An activated near-infrared second-zone fluorescent probe and its preparation method and application

Abstract

The present invention belongs to the field of biomedicine technology, and relates to an activated near-infrared zone II fluorescent probe and its preparation method and application. Intermediate a and alkoxy-substituted aryl boronic acid are subjected to Suzuki coupling reaction under the action of palladium catalyst and first base to obtain intermediate b; intermediate b is subjected to demethylation reaction under the action of BBr3 to obtain intermediate c; intermediate c and response structure compound are subjected to condensation reaction under the action of second base to obtain activated near-infrared zone II fluorescent probe I. The donor-acceptor type fluorophore skeleton in the activated near-infrared zone II fluorescent probe of the present invention can increase the light stability of the probe, has high optical activity, and the fluorescence intensity of the probe increases with the concentration after response, and the optical brightness is significantly improved. The activated small molecule near-infrared zone II optical probe prepared by the present invention for specific response to the microenvironment of major diseases has a deep penetration depth, which can effectively improve the signal-to-noise ratio.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to an activated near-infrared second-zone fluorescent probe and a preparation method and application thereof. Background Art

[0002] The incidence of major diseases such as tumors and cardiovascular diseases is increasing, showing a trend of younger people. Their occurrence and development seriously threaten human life and health, and bring unbearable impacts to patients. The rapid development of medical molecular imaging is playing an increasingly important role in the early diagnosis of diseases and real-time evaluation of treatment effects. Compared with traditional molecular imaging technology, optical imaging technology, especially fluorescence imaging technology, has many advantages such as high temporal and spatial resolution, high sensitivity, non-ionizing radiation, real-time imaging, and easy operation of imaging devices. As an important component of fluorescence imaging, the imaging effect of optical probes is a key factor affecting the accurate detection of diseases.

[0003] Near-infrared II (NIR-II, 900-1700nm) fluorescence imaging significantly reduces the absorption and scattering effects of biological tissues on light and the autofluorescence of tissues, greatly improves the tissue penetration depth and imaging signal-to-noise ratio, and can achieve deep-layer living tissue imaging. Most of the new near-infrared II optical probes reported so far are "constant-bright" fluorescent probes, which can only increase their signal intensity by passively or actively enriching at the lesion site. Compared with normal tissues, the occurrence and development of major diseases are almost always accompanied by changes in physiological and pathological microenvironments and overexpression of certain biological molecules. Taking the tumor microenvironment as an example, it has the characteristics of hypoxia, weak acidity outside the cell, multiple reactive oxygen species (ROS), reactive nitrogen species (RSS), reactive sulfur species (RSS), enzymes and biomacromolecules, etc., while the research on "responsive" near-infrared II optical probes that are specifically activated by the pathological biological environment of the lesion site such as the tumor microenvironment is very limited. Compared with "constantly bright" optical probes, "activated" optical probes have advantages such as high signal-to-noise ratio and low false positive signals in the accurate detection of tumors. Therefore, the construction of responsive near-infrared zone II optical probes with excellent imaging performance is helpful to achieve highly sensitive and accurate detection of major diseases. However, most of the current "activated" near-infrared zone II optical probes have the following problems: (1) Due to the π-π stacking effect, the optical brightness of molecules with strong planarity decreases sharply under certain conditions, limiting their application in in vivo disease diagnosis and treatment; (2) They are easily photobleached, which reduces the detection sensitivity and optical stability of the probes. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an activated near-infrared second-zone fluorescent probe in response to the shortcomings of the prior art, which can efficiently respond to a variety of reactive oxygen species, reactive nitrogen species, reactive sulfur species and biological macromolecules in the microenvironment of the lesion site of major diseases, thereby achieving early detection and accurate diagnosis of major diseases.

[0005] Another technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned activated near-infrared second-zone fluorescent probe.

[0006] The final technical problem to be solved by the present invention is to provide the application of the above-mentioned activated near-infrared second-zone fluorescent probe.

[0007] Invention idea: In view of the limitations of the above-mentioned activated near-infrared zone II fluorescent probes, the design strategy of the activated near-infrared zone II fluorescent probe of the present application is: on the basis of the donor-acceptor structure, the optical brightness and optical stability are improved by increasing the degree of molecular distortion, and at the same time, the recognition site of the response group is modified on the donor side to construct an activated near-infrared zone II optical probe skeleton, and the change of optical signal is realized through the ICT effect. The donor-acceptor structure activated near-infrared zone II probe is developed, which can efficiently respond to a variety of active oxygen species, active nitrogen species, active sulfur species and biological macromolecules in the microenvironment of the lesion site of major diseases, improve the imaging signal-to-noise ratio and detection sensitivity of the activated near-infrared zone II optical probe, and realize the early detection and accurate diagnosis of major diseases.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0009] The present invention discloses an activated near-infrared second-zone fluorescent probe, and the general structural formula of the activated near-infrared second-zone fluorescent probe is shown in Formula I:

[0010]

[0011] The structure of the activated near-infrared second-region fluorescent probe includes an electron acceptor structural unit A, an electron donor structural unit D, and a response structural unit R;

[0012] Wherein, the electron acceptor structural unit A is selected from

[0013] Wherein, the electron donor structural unit D is selected from

[0014] Among them, the response structure unit R is selected from

[0015] Among them, R 1 Selected from C 0 -C 20 Straight or branched alkyl; R 2 Selected from C 0-C 20 Straight or branched chain alkyl;

[0016] The oxygen atom end in the electron donor structural unit D is connected to the response structural unit R.

[0017] in, It is a π bridge.

[0018] In some embodiments, the activated near-infrared second region fluorescent probe is any one of the following structures:

[0019]

[0020] Furthermore, the present invention discloses a method for preparing the above-mentioned activated near-infrared second-zone fluorescent probe, comprising the following steps: intermediate a and alkoxy-substituted aryl boronic acid are subjected to Suzuki coupling reaction under the action of palladium catalyst and first base to obtain intermediate b; intermediate b is reacted with BBr 3 Under the action of the second base, the intermediate c undergoes a condensation reaction with the response structure compound to obtain the activated near-infrared second-zone fluorescent probe I;

[0021]

[0022] Wherein, the alkoxy substituted aryl boronic acid is selected from

[0023] Wherein, the responsive structure compound is selected from

[0024] The structure of the activated near-infrared second-region fluorescent probe includes an electron acceptor structural unit A, an electron donor structural unit D, and a response structural unit R;

[0025] Wherein, the electron acceptor structural unit A is selected from

[0026] Wherein, the electron donor structural unit D is selected from

[0027] Among them, the response structure unit R is selected from

[0028] Among them, R 1 Selected from C 0 -C 20 Straight or branched alkyl; R 2 Selected from C 0 -C 20 Straight or branched chain alkyl;

[0029] The oxygen atom end in the electron donor structural unit D is connected to the response structural unit R.

[0030] In some embodiments, the palladium catalyst is tetrakis(triphenylphosphine)palladium, palladium acetate, bis(diphenylphosphino)ferrocenepalladium dichloride, tris(dibenzylideneacetone)dipalladium or bistriphenylphosphinepalladium dichloride; the first base is potassium carbonate, cesium carbonate or potassium phosphate; the second base is potassium carbonate, cesium carbonate, potassium phosphate, triethylamine, N,N-diisopropylethylamine or 4-dimethylaminopyridine.

[0031] In some embodiments, preferably, the palladium catalyst is tetrakis(triphenylphosphine)palladium; the first base is potassium carbonate; and the second base is potassium carbonate or triethylamine.

[0032] In some embodiments, the molar ratio of the intermediate a to the alkoxy-substituted aryl boronic acid, the palladium catalyst, and the first base is 1:3.5-5:0.04-0.10:5-15; the reaction temperature of the Suzuki coupling reaction is 60°C-120°C, and the reaction time is 2h-24h.

[0033] In some embodiments, preferably, the molar ratio of the intermediate a to the alkoxy-substituted aryl boronic acid, the palladium catalyst, and the first base is 1:4:0.05:9; and the reaction temperature of the Suzuki coupling reaction is 60°C and the reaction time is 2h.

[0034] The solvent used in the Suzuki coupling reaction is any one or a combination of toluene, water, tetrahydrofuran and anhydrous ethanol, preferably a mixture of anhydrous ethanol and toluene in any proportion, more preferably a mixture of anhydrous ethanol and toluene in a volume ratio of 0.2:1; the volume amount of the solvent is such that the reaction raw materials in the reaction system are dissolved and the viscosity is moderate.

[0035] Wherein, the Suzuki coupling reaction is carried out under the protection of an inert gas; the inert gas is preferably argon.

[0036] In some embodiments, the intermediate b and BBr 3 The molar ratio is 1:5-10; the demethylation reaction has a reaction temperature of 0°C-25°C and a reaction time of 2h-6h.

[0037] In some embodiments, preferably, the intermediate b and BBr 3 The molar ratio is 1:8; the demethylation reaction has a reaction temperature of 0°C and a reaction time of 2h.

[0038] The solvent used in the demethylation reaction is ultra-dry dichloromethane; the volume amount of the solvent is such that the reaction raw materials in the reaction system are dissolved and the viscosity is moderate.

[0039] Wherein, the demethylation reaction is carried out under the protection of an inert gas; the inert gas is preferably nitrogen.

[0040] In some embodiments, the molar ratio of the intermediate c to the response structure compound and the second base is 1:4-6:3-9; the condensation reaction has a reaction temperature of 0°C-60°C and a reaction time of 10 min-24 h.

[0041] In some embodiments, preferably, the molar ratio of the intermediate c to the response structure compound and the second base is 1:4:3.5.

[0042] The solvent used in the condensation reaction is dichloromethane, acetonitrile or N,N-dimethylformamide; the volume amount of the solvent is such that the reaction raw materials in the reaction system are dissolved and the viscosity is moderate.

[0043] Furthermore, the present invention discloses the use of the above-mentioned activated near-infrared second-zone fluorescent probe in the preparation of a nano fluorescent probe that responds to active species in a disease microenvironment.

[0044] Specifically, the preparation method of the nano fluorescent probe is: mixing the activated near-infrared second zone fluorescent probe with the self-assembly packaging material, tetrahydrofuran and water, performing ultrasonic treatment, then removing the tetrahydrofuran and filtering to obtain the nano fluorescent probe.

[0045] Specifically, the self-assembly packaging material is F127, DSPE-PEG or PLGA-PEG; the mass ratio of the activated near-infrared zone II fluorescent probe to the self-assembly packaging material is 0.1:1 to 0.1:2; the mass volume ratio of the self-assembly packaging material to tetrahydrofuran is 1 mg:1 mL; the volume ratio of tetrahydrofuran to water is 1:9; the ultrasonic treatment has an ultrasonic frequency of 40 KHz, an ultrasonic temperature of 25°C, and an ultrasonic time of 10 to 30 min; the filtration is performed by filtering with a 0.22 μM filter membrane and then ultrafiltering 5 times with an ultrafiltration tube with a cut-off molecular weight of 100,000.

[0046] Specifically, preferably, the self-assembly packaging material is F127; the mass ratio of the activated near-infrared second zone fluorescent probe to the self-assembly packaging material is 0.1:1; the mass volume ratio of the self-assembly packaging material to tetrahydrofuran is 1 mg:1 mL; the volume ratio of tetrahydrofuran to water is 1:9; the ultrasonic treatment has an ultrasonic frequency of 40 KHz, an ultrasonic temperature of 25°C, and an ultrasonic time of 10 min; the filtration is filtered through a 0.22 μM filter membrane and then ultrafiltered 5 times using an ultrafiltration tube with a cut-off molecular weight of 100,000.

[0047] Specifically, the active species are active oxygen species, active nitrogen species, active sulfur species or biomacromolecules.

[0048] Specifically, the active oxygen species is O 2 ·- ; The active nitrogen species is ONOO - ; The active sulfur species is H 2 S; the biological macromolecule is glutathione.

[0049] Specifically, the disease is a tumor or a cardiovascular disease.

[0050] Wherein, when the activated near-infrared second-zone fluorescent probe is

[0051] When the active species is H 2 S.

[0052] Wherein, when the activated near-infrared second-zone fluorescent probe is

[0053] When , the corresponding active species is the active oxygen species O 2 ·- .

[0054] Wherein, when the activated near-infrared second-zone fluorescent probe is

[0055] When , the corresponding active species is the active nitrogen species ONOO - .

[0056] Wherein, when the activated near-infrared second-zone fluorescent probe is

[0057] When , the corresponding active species is the biological macromolecule glutathione (GSH).

[0058] The use of the above-mentioned activated near-infrared second-zone fluorescent probe in the preparation of nano fluorescent probes that respond to active species in disease microenvironments is also within the protection scope of the present invention.

[0059] Beneficial effects:

[0060] (1) Cyanine or fluoroboron dipyrrole probes are often designed as activated near-infrared zone II optical probes for detecting biomarkers of diseases in vivo and in vitro. However, cyanine and fluoroboron dipyrrole probes are prone to fluorescence quenching at high concentrations or in aggregated states, and cyanine fluorescent probes have the disadvantage of being easily photobleached, which reduces the optical brightness and optical stability of the probes. The activated small molecule near-infrared zone II optical probes prepared by the present invention that specifically respond to the microenvironment of major diseases have a deeper penetration depth and can effectively improve the signal-to-noise ratio.

[0061] (2) The present invention uses a donor-acceptor type fluorophore skeleton, increases the optical brightness by adjusting the planar configuration and electron cloud distribution of the luminophore, and designs an activated near-infrared second-zone fluorescent probe using the intramolecular charge transfer (ICT) effect. The activated near-infrared zone II fluorescent probe of the present invention: (1) Compared with cyanine probes, the donor-acceptor type fluorophore skeleton can increase the photostability of the probe; (2) The alkyl chain is modified at different positions on the π bridge side of the molecular structure to moderately increase the angle between the π bridge and the acceptor and reduce the planarity. On the one hand, it can maintain the effective transmission of electrons in the response process, and on the other hand, it can weaken the reduction in fluorescence brightness caused by π-π stacking. The experimental results show that the fluorescence intensity increases by 11.9 times after the response, and it has high optical activity. After the response, the fluorescence intensity of the probe increases with the concentration, and the optical brightness is significantly improved; (3) Overcoming the problem of limited response types of recognition groups modified from the electron acceptor side in the past, the present invention modifies the recognition group on the electron donor side of the molecular structure to broaden the types of response biomarkers in the process of major diseases. Taking malignant tumors as an example, it includes various biomarkers of the tumor microenvironment, such as reactive oxygen species, reactive nitrogen species, reactive sulfur species or biomacromolecules, etc., to achieve accurate detection at the tumor site. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0063] Figure 1 NIR-II-H in Example 1 of the present invention 2 Changes in the UV absorption spectrum of S nanofluorescent probe when NaHS is added.

[0064] Figure 2 NIR-II-H in Example 1 of the present invention 2 Changes in the fluorescence emission spectrum of S nanofluorescent probe when NaHS is added.

[0065] Figure 3 NIR-II-H in Example 1 of the present invention 2 Fluorescence change diagram of the S nano-fluorescent probe after adding NaHS.

[0066] Figure 4 NIR-II-H in Example 1 of the present invention 2 Response selectivity test diagram of S nanofluorescent probe.

[0067] Figure 5 NIR-II-H in Example 1 of the present invention 2 Optical brightness test chart of S nanofluorescent probe.

[0068] Figure 6 NIR-II-H in Example 1 of the present invention 2 Optical stability test diagram of S nanofluorescent probe.

[0069] Figure 7 NIR-II-0 in Example 2 of the present invention 2 ·- Nano fluorescent probes added to O 2 ·- Ultraviolet absorption spectrum changes.

[0070] Figure 8 NIR-II-ONOO in Example 3 of the present invention - Nano fluorescent probe added to ONOO - Ultraviolet absorption spectrum changes.

[0071] Fig. 9 This is a graph showing the change in the ultraviolet absorption spectrum of the NIR-II-GSH nanofluorescent probe when GSH is added in Example 4 of the present invention.

[0072] Fig.10 This is a schematic diagram of the molecular structure design principle of the activated near-infrared second-region optical probe of the present invention. DETAILED DESCRIPTION

[0073] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0074] The glutathione used in the embodiments of the present invention is abbreviated as GSH.

[0075] The molecular structure design principle diagram of the activated near-infrared second region optical probe of the present invention is as follows: Fig.10 As shown, the design ideas are as follows:

[0076] (1) Structural design of an activated near-infrared second-region optical probe with high optical brightness

[0077] Based on electron donors (phenol, naphthol, anthracenol, etc.), π bridge (R 1 , R 2 Alkylated thiophene) and electron acceptors (benzothiadiazole, naphthiadiazole, benzothiadiazole-quinoxaline, etc.) and other units are used to regulate the energy level matching of the corresponding electron donor and electron acceptor to construct a D-π-A structure near-infrared second region optical probe.

[0078] (2) Response performance and biological applications of high optical brightness activation type near-infrared region II optical probes

[0079] According to the microenvironment biomarkers of different major diseases, taking cancer as an example, it includes reactive oxygen species (O2 ·- etc.), reactive nitrogen species (ONOO - etc.), active sulfur species (H 2 S, etc.) and biomacromolecules (glutathione, GSH), etc., optical quenching groups that can specifically recognize the above biomarkers are selected, an activated near-infrared zone II optical probe library is constructed, and the application of activated near-infrared zone II optical probes in the precise diagnosis of cancer is investigated.

[0080] Based on electron donors (phenol, naphthol, anthracenol, etc.), π bridges (alkylated thiophene) and electron acceptors (benzothiadiazole, naphthiadiazole, benzothiadiazole and quinoxaline, etc.), the energy level matching of the corresponding electron donors and electron acceptors is regulated to construct a D-π-A structure near-infrared second-zone optical probe. The response group is modified on the electron donor side of the molecular structure to broaden the category of response species, including active oxygen species (O 2 ·- etc.), reactive nitrogen species (ONOO - etc.), active sulfur species (H 2 S, etc.) and biomacromolecules (glutathione, GSH), etc., overcoming the previous problem of limited types of modified responsive groups on the electron acceptor side, optimizing high optical brightness responsive near-infrared second-zone optical probes, and realizing early detection and accurate diagnosis of major diseases.

[0081] Example 1: Preparation of hydrogen sulfide activated near-infrared second region fluorescent probe and performance test of nano fluorescent probe

[0082] 1. Preparation of hydrogen sulfide activated near-infrared second region fluorescent probe I-1

[0083]

[0084] Synthesis of intermediate b-1: Compound a-1 (1.0 mmol, 1.0 equiv.), p-methoxyphenylboronic acid (4.0 mmol, 4.0 equiv.), and potassium carbonate (9.0 mmol, 9.0 equiv.) were dissolved in ethanol / toluene (20% V / V, 5 mL). After replacing argon three times, tetrakis(triphenylphosphine)palladium (0.05 mmol, 5% equiv.) was quickly added, and Suzuki coupling reaction was carried out at 60° C. under argon protection for 2 hours. After the reaction was completed, diatomaceous earth was used for filtration, and the filter residue was washed with dichloromethane. The combined filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography with petroleum ether / dichloromethane (100%-50% V / V) as the eluent to obtain a blue-green solid powder intermediate b-1 with a yield of 68%. The intermediate b-1 was stored at minus 20° C. in the dark.

[0085] NMR data of intermediate b-1: 1H NMR(400MHz,Chloroform-d)δ7.64(d,J=8.8Hz,4H),7.29(s,2H),6.95(d,J=8.8Hz,4H),3.86(s,6H),2 .57(d,J=7.0Hz,4H),1.36–1.27(m,2H),1.12–0.71(m,16H),0.72–0.57(m,6H),0.47(t,J=7.4Hz,6H).

[0086] Synthesis of intermediate c-1: The intermediate b-1 (1.0 mmol, 1.0 equiv.) was dissolved in 5 mL of ultra-dry dichloromethane, the nitrogen was replaced, and boron tribromide (8.0 mmol, 8.0 equiv.) was added dropwise at -15°C, and the mixture was stirred at 0°C for 2 hours under nitrogen protection for demethylation reaction; after the reaction was completed, ice water was added to the reaction solution to quench, and the pH was adjusted to 8 with saturated sodium bicarbonate solution. The mixture was extracted with dichloromethane, dried with sodium sulfate, and filtered. The filtrate was spin-dried and purified by column chromatography, and the eluent was dichloromethane. The blue-green solid powder intermediate c-1 was purified, recorded as NIR-II-OH, with a yield of 75%, and stored at -20°C in the dark.

[0087] NMR data of intermediate c-1: 1 H NMR(400MHz,Chloroform-d)δ7.58(d,J=8.6Hz,4H),7.27(s,2H),6.87(d,J=8.6Hz,4H),2.56(dt,J=7.2, 1.7Hz,4H),1.53(s,2H),1.36–1.27(m,2H),1.12–0.71(m,16H),0.68–0.59(m,6H),0.47(t,J=7.4Hz,6H).

[0088] Synthesis of hydrogen sulfide activated near-infrared zone II fluorescent probe I-1: The intermediate c-1 (1.0 mmol, 1.0 equiv.) was dissolved in dichloromethane, triethylamine (3.5 mmol, 3.5 equiv.) was added at 0°C and stirred for 10 minutes, followed by addition of 2,4-dinitrobenzenesulfonyl chloride (4.0 mmol, 4.0 equiv.) and condensation reaction at 0°C for 10 minutes; after the reaction, saturated ammonium chloride was used for quenching, dichloromethane was used for extraction, the organic phases were combined and dried over sodium sulfate, concentrated and purified by column chromatography, the eluent was petroleum ether / dichloromethane (90%-0% V / V), and a blue solid powder hydrogen sulfide activated near-infrared zone II fluorescent probe I-1 was obtained, namely compound NIR-II-H 2 S, yield 88%, stored at -20℃ away from light.

[0089] NMR data of activated near-infrared second-zone fluorescent probe I-1: 1 H NMR(400MHz,Chloroform-d)δ8.68(d,J=2.2Hz,2H),8.51(dd,J=8.7,2.2Hz,2H),8.22(d,J=8.6Hz,2H),7.69(d,J=8.8Hz,4H),7.37(s,2H ),7.26(d,J=8.8Hz,4H),2.56(d,J=7.0Hz,4H),1.36–1.27(m,2H),1.12–0.71(m,16H),0.63(td,J=7.0,1.5Hz,6H),0.46(t,J=7.3Hz,6H).

[0090] 2. NIR-II-H 2 Preparation of S nanofluorescent probe

[0091] Under an ultrasonic frequency of 40 KHz and an ultrasonic temperature of 25 °C, the compound NIR-II-H 2 S (100 μg) and F127 (MW = 12700, 1 mg) were dissolved in 1 mL of tetrahydrofuran to mix the components evenly, and then quickly injected into 9 mL of deionized water. After continuous ultrasonic treatment for 10 minutes, THF was evaporated by rotary evaporator, filtered through a 0.22 μM filter membrane, and ultrafiltered 5 times with an ultrafiltration tube with a molecular weight cutoff of 100,000 to finally obtain near infrared zone II NIR-II-H 2 S nano fluorescent probe, the encapsulation efficiency is 65%.

[0092] 3. Near infrared zone II NIR-II-H 2 Performance test of S nano fluorescent probe

[0093] Experimental Test 1: NIR-II-H 2 Changes in the UV absorption spectrum of S nanofluorescent probe after adding sodium hydrosulfide (NaHS)

[0094] At a final concentration of 50 μg / mL, NIR-II-H 2 NaHS (final concentration: 120 μM) was added to the PBS buffer system (10 mM, pH 7.4), and the mixture was incubated at 37° C. for 10 minutes, and then its UV absorption spectrum was measured.

[0095] from Figure 1 It can be seen that with the addition of NaHS, the maximum absorption wavelength of the ultraviolet absorption spectrum red-shifts, indicating that the nano-fluorescent probe reacts with NaHS and has good response performance.

[0096] Experimental Test 2: NIR-II-H 2The fluorescence emission change of the S nanofluorescent probe after adding sodium hydrosulfide (NaHS) at a final concentration of 50 μg / mL in NIR-II-H 2 NaHS (final concentration: 120 μM) was added to the PBS buffer system (10 mM, pH 7.4), and after incubation at 37° C. for 10 minutes, the fluorescence emission spectrum thereof was measured at an excitation wavelength of 808 nm.

[0097] like Figure 2 As shown, with the addition of NaHS, the maximum emission peak of the fluorescence emission spectrum red-shifted, and the fluorescence intensity at 1000nm~1200nm was significantly enhanced.

[0098] Experimental Test 3: NIR-II-H 2 Fluorescence emission changes of S nano-fluorescent probes after adding sodium hydrosulfide (NaHS)

[0099] At a final concentration of 50 μg / mL, NIR-II-H 2 Fluorescence titration experiments were performed in a PBS buffer system (10 mM, pH 7.4), NaHS (final concentration of 0-240 μM) was added sequentially, and after incubation at 37° C. for 10 minutes, the fluorescence emission spectrum was tested at an excitation wavelength of 808 nm.

[0100] like Figure 3 As shown, with the addition of NaHS, the maximum emission peak of the fluorescence emission spectrum red-shifts, and the fluorescence intensity at 1000nm~1200nm increases with the increase of NaHS concentration. The fluorescent probe has good response linearity with the NaHS solution and has high sensitivity.

[0101] Experimental Test 4: NIR-II-H 2 Response selectivity of S nanofluorescent probe to different compounds

[0102] At a final concentration of 50 μg / mL, NIR-II-H 2 Different ion solutions were added to the PBS buffer system (10 mM, pH 7.4) to conduct response selectivity test experiments. Different reagents were added, including: 1. Blank; 2. Na 2 CO 3 (10mM); 3. KCl (10mM); 4. CaCl 2 (10 mM); 5.MgCl 2 (10mM); 6.FeCl 2 (10mM); 7.NaCl (10mM); 8.NaHCO 3 (10mM); 9.Na 2 SO 4(10mM); 10.KNO 2 (10mM); 11.KNO 3 (10mM); 12.H 2 O 2 (500μM); 13.GSH (1mM); 14.L-Cys (1mM); 15.H 2 S (100 μM); (all final concentrations); then incubated at 37° C. for 10 minutes, and its fluorescence emission spectrum was tested at an excitation wavelength of 808 nm.

[0103] from Figure 4 It can be seen that NIR-II-H 2 The S nanofluorescent probe responds specifically to NaHS and will not be interfered by other substances.

[0104] Experimental Test 5: NIR-II-H 2 S Nano-fluorescent Probe Optical Brightness

[0105] The final concentration of NIR-II-H was 0-200 μg / mL. 2 Fluorescence emission spectrum of S PBS buffer system (10 mM, pH 7.4) at an excitation wavelength of 808 nm.

[0106] The results are as follows Figure 5 As shown, with the increase of concentration, NIR-II-H 2 The fluorescence intensity of the S nanofluorescent probe increases successively, indicating that NIR-II-H 2 S nanofluorescent probe has high optical brightness.

[0107] Experimental Test 6: NIR-II-H 2 S nanofluorescent probe photostability test

[0108] Using 808nm laser (power 80mW·cm -2 ) Continuously irradiate NIR-II-H with a final concentration of 50 μg / mL 2 SPBS buffer system (10mM, pH 7.4) was used for 0min, 5min, 10min, 30min, and 60min. At the same time, 50μg / mL ICG PBS buffer system (10mM, pH 7.4) was used as a control. The fluorescence emission spectra were tested at an excitation wavelength of 808nm.

[0109] The results are as follows Figure 6 As shown in Figure 2, with the increase of irradiation time, NIR-II-H 2The fluorescence intensity of the S nanofluorescent probe remained almost unchanged, while ICG underwent significant photobleaching, indicating that NIR-II-H 2 S nanofluorescent probe has high photostability.

[0110] Example 2: Preparation of superoxide anion activated near-infrared second region fluorescent probe and performance test of nano fluorescent probe

[0111] 1. Preparation of superoxide anion-activated near-infrared second-region fluorescent probe I-2

[0112]

[0113] NIR-II-O 2 ·- Synthesis of activated near-infrared zone II fluorescent probe I-2: The intermediate c-1 (1.0 mmol, 1.0 equiv., prepared in Example 1) was dissolved in 5 mL of dichloromethane, triethylamine (3.5 mmol, 3.5 equiv.) was added at 0°C and stirred for 10 minutes, followed by the addition of trifluoromethanesulfonic anhydride (4.0 mmol, 4.0 equiv.) and the mixture was placed at 0°C for condensation reaction for 30 minutes; after the reaction was completed, saturated ammonium chloride was used for quenching, dichloromethane was extracted, the organic phases were combined and dried over sodium sulfate, concentrated, and purified by column chromatography, the eluent was petroleum ether / dichloromethane (50%-0% V / V), and a blue solid powder superoxide anion-activated near-infrared zone II fluorescent probe I-2 was obtained, recorded as compound NIR-II-0. 2 ·- , yield 70%, stored at -20℃ away from light.

[0114] Compound NIR-II-O 2 ·- NMR data of fluorescent probes: 1 H NMR(400MHz,Chloroform-d)δ7.77(d,J=8.8Hz,4H),7.40(s,2H),7.33(d,J=8.8Hz,4H),2.58(d,J=7 .0Hz,4H),1.36–1.27(m,2H),1.12–0.71(m,16H),0.64(td,J=7.0,1.5Hz,6H),0.47(t,J=7.3Hz,6H).

[0115] 2. NIR-II-O 2 ·- Preparation of nanofluorescent probes

[0116] Under the condition of ultrasonic frequency of 40KHz and ultrasonic temperature of 25℃, compound NIR-II-O 2 ·-(100 μg) and F127 (MW = 12700, 1 mg) were dissolved in 1 mL of tetrahydrofuran to mix the components evenly, and then quickly injected into 9 mL of deionized water. After continuous ultrasonic treatment for 10 minutes, THF was evaporated by rotary evaporator, filtered with a 0.22 μM filter membrane, and ultrafiltered 5 times with an ultrafiltration tube with a molecular weight cutoff of 100000 to finally obtain near infrared zone II NIR-II-O 2 ·- Nano fluorescent probe, the encapsulation efficiency is 58%.

[0117] 3. Near infrared zone II NIR-II-O 2 ·- Performance test of nanofluorescent probe

[0118] Experimental Test 1: Test NIR-II-O 2 ·- And the UV absorption spectrum of NIR-II-OH

[0119] At a final concentration of 50 μg / mL NIR-II-O 2 ·- KO was added to PBS buffer solution (10 mM, pH 7.4). 2 (final concentration: 120 μM), incubated at 37°C for 30 minutes, and then tested for its UV absorption spectrum.

[0120] from Figure 7 It can be seen that with the KO 2 The addition of , the maximum absorption wavelength of the UV absorption spectrum red shifted, indicating that NIR-II-O 2 ·- Nano-fluorescent probes and O 2 ·- Reacts and has good response performance.

[0121] Example 3: Preparation of peroxynitrite anion-activated near-infrared second-region fluorescent probe and performance test of nano-fluorescent probe

[0122] 1. Preparation of peroxynitrite anion-activated near-infrared second-region fluorescent probe I-3

[0123]

[0124] Synthesis of peroxynitrite anion-activated near-infrared zone II fluorescent probe I-3: The intermediate c-1 (1.0 mmol, 1.0 equiv., prepared in Example 1) was dissolved in 5 mL of acetonitrile, potassium carbonate (3.5 mmol, 3.5 equiv.) was added at 0°C and stirred for 10 minutes, followed by addition of 4-bromomethylphenylboronic acid pinacol ester (4.0 mmol, 4.0 equiv.) and placed at 60°C for condensation reaction for 24 hours; after the reaction, saturated ammonium chloride was used for quenching, dichloromethane was used for extraction, the organic phases were combined and dried over sodium sulfate, concentrated, and purified by column chromatography, with dichloromethane as the eluent, to obtain a blue solid powder peroxynitrite anion-activated near-infrared zone II fluorescent probe I-3, recorded as compound NIR-II-ONOO - , yield 70%, stored at -20℃ away from light.

[0125] NIR-II-ONOO - NMR data of fluorescent probes: 1 H NMR(400MHz,Chloroform-d)δ8.37(d,J=9.3,4H),7.82(d,J=8.7Hz,4H),7.42(s,2H),7.20(d,J=8.8Hz,4H),7.14(d,J=9.3Hz,4H),5.30 (s,4H),2.60(d,J=7.1Hz,4H),1.36–1.27(m,2H),1.66(s,24H),1.12–0.71(m,16H),0.63(td,J=7.0,1.5Hz,6H),0.46(t,J=7.3Hz,6H).

[0126] 2. Preparation of NIR-II-ONOO-nanofluorescent probe

[0127] Under the condition of ultrasonic frequency of 40KHz and ultrasonic temperature of 25℃, the compound NIR-II-ONOO - (100 μg) and F127 (MW = 12700, 1 mg) were dissolved in 1 mL of tetrahydrofuran to mix the components evenly, and then quickly injected into 9 mL of deionized water. After continuous ultrasonic treatment for 10 minutes, THF was evaporated by rotary evaporator, filtered with a 0.22 μM filter membrane, and ultrafiltered 5 times with an ultrafiltration tube with a molecular weight cutoff of 100000 to finally obtain near-infrared II region NIR-II-ONOO - Nano fluorescent probe, the encapsulation efficiency is 60%.

[0128] 3. Near infrared zone II NIR-II-ONOO - Performance test of nanofluorescent probe

[0129] Experimental Test 1: NIR-II-ONOO - And the UV absorption spectrum of NIR-II-OH

[0130] ONOO - Solution preparation: ONOO used in this example - The solution was prepared in the laboratory, in which the cation was sodium ion. The specific synthesis method can refer to the existing technology, or it can be prepared according to the following method: 5 mL of NaNO 2 Solution (0.6 mol·L -1 ) and 350 μL of HO 2 O 2 Solution (0.7 mol·L -1 ) were mixed, and then 250 μL of HCl solution (0.6 mol·L -1 ) was pre-acidified and the concentration was determined to be 1 mM under ultraviolet light.

[0131] At a final concentration of 50 μg / mL NIR-II-ONOO - Add ONOO to PBS buffer solution (10 mM, pH 7.4). - The solution (final concentration: 120 μM) was incubated at 37° C. for 30 minutes, and then its UV absorption spectrum was tested.

[0132] from Figure 8 It can be seen that with ONOO - The addition of solution caused the maximum absorption wavelength of the UV absorption spectrum to red shift, indicating that NIR-II-ONOO - Nano-fluorescent probes and ONOO - Reacts and has good response performance.

[0133] Example 4: Preparation of glutathione-activated near-infrared second-region fluorescent probe and performance test of nano-fluorescent probe

[0134] 1. Preparation of glutathione-activated near-infrared region II fluorescent probe I-4

[0135]

[0136] Synthesis of glutathione-activated near-infrared zone II fluorescent probe I-4: The intermediate c-1 (1.0 mmol, 1.0 equiv., prepared in Example 1) was dissolved in 5 mL of dichloromethane, triethylamine (3.5 mmol, 3.5 equiv.) was added at 0°C and stirred for 10 minutes, followed by addition of 2,4-dinitrofluorobenzene (4.0 mmol, 4.0 equiv.) and the mixture was placed at 0°C for condensation reaction for 30 minutes; after the reaction was completed, saturated ammonium chloride was used to quench, dichloromethane was extracted, the organic phases were combined and dried over sodium sulfate, concentrated and purified by column chromatography, the eluent was petroleum ether / dichloromethane (50%-10% V / V), and a blue solid powder glutathione-activated near-infrared zone II fluorescent probe I-4 was obtained, recorded as NIR-II-GSH, with a yield of 65%, and stored at -20°C in the dark.

[0137] NIR-II-GSH fluorescence probe NMR data: 1 H NMR(400MHz,Chloroform-d)δ8.88(d,J=2.7Hz,2H),8.37(dd,J=9.3,2.7Hz,2H),7.81(d,J=8.7Hz,4H),7.42(s,2H),7.20(d,J=8.7Hz,4H ),7.13(d,J=9.3Hz,2H),2.60(d,J=7.2Hz,4H),1.36–1.27(m,2H),1.12–0.71(m,16H),0.63(td,J=7.0,1.5Hz,6H),0.46(t,J=7.3Hz,6H).

[0138] 2. Preparation of NIR-II-GSH nanofluorescent probe

[0139] At an ultrasonic frequency of 40 KHz and an ultrasonic temperature of 25°C, the compounds NIR-II-GSH (100 μg) and F127 (MW=12700, 1 mg) were dissolved in 1 mL of tetrahydrofuran to mix the components evenly, and then quickly injected into 9 mL of deionized water. After continuous ultrasonic treatment for 10 minutes, THF was evaporated by a rotary evaporator, filtered using a 0.22 μM filter membrane, and ultrafiltered 5 times using an ultrafiltration tube with a cutoff molecular weight of 100,000 to finally obtain the near-infrared II region NIR-II-GSH nanofluorescent probe with an encapsulation efficiency of 63%.

[0140] 3. Performance test of NIR-II-GSH nanofluorescence probe

[0141] Experimental Test 1: Testing the UV absorption spectra of NIR-II-GSH and NIR-II-OH

[0142] GSH (final concentration of 500 μM) was added to a NIR-II-GSH PBS buffer solution (10 mM, pH 7.4) with a final concentration of 50 μg / mL, and after incubation at 37° C. for 1 hour, its UV absorption spectrum was tested.

[0143] from Fig. 9 It can be seen that with the addition of GSH, the maximum absorption wavelength of the ultraviolet absorption spectrum red-shifts, indicating that the NIR-II-GSH nanofluorescent probe reacts with GSH and has good response performance.

[0144] The present invention provides an activated near-infrared second-zone fluorescent probe and its preparation method and application ideas and methods. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. An activated near-infrared second-region fluorescent probe, characterized in that: The general structural formula of the activated near-infrared second-region fluorescent probe is shown in Formula I: The structure of the activated near-infrared second-region fluorescent probe includes an electron acceptor structural unit A, an electron donor structural unit D, and a response structural unit R; Wherein, the electron acceptor structural unit A is selected from Wherein, the electron donor structural unit D is selected from Among them, the response structure unit R is selected from Wherein, R1 is selected from C0-C 20 Straight or branched alkyl; R2 is selected from C0C 20 Straight or branched chain alkyl; The oxygen atom end in the electron donor structural unit D is connected to the response structural unit R.

2. The activated near-infrared second-region fluorescent probe according to claim 1, characterized in that: The activated near-infrared second-region fluorescent probe is any one of the following structures:

3. The method for preparing the activated near-infrared second-region fluorescent probe according to claim 1, characterized in that: The method comprises the following steps: intermediate a and alkoxy substituted aryl boronic acid are subjected to Suzuki coupling reaction under the action of palladium catalyst and first base to obtain intermediate b; intermediate b is subjected to demethylation reaction under the action of BBr3 to obtain intermediate c; intermediate c and a response structure compound are subjected to condensation reaction under the action of second base to obtain activated near-infrared second zone fluorescent probe I; Wherein, the alkoxy substituted aryl boronic acid is selected from Wherein, the responsive structure compound is selected from The structure of the activated near-infrared second-region fluorescent probe includes an electron acceptor structural unit A, an electron donor structural unit D, and a response structural unit R; Wherein, the electron acceptor structural unit A is selected from Wherein, the electron donor structural unit D is selected from Among them, the response structure unit R is selected from Wherein, R1 is selected from C0-C 20 Straight or branched alkyl; R2 is selected from C0-C 20 Straight or branched chain alkyl; The oxygen atom end in the electron donor structural unit D is connected to the response structural unit R.

4. The preparation method according to claim 3, characterized in that: The palladium catalyst is tetrakis(triphenylphosphine)palladium, palladium acetate, bis(diphenylphosphino)ferrocenepalladium dichloride, tris(dibenzylideneacetone)dipalladium or bistriphenylphosphinepalladium dichloride; the first base is potassium carbonate, cesium carbonate or potassium phosphate; the second base is potassium carbonate, cesium carbonate, potassium phosphate, triethylamine, N,N-diisopropylethylamine or 4-dimethylaminopyridine.

5. The preparation method according to claim 3, characterized in that: The molar ratio of the intermediate a to the alkoxy-substituted aryl boronic acid, the palladium catalyst, and the first base is 1:3.5-5:0.04-0.10:5-15; the reaction temperature of the Suzuki coupling reaction is 60°C-120°C, and the reaction time is 2h-24h.

6. The preparation method according to claim 3, characterized in that: The molar ratio of the intermediate b to BBr3 is 1:5-10; the reaction temperature of the demethylation reaction is 0°C-25°C, and the reaction time is 2h-6h.

7. The preparation method according to claim 3, characterized in that: The molar ratio of the intermediate c to the response structure compound and the second base is 1:4-6:3-9; the reaction temperature of the condensation reaction is 0°C-60°C, and the reaction time is 10min-24h.

8. Use of the activated near-infrared second-region fluorescent probe according to claim 1 in preparing a nano fluorescent probe that responds to active species in a disease microenvironment; wherein, When the response structural unit R in the activated near-infrared second-zone fluorescent probe is selected from When , the active species in the disease-responsive microenvironment are active sulfur species; When the response structural unit R in the activated near-infrared second-zone fluorescent probe is selected from When , the active species in the disease-responsive microenvironment are reactive oxygen species; When the response structural unit R in the activated near-infrared second-zone fluorescent probe is selected from When , the active species in the disease-responsive microenvironment are reactive nitrogen species; When the response structural unit R in the activated near-infrared second-zone fluorescent probe is selected from When responding to disease, the active species in the microenvironment is glutathione.

9. The use according to claim 8, characterized in that: The preparation method of the nano fluorescent probe is as follows: the activated near-infrared second zone fluorescent probe is mixed with a self-assembly packaging material, tetrahydrofuran and water, and then ultrasonic treatment is performed, and then the tetrahydrofuran is removed and filtered to obtain the nano fluorescent probe.

10. The use according to claim 9, characterized in that: The self-assembly packaging material is F127, DSPE-PEG or PLGA-PEG; the mass ratio of the activated near-infrared second zone fluorescent probe to the self-assembly packaging material is 0.1:1-0.1:2; the mass volume ratio of the self-assembly packaging material to tetrahydrofuran is 1 mg:1 mL; the volume ratio of tetrahydrofuran to water is 1:9; the ultrasonic treatment has an ultrasonic frequency of 40 KHz, an ultrasonic temperature of 25°C, and an ultrasonic time of 10-30 min; the filtration is performed by filtering with a 0.22 μM filter membrane and then ultrafiltering 5 times with an ultrafiltration tube with a cut-off molecular weight of 100,000.

11. The use according to claim 8, characterized in that: The active species is an active oxygen species, an active nitrogen species, an active sulfur species or glutathione.

12. The use according to claim 11, characterized in that: The active oxygen species are The active nitrogen species is ONOO - ; The active sulfur species is H2S.

13. The use according to claim 8, characterized in that: The disease is a tumor or a cardiovascular disease.

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