Fluorescent compounds and probes

Abstract

The present application provides a fluorescent compound and a probe, the general structural formula of the fluorescent compound is shown in Formula I: #imgabs0# wherein X and Y are independently selected from a sulfur atom or an oxygen atom; R 11 ‑R 14 , R 21 ‑R 24 , R 31 ‑R 35 , R 41 ‑R 45 independently selected from hydrogen, a substituted or unsubstituted first alkyl group, a first substituent and a second substituent, the first substituent being OR a or SR b , R a , R b is independently selected from one of hydrogen, a substituted or unsubstituted second alkyl group and a substituted or unsubstituted first acyl group; the second substituent is N(R c )R d , R c , R d Independently selected from one of hydrogen, a substituted or unsubstituted third alkyl group and a substituted or unsubstituted second acyl group. The probe includes a fluorescent compound I. The fluorescent compound and the probe can be used for fluorescence imaging and photoacoustic imaging at the same time, achieving high sensitivity and high penetration of imaging, and have great potential in the fields of in vivo imaging, early tumor diagnosis and surgical navigation.

Description

Technical Field

[0001] The present application relates to the field of organic light-emitting materials, and in particular to a fluorescent compound and a probe. Background Art

[0002] Bioimaging is an important research method to understand the structure of biological tissues and to clarify the various physiological functions of organisms. Bioimaging technologies mainly include magnetic resonance imaging, tomography, X-ray imaging, fluorescence imaging and photoacoustic imaging. Fluorescence imaging is widely used in biotechnology and life sciences due to its advantages of non-invasiveness, high spatiotemporal resolution and high resolution. Photoacoustic imaging is an emerging non-radiation, non-destructive biomedical imaging technology with the advantages of high contrast, high resolution and high penetration of biological tissues. It has great application prospects in the field of real-time medical imaging.

[0003] However, due to the influence of tissue autofluorescence and tissue light scattering, fluorescence imaging has poor penetration ability; photoacoustic imaging achieves imaging by detecting ultrasonic signals, which has disadvantages such as low sensitivity. Using a single imaging probe cannot meet the requirements of strong penetration ability and high sensitivity at the same time, and using multiple imaging probes may interfere with each other and have great toxic side effects on biological tissues. Summary of the invention

[0004] The present application provides a fluorescent compound and a probe that can be used for fluorescence imaging and photoacoustic imaging at the same time, achieving high sensitivity and high penetration of imaging, and has great potential in the fields of in vivo imaging, early tumor diagnosis and surgical navigation.

[0005] In a first aspect, the present application provides a fluorescent compound, the general structural formula of the fluorescent compound is shown in Formula I:

[0006]

[0007] Wherein, X and Y are independently selected from sulfur atoms or oxygen atoms; 11 -R 14 , R 21 -R 24 , R 31 -R 35 , R 41 -R 45 independently selected from hydrogen, a substituted or unsubstituted first alkyl group, a first substituent and a second substituent, wherein the first substituent is OR a or SR b , the R a , R b is independently selected from one of hydrogen, a substituted or unsubstituted second alkyl group and a substituted or unsubstituted first acyl group; the second substituent is N(R c )R d, the R c , R d Independently selected from one of hydrogen, a substituted or unsubstituted third alkyl group and a substituted or unsubstituted second acyl group.

[0008] The fluorescent compound can generate fluorescence and photoacoustic signals at the same time, and is used for fluorescence imaging and photoacoustic imaging. The fluorescent compound can organically combine the sensitivity of fluorescence imaging and the advantage of large imaging depth of photoacoustic imaging. In one embodiment, the absorption band of the fluorescent compound I is 900-1200 nanometers, and the emission wavelength is 1000-1700 nanometers (near infrared region II). The absorption value of biological tissues to the 900-1200 nanometer band is low, and the background of fluorescence and photoacoustic imaging is low, with higher sensitivity, and the laser tissue penetration ability of this wavelength is strong, and its fluorescence imaging has a deeper penetration depth, which makes the fluorescent compound I have a more outstanding imaging advantage.

[0009] In a possible implementation, the substituted or unsubstituted first alkyl group, the substituted or unsubstituted second alkyl group, the substituted or unsubstituted first acyl group, the substituted or unsubstituted third alkyl group and the substituted or unsubstituted second acyl group have 1-18 carbon atoms.

[0010] In one possible implementation, the substituted or unsubstituted first alkyl group, the substituted or unsubstituted second alkyl group, and the substituted or unsubstituted third alkyl group include alkyl groups substituted by at least one of a carboxylic acid group, a sulfonic acid group, a sulfate group, a phosphoric acid group, an amino group, and a quaternary ammonium group; the substituted or unsubstituted first acyl group and the substituted or unsubstituted second acyl group include acyl groups substituted by at least one of a carboxylic acid group, a sulfonic acid group, a sulfate group, a phosphoric acid group, an amino group, and a quaternary ammonium group.

[0011] In a possible implementation, the general structural formula of the fluorescent compound is as shown in Formula II:

[0012]

[0013] The R 1 , R 2 , R 3 , R 4 are independently selected from hydrogen, the substituted or unsubstituted first alkyl group, the first substituent and the second substituent.

[0014] In a possible implementation, the R 1 , R 2 , R 3 , R 4 is independently selected from the first substituent or the second substituent, in the first substituent, the R a , R bindependently represent one of hydrogen, an alkyl group having 1 to 4 carbon atoms, and an acyl group having 1 to 4 carbon atoms; in the second substituent, the R c , R d independently represent hydrogen, an alkyl group having 1 to 4 carbon atoms, or the R c and the R d One of them is hydrogen, the R c and the R d The other is an acyl group having 1 to 4 carbon atoms.

[0015] In a possible implementation, the method for preparing the fluorescent compound I comprises the following steps:

[0016] (1) providing or preparing compound III-1 and compound III-2 as shown below, and providing or preparing compound IV;

[0017]

[0018] (2) mixing the compound III-1, the compound IV and a strong base catalyst, and reacting to obtain the compound V-1 shown below; mixing the compound III-2, the compound IV and a strong base catalyst, and reacting to obtain the compound V-2 shown below;

[0019]

[0020] (3) Compound V-1, compound VI-1 provided or prepared as shown below, and polyphosphoric acid are mixed to obtain compound VII-1 as shown below; Compound V-2, compound VI-2 provided or prepared as shown below, and polyphosphoric acid are mixed to obtain compound VII-2 as shown below;

[0021]

[0022] (4) mixing the compound VII-1 and a methyl Grignard reagent, and after the reaction is completed, pouring the resulting mixture into a perchloric acid aqueous solution to obtain the compound VIII-1 shown below; mixing the compound VII-2 and a methyl Grignard reagent, and after the reaction is completed, pouring the resulting mixture into a perchloric acid aqueous solution to obtain the compound VIII-2 shown below;

[0023]

[0024] (5) mixing the compound VIII-1, the compound VIII-2, and the provided or prepared compound IX shown below, and reacting them to obtain the fluorescent compound I,

[0025]

[0026] In a second aspect, the present application provides a probe, wherein the probe comprises the fluorescent compound as described in any one of the above items.

[0027] After light excitation, the probe can simultaneously generate fluorescence signals and photoacoustic signals, which can be used for fluorescence imaging and photoacoustic imaging, achieving high sensitivity and high penetration of imaging. It is expected to play a significant role in future fields such as in vivo imaging, early tumor diagnosis and surgical navigation.

[0028] In a possible implementation, the probe further comprises an amphiphilic excipient, one end of the amphiphilic excipient or the hydrolysate of the amphiphilic excipient is a hydrophilic group, and the other end of the amphiphilic excipient or the hydrolysate of the amphiphilic excipient is a hydrophobic group.

[0029] In a possible implementation, the amphiphilic excipient is a maleic anhydride-1-octadecene alternating copolymer, the weight average molecular weight of the maleic anhydride-1-octadecene alternating copolymer is 30,000-50,000, and the mass ratio of the fluorescent compound to the maleic anhydride-1-octadecene alternating copolymer is 1:(5-1000).

[0030] In a possible implementation, the method for preparing the probe comprises the following steps:

[0031] (1) dissolving the fluorescent compound and the amphiphilic excipient in an organic solvent to obtain a clear solution;

[0032] (2) removing the organic solvent in the clarified solution of step (1) to obtain a film-like mixture;

[0033] (3) adding water to the film-like mixture in step (2), and obtaining a probe aqueous solution after ultrasonic treatment;

[0034] (4) Removing the water in step (3) to obtain a probe.

[0035] In the present application, under light excitation, the fluorescent compounds and probes can simultaneously generate fluorescent signals and photoacoustic signals. The generated fluorescent signals are used for fluorescent imaging, and the generated photoacoustic signals are used for photoacoustic imaging, thereby achieving high sensitivity and high penetration of imaging, and has great potential in the fields of in vivo imaging, early tumor diagnosis, and surgical navigation. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments of the present application will be described below.

[0037] Figure 1 is an absorption spectrum of the fluorescent compound XI in dichloromethane provided in one embodiment of the present application;

[0038] Figure 2is a fluorescence spectrum of the fluorescent compound XI in dichloromethane provided in one embodiment of the present application;

[0039] Figure 3 is an absorption spectrum of a probe in an aqueous solution provided by an embodiment of the present application;

[0040] Figure 4 is a fluorescence spectrum of a probe provided in an aqueous solution according to an embodiment of the present application;

[0041] Figure 5 This is a fluorescence imaging diagram of the probe provided in one embodiment of the present application;

[0042] Figure 6 is a photoacoustic imaging diagram of a probe provided in one embodiment of the present application;

[0043] Figure 7a It is a fluorescence imaging diagram of probes of different concentrations provided in one embodiment of the present application;

[0044] Figure 7b is a graph showing the relationship between probe concentration and fluorescence imaging signal intensity provided in one embodiment of the present application;

[0045] Figure 8a This is a photoacoustic imaging diagram of probes of different concentrations provided in one embodiment of the present application;

[0046] Figure 8b is a graph showing the relationship between probe concentration and photoacoustic imaging signal intensity provided in one embodiment of the present application;

[0047] Fig. 9 This is a schematic diagram of probe stability provided in one embodiment of the present application. DETAILED DESCRIPTION

[0048] The technical solutions in the embodiments of the present application will be described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all of the embodiments.

[0049] In this document, the terms "first", "second", etc. are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, unless otherwise specified, "plurality" means two or more.

[0050] For ease of understanding, the English abbreviations and related technical terms involved in the embodiments of the present application are explained and described below.

[0051] Fluorescent compound: refers to a substance that, under the stimulation of light of sufficient intensity, electrons transition from the ground state of the molecular orbital energy level to the excited state, and the excited state molecules release energy by emitting fluorescent photons to return to the ground state, emitting fluorescence of a certain wavelength.

[0052] Fluorescence imaging: Imaging is achieved by detecting fluorescent photons.

[0053] Photoacoustic imaging: a non-invasive optical imaging technology that uses optical excitation and ultrasonic detection.

[0054] Near infrared region 2: 1000-1700nm.

[0055] Near infrared region 1: 650-900nm.

[0056] PPA: Polyphosphoric acid;

[0057] THF: Tetrahydrofuran;

[0058] One embodiment of the present application provides a fluorescent compound, and the general structural formula of the fluorescent compound is shown in Formula I:

[0059]

[0060] Wherein, X and Y are independently selected from a sulfur atom or an oxygen atom; R 11 -R 14 , R 21 -R 24 , R 31 -R 35 , R 41 -R 45 independently selected from hydrogen, a substituted or unsubstituted first alkyl group, a first substituent and a second substituent, the first substituent being OR a or SR b , R a , R b is independently selected from one of hydrogen, a substituted or unsubstituted second alkyl group and a substituted or unsubstituted first acyl group; the second substituent is N(R c )R d , the R c , R d Independently selected from one of hydrogen, a substituted or unsubstituted third alkyl group and a substituted or unsubstituted second acyl group.

[0061] After being excited by laser, fluorescent compound I can generate fluorescence and photoacoustic signals, which can be used for fluorescence imaging and photoacoustic imaging.

[0062] First of all, fluorescence imaging has the advantages of being non-invasive, real-time, and having high resolution. Photoacoustic imaging is an emerging non-radiative, non-destructive biomedical imaging technology that achieves real-time imaging by collecting ultrasonic signals that carry optical information of matter. Compared with optical signals, the scattering of ultrasonic signals in biological tissues is about 100 times weaker, making photoacoustic imaging have a greater imaging depth than traditional high-resolution fluorescence imaging methods. The resolution and imaging depth of photoacoustic imaging can be flexibly designed according to needs. It can perform high ultrasonic resolution imaging of centimeter-deep tissues, as well as high-precision imaging of superficial (about 1-2mm) tissues with optical diffraction-limited resolution. Photoacoustic imaging can be applied to the fields of biotechnology and life sciences, and has great application prospects in the field of real-time medical imaging.

[0063] In one embodiment, the absorption band of fluorescent compound I is 900-1200 nanometers, and the emission wavelength is 1000-1700 nanometers (near infrared region II). The absorption value of biological tissue in the 900-1200 nanometer band is low, and the background of fluorescence and photoacoustic imaging is low, with higher sensitivity. The laser tissue penetration ability of this wavelength is strong, and its fluorescence imaging has a deeper penetration depth, which makes the fluorescent compound I have a more outstanding imaging advantage.

[0064] Secondly, fluorescent compound I has both fluorescence imaging and photoacoustic imaging capabilities, which can achieve complementary advantages between the two imaging modes. Fluorescence imaging has high sensitivity but poor penetration, while photoacoustic imaging has strong penetration but low sensitivity. Fluorescent compound I has both fluorescence imaging and photoacoustic imaging capabilities, which can organically combine the sensitivity of fluorescence imaging and the advantages of large imaging depth of photoacoustic imaging, and has very broad application prospects.

[0065] Thirdly, fluorescent compound I has low toxicity and good biocompatibility, and is expected to play a significant role in future fields such as in vivo imaging, early tumor diagnosis, and surgical navigation.

[0066] In one possible implementation, the substituted or unsubstituted first alkyl group has 1 to 18 carbon atoms, and the substituted or unsubstituted second alkyl group, the substituted or unsubstituted first acyl group, the substituted or unsubstituted third alkyl group, and the substituted or unsubstituted second acyl group have 1 to 18 carbon atoms. 11 -R 14 , R 21 -R 24 , R 31 -R 35 , R 41 -R 45 The number of carbon atoms is 1-12. In one embodiment, R 11 -R 14 , R 21 -R 24 , R31 -R 35 , R 41 -R 45 The number of carbon atoms is 1-4.

[0067] In a possible implementation, the substituted or unsubstituted first alkyl group, the substituted or unsubstituted second alkyl group, and the substituted or unsubstituted third alkyl group include alkyl groups substituted by at least one of carboxylic acid group, sulfonic acid group, sulfuric acid group, phosphoric acid group, amino group, and quaternary ammonium group; the substituted or unsubstituted first acyl group and the substituted or unsubstituted second acyl group include acyl groups substituted by at least one of carboxylic acid group, sulfonic acid group, sulfuric acid group, phosphoric acid group, amino group, and quaternary ammonium group. Among them, the carboxylic acid group, sulfonic acid group, sulfuric acid group, phosphoric acid group, amino group, and quaternary ammonium group are hydrophilic groups, which can increase the solubility of the fluorescent compound I in water.

[0068] In a possible implementation, the general structural formula of the fluorescent compound is as shown in Formula II:

[0069]

[0070] Among them, R 1 , R 2 , R 3 , R 4 independently selected from hydrogen, a substituted or unsubstituted first alkyl group, a first substituent and a second substituent, wherein the first substituent is OR a or SR b , R a , R b is independently selected from one of hydrogen, a substituted or unsubstituted second alkyl group and a substituted or unsubstituted first acyl group; the second substituent is N(R c )R d , R c , R d R is independently selected from one of hydrogen, a substituted or unsubstituted third alkyl group and a substituted or unsubstituted second acyl group. 1 Any position on the six-membered ring can be replaced by R 1 Replacement, R 2 Any position on the six-membered ring can be replaced by R 2 Replacement, R 3 Any position on the six-membered ring can be replaced by R 3 Replacement, R 4 Any position on the six-membered ring can be replaced by R 4 For example, in one embodiment, R 11 By R 1 Replacement, R 12 , R 13 , R 14 In one embodiment, R 12 By R1 Replacement, R 11 , R 13 , R 14 In one embodiment, R 13 By R 1 Replacement, R 11 , R 12 , R 14 In one embodiment, R 14 By R 1 Replacement, R 11 , R 12 , R 13 For hydrogen.

[0071] In one possible implementation, R 1 , R 2 , R 3 , R 4 independently selected from the first substituent, R a , R b Each of them independently represents hydrogen, an alkyl group having 1 to 4 carbon atoms, or an acyl group having 1 to 4 carbon atoms.

[0072] In one possible implementation, R 1 , R 2 , R 3 , R 4 is independently selected from a second substituent, R c , R d independently represent hydrogen, an alkyl group having 1 to 4 carbon atoms, or R c and R d One of them is hydrogen, R c and R d The other is an acyl group having 1 to 4 carbon atoms.

[0073] One embodiment of the present application also provides a method for preparing a fluorescent compound I, comprising step A1, step A2, step A3 and step A4. The detailed steps are as follows.

[0074] Step A1, providing or preparing compound III-1 and compound III-2 as shown below, and providing or preparing compound IV;

[0075]

[0076] Step A2, mixing compound III-1, compound IV and a strong base catalyst to react to obtain compound V-1; mixing compound III-2, compound IV and a strong base catalyst to react to obtain compound V-2; in one embodiment, the strong base catalyst is sodium hydride (NaH);

[0077]

[0078] Step A3, compound V-1, provided or prepared compound VI-1, and polyphosphoric acid (PPA) are mixed to obtain compound VII-1; compound V-2, provided or prepared compound VI-2, and polyphosphoric acid are mixed to obtain compound VII-2;

[0079]

[0080] Step A4, mixing compound VII-1 and methyl Grignard reagent (CH3MgBr), and after the reaction is completed, pouring the resulting mixture into a perchloric acid (HClO4) aqueous solution to obtain compound VIII-1; mixing compound VII-2 and methyl Grignard reagent, and after the reaction is completed, pouring the resulting mixture into a perchloric acid (HClO4) aqueous solution to obtain compound VIII-2;

[0081]

[0082] Step A5, compound VIII-1, compound VIII-2, and the provided or prepared compound IX are mixed and reacted to obtain the fluorescent compound I,

[0083]

[0084] In one possible implementation, R 11 , R 13 , R 14 , R 21 , R 23 , R 24 , R 31 , R 32 , R 34 , R 35 , R 41 , R 42 , R 44 , R 45 All are hydrogen, R 12 and R 22 All are N(CH2CH3)2, R 33 , R 43 are both N(CH3)2, X and Y are both selected from sulfur atoms, and the structure of the fluorescent compound is shown in Formula XI:

[0085]

[0086] Among them, the preparation method of the fluorescent compound XI includes step A1-1, step A2-1, step A3-1 and step A4-1, and the detailed steps are as follows.

[0087] Step A1-1, as shown below, under a nitrogen atmosphere, 25mmol sodium hydride (NaH), 10mL toluene and 18mmol compound IV are added to a 100mL round-bottom flask in sequence, and during stirring, 10mL of a toluene solution of compound III-3 with a concentration of 0.9mol / L is added dropwise to the round-bottom flask, refluxed for 2 hours, and cooled to room temperature. The reaction system is poured into ice water, 10mL of hydrochloric acid with a concentration of 1mol / L is added to neutralize the strong base, the pH is adjusted to neutral, and then ethyl acetate is added to extract, the organic phase is collected, dried over anhydrous sodium sulfate, and the organic phase is concentrated by vacuum distillation, and finally purified by silica gel column chromatography to obtain a white solid product V-3 with a yield of 78%.

[0088]

[0089] Step A2-1, as shown below, add 20 mL of polyphosphoric acid (PPA) into a round-bottom flask, stir and heat to 95°C, then add 20 mmol of compound Ⅴ-3 and 20 mmol of compound Ⅵ-3 in sequence, react for 3 hours, slowly add ice water, then add dichloromethane for extraction, collect the organic phase, dry it with anhydrous sodium sulfate, concentrate the organic phase by distillation under reduced pressure, and finally purify it by silica gel column chromatography to obtain a yellow-green solid product Ⅶ-3.

[0090]

[0091] In one embodiment, VII-3 can also be prepared by the following method. In the first step, 20 mL of polyphosphoric acid (PPA) is added to a round-bottom flask, stirred and heated to 95° C., and then 20 mmol of compound V-3 and 20 mmol of compound VI-4 are added in sequence, reacted for 3 hours, ice water is slowly added, and then dichloromethane is added for extraction, the organic phase is collected, dried over anhydrous sodium sulfate, concentrated by rotary evaporation under reduced pressure, and purified by silica gel column chromatography to obtain a yellow solid product VII-4 with a yield of 38%;

[0092] In the second step, under a nitrogen atmosphere, 8 mmol of compound Ⅶ-4, 0.2 mmol of tris(dibenzylideneacetone)dipalladium (Pd2(dba)3), 0.2 mmol of 2-dicyclohexylphosphino-2'-(N,N-dimethylamino)-biphenyl (Davephos), 20 mmol of cesium carbonate (Cs2CO3), 40 mL of anhydrous dioxane (dioxane) and 4 mL of diethylamine (HNEt2) were stirred and heated to reflux, and reacted for 18 hours. When the temperature dropped to room temperature, the filtrate was filtered and collected, and the filtrate was concentrated by distillation under reduced pressure. Finally, it was purified by silica gel column chromatography to obtain a yellow-green solid product Ⅶ-3 with a yield of 73%.

[0093]

[0094] Step A3-1, under a nitrogen atmosphere, add 2.9 mmol of compound Ⅶ-3 and 30 mL of anhydrous tetrahydrofuran (THF) into a round-bottom flask, place the reaction system in an ice-water bath, add 8.7 mL of 1 mol / L methyl Grignard reagent (CH3MgBr) dropwise while stirring, stir at room temperature for 2 hours, pour the reaction system into a 10% HClO4 aqueous solution, add dichloromethane for extraction, collect the organic phase, add anhydrous sodium sulfate for drying, and concentrate the organic phase by distillation under reduced pressure to obtain a blue solid product Ⅷ-3 with a yield of 96%.

[0095]

[0096] Step A4-1, under nitrogen atmosphere, 0.2 mmol of compound VIII-3, 0.1 mmol of compound IX, 8 mL of toluene and 2 mL of n-butanol were sequentially added. Heat to reflux and stir for 22 hours. Concentrate by rotary evaporation under reduced pressure, add 5 mL of acetone, stir for 5 minutes, filter, and collect black solid product XI, with a yield of 61%.

[0097]

[0098] See also Figure 1 , dissolve the fluorescent compound XI in dichloromethane and measure its absorption spectrum. The absorption spectrometer used for the test is Shimadzu UV-3600Plus. The absorption band of the fluorescent compound XI is between 900 and 1200 nanometers, and the maximum absorption wavelength is 1082 nanometers. The half-peak width of the absorption spectrum of the fluorescent compound XI is relatively narrow, and the selectivity for light is high. In the 900-1200 nanometer band, the absorption value of biological tissue is low, the background of fluorescence and photoacoustic imaging is low, and it has higher sensitivity.

[0099] See also Figure 2 , the fluorescent compound XI was dissolved in dichloromethane, excited with a 980-nanometer laser, and its fluorescence spectrum was measured. The fluorescence spectrometer used for the test was Edinburgh FLS920. The emission band of the fluorescent compound XI is 1000-1400 nanometers, and the maximum emission wavelength is 1116 nanometers, which falls in the near-infrared region II. The autofluorescence of biological tissues is weak, and the fluorescence imaging of the fluorescent compound XI has a deeper penetration depth, which gives the fluorescent compound XI a more outstanding imaging advantage.

[0100] In one embodiment, under irradiation of near-infrared zone II laser, the fluorescent compound XI transitions from the ground state to the excited state, and then releases energy in the form of light and heat to return to the ground state, wherein the part of energy released in the form of light generates a fluorescent signal, and the part of energy released in the form of heat generates a photoacoustic signal.

[0101] One embodiment of the present application provides a probe, which includes any one of the fluorescent compounds described above. After being excited by laser, the probe can generate fluorescence and photoacoustic signals for use in fluorescence imaging and photoacoustic imaging.

[0102] In a possible implementation, the probe further includes an amphiphilic excipient, one end of the amphiphilic excipient is a hydrophilic group, and the other end of the amphiphilic excipient is a hydrophobic group. Amphiphilic excipients include maleic anhydride-1-octadecene alternating copolymer (PMAO), poloxamer (polyoxyethylene polyoxypropylene ether block copolymer), distearoyl phosphatidylethanolamine-polyethylene glycol (DSPE-mPEG), polypropylene glycol-polyethylene glycol-polypropylene glycol triblock copolymer (PEG-b-PPG-b-PEG), dioleoyl phosphatidylethanolamine-polyethylene glycol (DOPE-mPEG), benzyl (Brij) surfactant, bovine serum albumin (BSA). Amphiphilic excipients can increase the solubility of the probe in water, so that the probe can be better used in the fields of biotechnology and life sciences.

[0103] In one embodiment, the amphiphilic excipient is maleic anhydride-1-octadecene alternating copolymer (PMAO). The structure of PMAO is shown in Formula XII, where n is a positive integer. Fluorescent compound I has poor water solubility and poor compatibility with biological bodies. After adding PMAO, on the one hand, the long alkyl chains in the PMAO structure wrap around fluorescent compound I to form a hydrophobic environment around the fluorescent compound I molecule; on the other hand, the anhydride structure of PMAO is hydrolyzed under alkaline conditions to obtain a carboxylate as shown in Formula XIII, thereby obtaining a hydrophilic end and achieving water solubility. The alkyl chains of PMAO after hydrolysis form a hydrophobic environment, which enhances the stability of fluorescent compound I and maintains the good optical properties of fluorescent compound I; the hydrophilic carboxylate structure increases the water solubility of the probe, thereby improving the biocompatibility of the probe. In one embodiment, the weight average molecular weight (M w ) is 30000-50000.

[0104]

[0105] In one possible implementation, the mass ratio of fluorescent compound I to PMAO is 1:(5-1000). In one embodiment, the mass ratio of fluorescent compound I to PMAO is 1:(20-1000). In one embodiment, the mass ratio of fluorescent compound I to PMAO is 1:(100-1000). In one embodiment, the mass ratio of fluorescent compound I to PMAO is 1:(100-600). In one embodiment, the mass ratio of fluorescent compound I to PMAO is 1:(200-800). In one embodiment, the mass ratio of fluorescent compound I to PMAO is 1:(400-1000). In one embodiment, the mass ratio of fluorescent compound I to PMAO may be 1:20, 1:50, 1:100, 1:200, 1:400, 1:600, 1:800, 1:1000.

[0106] In a possible implementation, the method for preparing the probe includes step B1, step B2, step B3, and step B4. The specific steps are as follows.

[0107] Step B1, dissolving the fluorescent compound I and the amphiphilic excipient in an organic solvent to obtain a clear solution; in one embodiment, the organic solvent is selected from at least one of dichloromethane and chloroform; this step allows the fluorescent compound I and the amphiphilic excipient to be uniformly mixed;

[0108] Step B2, removing the organic solvent in the clarified solution of step B1 to obtain a film-like mixture;

[0109] Step B3, adding water to the film-like mixture in step B2, and obtaining a probe aqueous solution after ultrasonication; in one embodiment, the probe aqueous solution can be directly used for fluorescence and photoacoustic imaging; the alkyl long chain in the PMAO structure wraps around the fluorescent compound I in water to obtain a nanoparticle aqueous solution;

[0110] Step B4, removing the water in step B3 to obtain a probe; the probe is a solid nanoparticle, which is easy to store and can be used for fluorescence and photoacoustic imaging after being dissolved in water.

[0111] In a possible implementation, the probe includes a fluorescent compound XI and PMAO (M w =30000-50000), the mass ratio of the fluorescent compound XI to PMAO is 1:100, and the preparation method of the probe includes step B1-1, step B2-1, step B3-1, and step B4-1. The specific steps are as follows.

[0112] Step B1-1, dissolving 1 mg of fluorescent compound XI and 100 mg of PMAO in 10-50 ml of dichloromethane or chloroform to obtain a clear solution;

[0113] Step B2-1, removing the organic solvent in the solution of step B1-1 by distillation under reduced pressure to obtain a film-like mixture;

[0114] Step B3-1, adding 10-50 ml of water to the film-like mixture in step B2-1, and obtaining a probe aqueous solution after ultrasonic treatment; or adding 0.1 mol / L sodium hydroxide aqueous solution or 0.1 mol / L 4-dimethylaminopyridine aqueous solution to the film-like mixture in step B2-1, and obtaining a clear solution after ultrasonic treatment, and removing the sodium hydroxide or 4-dimethylaminopyridine in the solution by ultrafiltration or dialysis to obtain a probe aqueous solution;

[0115] Step B4-1, remove the water in step B3-1 by distillation under reduced pressure or freeze drying to obtain the probe.

[0116] In one embodiment, under the irradiation of near-infrared zone II laser, the fluorescent compound XI in the probe transitions from the ground state to the excited state, and then releases energy in the form of light and heat to return to the ground state, wherein the part of energy released in the form of light generates a fluorescent signal, and the part of energy released in the form of heat generates a photoacoustic signal.

[0117] In one embodiment, the response wavelength of the probe is in the near-infrared region II (1000-1700 nm), which has strong tissue penetration capability. In this band, the spontaneous fluorescence and light scattering of blood and tissue are weak, thus having high sensitivity and large imaging depth.

[0118] In a possible implementation, the probe described in any one of the above items can be used for fluorescence imaging.

[0119] In a possible implementation, the probe described in any one of the above items can be used for photoacoustic imaging.

[0120] In a possible implementation, the probe described in any one of the above items can be used for fluorescence and photoacoustic imaging.

[0121] In order to illustrate the imaging effect of the fluorescent compound of the present application, the present application also makes an example of the effect of different mass ratios of the fluorescent compound XI and PMAO in the probe. Specifically, the concentration of the fluorescent compound XI is controlled to remain unchanged, and the mass ratios of the fluorescent compound XI and PMAO are 1:100, 1:200, 1:400, 1:600, 1:800, and 1:1000 to prepare probes P1, P2, P3, P4, P5, and P6, respectively. The absorption spectra of probes P1-P6 in water are shown in FIG. Figure 3As shown in the figure, the model of the absorption spectrometer used in the test is INESAL3S. The absorption peak at 961 nanometers is the aggregation peak of the fluorescent compound XI. As the mass proportion of PMAO increases, the absorption value of the probe at 961 nanometers gradually decreases, indicating that the addition of PMAO makes the fluorescent compound XI more dispersed in the solution. As the mass proportion of PMAO increases, the aggregation degree of the fluorescent compound XI decreases, and the higher the dispersion degree.

[0122] The control probes P1-P6 have the same absorption value at 980nm. With 980nm as the excitation wavelength, the fluorescence spectrum of the probes P1-P6 is tested. The fluorescence spectrum of the probes P1-P6 in water is shown in the figure below: Figure 4 As shown, the fluorescence intensity of probe P1 is the smallest, and the fluorescence intensity of probe P6 is the largest. The fluorescence intensities of probe P1, probe P2, probe P3, probe P4, probe P5, and probe P6 increase in sequence.

[0123] The concentration of fluorescent compound XI in water was kept constant (50 ug / ml), and aqueous solutions of probes with different ratios were prepared. Fluorescence imaging was performed under 1064 nm laser excitation (e.g. Figure 5 As shown in the figure, the laser model used for fluorescence imaging is Beijing Razorway LWIRPD-1F. When the mass ratio of fluorescent compound XI to PMAO is in the range of 1:(100-600), the fluorescence intensity increases with the increase of PMAO usage ratio; in the range of 1:(600-1000), the fluorescence intensity has no obvious enhancement. The addition of PMAO enhances the fluorescence intensity of the probe during imaging. When the mass ratio of PMAO to fluorescent compound XI exceeds 1:600, the enhancement effect of PMAO on the fluorescence intensity of the probe imaging is not obvious.

[0124] The concentration of fluorescent compound XI in water was kept constant (50ug / ml), and aqueous solutions with different ratios of probes were prepared. Photoacoustic imaging was performed under 1064nm pulsed laser excitation. The laser model used for photoacoustic imaging was Innolas EVOs OPO-532. The intensity of the photoacoustic signal was the strongest when the mass ratio of fluorescent compound XI to PMAO was 1:100 (e.g. Figure 6 As shown), there is no obvious difference in fluorescence intensity when the mass ratio of fluorescent compound XI and PMAO is 1:(200-1000).

[0125] Taking the probe prepared with the mass ratio of fluorescent compound Ⅺ and PMAO as 1:1000 as an example, under the same excitation conditions, the intensity of the probe fluorescence signal has a linear relationship with the probe concentration (e.g. Figure 7a and Figure 7b As shown in the figure, the concentration of the probe increases, and the fluorescence signal of the probe becomes stronger.

[0126] Taking the probe prepared with the mass ratio of fluorescent compound Ⅺ and PMAO as 1:1000 as an example, under the same excitation conditions, the intensity of the probe photoacoustic signal has a linear relationship with the probe concentration (e.g. Figure 8a and Figure 8b As shown in the figure, the photoacoustic signal of the probe increases as the concentration of the probe increases.

[0127] The probe has good stability. Take the probe prepared with the mass ratio of fluorescent compound Ⅺ and PMAO as 1:1000 as an example (e.g. Fig. 9 As shown, the absorption spectrum of the probe's aqueous solution at 4°C in the dark basically overlaps with that of the first day on the third day; compared with the absorption spectrum on the first day, the absorption spectrum on the eighth day has no obvious change, and the maximum absorption peak value changes by less than 5%, indicating that the probe has good stability.

[0128] The fluorescent compounds and probes provided in the embodiments of the present application are introduced in detail above. Specific examples are used in this article to illustrate the principles and embodiments of the present application. The description of the above embodiments is only used to help understand the method of the present application and its core idea. At the same time, for general technicians in this field, according to the idea of ​​the present application, there will be changes in the specific embodiments and application scope. In summary, the content of this specification should not be understood as a limitation on the present application.

Claims

1. A fluorescent compound, characterized in that: The absorption band of the fluorescent compound is between 900 nanometers and 1200 nanometers, and the emission band is between 1000 nanometers and 1700 nanometers. The general structural formula of the fluorescent compound is shown in Formula I: , Wherein, X and Y are independently selected from a sulfur atom or an oxygen atom; The R 11 -R 14 , R 21 -R 24 , R 31 -R 35 , R 41 -R 45 are independently selected from hydrogen or a second substituent; The second substituent is N(R c )R d , the R c , R d is independently selected from hydrogen or an unsubstituted third alkyl group; The number of carbon atoms in the unsubstituted third alkyl group is 1-4.

2. The fluorescent compound according to claim 1, characterized in that The general structural formula of the fluorescent compound is shown in Formula II: ， The R 1 , R 2 , R 3 , R 4 are independently selected from hydrogen and the second substituent.

3. The fluorescent compound according to claim 1, characterized in that The preparation method of the fluorescent compound I comprises the following steps: (1) Providing or preparing compound III-1 and compound III-2 as shown below, and providing or preparing compound IV; ， , ; (2) mixing the compound III-1, the compound IV and a strong base catalyst, and reacting to obtain the compound V-1 shown below; mixing the compound III-2, the compound IV and a strong base catalyst, and reacting to obtain the compound V-2 shown below; , ; (3) Compound V-1, compound VI-1 provided or prepared as shown below, and polyphosphoric acid are mixed to obtain compound VII-1 as shown below; Compound V-2, compound VI-2 provided or prepared as shown below, and polyphosphoric acid are mixed to obtain compound VII-2 as shown below; , ， ， ； (4) mixing the compound VII-1 and a methyl Grignard reagent, and after the reaction is completed, pouring the resulting mixture into a perchloric acid aqueous solution to obtain the compound VIII-1 shown below; mixing the compound VII-2 and a methyl Grignard reagent, and after the reaction is completed, pouring the resulting mixture into a perchloric acid aqueous solution to obtain the compound VIII-2 shown below; ， ； (5) Compound VIII-1, compound VIII-2, and compound IX provided or prepared as shown below are mixed and reacted to obtain the fluorescent compound I. 。 4. A probe, characterized in that: The probe comprises the fluorescent compound as described in any one of claims 1 to 3.

5. The probe according to claim 4, characterized in that The probe further comprises an amphiphilic excipient, one end of the amphiphilic excipient or the hydrolyzate of the amphiphilic excipient is a hydrophilic group, and the other end of the amphiphilic excipient or the hydrolyzate of the amphiphilic excipient is a hydrophobic group.

6. The probe according to claim 5, characterized in that The amphiphilic auxiliary material is maleic anhydride-1-octadecene alternating copolymer, the weight average molecular weight of the maleic anhydride-1-octadecene alternating copolymer is 30000-50000, and the mass ratio of the fluorescent compound to the maleic anhydride-1-octadecene alternating copolymer is 1: (5-1000).

7. The probe according to claim 5 or 6, characterized in that: The method for preparing the probe comprises the following steps: (1) dissolving the fluorescent compound and the amphiphilic excipient in an organic solvent to obtain a clear solution; (2) removing the organic solvent in the clarified solution of step (1) to obtain a film-like mixture; (3) adding water to the film-like mixture in step (2), and obtaining a probe aqueous solution after ultrasonication; (4) Remove the water in step (3) to obtain the probe.

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