indicating that cancer cells may be less polar than normal cells.
Yu Xiao the subject group invents coumarin-pyridine derivatives obtained by covalently linking 4-methylpyridine via double bonds with coumarin as a parent as a reagent for detecting endogenous SO in cells 2 The structure of the dye is shown as c. The organic matter primarily stains the cytoplasmic fraction.
The cell membrane is composed of a phospholipid bilayer and various proteins (glycoprotein, channel and receptor) embedded on the phospholipid bilayer, is a dynamic structure and plays an important role in cell communication and regulating the relationship between cells and the external environment. Although cell membranes are important, their properties in various aspects of the primordial cellular microenvironment are not yet clear. Therefore, intensive studies on the structure-function relationship of cell membranes are urgently required. In response to this need, small molecule fluorescence imaging techniques with many advantages, such as non-invasiveness, high spatial-temporal resolution, high signal-to-noise ratio (S/N), are ideal choices for observing cell structures. Despite the progress made in the research of cell membrane fluorescent dyes, there is still a need in the art for a solution.
Patent application publication No. CN110684370A discloses a fluorescent dye for staining cell membranes, but the membrane retention capacity of the fluorescent dye is poor, and part of the dye enters cytoplasm after 10 minutes of staining. Meanwhile, cell membrane dyes which are reported or sold at present, such as various cell membrane fluorescent dyes taking fluorescein, cyanine, rhodamine and dioxazine as skeletons, all have the problem of smaller Stokes shift (less than 30 nm). This means that the excitation and emission spectra can generate serious light channeling, which brings the defects of low imaging signal-to-noise ratio and fluorescence self-quenching; (2) Most staining reagents have absorption and emission wavelengths in the visible light range (400-700 nm), light in the wave band is easily interfered by biological background fluorescence, and biological tissues of the staining reagents have weak penetrability, so that the application of cell membrane dyes in vivo imaging is limited; (3) Cell membrane dyes with unbalanced hydrophobicity and hydrophilicity are easy to spontaneously aggregate into micelles in aqueous solution due to over-strong lipophilicity, so that imaging effect is affected, or the cell membrane dyes can rapidly enter cells or fall off from the membrane due to poor retention on the membrane, and finally, the cell membrane state cannot be accurately observed.
Disclosure of Invention
In order to solve the problems, the invention provides a near infrared cell membrane targeting dye with large Stokes shift based on a coumarin skeleton, and a preparation method and application thereof.
The present invention provides a compound of formula I, a salt thereof, a stereoisomer thereof, a solvate thereof, or a hydrate thereof:
wherein,
R 1 、R 2 independently selected from hydrogen, substituted or unsubstituted C 1 ～C 12 An alkyl group;
R 3 selected from hydrogen, substituted or unsubstituted C 1 ～C 6 Alkyl, halogen, hydroxy, amino, carboxy, nitro, cyano, monovalent anions, quaternary ammonium salts;
Z - selected from the group consisting of non-or monovalent anions; when R is 3 Z is a monovalent anion - Selected from none;
n is an integer of 1 to 10;
the substituent of the alkyl is selected from halogen, hydroxy, amino, carboxyl, nitro and cyano.
Further, the method comprises the steps of,
R 1 、R 2 selected from the same or different substituted or unsubstituted C 1 ～C 12 An alkyl group;
R 3 selected from hydroxyl, monovalent anions, quaternary ammonium salts;
Z - selected from the group consisting of no or halogen ions; when R is 3 Z is a monovalent anion - Selected from none;
n is an integer of 1 to 5;
the substituent of the alkyl is selected from halogen, hydroxy, amino, carboxyl, nitro and cyano.
Further, the compound is represented by formula II:
wherein,
R 1 、R 2 selected from identical or different C 1 ～C 10 An alkyl group;
R 3 selected from the group consisting of hydroxyl groups,
Y - Selected from halogen ions;
Z - selected from halogen ions.
Further, the method comprises the steps of,
R 1 、R 2 selected from identical or different C 1 ～C 8 An alkyl group;
Y - selected from chloride, bromide, fluoride, iodide;
Z - selected from chloride, bromide, fluoride, iodide.
Further, the compound is represented by formula III:
wherein,
R 1 、R 2 selected from identical or different C 1 ～C 8 An alkyl group;
X - selected from sulfite ions and acetate ions.
Further, the compounds were selected as follows:
the invention also provides a method for preparing the compound, which comprises the following steps:
(1) Dissolving 3-aminophenol and 1-bromooctane in an organic solvent, and carrying out heating reflux reaction to obtain an intermediate CP-S1;
(2) Adding the intermediate CP-S1 into an organic solvent containing phosphorus oxychloride, and reacting to obtain an intermediate CP-S2;
(3) Dissolving an intermediate CP-S2, diethyl malonate and alkali in an organic solvent, removing the solvent after reflux reaction, adding acid for reaction, and adjusting the pH value to 5 to obtain an intermediate CP-S3;
(4) Adding the intermediate CP-S3 into an organic solvent containing phosphorus oxychloride, and reacting to obtain an intermediate CP-S4;
(5) Dissolving 4-methylpyridine and 1, 3-propane sultone in an organic solvent, and heating for reaction to obtain CP-S5;
(6) Dissolving 3-bromo-1-propanol and 4-methylpyridine in an organic solvent, and heating for reaction to obtain CP-O1;
(7) Dissolving (3-bromopropyl) -trimethyl ammonium bromide in 4-methylpyridine, and heating for reaction to obtain CP-N1;
(8) Dissolving CP-S4 and CP-S5 in an organic solvent, and adding alkali for reflux reaction to obtain CP-S;
(9) Dissolving CP-S4 and CP-O1 in an organic solvent, and adding alkali for reflux reaction to obtain CP-O;
(10) Dissolving CP-S4 and CP-N1 in an organic solvent, and adding alkali for reflux reaction to obtain CP-N.
Further, the method comprises the steps of,
in the step (1), the organic solvent is ethanol;
and/or, in the step (2), the organic solvent is N, N-dimethylformamide; and/or, the temperature is 0-4 ℃ when the intermediate CP-S1 is added; and/or, the temperature of the reaction is 70-80 ℃;
and/or, in the step (3), the organic solvent is ethanol; and/or, the base is piperidine; and/or the acid is a mixed solution of concentrated sulfuric acid and acetic acid; and/or, the adjusting the pH uses sodium hydroxide; and/or the temperature of the acid adding reaction is 110-120 ℃;
and/or, in the step (4), the organic solvent is N, N-dimethylformamide; and/or, the temperature is 0-4 ℃ when the intermediate CP-S3 is added; and/or, the temperature of the reaction is 70-80 ℃;
and/or, in the step (5), the organic solvent is acetonitrile; and/or, the temperature of the reaction is 80-100 ℃;
and/or, in the step (6), the organic solvent is ethanol; and/or, the temperature of the reaction is 60-80 ℃; and/or, the reaction is carried out under an inert gas atmosphere;
and/or, in the step (7), the temperature of the reaction is 120-130 ℃;
and/or, in the step (8), the organic solvent is ethanol; and/or, the base is piperidine;
and/or, in the step (9), the organic solvent is ethanol; and/or, the base is piperidine;
and/or, in the step (10), the organic solvent is ethanol; and/or, the base is piperidine.
The invention also provides application of the compound, the salt, the stereoisomer, the solvate or the hydrate thereof in preparing fluorescent dye;
preferably, the fluorescent dye is a cell membrane dye.
The present invention also provides a fluorescent dye comprising the aforementioned compound, a salt thereof, a stereoisomer thereof, a solvate thereof or a hydrate thereof;
preferably, the fluorescent dye is a cell membrane dye.
The compounds and derivatives provided in the present invention may be named according to IUPAC (international union of pure and applied chemistry) or CAS (chemical abstract service, columbus, OH) naming system.
Definition of terms used in connection with the present invention: unless otherwise indicated, the initial definitions provided for groups or terms herein apply to the groups or terms throughout the specification; for terms not specifically defined herein, the meanings that one skilled in the art can impart based on the disclosure and the context.
"substituted" means that a hydrogen atom in a molecule is replaced by a different atom or molecule.
The minimum and maximum values of the carbon atom content of the hydrocarbon groups are indicated by a prefix, e.g. prefix C a ～C b Alkyl indicates any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, "C 1 ～C 6 Alkyl "refers to an alkyl group containing 1 to 6 carbon atoms.
"alkyl" refers to a saturated hydrocarbon chain having the indicated number of carbon atoms. For example, C 1 ～C 8 Alkyl means an alkyl group having 1 to 8 carbon atoms, i.e. having 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms. The alkyl group may be linear or branched. Representative branched alkyl groups have one, two or three branches. Alkyl groups include methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl and tert-butyl), pentyl (n-pentyl, isopentyl and neopentyl), hexyl and the like.
"halogen" is fluorine, chlorine, bromine or iodine.
According to the invention, a coumarin structure which is simple and easy to synthesize, easy to modify and suitable for biological imaging is selected as a fluorescent parent, a conjugated double bond is used for connecting an electricity-absorbing group at the 3 rd position of the coumarin structure, and a long-chain alkyl amino group is used as a power supply group at the 7 th position of the coumarin structure, so that modification of the coumarin parent is completed, and the cell membrane targeting fluorescent dye CP-S, CP-O, CP-N which has the advantages of large Stokes displacement, near infrared emission, high fluorescence quantum yield, good biocompatibility, strong cell membrane targeting capability, uniform dyeing and good retention is obtained. The modification of the number 3 and the number 7 leads the conjugated system of the dye mother nucleus to be enlarged, and simultaneously forms a push-pull electronic structure, so that the fluorescence red shift of the dye reaches the near infrared region, and the Stokes shift of the dye is also enlarged. In addition, the long alkyl chain at the 7 th position can be combined with the long hydrophobic chain at the tail part of the small She Linzhi molecule outside the cell membrane through hydrophobic interaction, and the polar hydrophilic pyridinium structure modified at the 3 rd position can be combined with the phosphate group at the hydrophilic head through electrostatic interaction, so that the substituents on two sides can help the dye to be more firmly positioned on the cell membrane, and the cell membrane imaging which is stable for a longer time can be realized. Thereby solving the three-point problem mentioned above.
Compared with the prior art, the series of cell membrane targeting fluorescent dyes CP-S, CP-O, CP-N developed by the invention has the advantages of simple and mild preparation process, high efficiency, near infrared emission, large Stokes shift, high fluorescence quantum yield, good biocompatibility and strong cell membrane targeting capability, and particularly has obviously better dyeing uniformity and detention than commercial dyes, and has good application prospect.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1 is a synthetic scheme of the preparation method of the staining reagent of the present invention.
FIG. 2 is a hydrogen spectrum of CP-S prepared in example 1.
FIG. 3 is a carbon spectrum of CP-S prepared in example 1.
FIG. 4 is a hydrogen spectrum of CP-O prepared in example 2.
FIG. 5 is a carbon spectrum of CP-O prepared in example 2.
FIG. 6 is a hydrogen spectrum of CP-N prepared in example 3.
FIG. 7 is a carbon spectrum of CP-N prepared in example 3.
FIG. 8 is an ultraviolet absorption spectrum of the staining reagent CP-S of example 1 in PBS solution: the left plot is the ultraviolet absorbance spectrum and the right plot is the absorbance-concentration curve at the maximum absorbance wavelength.
FIG. 9 is an ultraviolet absorption spectrum of the staining reagent CP-O of example 2 in PBS solution: the left plot is the ultraviolet absorbance spectrum and the right plot is the absorbance-concentration curve at the maximum absorbance wavelength.
FIG. 10 is an ultraviolet absorption spectrum of the staining reagent CP-N of example 3 in PBS solution: the left plot is the ultraviolet absorbance spectrum and the right plot is the absorbance-concentration curve at the maximum absorbance wavelength.
FIG. 11 is a fluorescence emission spectrum of the staining reagent CP-S of example 1 in PBS solution.
FIG. 12 is a fluorescence emission spectrum of the staining reagent CP-O of example 2 in PBS solution.
FIG. 13 is a fluorescence emission spectrum of the staining reagent CP-N of example 3 in PBS solution.
FIG. 14 shows the results of MTT cytotoxicity experiments on the staining reagents CP-S, CP-O and CP-N of examples 1, 2 and 3.
FIG. 15 shows the results of cell membrane staining confocal experiments of the staining reagents CP-S, CP-O and CP-N of examples 1, 2, 3 in HepG2 cells.
FIG. 16 shows the results of laser confocal co-localization experiments of cell membrane staining of the staining reagents CP-S, CP-O and CP-N of examples 1, 2, 3 with the commercial dye DID in HepG2 cells.
Detailed Description
The materials and equipment used in the embodiments of the present invention are all known products and are obtained by purchasing commercially available products.
1. Experimental materials
In the specific embodiment of the invention, 3-aminophenol, 1-bromooctane, 4-methylpyridine and diethyl malonate are purchased from Chengdu moisturizing local chemical Co., ltd, and N, N-dimethylformamide, phosphorus oxychloride, absolute ethyl alcohol, concentrated hydrochloric acid, glacial acetic acid and piperidine are purchased from Chengdu Colon chemical Co., ltd. 3-bromo-1-propanol, (3-bromopropyl) -trimethylammonium bromide was purchased from Anhuizhen technologies Co. Cell lines were purchased from ATCC (AmericanType Culture Collection), 10% Fetal Bovine Serum (FBS) from Hyclone,1640 medium from Gibco, usa.
2. Experimental method
The synthetic route of the dye reagent of the invention is shown in figure 1, and the process comprises the following steps:
(1) Dissolving 3-aminophenol and 1-bromooctane in a first organic solvent, and heating, refluxing and stirring to obtain a first intermediate (CP-S1) of CP-S;
(2) Adding the first intermediate (CP-S1) of the CP-S into a second organic solvent containing phosphorus oxychloride, and stirring to obtain a second intermediate (CP-S2) of the CP-S;
(3) Dissolving a second intermediate (CP-S2) of the CP-S, diethyl malonate and a first weak alkali into a first organic solvent, stirring and refluxing, then removing the solvent, adding a first strong acid, heating and stirring, cooling to room temperature, and adding the first strong alkali into an ice bath to obtain a third intermediate (CP-S3) of the CP-S;
(4) Adding the third intermediate (CP-S3) of the CP-S into a second organic solvent containing phosphorus oxychloride, and stirring to obtain a fourth intermediate (CP-S4) of the CP-S;
(5) Dissolving 4-methylpyridine and 1, 3-propane sultone in a third organic solvent, heating and stirring to obtain a fifth intermediate (CP-S5) of CP-S;
(6) Dissolving a fourth intermediate (CP-S4) and a fifth intermediate (CP-S5) of the CP-S in a first organic solvent, adding a first weak base, and heating, refluxing and stirring to obtain the CP-S;
(7) Adding a first organic solvent, vacuumizing and filling nitrogen, placing the system in a nitrogen atmosphere, adding 3-bromo-1-propanol and 4-methylpyridine into the system, and heating and stirring to obtain a first intermediate (CP-O1) of CP-O;
(8) Dissolving a fourth intermediate (CP-S4) of the CP-S and a first intermediate (CP-O1) of the CP-O in a first organic solvent, adding a first weak base, heating, refluxing and stirring to obtain the CP-O;
(9) Dissolving (3-bromopropyl) -trimethylammonium bromide in 4-methylpyridine, and heating and stirring to obtain a first intermediate (CP-N1) of CP-N;
(10) Dissolving a fourth intermediate (CP-S4) of the CP-S and a first intermediate (CP-N1) of the CP-N in a first organic solvent, adding a first weak base, heating, refluxing and stirring to obtain the CP-N.
In the steps (1), (3), (6), (7), (8) and (10), the first organic solvent is ethanol; the second organic solvent in the steps (2) and (4) is N, N-dimethylformamide; and (3) the third organic solvent in the step (5) is acetonitrile.
The first strong acid in the step (3) is a combination of concentrated hydrochloric acid and glacial acetic acid; the first strong base is sodium hydroxide; the first weak base in the steps (3), (6), (8) and (10) is piperidine.
EXAMPLE 1 preparation of CP-S
(1) Synthesis of first intermediate CP-S1 for synthesizing CP-S
The synthesis method comprises the following steps:
6.00g of 3-aminophenol (54.98 mmol,1.0 equiv) and 26.55g of 1-bromooctane (137.45 mmol,2.5 equiv) were charged under air to a 250mL round bottom flask, to which 45mL of ethanol solvent was added. The system was then refluxed with stirring for 12 hours, and after the progress of the reaction was monitored by thin layer chromatography to the end of the reaction, the solvent in the system was removed by rotary distillation under reduced pressure. The upper excess 1-bromooctane starting material was then removed by standing for delamination, and the resulting crude product was then purified by silica gel chromatography with a developing solvent in a ratio of methylene chloride: ethanol=200:1 (v/v), to thereby isolate a first intermediate CP-S1 (8.82 g, yield 48%) as a brown oily compound.
1 H NMR(400MHz,Chloroform-d)δ(ppm):7.12(t,J＝7.8Hz,1H),6.38-6.25(m,3H),3.29(t,J＝7.4Hz,4H),1.68-1.59(m,4H),1.38-1.22(m,20H),0.90(t,J＝7.0Hz,6H).
(2) Synthesis of second intermediate CP-S2 for synthesizing CP-S
The synthesis method comprises the following steps:
8.8g of N, N-dimethylformamide (120 mmol,6.0 equiv) was charged under air conditions into a 100mL round bottom flask, 2mL of phosphorus oxychloride (20 mmol,1.0 equiv) was added dropwise to the flask after cooling to 0deg.C by ice water bath, the system was then heated and stirred at 40deg.C for 30 minutes, then cooled by ice water bath, 5.67g of CP-S1 (17 mmol,0.85 equiv) as the first intermediate of CP-S was added dropwise to the system, and after stirring for 30 minutes, the temperature was raised to 70deg.C and heated and stirred for 6 hours, and the reaction was monitored by thin layer chromatography. After the reaction was completed, the system was cooled to room temperature, and then the system was poured into 100mL of ice water, and extraction operation was performed using ethyl acetate. The collected organic phase was washed with 100mL of saturated sodium bicarbonate solution, the organic phase was dried over anhydrous sodium sulfate, the solvent in the system was removed by rotary distillation under reduced pressure, and then purified by silica gel chromatography with a developing solvent at a ratio of ethyl acetate: n-hexane=25:1 (v/v), and the second intermediate CP-S2 (4.23 g, yield 36%) was obtained as a brown oily compound by separation and purification.
1 H NMR(400MHz,Chloroform-d)δ(ppm):9.50(s,1H),7.24(d,J＝8.8Hz,1H),6.22(dd,J＝8.8Hz,1.6Hz,1H),6.01(s,J＝1.6Hz,1H),3.35-3.25(m,4H),1.66-1.52(m,4H),1.37-1.20(m,20H),0.89(t,J＝7.5Hz,6H).
(3) Synthesis of third intermediate CP-S3 for synthesizing CP-S
The synthesis method comprises the following steps:
under air conditions, 1.81g of CP-S2 (5 mmol,1.0 equiv), 1.60g of diethyl malonate (10 mmol,2.0 equiv), 0.5mL of piperidine, and 30mL of absolute ethanol solvent were added to a 250mL round bottom flask. The system was stirred and refluxed for 6 hours, monitored by thin layer chromatography to end the reaction, the solvent in the system was removed by rotary distillation under reduced pressure, then 20mL of concentrated hydrochloric acid and 20mL of glacial acetic acid were added thereto, the system was further placed at 115 ℃ and heated and stirred for 8 hours, after monitoring by thin layer chromatography to end the reaction, the system was cooled to room temperature, poured into 100mL of ice water prepared in advance, and then 40% sodium hydroxide solution was slowly added dropwise thereto to adjust the pH of the system to 5. Then, ethyl acetate is used for extraction operation, 10mL of saturated sodium bicarbonate solution and 10mL of pure water are used for washing the combined organic phases, anhydrous sodium sulfate is used for drying the organic phases, and solvent in the system is removed through reduced pressure rotary distillation to obtain a yellowish green solid crude product, namely a third intermediate CP-S3, and the crude product can be directly used for the next step of synthesis without further purification.
(4) Synthesis of fourth intermediate CP-S4 for synthesizing CP-S
The synthesis method comprises the following steps:
4.38g of N, N-dimethylformamide (60 mmol,6.0 equiv) was charged under air conditions into a 100mL round bottom flask, after cooling to 0deg.C by ice-water bath, 1mL of phosphorus oxychloride (10 mmol,1.0 equiv) was added dropwise to the flask, followed by heating and stirring the system at 40deg.C for 30 minutes, followed by cooling by ice-water bath, and then a third intermediate of CP-S2.31 g of CP-S3 (6 mmol,0.6 equiv) dissolved in 5mL of N, N-dimethylformamide was added dropwise to the system, followed by stirring for 30 minutes, heating and stirring to 70deg.C for 6 hours, and the reaction was monitored by thin layer chromatography. After the reaction was completed, the system was cooled to room temperature, and then the system was poured into 100mL of ice water, and extraction operation was performed using ethyl acetate. The collected organic phase was washed with 100mL of saturated sodium bicarbonate solution, the organic phase was dried over anhydrous sodium sulfate, the solvent in the system was removed by rotary distillation under reduced pressure, and then purified by silica gel chromatography with a developing solvent at a ratio of petroleum ether: ethyl acetate=9:1 (v/v), and separated and purified to give a fourth intermediate CP-S4 (1.76 g, yield 71%) as a yellowish green solid.
1 H NMR(400MHz,Chloroform-d)δ(ppm):10.13(s,1H),8.25(s,1H),7.40(d,J＝9.0Hz,1H),6.60(dd,J＝9.0,2.4Hz,1H),6.45(d,J＝2.4Hz,1H),3.37(t,J＝7.5Hz,4H),1.68-1.60(m,4H),1.35-1.21(m,20H),0.92-0.86(m,6H). 13 C NMR(101MHz,Chloroform-d)δ(ppm):187.94,161.99,158.83,153.79,145.35,132.39,114.12,110.32,108.17,97.26,51.59,31.75,29.36,29.23,27.21,26.97,22.61,14.09.HRMS(ESI)m/z:Calcd.for C 26 H 40 NO 3 + :414.3003[M+H] + ,Found:414.3003.
(5) Synthesis of fifth intermediate CP-S5 for synthesizing CP-S
The synthesis method comprises the following steps:
1.92g of 4-methylpyridine (20 mmol,1.0 equiv), 3.72g of 1, 3-propane sultone (31 mmol,1.55 equiv) and 15mL of acetonitrile solvent were charged into a 50mL pressure-resistant tube. The system was left at 80℃for 4 hours with stirring and after completion of the reaction, which was monitored by thin layer chromatography, the system was cooled to room temperature, and a white solid was observed to precipitate, which was suction-filtered and the solid product was washed with diethyl ether to give a fifth intermediate CP-S5 (4.17 g, yield 93%) as a white solid.
1 H NMR(400MHz,DMSO-d 6 )δ(ppm):8.92(d,J＝6.2Hz,2H),7.98(d,J＝6.2Hz,2H),4.65(t,J＝7.0Hz,2H),2.60(s,3H),2.39(t,J＝7.0Hz,2H),2.19(p,J＝7.0Hz,2H).
(6) Synthesis of CP-S
The synthesis method comprises the following steps:
100mg of the fourth intermediate CP-S4 (0.24 mmol,1.0 equiv), 52mg of the fifth intermediate CP-S5 (0.24 mmol,1.0 equiv), 100. Mu.L of piperidine and 1.5mL of absolute ethanol solvent were added to a 15mL pressure-resistant tube, the system was placed in a reflux reaction at 80℃for 1 hour, after monitoring to the end of the reaction by thin layer chromatography, the solvent in the system was removed by rotary distillation under reduced pressure, and then purified by silica gel chromatography with a developing solvent in a ratio of dichloromethane: methanol=5:1 (v/v), and the product CP-S was isolated and purified as a dark red solid (93 mg, yield 62%).
1 H NMR(400MHz,Chloroform-d)δ(ppm):8.88(d,J＝4.7Hz,2H),8.08(s,1H),7.94(d,J＝5.3Hz,2H),7.71-7.50(m,2H),7.40(d,J＝8.5Hz,1H),6.49(d,J＝8.5Hz,1H),6.32(s,1H),4.90(s,2H),3.26(s,4H),2.97(s,2H),2.57-2.42(s,2H),1.67-1.48(m,4H),1.39-1.15(m,20H),0.94-0.81(m,6H).
13 C NMR(101MHz,Chloroform-d)δ(ppm):160.20,156.44,153.97,152.25,146.40,144.16,137.64,130.89,123.70,122.63,114.21,109.76,109.21,96.57,58.81,51.43,47.23,31.79,29.70,29.41,29.32,27.24,27.04,22.64,14.11.HRMS(ESI)m/z:Calcd.for C 35 H 51 N 2 O 5 S + :611.3513[M+H] + ,Found:611.3514.
The hydrogen spectrum and the carbon spectrum of the sulfonate cell membrane targeted staining reagent (CP-S) based on coumarin skeleton prepared in the embodiment are shown in figures 2 and 3 respectively.
EXAMPLE 2 preparation of CP-O
The process for preparing CP-O is essentially the same as in example 1, except that the intermediate CP-S5 is replaced with intermediate CP-O1 in the preparation of CP-O, the synthetic route is as follows:
(1) Synthesis of CP-O1, an intermediate for CP-O synthesis
10mL of an absolute ethanol solvent was charged into a 50mL two-necked flask, and after repeating the vacuum and nitrogen charging three times, 2.00g of 3-bromo-1-propanol (14.4 mmol,1.0 equiv) and 1.46g of 4-methylpyridine (15.7 mmol,1.1 equiv) were added to the flask via syringe from the side port. The system was then heated and stirred at 60℃for 24 hours, and after completion of the reaction, the solvent was removed from the system by rotary distillation under reduced pressure. The solid was then washed four times with diethyl ether and the diethyl ether was distilled off under reduced pressure to give first intermediate CP-O1 (3.17 g, yield 95%) as a brown oil.
1 H NMR(400MHz,DMSO-d 6 )δ(ppm):8.99(d,J＝6.5Hz,2H),8.01(d,J＝6.4Hz,2H),4.68(t,J＝7.0Hz,2H),4.18(s,1H),3.44(t,J＝5.8Hz,2H),2.63(s,3H),2.18-2.09(m,2H).
(2) Synthesis of CP-O
100mg of intermediate CP-S4 (0.24 mmol,1.0 equiv), 56mg of intermediate CP-O1 (0.24 mmol,1.0 equiv), 100. Mu.L of piperidine and 1.5mL of absolute ethanol solvent were added to a 15mL pressure-resistant tube, the system was left to reflux-react at 80℃for 1 hour, after monitoring to the end of the reaction by thin layer chromatography, the solvent in the system was removed by rotary distillation under reduced pressure, and then purified by silica gel chromatography with a developing solvent in a ratio of dichloromethane: methanol=5:1 (v/v), and the compound CP-O was isolated and purified as a dark red solid (98 mg, yield 65%).
1 H NMR(400MHz,Chloroform-d)δ(ppm):9.00(d,J＝6.3Hz,2H),8.12(s,1H),8.00(d,J＝6.5Hz,2H),7.67(q,J＝15.9Hz,2H),7.43(d,J＝9.0Hz,1H),6.54(dd,J＝9.0,2.0Hz,1H),6.36(d,J＝1.9Hz,1H),4.85(t,J＝6.5Hz,2H),3.75(t,J＝5.2Hz,2H),3.30(t,J＝7.2Hz,4H),2.63(s,1H),2.30(p,J＝6.6Hz,2H),1.60-1.54(m,4H),1.36-1.20(m,20H),0.93-0.84(m,6H).
13 C NMR(101MHz,Chloroform-d)δ(ppm):160.36,156.58,154.15,152.48,146.23,144.02,137.80,130.75,123.74,122.60,114.08,109.94,109.10,57.98,57.71,51.48,33.78,31.77,29.69,29.40,29.29,27.24,27.03,22.63,14.11.HRMS(ESI)m/z:Calcd.for C 35 H 51 N 2 O 3 + :547.3894[M-Br] + ,Found:547.3895.
The hydrogen spectrum and the carbon spectrum of the hydroxyl-substituted cell membrane targeting staining reagent (CP-O) based on the coumarin skeleton prepared in the example are shown in fig. 4 and 5 respectively.
EXAMPLE 3 preparation of CP-N
The process for preparing CP-N is essentially the same as in example 1, except that the intermediate CP-S5 is replaced by intermediate CP-N1 in the preparation of CP-N, the synthetic route is as follows:
(1) Synthesis of intermediate CP-N1 for synthesizing CP-N
1.31g of (3-bromopropyl) -trimethylammonium bromide was charged into a 25mL two-necked flask, and after repeating the vacuum-charging with nitrogen three times, 460 mg of 4-methylpyridine (5 mmol,1.0 equiv) was added to the flask via a syringe from a side port. Subsequently, the system was placed at 125℃and stirred for 0.9h with heating, after the reaction was completed as monitored by thin layer chromatography, 1.5mL of boiling methanol was poured into a two-necked flask, and after complete dissolution of the solid was observed, 5mL of tetrahydrofuran was slowly dropped along the wall with a pipette, and the solid was gradually precipitated. Suction filtration and washing of the solid with cold tetrahydrofuran gave first intermediate CP-N1 (1.60 g, 90% yield) as a white solid.
1 H NMR(400MHz,Methanol-d 4 )δ(ppm):8.95(d,J＝6.4Hz,2H),7.99(d,J＝6.4Hz,2H),4.74-4.68(m,2H),3.63-3.56(m,2H),3.30(p,J＝1.7Hz,2H),3.22(s,9H),2.70(s,3H).
(2) Synthesis of CP-N
100mg of intermediate CP-S4 (0.24 mmol,1.0 equiv), 85mg of intermediate CP-N1 (0.24 mmol,1.0 equiv), 100. Mu.L of piperidine and 1.5mL of absolute ethyl alcohol solvent were added to a 15mL pressure-resistant tube, the system was placed at 80℃for reflux reaction for 1 hour, after the reaction was monitored by thin layer chromatography, the solvent in the system was removed by rotary distillation under reduced pressure, 0.5mL of methanol was added, and after complete dissolution of the solid was observed, 4mL of absolute ethyl ether was slowly dropped along the wall with a suction tube, stirred for 5 hours, and the solid was gradually precipitated. The solid product was suction filtered and dried to give compound CP-N (86.6 mg, 48% yield) as a dark red solid.
1 H NMR(400MHz,Chloroform-d:Methanol-d4＝5:1)δ8.94(d,J＝6.5Hz,2H),7.84(s,1H),7.79(d,J＝6.1Hz,2H),7.53(dd,J＝28.3,15.8Hz,2H),7.24(s,1H),6.49(d,J＝9.7Hz,1H),6.31(s,1H),4.64-4.55(t,2H),3.67-3.51(t,4H),3.23(m,4H),3.04(s,9H),2.50(t,2H),1.48(s,4H),1.11(m,20H),0.72(t,6H).
13 C NMR(101MHz,Chloroform-d)δ(ppm):160.22,156.51,154.18,152.41,146.12,144.11,137.63,130.82,123.86,122.61,114.01,109.88,109.08,96.61,62.53,54.02,51.42,44.49,31.78,29.69,29.40,29.31,27.25,27.04,22.63,14.14.HRMS(ESI)m/z:Calcd.for C 38 H 59 N 3 O 2 2+ :294.7298[M-2Br] 2+ ,Found:294.7297.
The nuclear magnetic hydrogen spectrum and the nuclear magnetic carbon spectrum of the quaternary ammonium salt cell membrane targeted staining reagent (CP-N) based on the coumarin skeleton prepared in the embodiment are shown in figures 6 and 7.
The beneficial effects of the present invention are demonstrated by specific test examples below.
Test example 1, ultraviolet absorption Spectrum
The coumarin skeleton-based cell membrane targeted staining reagents (CP-S, CP-O and CP-N) prepared in examples 1-3 above were each formulated as 10mM DMSO stock solution. The stock solution was diluted with PBS to 1. Mu.M, 2. Mu.M, 4. Mu.M, 6. Mu.M, 8. Mu.M, 10. Mu.M PBS. The ultraviolet absorption value in the wavelength range of 300-700nm is scanned, the data is used for plotting, and the corresponding molar extinction coefficient is calculated through the maximum absorption.
The ultraviolet absorption spectrum and absorbance-concentration curve at the maximum absorption wavelength of the example 1CP-S staining reagent are shown in fig. 8, the ultraviolet absorption spectrum and absorbance-concentration curve at the maximum absorption wavelength of the example 2CP-O staining reagent are shown in fig. 9, and the ultraviolet absorption spectrum and absorbance-concentration curve at the maximum absorption wavelength of the example 3CP-N staining reagent are shown in fig. 10. As shown, the absorption peak of example 1 is at lambda abs =456 nm, molar extinction coefficient at maximum uv-visible absorption wavelength of 1.05X10 4 M -1 cm -1 The method comprises the steps of carrying out a first treatment on the surface of the Example 2 has two absorption peaks, each at lambda abs Molar extinction coefficient at maximum uv-visible absorption wavelength of 1.57×10 =415 nm and 450nm 4 M -1 cm -1 The method comprises the steps of carrying out a first treatment on the surface of the The molar extinction coefficient at the maximum UV-visible absorption wavelength of the absorption peak of example 3 at 468nm is 1.35×10 4 M -1 cm -1 The three staining reagents are shown to have a strong light absorption capacity.
Test example 2, fluorescence Spectrum
The CP-S, CP-O, CP-N staining reagents prepared in examples 1, 2 and 3 were prepared as a 10mM DMSO stock solution. The mother solution was diluted into 10. Mu.M PBS solution by PBS, and fluorescence spectra thereof were measured, respectively, to thereby obtain fluorescence emission curves.
In PBS solution, examples 1, 2, 3 each had a maximum emission wavelength of λ em ＝708nm、λ em ＝715nm、λ em 664nm, the maximum emission wavelength is significantly redThe shift was achieved in the near infrared emission region, with the red shift being most pronounced for examples 1 and 2. The fluorescent spectrum of the staining reagent of example 1 is shown in FIG. 11, the fluorescent spectrum of the staining reagent of example 2 is shown in FIG. 12, and the fluorescent spectrum of the staining reagent of example 3 is shown in FIG. 13. The dye prepared by the invention can be red shifted to a near infrared emission region in PBS, so that the dye has excellent deep tissue penetration capability, lower background signal and phototoxicity, and has very broad application prospect in the field of bioluminescence imaging.
Test example 3, absolute fluorescence Quantum yield test
The 10mM DMSO stock solutions of CP-S, CP-O, CP-N prepared in examples 1, 2, and 3 were diluted with DCM, DMSO, PBS as a solvent to a test solution having a maximum absorbance of less than 0.1, respectively, and measured in a quantum yield-specific cuvette by a HORIBAFluolog-3 fluorescence spectrometer.
The absolute fluorescence quantum yields of CP-S, CP-O, CP-N are shown in Table 1. The quantum yield of the three in DCM solvent is very high. The dye reagent prepared by the invention has the highest quantum yield in a DCM (DCM) aprotic solvent with low polarity, and the solvent effect of the dye is laterally verified, namely the TICT process exists. And its absolute quantum yield in aqueous solution is also sufficient for fluorescence imaging of the dye in vivo.
TABLE 1 absolute fluorescence quantum yields of the dye reagents in different solvents
Test example 4 MTT cytotoxicity test
HepG2 cells in the logarithmic growth phase were seeded in 96-well plates, with approximately 5000 cells per well. 37 ℃,5% CO 2 The culture was carried out overnight under conditions with DMEM high-glucose medium containing 10% Fetal Bovine Serum (FBS), 1% diabody (100 kU/L streptomycin and 100kU/L penicillin). After confirming complete cell attachment, CP-S, CP-O, CP-N staining reagents prepared in examples 1, 2, 3 were added at different concentration gradients (1.25. Mu.M, 2.5. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M), each concentrated5 duplicate wells were set, and a blank (0. Mu.M) was set. The incubation was continued for 24 hours after the staining reagent was added, and then the viability of the cells was checked by MTT method.
The results are shown in FIG. 14. At a high concentration of 20. Mu.M, example 3 was highly cytotoxic to HepG2 cells, and examples 1 and 2 were poorly toxic. However, at concentrations below 20. Mu.M, cell survival is not substantially affected. The dye of the invention has low cytotoxicity.
Experimental example 5 laser confocal imaging of HepG2 cell membrane staining
HepG2 cells in the logarithmic growth phase were transplanted into 35 mm glass bottom confocal dishes and cultured overnight. At 37℃with a concentration of 5. Mu.M (in DMSO<0.1vol% of 2mL of DMEM high-sugar medium was added with 1. Mu.L of 10mM dye stock solution of CP-S, CP-O, CP-N staining reagent prepared using DMSO) and incubated for 15 minutes, the medium with the staining reagent was aspirated, and 1mL of DMEM high-sugar medium containing no serum was added to the dish. The dye in the cells is then imaged under a confocal laser microscope with appropriate excitation and emission filters: example 1, lambda ex ＝488nm，λ em =650-750 nm; example 2, lambda ex ＝488nm，λ em =650-750 nm; example 3, lambda ex ＝488nm，λ em ＝620-680nm。
As shown in FIG. 15, the staining reagents CP-S, CP-O, CP-N were all able to target the cell membrane and were uniformly distributed on the membrane. Wherein, the staining reagent CP-S can clearly image the micro vesicles of the cell membrane, so as to conveniently track the vesicles on the cell membrane, thereby analyzing the cell membrane state.
Meanwhile, in order to verify the targeting effect of the dye on cell membranes, a co-localization imaging experiment is carried out by using a commercial reagent DID: after incubation of HepG2 cells with 5 μm DID (diluted in serum-free DMEM high-sugar medium) at 37 ℃ for 10 min, the medium was aspirated, gently rinsed with 1mL PBS buffer, then aspirated, and then concentrated with 5 μm (in DMSO<0.1vol% of 2mL of DMEM high-sugar medium added with 1. Mu.L of 10mM dye mother liquor of CP-S, CP-O, CP-N staining reagent prepared using DMSO) was incubated for 15 minutes, the solution was incubated with the staining reagentTo the dish, 1mL of DMEM high sugar medium without serum was added. The dye in the cells is then imaged under a confocal laser microscope using appropriate excitation and emission filters, the imaging conditions of the staining reagents being identical to those of the previous, lambda of DID ex ＝633nm，λ em ＝620-680nm。
As a result, as shown in FIG. 16, the imaging position of the staining reagent CP-S, CP-O, CP-N was substantially overlapped with the commercially available dye DID, thereby further confirming the imaging ability of the series of staining reagents on cell membranes. Meanwhile, for cells stacked inside, CP-S, CP-O, CP-N can be uniformly stained, but DID fails to stain, indicating that the series of staining reagents are significantly better than commercially available reagents.
The invention takes coumarin skeleton as a parent nucleus of cell membrane dye, and dioctyl amino and pyridinium groups are respectively introduced into the 7 th and 3 rd positions of the parent nucleus to construct the cell membrane dye CP-S, CP-O, CP-N. The introduction of the modification group enlarges the dye conjugated system, forms a push-pull electronic structure, leads fluorescence emission to red shift to reach the near infrared region, and increases Stokes shift. The long alkyl chain of the dye is combined with the phospholipid molecules of the cell membrane through hydrophobic action, and the hydrophilic end of the pyridinium is combined with the phosphate group through electrostatic action, so that the dye can quickly, stably and uniformly dye the cell membrane.
Compared with the prior art, the series of cell membrane targeting fluorescent dyes CP-S, CP-O, CP-N developed by the invention has the advantages of simple and mild preparation process, high efficiency, near infrared emission, large Stokes shift, high fluorescence quantum yield, good biocompatibility and strong cell membrane targeting capability, and particularly has the dyeing uniformity and detention which are obviously superior to those of commercial dyes.
Claims (10)
1. A compound of formula I, a salt thereof, a stereoisomer thereof, a solvate thereof, or a hydrate thereof:
wherein,
R 1 、R 2 independently selected from hydrogen, substituted or unsubstituted C 1 ～C 12 An alkyl group;
R 3 selected from hydrogen, substituted or unsubstituted C 1 ～C 6 Alkyl, halogen, hydroxy, amino, carboxy, nitro, cyano, monovalent anions, quaternary ammonium salts;
Z - selected from the group consisting of non-or monovalent anions; when R is 3 Z is a monovalent anion - Selected from none;
n is an integer of 1 to 10;
the substituent of the alkyl is selected from halogen, hydroxy, amino, carboxyl, nitro and cyano.
2. The compound, salt thereof, stereoisomer thereof, solvate thereof, or hydrate thereof according to claim 1, wherein:
R 1 、R 2 selected from the same or different substituted or unsubstituted C 1 ～C 12 An alkyl group;
R 3 selected from hydroxyl, monovalent anions, quaternary ammonium salts;
Z - selected from the group consisting of no or halogen ions; when R is 3 Z is a monovalent anion - Selected from none;
n is an integer of 1 to 5;
the substituent of the alkyl is selected from halogen, hydroxy, amino, carboxyl, nitro and cyano.
3. The compound, salt thereof, stereoisomer thereof, solvate thereof, or hydrate thereof according to claim 1, wherein: the compound is shown in a formula II:
wherein,
R 1 、R 2 selected from identical or different C 1 ～C 10 An alkyl group;
R 3 selected from the group consisting of hydroxyl groups,
Y-is selected from halogen ions;
Z - selected from halogen ions.
4. A compound according to claim 3, a salt thereof, a stereoisomer thereof, a solvate thereof or a hydrate thereof, wherein:
R 1 、R 2 selected from identical or different C 1 ～C 8 An alkyl group;
Y - selected from chloride, bromide, fluoride, iodide;
Z - selected from chloride, bromide, fluoride, iodide.
5. The compound, salt thereof, stereoisomer thereof, solvate thereof, or hydrate thereof according to claim 1, wherein: the compound is shown in a formula III:
wherein,
R 1 、R 2 selected from identical or different C 1 ～C 8 An alkyl group;
X - selected from sulfite ions and acetate ions.
6. A compound according to any one of claims 1 to 5, a salt thereof, a stereoisomer thereof, a solvate thereof or a hydrate thereof, wherein: the compounds were selected as follows:
7. a process for preparing the compound of claim 6, characterized by: it comprises the following steps:
(1) Dissolving 3-aminophenol and 1-bromooctane in an organic solvent, and carrying out heating reflux reaction to obtain an intermediate CP-S1;
(2) Adding the intermediate CP-S1 into an organic solvent containing phosphorus oxychloride, and reacting to obtain an intermediate CP-S2;
(3) Dissolving an intermediate CP-S2, diethyl malonate and alkali in an organic solvent, removing the solvent after reflux reaction, adding acid for reaction, and adjusting the pH value to 5 to obtain an intermediate CP-S3;
(4) Adding the intermediate CP-S3 into an organic solvent containing phosphorus oxychloride, and reacting to obtain an intermediate CP-S4;
(5) Dissolving 4-methylpyridine and 1, 3-propane sultone in an organic solvent, and heating for reaction to obtain CP-S5;
(6) Dissolving 3-bromo-1-propanol and 4-methylpyridine in an organic solvent, and heating for reaction to obtain CP-O1;
(7) Dissolving (3-bromopropyl) -trimethyl ammonium bromide in 4-methylpyridine, and heating for reaction to obtain CP-N1;
(8) Dissolving CP-S4 and CP-S5 in an organic solvent, and adding alkali for reflux reaction to obtain CP-S;
(9) Dissolving CP-S4 and CP-O1 in an organic solvent, and adding alkali for reflux reaction to obtain CP-O;
(10) Dissolving CP-S4 and CP-N1 in an organic solvent, and adding alkali for reflux reaction to obtain CP-N.
8. The method according to claim 7, wherein:
in the step (1), the organic solvent is ethanol;
and/or, in the step (2), the organic solvent is N, N-dimethylformamide; and/or, the temperature is 0-4 ℃ when the intermediate CP-S1 is added; and/or, the temperature of the reaction is 70-80 ℃;
and/or, in the step (3), the organic solvent is ethanol; and/or, the base is piperidine; and/or the acid is a mixed solution of concentrated sulfuric acid and acetic acid; and/or, the adjusting the pH uses sodium hydroxide; and/or the temperature of the acid adding reaction is 110-120 ℃;
and/or, in the step (4), the organic solvent is N, N-dimethylformamide; and/or, the temperature is 0-4 ℃ when the intermediate CP-S3 is added; and/or, the temperature of the reaction is 70-80 ℃;
and/or, in the step (5), the organic solvent is acetonitrile; and/or, the temperature of the reaction is 80-100 ℃;
and/or, in the step (6), the organic solvent is ethanol; and/or, the temperature of the reaction is 60-80 ℃; and/or, the reaction is carried out under an inert gas atmosphere;
and/or, in the step (7), the temperature of the reaction is 120-130 ℃;
and/or, in the step (8), the organic solvent is ethanol; and/or, the base is piperidine;
and/or, in the step (9), the organic solvent is ethanol; and/or, the base is piperidine;
and/or, in the step (10), the organic solvent is ethanol; and/or, the base is piperidine.
9. Use of a compound according to any one of claims 1 to 6, a salt thereof, a stereoisomer thereof, a solvate thereof or a hydrate thereof, for the preparation of a fluorescent dye;
preferably, the fluorescent dye is a cell membrane dye.
10. A fluorescent dye, characterized in that: comprising a compound according to any one of claims 1 to 6, a salt thereof, a stereoisomer thereof, a solvate thereof or a hydrate thereof;
preferably, the fluorescent dye is a cell membrane dye.
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