CN109369633B - Near-infrared two-region fluorescent compound capable of targeting mitochondria and preparation method and application thereof - Google Patents
Near-infrared two-region fluorescent compound capable of targeting mitochondria and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a near-infrared two-region fluorescent compound capable of targeting mitochondria and a preparation method and application thereof. The emission wavelength is from 600 to 1200nm, and the emission wavelength can reach a near infrared two-region (900-1200 nm) through the modification of different functional groups. The compound can be used in the biological fields of mitochondrial imaging, photothermal therapy, osteosarcoma, whole body bone imaging and the like. The added modifiable sites can be used for connecting different bioactive groups, so as to improve the water solubility, biocompatibility and targeting property of the modified sites, and can also be used for in vitro detection of various disease markers and in-vivo early diagnosis of tumors such as breast cancer, brain glioma, colon cancer, liver cancer and the like. The fluorescent compound has the advantages of novel structure, simple synthesis steps, photobleaching resistance, no toxicity, good biocompatibility and the like, and has excellent industrial production value and biomedical application prospect.
Description
Technical Field
The invention belongs to the field of near-infrared two-region fluorescent probe dyes, and particularly relates to a novel near-infrared fluorescent compound with a thiopyrylium structure, wherein the emission wavelength of the near-infrared fluorescent compound is 600-1200 nm. Through modification of different functional groups, the emission wavelength can reach a near infrared two-region (900-.
Background
Cancer (also known as malignant tumor) seriously threatens human health. Because of the limitations of medical technology, there is no effective treatment means for advanced cancer, so early diagnosis of cancer is particularly important for patients, and if the cancer can be found as early as possible and treated in time, the survival rate of cancer patients can be significantly improved. The advent of molecular imaging techniques such as non-invasive in vivo animal fluorescence imaging techniques opens new avenues for the early diagnosis of cancer.
Fluorescence imaging in the near infrared two region (NIR-II, 1000-1700nm) has been widely used for biological and biomedical research because of the advantages of deep penetration depth, high spatial resolution and low biological autofluorescence of light in this wavelength region. The initial near infrared two-region small molecule fluorescent probes are constructed by taking benzodithiadiazole (BBTD) as an electron acceptor (A) and triphenylamine as an electron donor (D) to form a donor-acceptor-donor (D-A-D) molecular skeleton, although a series of near infrared two-region small molecule fluorescent probes have been discovered recently, the reported near infrared two-region small molecule probes are basically constructed by taking benzodithiadiazole as a mother nucleus and have a single structure (nat. Mater.,2016,15, 235-242; chem. Sci.,2016,7, 6203-6207). Therefore, the skeleton of a novel near-infrared two-region small molecule probe with good biocompatibility, low toxicity and high light stability is imperative to be searched.
As an important structural component of heterocyclic type light-emitting compounds having positive charges, pyrylium salts are widely used as photosensitizers, molecular switching agents and near-infrared dyes. At present, no report is available on the substitution of an oxygen atom in a pyrylium salt with a sulfur atom to obtain a thiopyrylium salt.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a near-infrared two-region fluorescent organic small-molecule dye with a novel D-A structure, which is modifiable, high in light stability and good in biocompatibility, and the near-infrared two-region fluorescent organic small-molecule dye is used for early diagnosis and photothermal therapy of cancers such as mitochondrial imaging and osteosarcoma.
In order to achieve the above purpose, the invention provides the following technical scheme:
in a first aspect, there is provided a modifiable near-infrared two-domain fluorescent compound having the structure of formula (I):
wherein: n is 0 to 1;
R1comprises the following steps: h, NH2，OH，
R2Comprises the following steps:
n＝0～18；m＝1～200；X＝F，Cl，Br，I，N3；
R3comprises the following steps:
R4comprises the following steps:
R5comprises the following steps:
R6comprises the following steps:
the fluorescent compound with the structure shown in the general formula (I) has a fluorescence emission wavelength of 600-1200 nm.
In a second aspect, a method for preparing a fluorescent compound of formula (I) is provided, the reaction scheme is as follows:
wherein: n is 0 to 1;
R1comprises the following steps: h, NH2，OH，
R2Comprises the following steps:
n＝0～18；m＝1～200；X＝F，Cl，Br，I，N3；
R3comprises the following steps:
R4comprises the following steps:
R5comprises the following steps:
R6comprises the following steps:
the reaction conditions are as follows:
(1) dissolving compound 2 in 20M L ethanol, adding 20% KOH solution, reacting at room temperature for 10 minutes, then adding compound 3 to the mixture, and stirring the reaction at room temperature overnight, after cooling to room temperature, adjusting the reaction solution to pH 3 with 2M HCl solution, and collecting the formed yellow intermediate compound 4 by filtration;
(2) compound 5 and pyrrolidine were dissolved in benzene and the solution was heated to 100 ℃ and stirred for 4 hours. After cooling to room temperature, benzene was removed under reduced pressure. Then compound 4 and anhydrous 1, 4-dioxane were added to the above reaction solution, the solution was heated under reflux for 6 hours, after completion of the reaction, water was added to the mixture and extracted with ethyl acetate 3 times, after which the organic layer was washed with saturated brine. The combined organic layers were dried over anhydrous magnesium sulfate, filtered and concentrated. Then removing the solvent in vacuum, and purifying the crude product by silica gel column chromatography to obtain an intermediate compound 6;
(3) compound 6 was dissolved in diethyl ether and stirred for 10 minutes, and thioacetic acid and boron trifluoride ether were added to the mixture to react under reflux for 3 hours. After cooling to room temperature, the reaction mixture was quenched with water, an excess of diethyl ether was added to the solution, and then the mixture was stirred at room temperature to precipitate a pale yellow intermediate compound 7;
(4) adding the compound 7, the raw material 8 and acetic anhydride into a reaction vessel, stirring for 2h at 70 ℃, adding a large amount of diethyl ether, separating out a crude product, and purifying by a silica gel chromatographic column to obtain a final product 1, namely the fluorescent compound shown in the general formula (I).
Preferably, the molar ratio of the compounds 2 and 3 in the step (1) is 1: 1-3; the molar ratio of the compound 4 to the pyrrolidine to the compound 5 in the step (2) is 1-5: 2-5: 1-10; the molar ratio of the compound 6, the thioacetic acid and the boron trifluoride diethyl etherate in the step (3) is 1-5: 3-10: 4-20; the molar ratio of the compound 7 to the raw material 8 in the step (4) is 1-3: 2-10.
In a third aspect, a near-infrared two-region fluorescence imaging probe for in vivo imaging is provided, and specifically polyethylene glycol, polypeptide, protein, aptamer, folic acid and derivatives thereof are modified at a site where a compound shown in a general formula (I) can be modified.
In a fourth aspect, an application of the near-infrared two-region fluorescence imaging probe in preparation of a reagent for cell mitochondrial imaging is provided.
In a fifth aspect, an application of the near-infrared two-zone fluorescence imaging probe in preparing a reagent for tumor diagnosis is provided.
Preferably, the tumor is osteosarcoma, colon cancer, liver cancer, brain glioma, breast cancer, prostate cancer, melanoma, esophageal cancer, cervical cancer, ovarian cancer.
In a sixth aspect, an application of the near-infrared two-region fluorescence imaging probe in preparing a reagent for imaging osteosarcoma is provided.
In a seventh aspect, the application of the near-infrared two-region fluorescence imaging probe in preparing a medicine for osteosarcoma photothermal therapy is provided.
In an eighth aspect, an application of the near-infrared two-zone fluorescence imaging probe in preparing a reagent for in vivo imaging is provided.
According to the near-infrared two-zone fluorescent compound, compounds 2 and 3 sold in the market are subjected to aldol condensation reaction under an alkaline condition to obtain a compound 4, the compound 4 and 5 are subjected to Michael addition reaction under the action of tetrahydropyrrole to obtain a compound 6, then under the combined action of thioacetic acid and boron trifluoride diethyl etherate, a main molecular structure 7 is generated in a ring closing manner, and finally the compound reacts with a raw material 8 to obtain a final product 1, namely the final product shown in the general formula (I), the final product is a brand-new fluorescent compound with a D-A structure, the fluorescence emission wavelength of the brand-new fluorescent compound is 600-1200 nanometers, and the scheme is simple in synthetic route, high in reaction efficiency, high in yield, simple in post-treatment, low in toxicity, good in biocompatibility and easy to absorb and metabolize by organisms. Different tumors can be diagnosed and detected through different post-modification, and the method has higher industrial production value.
The invention has the creativity that a thiopyran onium salt structure mother nucleus is introduced, the fluorescence can be red-shifted by changing different groups, and finally the near-infrared two-region fluorescent compound which is easy to prepare is obtained. Furthermore, it is the first near infrared two-zone dye that can be used for mitochondrial imaging. The modified group is introduced into the left side ring, and the increased modified sites can be used for connecting different bioactive functional groups or targeting groups, so that the application prospect of the fluorescent probe is increased, the water solubility and the biocompatibility of the fluorescent probe are improved, and the targeting property to different tumors is improved. The fluorescent probe is found to have very good imaging effect in biomedical imaging experiments and has wide application prospect.
Drawings
FIG. 1 is a synthetic route of near-infrared two-region fluorescence imaging probes 1aa to 1ag in the embodiment.
FIG. 2 is an H spectrum of a near-infrared two-region fluorescent compound 1 ag.
FIG. 3 is a C spectrum of a near-infrared two-region fluorescent compound 1 ag.
FIG. 4 is a diagram of energy levels of a graph of the highest occupied molecular orbitals and the lowest unoccupied molecular orbitals of the near-infrared two-zone fluorescence imaging probes 1aa to 1 ag.
FIG. 5 is an absorption spectrum chart of compounds 1aa to 1 af.
FIG. 6 is a graph showing emission spectra of compounds 1aa to 1 af.
FIG. 7 shows the absorption spectra of the near infrared two-region fluorescent compound 1ag in different solvents.
FIG. 8 is an emission spectrum of a near-infrared two-region fluorescent compound 1ag in different solvents.
FIG. 9 is an image of mitochondria in 143B cells of a near-infrared two-zone imaging probe 1ag (10 nM-100. mu.M).
FIG. 10 shows the synthetic route of near-infrared two-region fluorescence imaging probe 1ag-PEG-PT modifier.
FIG. 11 is a Maldi TOF mass spectrum of a near-infrared two-zone fluorescence imaging probe 1ag-PEG-PT modification.
FIG. 12 shows the near-infrared two-zone imaging effect of the tail vein injection compound 1ag-PEG-PT (150. mu.g) modifier in tumor-bearing mice inoculated with tumor cells on the left hind limb on the upper side, and the near-infrared two-zone imaging effect of the mixed solution of the tail vein injection compound 1ag-PEG-PT (150. mu.g) modifier and polypeptide PT (2mg) in tumor-bearing mice inoculated with tumor cells on the left hind limb on the lower side.
FIG. 13 is a photo-thermal image of the modified compound 1ag-PEG-PT (150. mu.g) injected into the tail vein of tumor-bearing mice under the laser of 808nm, and the laser irradiation time is 5 minutes.
FIG. 14 shows the isolated tumor size after 14 days after continuous irradiation with 808nm laser for 5 minutes after intravenous injection of compound 1ag-PEG-PT (150. mu.g) modifier at the tail of tumor-bearing mice.
Detailed Description
The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.
Example 1: preparation of Compound 4a
The data for the structural determination of compound 4a are as follows:
1H NMR(400MHz,Acetone-d6)8.24–8.18(m,2H),7.92(d,J＝15.6Hz,1H),7.88–7.83(m,2H),7.79(d,J＝15.6Hz,1H),7.52–7.43(m,3H),7.21–7.15(m,2H),4.95(d,J＝2.4Hz,2H),3.19(t,J＝2.4Hz,1H).
13C NMR(101MHz,Acetone-d6)187.3,161.5,143.3,135.3,131.7,130.7,130.3,128.9,128.6,121.9,114.7,78.3,76.7,55.6.
example 2: preparation of Compound 6a
Cyclopentanone (1.2g,14.3mmol) and tetrahydropyrrole (1.02g,14.3mmol) were dissolved in benzene (20m L), the solution was heated to 100 ℃ and stirred for 4 hours, after cooling to room temperature, benzene was removed under reduced pressure, then compound 4a (2.5g,9.53mmol) and anhydrous 1, 4-dioxane (20m L) were added to the above reaction solution, the solution was heated under reflux for 6 hours, after completion of the reaction, water (40m L) was added to the mixture and extracted with ethyl acetate (50m L× 3), after which the organic layer was washed with saturated brine, the combined organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated, then the solvent was removed in vacuo, and the crude product was purified by silica gel column chromatography (2.17g, yield 66%).
The data for the structural determination of compound 6a are as follows:
1H NMR(400MHz,Chloroform-d)8.02–7.95(m,2H),7.28(m,1H),7.25(m,2H),7.25–7.16(m,2H),7.03–6.98(m,2H),4.74(d,J＝2.5Hz,2H),3.82–3.68(m,2H),3.48–3.35(m,1H),2.55(t,J＝2.4Hz,1H),2.29–2.19(m,1H),2.13(m,1H),1.92(m,1H),1.77(m,2H),1.71–1.58(m,2H).
13C NMR(101MHz,Chloroform-d)220.8,197.7,161.4,142.5,131.0,130.5,128.6,128.6,126.8,114.7,77.9,76.3,55.9,53.2,41.3,40.7,39.8,27.2,20.7.
example 3: preparation of Compound 7a
The data for the structural determination of compound 7a are as follows:
1H NMR(400MHz,Acetonitrile-d3)8.63(s,1H),8.01(d,J＝9.1Hz,2H),7.85–7.76(m,2H),7.76–7.63(m,3H),7.29(d,J＝8.9Hz,2H),4.92(d,J＝2.4Hz,2H),3.67(t,J＝7.6Hz,2H),3.34(t,J＝7.4Hz,3H),2.95(t,J＝2.5Hz,1H),2.37(p,J＝7.5Hz,2H).
13C NMR(101MHz,Acetonitrile-d3)174.5,165.7,161.9,159.4,149.5,137.2,132.5,131.7,130.3,129.3,129.2,127.3,116.6,77.8,76.8,56.2,38.2,34.1,24.6.
HRMS(ESI)Calcd for:C23H19OS+([M-BF4]+):343.1151,found:343.1155.
example 4: preparation of Compounds 1aa-1ag
1aa 1H NMR(400MHz,Acetonitrile-d3)8.47(s,1H),8.03(d,J＝8.9Hz,2H),7.77(d,J＝7.0Hz,2H),7.76(d,J＝7.8Hz,2H),7.72(d,J＝7.2Hz,2H),7.70–7.68(m,2H),7.54(d,J＝7.5Hz,2H),7.29(d,J＝8.8Hz,2H),5.47(s,1H),4.92(d,J＝2.4Hz,2H),3.50–3.43(m,2H),3.39–3.33(m,2H),2.95(t,J＝2.4Hz,1H).
HRMS(ESI)Calcd for:C30H23OS+([M-BF4]+):431.15,found:431.21.
1ab 1H NMR(600MHz,Acetonitrile-d3)8.48(s,1H),8.04(d,J＝8.8Hz,2H),7.80(s,1H),7.80–7.74(m,4H),7.69(d,J＝7.0Hz,2H),7.31(dd,J＝8.8,1.9Hz,4H),4.93(d,J＝2.5Hz,2H),3.52–3.45(m,2H),3.41–3.34(m,2H),2.94(t,J＝2.5Hz,1H),2.32(s,3H).13C NMR(151MHz,Acetonitrile-d3)171.0,163.9,154.2,143.9,138.8,135.1,134.6,133.7,133.3,133.0,132.7,132.0,131.6,131.1,130.6,130.5,129.2,124.5,124.4,118.4,79.5,78.5,58.0,31.6,31.1,22.0.
HRMS(ESI)Calcd for:C31H25O3S+([M-BF4]+):489.1519,found:489.1509.
1ac 1H NMR(600MHz,Acetonitrile-d3)8.37(s,1H),8.00(d,J＝8.9Hz,2H),7.78–7.73(m,3H),7.71(d,J＝8.9Hz,2H),7.69–7.66(m,3H),7.28(d,J＝8.9Hz,2H),7.09(d,J＝8.8Hz,2H),4.91(d,J＝2.4Hz,2H),3.89(s,3H),3.46–3.43(m,2H),3.33–3.30(m,2H),2.94(t,J＝2.4Hz,1H).13C NMR(151MHz,Acetonitrile-d3)163.8,163.7,141.5,138.9,136.8,135.3,134.8,133.1,132.1,131.8,131.6,131.3,131.2,131.0,130.6,129.8,129.2,118.3,116.7,79.5,78.5,58.0,57.2,34.3,31.6.
HRMS(ESI)Calcd for:C31H25O2S+([M-BF4]+):461.1570,found:461.1555.
1ad 1H NMR(600MHz,Acetonitrile-d3)8.50(s,1H),8.06(d,J＝8.9Hz,2H),7.82(s,1H),7.81–7.76(m,4H),7.73–7.68(m,3H),7.47(d,J＝8.5Hz,2H),7.31(d,J＝8.9Hz,2H),4.94(d,J＝2.3Hz,2H),3.55–3.49(m,2H),3.42–3.38(m,2H),3.29(s,3H),2.95(t,J＝2.4Hz,1H),1.30(s,3H).13C NMR(151MHz,Acetonitrile-d3)174.6,163.6,147.8,143.9,138.5,135.2,134.9,133.8,133.5,133.1,133.0,132.2,131.7,131.1,131.0,130.8,130.4,128.9,118.1,117.7,79.2,78.3,57.7,34.2,31.4,30.8,23.4.
HRMS(ESI)Calcd for:C33H28NO2S+([M-BF4]+):502.1835,found:502.1855.
1ae 1H NMR(500MHz,Acetonitrile-d3)8.40(s,1H),8.00(d,J＝8.9Hz,2H),7.76–7.71(m,3H),7.69–7.65(m,3H),7.63(d,J＝8.5Hz,2H),7.37(d,J＝8.5Hz,2H),7.27(d,J＝8.9Hz,2H),4.90(d,J＝2.4Hz,2H),3.46–3.42(m,2H),3.34–3.30(m,2H),2.91(t,J＝2.4Hz,1H),2.53(s,3H).13C NMR(126MHz,Acetonitrile-d3)162.1,143.8,141.2,137.2,134.4,131.5,131.4,131.2,130.5,130.3,130.1,129.6,129.5,129.3,129.2,128.8,127.5,125.7,116.6,77.8,76.8,56.2,32.7,30.0,19.6.
HRMS(ESI)Calcd for:C31H25OS2+([M-BF4]+):477.1341,found:477.1330.
1af 1H NMR(500MHz,Acetonitrile-d3)8.05(s,1H),7.90(d,J＝8.8Hz,2H),7.72(s,1H),7.71–7.68(m,2H),7.66–7.62(m,5H),7.24(d,J＝8.8Hz,2H),6.84(d,J＝9.0Hz,2H),4.89(d,J＝2.4Hz,2H),3.38–3.33(m,2H),3.28–3.22(m,2H),3.11(s,6H),2.93(t,J＝2.4Hz,1H).13C NMR(126MHz,Acetonitrile-d3)170.4,161.1,156.0,154.8,153.0,147.2,138.2,137.6,136.4,134.5,130.8,129.4,129.3,129.2,128.6,127.7,123.5,116.2,112.6,77.9,76.7,56.1,39.5,31.9,30.2.
HRMS(ESI)Calcd for:C32H28NOS+([M-BF4]+):474.1886,found:474.1895.
1ag 1H NMR(800MHz,Acetonitrile-d3)8.12(s,1H),7.95(s,1H),7.91(d,J＝8.9Hz,2H),7.70(dd,J＝6.8,2.9Hz,2H),7.68–7.66(m,3H),7.58(d,J＝3.9Hz,1H),7.56(d,J＝8.7Hz,2H),7.44(d,J＝3.9Hz,1H),7.25(d,J＝8.9Hz,2H),6.68(d,J＝8.7Hz,2H),4.91(d,J＝2.4Hz,2H),3.48–3.28(m,2H),3.23–3.11(m,2H),2.95(s,6H),2.92(t,J＝2.4Hz,1H).13C NMR(151MHz,Acetonitrile-d3)168.1,161.3,158.1,156.0,155.5,151.2,148.4,137.7,137.0,130.8,129.8,129.5,129.3,129.2,129.0,129.0,128.5,127.2,126.9,122.8,120.0,116.1,111.9,77.6,76.5,55.9,34.9,31.3,29.2.
HRMS(ESI)Calcd for:C23H19OS+([M-BF4]+):556.18,found:556.42.
The synthetic route of compound 1a is shown in figure 1. The NMR spectrum of the compound 1ag is shown in FIG. 2, and the NMR spectrum of the compound is shown in FIG. 3, which proves that the structure is correct.
Example 5: mitochondrial imaging of fluorescent compound 1ag in 143B cells
The preparation method comprises the steps of digesting U87-MG cells growing in an adherent manner in a culture bottle, wherein the total amount of the U87-MG cells is 3M L, taking 0.5M L cell suspension into an EP tube, adding 1.5M L fresh culture medium for dilution, swirling to uniformly disperse the cells, adding 1M L cell suspension into a small fluorescent dish, culturing for 24 hours, allowing the cells to grow in an adherent manner, adding 1g (10nM-100 mu M) of fluorescent compound, incubating for 6 hours, removing the culture medium containing the compound after 6 hours, repeatedly cleaning the cells for 3 times by using PBS, preparing 100nM Mito-Tracker Green solution, taking 1 mu L-1M L culture medium, shaking uniformly, adding the mixture into the small fluorescent dish, dyeing for 30 minutes, cleaning the cells for 3 times by using PBS again after dyeing, removing other substances except the adherent cells, adding the fresh culture medium, directly using a laser confocal microscope for observation, and respectively discovering the Mito completely dye the cells by using the Mito-Tracker and finding that the Mito dye the mitochondria of the cells, wherein the Mito have a good prospect of near infrared staining the mitochondria.
Example 6: preparation of compound PEG-PT
The compounds COOH-PEG-N3(2.0mg,0.0024mmol), EDCI (0.55mg,0.00288mmol) and NHS (0.33mg,0.00288mmol) were dissolved in 500. mu. L distilled DMF under argon atmosphere and stirred at room temperature for 30 minutes, then the peptides PPSHTPT (2.1mg,0.00288mmol) and DIPEA (50. mu. L) were added to the above reaction mixture and the solution was stirred at room temperature overnight the crude product was precipitated in cold ether the crude product was dissolved in water and purified by HP L C the structural determination data of the compound PEG-PT modification was MA L DI-TOF-MS Calcd for 1537.3([ M-BF4] +), Measured M.W.1538.9.
Example 7: preparation of compound 1ag-PEG-PT modifier and tumor imaging effect
1ag (1.2mg,0.00186mmol), PEG-PT (2.86mg,0.00186mmol) and DMF/PBS (300. mu. L/300. mu. L) were added to the reaction vessel, followed by the addition of 50. mu. L catalyst solution (10mM CuSO 4.5H 2O, 50mM sodium ascorbate in 20mM PBS) to the above solution, and the mixture was stirred overnight at room temperature under argon protection.
150 mu g of the near-infrared two-region fluorescent dye 1ag-PEG-PT prepared as described above is injected into a tumor-bearing mouse body inoculated with tumor cells on the left hind limb through tail vein injection, a near-infrared two-region camera shoots a tumor position picture of the mouse, and the tumor position of the mouse is clearly visible as shown in figure 12. The material of the invention has better application prospect in the aspect of early diagnosis of tumors.
200 mu g of the near-infrared two-region fluorescent dye 1ag-PEG-PT prepared as described above is injected into a tumor-bearing mouse body inoculated with tumor cells in the left hind limb through tail vein injection, after 12 hours, the tumor part of the tumor-bearing mouse is continuously irradiated by laser with the wavelength of 808nm for 5 minutes, and the change of the temperature of the tumor part along with the time is clearly seen, as shown in FIG. 13. Photothermal therapy imaging effect referring to fig. 14, tumor-bearing mice injected with 1ag-PEG-PT dye and tumors irradiated with laser for 5 minutes had significantly reduced or even disappeared after 14 days. The material of the invention has better application prospect in the aspect of tumor photothermal treatment.
Claims (10)
1. A modifiable near-infrared two-domain fluorescent compound having the structure:
2. a method of preparing a fluorescent compound according to claim 1, wherein the reaction scheme is as follows:
the reaction conditions are as follows:
(1) dissolving the compound 2a in 20M L ethanol, adding 20% KOH solution, reacting at room temperature for 10 minutes, then adding the compound 3a to the mixture, stirring the reaction at room temperature overnight, cooling to room temperature, adjusting the pH of the reaction solution to 3 with 2M HCl solution, filtering and collecting a formed yellow intermediate, then dissolving the obtained intermediate in acetone, adding potassium carbonate, and carrying out reflux reaction with a raw material bromopropyne under an alkaline condition for 4 hours to obtain a compound 4 a;
(2) dissolving compound 5a and tetrahydropyrrole in benzene, heating the solution to 100 ℃ and stirring for 4 hours, cooling to room temperature, removing benzene under reduced pressure, then adding compound 4a and anhydrous 1, 4-dioxane to the reaction solution, heating the solution under reflux for 6 hours, after the reaction is completed, adding water to the mixture, extracting with ethyl acetate for 3 times, then washing the organic layer with saturated saline, drying the combined organic layer with anhydrous magnesium sulfate, filtering and concentrating, then removing the solvent in vacuum, and purifying the crude product by silica gel column chromatography to obtain intermediate compound 6 a;
(3) dissolving compound 6a in diethyl ether and stirring for 10 minutes, adding thioacetic acid and boron trifluoride diethyl etherate to the mixture to react under reflux for 3 hours, after cooling to room temperature, quenching the reaction mixture with water, adding excess diethyl ether to the solution, and then stirring the mixture at room temperature to precipitate light yellow intermediate compound 7 a;
(4) adding the compound 7a, the raw material 8a and acetic anhydride into a reaction vessel, stirring for 2h at 70 ℃, adding a large amount of diethyl ether, separating out a crude product, and purifying by a silica gel chromatographic column to obtain a final product 1ag fluorescent compound.
3. The preparation method according to claim 2, wherein the molar ratio of the compounds 2a and 3a in the step (1) is 1: 1-3; the molar ratio of the compound 4a to the tetrahydropyrrole in the step (2) to the compound 5a is 1-5: 2-5: 1-10; the molar ratio of the compound 6a, the thioacetic acid and the boron trifluoride diethyl etherate in the step (3) is 1-5: 3-10: 4-20; the molar ratio of the compound 7a to the raw material 8a in the step (4) is 1-3: 2-10.
4. A near-infrared two-region fluorescence imaging probe for in vivo imaging, which is characterized in that the probe is the near-infrared two-region fluorescence compound of claim 1 which can modify the site with polyethylene glycol, polypeptide, protein, aptamer and folic acid.
5. Use of the near-infrared two-zone fluorescence imaging probe of claim 4 in the preparation of a reagent for cellular mitochondrial imaging.
6. Use of the near-infrared two-zone fluorescence imaging probe of claim 4 for preparing a reagent for tumor diagnosis.
7. The use of claim 6, wherein the tumor is osteosarcoma, colon carcinoma, liver carcinoma, brain glioma, breast carcinoma, prostate carcinoma, melanoma, esophageal carcinoma, cervical carcinoma, ovarian carcinoma.
8. Use of the near-infrared two-zone fluorescence imaging probe of claim 4 in the preparation of an agent for imaging osteosarcoma.
9. Use of the near-infrared two-zone fluorescence imaging probe of claim 4 in the preparation of a medicament for photothermal therapy of osteosarcoma.
10. Use of the near-infrared two-zone fluorescent imaging probe of claim 4 in the preparation of a reagent for in vivo imaging.
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