WO2023040089A1 - 荧光化合物和探针 - Google Patents

荧光化合物和探针 Download PDF

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WO2023040089A1
WO2023040089A1 PCT/CN2021/137625 CN2021137625W WO2023040089A1 WO 2023040089 A1 WO2023040089 A1 WO 2023040089A1 CN 2021137625 W CN2021137625 W CN 2021137625W WO 2023040089 A1 WO2023040089 A1 WO 2023040089A1
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compound
substituted
unsubstituted
group
probe
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PCT/CN2021/137625
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French (fr)
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常宗
孙钦超
刘郴郴
刘良检
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中国科学院深圳先进技术研究院
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/18—Halogen atoms or nitro radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D335/00—Heterocyclic compounds containing six-membered rings having one sulfur atom as the only ring hetero atom
    • C07D335/04—Heterocyclic compounds containing six-membered rings having one sulfur atom as the only ring hetero atom condensed with carbocyclic rings or ring systems
    • C07D335/06—Benzothiopyrans; Hydrogenated benzothiopyrans
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

Definitions

  • This application relates to the field of organic luminescent materials, in particular to a fluorescent compound and a probe.
  • Bioimaging is an important research method to understand the tissue structure of organisms and clarify various physiological functions of organisms.
  • Bioimaging technologies mainly include magnetic resonance imaging, tomographic imaging, X-ray imaging, fluorescence imaging and photoacoustic imaging.
  • Fluorescence imaging is widely used in the fields of biotechnology and life science due to its advantages of non-invasiveness, high temporal and spatial resolution, and high resolution.
  • Photoacoustic imaging is an emerging non-radiative and non-invasive biomedical imaging technology, which has the advantages of high contrast, high resolution, and high penetration of biological tissues, and has great application prospects in the field of real-time medical imaging.
  • This application provides a fluorescent compound and probe, which can be used for both fluorescence imaging and photoacoustic imaging to achieve high sensitivity and high penetration of imaging, and have great potential in the fields of in vivo imaging, early diagnosis of tumors, and surgical navigation. .
  • the present application provides a fluorescent compound, the general structural formula of which is shown in Formula I:
  • 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 advantages of the sensitivity of fluorescence imaging and the large imaging depth of photoacoustic imaging.
  • the absorption band of fluorescent compound I is 900-1200 nanometers, and the emission wavelength is 1000-1700 nanometers (the second near-infrared region).
  • the absorption value of biological tissues in the 900-1200 nanometer band is low, and the background of fluorescence and photoacoustic imaging is low. It has higher sensitivity, and the laser light of this wavelength has strong tissue penetration ability, and its fluorescence imaging has a deeper penetration depth, which makes the fluorescent compound I have more excellent imaging advantages.
  • 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 The number of carbon atoms in the third alkyl group and the substituted or unsubstituted second acyl group is 1-18.
  • the substituted or unsubstituted first alkyl group, the substituted or unsubstituted second alkyl group, and the substituted or unsubstituted third alkyl group include carboxylic acid groups, Sulfonic acid group, sulfate group, phosphoric acid group, amino group, substituted alkyl group in at least one of quaternary ammonium groups;
  • the substituted or unsubstituted first acyl group, the substituted or unsubstituted second acyl group include carboxylic acid group, An acyl group substituted by at least one of a sulfonic acid group, a sulfate group, a phosphoric acid group, an amino group, and a quaternary ammonium group.
  • R 1 , R 2 , R 3 , R 4 are independently selected from hydrogen, said substituted or unsubstituted first alkyl group, said first substituent and said second substituent.
  • the R 1 , R 2 , R 3 , and R 4 are independently selected from the first substituent or the second substituent, and in the first substituent, the R a and R b independently represent one of hydrogen, an alkyl group with 1-4 carbon atoms, and an acyl group with 1-4 carbon atoms; in the second substituent, the R c and R d independently represent hydrogen, an alkyl group with a carbon number of 1-4, or one of the R c and the R d is hydrogen, and the other of the R c and the R d is a carbon atom number of 1-4 4 acyl.
  • the preparation method of the fluorescent compound I includes the following steps:
  • the present application provides a probe, which includes the fluorescent compound described in any one of the above.
  • the probe After the probe is excited by light, it can simultaneously generate fluorescence signals and photoacoustic signals, which can be used for fluorescence imaging and photoacoustic imaging to achieve high sensitivity and high penetration of imaging. Surgical navigation and other fields play a major role.
  • the probe further includes an amphiphilic excipient, one end of the amphiphilic excipient or the hydrolyzate of the amphiphilic excipient is a hydrophilic group, and the amphiphilic excipient or the amphiphilic excipient The other end of the hydrolyzate of sexual excipients is a hydrophobic group.
  • 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
  • the mass ratio of the fluorescent compound to the maleic anhydride-1-octadecene alternating copolymer is 1:(5-1000).
  • the preparation method of the probe includes the following steps:
  • step (3) adding water to the membranous mixture described in step (2), and obtaining the probe aqueous solution after ultrasonication;
  • step (3) (4) removing the water in step (3) to obtain the probe.
  • 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, achieving high sensitivity and With high penetration, it has great potential in the fields of in vivo imaging, early tumor diagnosis and surgical navigation.
  • Figure 1 is an absorption spectrum diagram of fluorescent compound XI in dichloromethane provided by an embodiment of the present application
  • Figure 2 is a fluorescence spectrum diagram of fluorescent compound XI in dichloromethane provided by an embodiment of the present application
  • Fig. 3 is the absorption spectrogram of the probe provided in an embodiment of the present application in aqueous solution
  • Fig. 4 is the fluorescence spectrogram of the probe provided in an embodiment of the present application in an aqueous solution
  • Fig. 5 is a fluorescence imaging diagram of a probe provided by an embodiment of the present application.
  • Fig. 6 is a photoacoustic imaging diagram of a probe provided by an embodiment of the present application.
  • Figure 7a is a fluorescence imaging diagram of probes with different concentrations provided by an embodiment of the present application.
  • Figure 7b is a diagram of the relationship between probe concentration and fluorescence imaging signal intensity provided by an embodiment of the present application.
  • Figure 8a is a photoacoustic imaging diagram of probes with different concentrations provided by an embodiment of the present application.
  • Figure 8b is a diagram of the relationship between probe concentration and photoacoustic imaging signal intensity provided by an embodiment of the present application.
  • Fig. 9 is a schematic diagram of probe stability provided by an embodiment of the present application.
  • first, second, etc. are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features. Thus, a feature defined as “first” and “second” may explicitly or implicitly include one or more of these features. In the description of the present application, unless otherwise specified, "plurality" means two or more.
  • Fluorescent compound refers to a substance that, under the excitation of sufficient intensity of light, electrons transition from the ground state of the molecular orbital energy level to the excited state, and the molecules in the excited state release energy by emitting fluorescent photons to return to the ground state, emitting fluorescence of a certain wavelength.
  • Fluorescence imaging the purpose of imaging is achieved by detecting fluorescent photons.
  • Photoacoustic imaging It is a non-invasive optical imaging technology with optical excitation and ultrasonic detection.
  • PPA Polyphosphoric acid, polyphosphoric acid
  • THF Tetrahydrofuran, tetrahydrofuran
  • One embodiment of the present application provides a fluorescent compound.
  • the general structural formula of the fluorescent compound is shown in Formula I:
  • X, Y are independently selected from sulfur atom or oxygen atom;
  • R 11 -R 14 , R 21 -R 24 , R 31 -R 35 , R 41 -R 45 are independently selected from hydrogen, substituted or unsubstituted An alkyl group, a first substituent and a second substituent, the first substituent is OR a or SR b , R a and R b are independently selected from hydrogen, substituted or unsubstituted second alkyl and substituted or unsubstituted One of the first acyl groups; the second substituent is N(R c ) R d , said R c , R d are independently selected from hydrogen, substituted or unsubstituted third alkyl groups and substituted or unsubstituted One of the second acyl groups.
  • Fluorescent compound I can generate fluorescence and photoacoustic signals after being excited by laser light, which can be used for fluorescence imaging and photoacoustic imaging.
  • fluorescence imaging has the advantages of non-invasiveness, real-time, and high resolution; photoacoustic imaging is an emerging non-radiation, non-invasive biomedical imaging technology, which realizes real-time imaging by collecting ultrasonic signals carrying optical information of substances, and photoacoustic imaging Compared with ultrasonic 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 customized according to requirements.
  • Photoacoustic imaging can be applied to biological In the field of technology and life sciences, it has great application prospects in the field of real-time medical imaging.
  • the absorption band of fluorescent compound I is 900-1200 nanometers, and the emission wavelength is 1000-1700 nanometers (the second near-infrared region).
  • the absorption value of biological tissues in the 900-1200 nanometer band is low, and the background of fluorescence and photoacoustic imaging is low. It has higher sensitivity, and the laser light of this wavelength has strong tissue penetration ability, and its fluorescence imaging has a deeper penetration depth, which makes the fluorescent compound I have more excellent imaging advantages.
  • fluorescent compound I has both fluorescence imaging and photoacoustic imaging capabilities, which can realize the complementary advantages between the two imaging modes. Fluorescence imaging has high sensitivity but poor penetration ability, while photoacoustic imaging has strong penetration ability but low sensitivity. I It has both fluorescence imaging and photoacoustic imaging capabilities, and can organically combine the advantages of the sensitivity of fluorescence imaging and the large imaging depth of photoacoustic imaging, and has a very broad application prospect.
  • fluorescent compound I has low toxicity and good biocompatibility, and is expected to play an important role in the fields of in vivo imaging, early tumor diagnosis and surgical navigation in the future.
  • the substituted or unsubstituted first alkyl group has 1-18 carbon atoms
  • the substituted or unsubstituted second alkyl group has 1-18 carbon atoms
  • the substituted or unsubstituted first acyl group has 1-18 carbon atoms
  • the number of carbon atoms of the third alkyl group and the substituted or unsubstituted second acyl group is 1-18.
  • R 11 -R 14 , R 21 -R 24 , R 31 -R 35 , R 41 -R 45 have 1-12 carbon atoms.
  • R 11 -R 14 , R 21 -R 24 , R 31 -R 35 , R 41 -R 45 have 1-4 carbon atoms.
  • the substituted or unsubstituted first alkyl group, the substituted or unsubstituted second alkyl group, and the substituted or unsubstituted third alkyl group include carboxylic acid groups, sulfonic acid groups, and sulfuric acid groups , phosphoric acid group, amino group, quaternary ammonium group at least one substituted alkyl group; substituted or unsubstituted first acyl group, substituted or unsubstituted second acyl group includes carboxylic acid group, sulfonic acid group, sulfuric acid group, phosphoric acid group, Acyl group substituted by at least one of amino group and quaternary ammonium group.
  • 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 fluorescent compound I in water.
  • R 1 , R 2 , R 3 , R 4 are independently selected from hydrogen, a substituted or unsubstituted first alkyl, a first substituent and a second substituent, and the first substituent is OR a or SR b , R a , R b are independently selected from one of hydrogen, substituted or unsubstituted second alkyl and substituted or unsubstituted first acyl; 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.
  • R 11 is substituted by R 1
  • R 12 , R 13 , R 14 are hydrogen
  • R 12 is substituted by R 1
  • R 11 , R 13 , R 14 are hydrogen
  • R 13 is substituted by R 1
  • R 11 , R 12 , R 14 are hydrogen
  • R 14 is substituted by R 1 , and R 11 , R 12 , R 13 are hydrogen.
  • R 1 , R 2 , R 3 , and R 4 are independently selected from the first substituent, R a and R b independently represent hydrogen, an alkyl group with 1-4 carbon atoms, or One of the acyl groups having 1 to 4 carbon atoms.
  • R 1 , R 2 , R 3 , and R 4 are independently selected from the second substituent, R c and R d independently represent hydrogen and an alkyl group with 1-4 carbon atoms, Or one of R c and R d is hydrogen, and the other of R c and R d is an acyl group with 1-4 carbon atoms.
  • One embodiment of the present application also provides a preparation method of fluorescent compound I, comprising step A1, step A2, step A3 and step A4. Detailed steps are described below.
  • Step A1 providing or preparing compound III-1 and compound III-2 as shown below, and providing or preparing compound IV;
  • 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);
  • Step A3 compound V-1, provide or prepare compound VI-1, polyphosphoric acid (PPA) mixed reaction to obtain compound VII-1;
  • compound V-2 provide or prepare compound VI-2, polyphosphoric acid mixed reaction Obtain compound VII-2;
  • Step A4 mix compound VII-1 and methyl Grignard reagent (CH 3 MgBr), after the reaction is completed, pour the resulting mixture into aqueous perchloric acid (HClO 4 ) solution to obtain compound VIII-1;
  • compound VII -2 is mixed with methyl Grignard reagent, and after the reaction is completed, the obtained mixed solution is poured into an aqueous solution of perchloric acid (HClO 4 ) to obtain compound VIII-2;
  • Step A5 mixing compound VIII-1, compound VIII-2, provided or prepared compound IX, and reacting to obtain the fluorescent compound I,
  • Both R 12 and R 22 are N(CH 2 CH 3 ) 2
  • R 33 and R 43 are both N(CH 3 ) 2
  • X and Y are both selected from sulfur atoms.
  • the structure of the fluorescent compound is as follows: As shown in XI:
  • the preparation method of fluorescent compound XI includes step A1-1, step A2-1, step A3-1 and step A4-1, and the detailed steps are as follows.
  • Step A1-1 as shown below, under a nitrogen atmosphere, sequentially add 25mmol of sodium hydride (NaH), 10mL of toluene and 18mmol of compound IV into a 100mL round bottom flask. During stirring, add 10mL of 0.9mol/L The toluene solution of compound III-3 was added dropwise into a round bottom flask, refluxed for 2 hours, and cooled to room temperature.
  • NaH sodium hydride
  • Step A2-1 as shown below, add 20mL polyphosphoric acid (PPA) into a round bottom flask, stir and heat to 95°C, then add 20mmol compound V-3 and 20mmol compound VI-3 in turn, react for 3 hours, slowly Add ice water, then add dichloromethane for extraction, collect the organic phase, dry over anhydrous sodium sulfate, concentrate the organic phase by distillation under reduced pressure, and finally use silica gel column chromatography to obtain yellow-green solid product VII-3.
  • PPA polyphosphoric acid
  • VII-3 can also be prepared by the following method.
  • PPA polyphosphoric acid
  • first step 20 mL of polyphosphoric acid (PPA) is added into a round bottom flask, stirred and heated to 95° C., followed by adding 20 mmol of compound V-3 and 20 mmol of compound VI-4, reacted for 3 hours, slowly added ice water, then added dichloromethane for extraction, collected the organic phase, dried with anhydrous sodium sulfate, concentrated by rotary evaporation under reduced pressure, purified by silica gel column chromatography to obtain the yellow solid product VII- 4. The yield is 38%;
  • 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, and add 8.7 mL of 1 mol/L tetrahydrofuran (THF) dropwise while stirring.
  • Methyl Grignard reagent CH 3 MgBr
  • 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, and filter to collect the black solid product XI with a yield of 61%.
  • the model of the absorption spectrometer used in the test is Shimadzu UV-3600Plus.
  • the absorption band of the fluorescent compound XI is 900-1200 nanometers, and the maximum absorption wavelength is 1082 nanometers.
  • the half-maximum width of the absorption spectrum of the fluorescent compound XI is relatively narrow, and the selectivity to light is high.
  • the absorption value of biological tissue is low, the background of fluorescence and photoacoustic imaging is low, and it has higher sensitivity
  • the fluorescent compound XI was dissolved in dichloromethane, excited with a 980 nm laser, and its fluorescence spectrum was measured.
  • the model of the fluorescence spectrometer used in the test was Edinburgh FLS920.
  • the emission band of fluorescent compound XI is 1000-1400 nanometers, and the maximum emission wavelength is 1116 nanometers, which falls in the second near-infrared region.
  • Compound XI has more excellent imaging advantages.
  • 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 that releases energy in the form of light produces fluorescence signal; the part that releases energy in the form of heat, producing a photoacoustic signal.
  • One embodiment of the present application provides a probe, which includes any one of the above-mentioned fluorescent compounds. After the probe is excited by laser light, it can generate fluorescence and photoacoustic signals for fluorescence imaging and photoacoustic imaging.
  • the probe further includes an amphiphilic auxiliary material, one end of the amphiphilic auxiliary material is a hydrophilic group, and the other end of the amphiphilic auxiliary material is a hydrophobic group.
  • Amphiphilic excipients include maleic anhydride-1-octadecene alternating copolymer (PMAO), poloxamer (polyoxyethylene polyoxypropylene ether block copolymer), distearoylphosphatidylethanolamine-polyethylene glycol alcohol (DSPE-mPEG), polypropylene glycol-polyethylene glycol-polypropylene glycol triblock copolymer (PEG-b-PPG-b-PEG), dioleoylphosphatidylethanolamine-polyethylene glycol (DOPE-mPEG) , Benze (Brij) class surface activity, bovine serum albumin (BSA). Amphiphilic excipients can increase the solubility of the probe in water, making the probe
  • the amphiphilic excipient is maleic anhydride-1-octadecene alternating copolymer (PMAO).
  • PMAO maleic anhydride-1-octadecene alternating copolymer
  • the structure of PMAO is shown in Formula XII, wherein n is a positive integer.
  • Fluorescent compound I has poor water solubility and poor compatibility with organisms.
  • the long chain of alkyl in the PMAO structure wraps fluorescent compound I, forming a hydrophobic environment around the molecule of fluorescent compound I;
  • the acid anhydride structure of PMAO is hydrolyzed under alkaline conditions to obtain a carboxylate as shown in formula XIII, thereby obtaining a hydrophilic end and realizing water solubility.
  • the alkyl chain of the hydrolyzed PMAO forms a hydrophobic environment, which enhances the stability of the fluorescent compound I and maintains the good optical properties of the fluorescent compound I; the hydrophilic carboxylate structure increases the water solubility of the probe, thereby improving the Probe biocompatibility.
  • the weight average molecular weight (M w ) of PMAO is 30000-50000.
  • 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 can be 1:20, 1:50, 1:100, 1:200, 1:400, 1:600, 1:800, 1:1000.
  • the method for preparing the probe includes step B1, step B2, step B3, and step B4, and the specific steps are as follows.
  • Step B1 dissolving fluorescent compound I and amphiphilic excipients in an organic solvent to obtain a clear solution;
  • the organic solvent is at least one selected from dichloromethane or chloroform; this step makes fluorescent compound I and amphiphilic Sexual excipients are mixed evenly;
  • Step B2 removing the organic solvent in the clarified solution of step B1 to obtain a film-like mixture
  • Step B3 add water to the membranous mixture in step B2, obtain probe aqueous solution after ultrasonic;
  • this probe aqueous solution can be directly used for fluorescence and photoacoustic imaging;
  • step B4 the water in step B3 is removed to obtain the probe; the probe is a solid nanoparticle, which is convenient for storage, and can be used for fluorescence and photoacoustic imaging after being dissolved in water.
  • 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;
  • 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
  • Step B3-1 add 10-50 ml of water to the filmy mixture in step B2-1, and obtain an aqueous probe solution after ultrasonication; or add 0.1 moles per liter of sodium hydroxide aqueous solution or 0.1 Mole per liter of 4-dimethylaminopyridine aqueous solution, obtain a clear solution after ultrasonication, remove sodium hydroxide or 4-dimethylaminopyridine in the solution by ultrafiltration or dialysis, and obtain an aqueous probe solution;
  • step B4-1 the water in step B3-1 is removed by distillation under reduced pressure or freeze-drying to obtain the probe.
  • 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 energy released in the form of light
  • the part that produces a fluorescent signal; the part that releases energy in the form of heat produces a photoacoustic signal.
  • the response wavelength of the probe is in the second near-infrared region (1000-1700nm), which has a strong ability to penetrate laser tissue, and under this wavelength band, blood and tissue autofluorescence and light scattering are weak, so it has high sensitivity and Large imaging depth.
  • the probe as described in any one of the above can be used for fluorescence imaging.
  • the probe as described in any one of the above can be used for photoacoustic imaging.
  • the probe as described in any one of the above can be used for fluorescence and photoacoustic imaging.
  • the present application also made an example of the effect of different mass ratios of the fluorescent compound XI and PMAO in the probe.
  • concentration of the fluorescent compound XI is controlled to be constant, 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.
  • the absorption spectra of probes P1-P6 in water are shown in Figure 3, and the model of the absorption spectrometer used in the test is INESAL3S.
  • the absorption peak at 961 nanometers is the aggregation peak of fluorescent compound XI.
  • the control probes P1-P6 have the same absorption value at 980nm. With 980nm as the excitation wavelength, the fluorescence spectrum test is carried out on the probes P1-P6.
  • the fluorescence spectrum diagram of the probes P1-P6 in water is shown in Figure 4. The fluorescence intensity of probe P1 is the smallest, the fluorescence intensity of probe P6 is the highest, and the fluorescence intensity of probe P1, probe P2, probe P3, probe P4, probe P5, and probe P6 increases in turn.
  • the concentration of fluorescent compound XI in water was kept constant (50ug/ml), and aqueous solutions with different proportions of probes were prepared, and photoacoustic imaging was performed under 1064 nm pulsed laser excitation.
  • the laser model used for photoacoustic imaging was Innolas EVOs OPO-532.
  • the photoacoustic signal intensity was the strongest when the mass ratio of fluorescent compound XI to PMAO was 1:100 (as shown in Figure 6), and there was no significant difference in fluorescence intensity between the mass ratio of fluorescent compound XI and PMAO at 1: (200-1000).
  • the fluorescence signal intensity of the probe has a linear relationship with the probe concentration (as shown in Figure 7a and Figure 7b), the probe The higher the concentration of the probe, the stronger the fluorescence signal of the probe.
  • the photoacoustic signal intensity of the probe has a linear relationship with the probe concentration (as shown in Figure 8a and Figure 8b). As the concentration of needles increases, the photoacoustic signal of the probe becomes stronger.
  • the probe has good stability. Take the probe prepared by fluorescent compound XI and PMAO with a mass ratio of 1:1000 as an example (as shown in Figure 9).
  • the absorption spectrum basically overlapped with the absorption spectrum of the first day; compared with the absorption spectrum of the first day, the absorption spectrum of the eighth day did not change significantly, and the peak value of the maximum absorption peak changed less than 5%, indicating that the probe had good stability.

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Abstract

本申请提供一种荧光化合物和探针，荧光化合物的结构通式如式Ⅰ所示：其中，X、Y独立地选自硫原子或氧原子；R11-R14、R21-R24、R31-R 35、R41-R45独立地选自氢、取代或未取代的第一烷基、第一取代基和第二取代基，第一取代基为ORa或SRb，Ra、Rb独立地选自氢、取代或未取代的第二烷基和取代或未取代的第一酰基中的一种；第二取代基为N(Rc)R d，Rc、Rd独立地选自氢、取代或未取代的第三烷基和取代或未取代的第二酰基中的一种。探针包括荧光化合物Ⅰ。该荧光化合物和探针可同时用于荧光成像和光声成像，实现成像的高灵敏性和高穿透性，在活体成像、肿瘤早期诊断和手术导航等领域有较大的潜力。

Description

荧光化合物和探针 技术领域
本申请涉及有机发光材料领域，特别涉及一种荧光化合物和探针。
背景技术
生物成像是了解生物体组织结构，阐明生物体各种生理功能的一种重要研究手段。生物成像技术主要包括磁共振成像、断层扫描成像、X-射线成像、荧光成像和光声成像技术。荧光成像由于具有非入侵性、高时空分辨率、高分辨率等优点，被广泛应用于生物技术及生命科学领域。光声成像是一种新兴的非辐射、无损伤生物医学影像技术，具有高对比度、高分辨率及生物组织高穿透性等优点，在实时医学成像领域有着巨大的应用前景。
但是，受组织自发荧光和组织的光散射的影响，荧光成像的穿透能力差；光声成像通过探测超声信号实现成像，具有灵敏度低等缺点。使用单一的成像探针无法同时满足穿透能力强、灵敏度高的需求，而采用多种成像探针可能会互相干扰、对生物组织毒副作用大。
发明内容
本申请提供一种荧光化合物和探针，可同时用于荧光成像和光声成像，实现成像的高灵敏性和高穿透性，在活体成像、肿瘤早期诊断和手术导航等领域有较大的潜力。
第一方面，本申请提供一种荧光化合物，所述荧光化合物的结构通式如式Ⅰ所示：
Figure PCTCN2021137625-appb-000001
其中，所述X、Y独立地选自硫原子或氧原子；所述R 11-R 14、R 21-R 24、R 31-R 35、R 41-R 45独立地选自氢、取代或未取代的第一烷基、第一取代基和第二取代基，所述第一取代基为OR a或SR b，所述R a、R b独立地选自氢、取代或未取代的第二烷基和取代或未取代的第一酰基中的一种；所述第二取代基为N(R c)R d，所述R c、R d独立地选自氢、取代或未取代的第三烷基和取代或未取代的第二酰基中的一种。
所述荧光化合物同时可产生荧光和光声信号，用于荧光成像和光声成像，所述荧光化合物能够有机的组合荧光成像的灵敏度和光声成像的大成像深度的优势。在一实施方式中，荧光化合物Ⅰ吸收波段在900-1200纳米，发射波长在1000-1700纳米(近红外二区)，生物组织对900-1200纳米波段吸收值低，荧光和光声成像背景低，具有更高的灵敏度，且该波长的激光组织穿透能力强，其荧光成像具有较深的穿透深度，使荧光化合物Ⅰ具有更卓越的成像优势。
在一种可能的实现方式中，所述取代或未取代的第一烷基、所述取代或未取代的第二烷基、所述取代或未取代的第一酰基、所述取代或未取代的第三烷基和所述取代或未取代的第二酰基的碳原子数为1-18。
在一种可能的实现方式中，所述取代或未取代的第一烷基、所述取代或未取代的第二烷基、所述取代或未取代的第三烷基包括被羧酸基、磺酸基、硫酸基、磷酸基、氨基、季铵基中的至少一种取代的烷基；所述取代或未取代的第一酰基、所述取代或未取代的第二酰基包括被羧酸基、磺酸基、硫酸基、磷酸基、氨基、季铵基中的至少一种取代的酰基。
在一种可能的实现方式中，所述荧光化合物的结构通式如式Ⅱ所示：
Figure PCTCN2021137625-appb-000002
所述R 1、R 2、R 3、R 4独立地选自氢、所述取代或未取代的第一烷基、所述第一取代基和所述第二取代基。
在一种可能的实现方式中，所述R 1、R 2、R 3、R 4独立地选自所述第一取代基或所述第二取代基，所述第一取代基中，所述R a、R b独立地表示氢、碳原子数为1-4的烷基、碳原子数为1-4的酰基中的一种；所述第二取代基中，所述R c、R d独立地表示氢、碳原子数为1-4的烷基，或所述R c和所述R d其中一个为氢，所述R c和所述R d中另一个为碳原子数为1-4的酰基。
在一种可能的实现方式中，所述荧光化合物Ⅰ的制备方法，包括以下步骤：
(1)提供或制备如下所示的化合物Ⅲ-1、化合物Ⅲ-2，以及提供或制备化合物Ⅳ；
Figure PCTCN2021137625-appb-000003
(2)将所述化合物Ⅲ-1、所述化合物Ⅳ和强碱催化剂混合，反应得到如下所示的化合物Ⅴ-1；将所述化合物Ⅲ-2、所述化合物Ⅳ和强碱催化剂混合，反应得到如下所示的化合物Ⅴ-2；
Figure PCTCN2021137625-appb-000004
(3)将所述化合物Ⅴ-1、提供或制备如下所示的化合物Ⅵ-1、多聚磷酸混合反应得到如下所示的化合物Ⅶ-1；将所述化合物Ⅴ-2、提供或制备如下所示的化合物Ⅵ-2、多聚磷酸混合反应得到如下所示的化合物Ⅶ-2；
Figure PCTCN2021137625-appb-000005
(4)将所述化合物Ⅶ-1和甲基格氏试剂混合，反应完毕后将得到的混合液倒入高氯酸水溶液中，得到如下所示的化合物Ⅷ-1；将所述化合物Ⅶ-2和甲基格氏试剂混合，反应完毕后将得到的混合液倒入高氯酸水溶液中，得到如下所示的化合物Ⅷ-2；
Figure PCTCN2021137625-appb-000006
(5)将所述化合物Ⅷ-1、所述化合物Ⅷ-2、提供或制备化合物如下所示的Ⅸ混合，反应制得所述荧光化合物Ⅰ，
Figure PCTCN2021137625-appb-000007
第二方面，本申请提供一种探针，所述探针包括如上任意一项所述的荧光化合物。
所述探针在光激发后，可同时产生荧光信号和光声信号，用于荧光成像和光声成像，实现成像的高灵敏性和高穿透性，有希望在未来的活体成像、肿瘤早期诊断和手术导航等领域发挥重大作用。
在一种可能的实现方式中，所述探针还包括双亲性辅料，所述双亲性辅料或所述双亲性辅料的水解产物的一端为亲水基团，所述双亲性辅料或所述双亲性辅料的水解产物的另一端为疏水基团。
在一种可能的实现方式中，所述双亲性辅料为马来酸酐-1-十八碳烯交替共聚物，所述马 来酸酐-1-十八碳烯交替共聚物的重均分子量为30000-50000，所述荧光化合物和所述马来酸酐-1-十八碳烯交替共聚物的质量比为1∶(5-1000)。
在一种可能的实现方式中，所述探针的制备方法包括以下步骤：
(1)将所述荧光化合物和所述双亲性辅料溶于有机溶剂中得到澄清溶液；
(2)除去步骤(1)所述澄清溶液中的有机溶剂得到膜状混合物；
(3)向步骤(2)中所述膜状混合物加入水，超声后得到探针水溶液；
(4)除去步骤(3)中的水，得到探针。
本申请中，在光激发下，荧光化合物和探针可同时产生荧光信号和光声信号，产生的荧光信号用于荧光成像，产生的光声信号用于光声成像，实现成像的高灵敏性和高穿透性，在活体成像、肿瘤早期诊断和手术导航等领域有较大的潜力。
附图说明
为了更清楚地说明本申请实施例中的技术方案，下面将对本申请实施例中所需要使用的附图进行说明。
图1是本申请一实施方式提供的荧光化合物Ⅺ在二氯甲烷中的吸收光谱图；
图2是本申请一实施方式提供的荧光化合物Ⅺ在二氯甲烷中的荧光光谱图；
图3是本申请一实施方式提供的探针在水溶液中的吸收光谱图；
图4是本申请一实施方式提供的探针在水溶液中的荧光光谱图；
图5是本申请一实施方式提供的探针的荧光成像图；
图6是本申请一实施方式提供的探针的光声成像图；
图7a是本申请一实施方式提供的不同浓度探针的荧光成像图；
图7b是本申请一实施方式提供的探针浓度与荧光成像信号强度关系图；
图8a是本申请一实施方式提供的不同浓度探针的光声成像图；
图8b是本申请一实施方式提供的探针浓度与光声成像信号强度关系图；
图9是本申请一实施方式提供的探针稳定性示意图。
具体实施方式
下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行描述，显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。
本文中，术语“第一”、“第二”等仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。在本申请的描述中，除非另有说明，“多个”的含义是两个或两个以上。
为方便理解，下面先对本申请实施例所涉及的英文简写和有关技术术语进行解释和描述。
荧光化合物：是指在足够强度的光照的激发下，电子从分子轨道能级基态跃迁到激发态，激发态分子通过发射荧光光子释放能量返回基态，发出一定波长的荧光的物质。
荧光成像：是通过检测荧光光子实现成像目的。
光声成像：是一种光学激发、超声探测无创光学成像技术。
近红外二区：1000-1700nm。
近红外一区：650-900nm。
PPA：Polyphosphoric acid，多聚磷酸；
THF：Tetrahydrofuran，四氢呋喃；
本申请一实施方式提供一种荧光化合物，荧光化合物的结构通式如式Ⅰ所示：
Figure PCTCN2021137625-appb-000008
其中，X、Y独立地选自硫原子或氧原子；R 11-R 14、R 21-R 24、R 31-R 35、R 41-R 45独立地选自氢、取代或未取代的第一烷基、第一取代基和第二取代基，第一取代基为OR a或SR b，R a、R b独立地选自氢、取代或未取代的第二烷基和取代或未取代的第一酰基中的一种；第二取代基为N(R c)R d，所述R c、R d独立地选自氢、取代或未取代的第三烷基和取代或未取代的第二酰基中的一种。
荧光化合物Ⅰ受激光激发后，可产生荧光和光声信号，用于荧光成像和光声成像。
首先，荧光成像具有非入侵性、实时、分辨率高等优点；光声成像是一种新兴的非辐射、无损伤生物医学影像技术，该技术通过采集携带物质光学信息的超声波信号实现实时成像，和光信号相比，超声波信号在生物组织中的散射要弱约100倍，使得光声成像比传统高分辨荧光成像方法具有更大的成像深度，光声成像的分辨率和成像深度，可根据需求，灵活设计，既能对厘米深的组织进行高超声分辨率的成像，也可对浅表(约1-2mm)组织进行具备光学衍射极限分辨率的高精度成像，光声成像可被应用于生物技术及生命科学领域，在实时医学成像领域有着巨大的应用前景。
在一实施方式中，荧光化合物Ⅰ吸收波段在900-1200纳米，发射波长在1000-1700纳米(近红外二区)，生物组织对900-1200纳米波段吸收值低，荧光和光声成像背景低，具有更高的灵敏度，且该波长的激光组织穿透能力强，其荧光成像具有较深的穿透深度，使荧光化合物Ⅰ具有更卓越的成像优势。
其次，荧光化合物Ⅰ同时具备荧光成像和光声成像能力，可实现两种成像模式之间的优势互补，荧光成像灵敏度高但穿透能力差，而光声成像穿透能力强但灵敏度低，荧光化合物Ⅰ同时具备荧光成像和光声成像能力，能够有机的组合荧光成像的灵敏度和光声成像的大成像深度的优势，具有非常广阔的应用前景。
再次，荧光化合物Ⅰ毒性小、生物相容性好，有希望在未来的活体成像、肿瘤早期诊断和手术导航等领域发挥重大作用。
在一种可能的实现方式中，取代或未取代的第一烷基的碳原子数为1-18，取代或未取代的第二烷基、取代或未取代的第一酰基、取代或未取代的第三烷基和取代或未取代的第二酰基的碳原子数为1-18。在一实施方式中，R 11-R 14、R 21-R 24、R 31-R 35、R 41-R 45的碳原子数为1-12。在一实施方式中，R 11-R 14、R 21-R 24、R 31-R 35、R 41-R 45的碳原子数为1-4。
在一种可能的实现方式中，取代或未取代的第一烷基、取代或未取代的第二烷基、取代或未取代的第三烷基包括被羧酸基、磺酸基、硫酸基、磷酸基、氨基、季铵基中的至少一种取代的烷基；取代或未取代的第一酰基、取代或未取代的第二酰基包括被羧酸基、磺酸基、硫酸基、磷酸基、氨基、季铵基中的至少一种取代的酰基。其中，羧酸基、磺酸基、硫酸基、磷酸基、氨基、季铵基为亲水基团，可增加荧光化合物Ⅰ在水中的溶解度。
在一种可能的实现方式中，荧光化合物的结构通式如式Ⅱ所示：
Figure PCTCN2021137625-appb-000009
其中，R 1、R 2、R 3、R 4独立地选自氢、取代或未取代的第一烷基、第一取代基和第二取代基，所述第一取代基为OR a或SR b，R a、R b独立地选自氢、取代或未取代的第二烷基和取代或未取代的第一酰基中的一种；第二取代基为N(R c)R d，R c、R d独立地选自氢、取代或未取代的第三烷基和取代或未取代的第二酰基中的一种。R 1所在六元环上的任意位置均可被R 1取代，R 2所在六元环上的任意位置均可被R 2取代，R 3所在六元环上的任意位置均可被R 3取代，R 4所在六元环上的任意位置均可被R 4取代。例如，在一实施方式中，R 11被R 1取代，R 12、R 13、R 14为氢。在一实施方式中，R 12被R 1取代，R 11、R 13、R 14为氢。在一实施方式中，R 13被R 1取代，R 11、R 12、R 14为氢。在一实施方式中，R 14被R 1取代，R 11、R 12、R 13为氢。
在一种可能的实现方式中，R 1、R 2、R 3、R 4独立地选自第一取代基，R a、R b独立地表示氢、碳原子数为1-4的烷基或碳原子数为1-4的酰基中的一种。
在一种可能的实现方式中，R 1、R 2、R 3、R 4独立地选自第二取代基，R c、R d独立地表示氢、碳原子数为1-4的烷基，或R c和R d其中一个为氢，R c和R d中另一个为碳原子数为1-4的酰基。
本申请一实施方式还提供一种荧光化合物Ⅰ的制备方法，包括步骤A1、步骤A2、步骤A3和步骤A4。详细步骤如下所述。
步骤A1，提供或制备如下所示的化合物Ⅲ-1、化合物Ⅲ-2，以及提供或制备化合物Ⅳ；
Figure PCTCN2021137625-appb-000010
步骤A2，将化合物Ⅲ-1、化合物Ⅳ和强碱催化剂混合，反应得到化合物Ⅴ-1；将化合物 Ⅲ-2、化合物Ⅳ和强碱催化剂混合，反应得到化合物Ⅴ-2；在一实施方式中，强碱催化剂为氢化钠(NaH)；
Figure PCTCN2021137625-appb-000011
步骤A3，将化合物Ⅴ-1、提供或制备化合物Ⅵ-1、多聚磷酸(PPA)混合反应得到化合物Ⅶ-1；将化合物Ⅴ-2、提供或制备化合物Ⅵ-2、多聚磷酸混合反应得到化合物Ⅶ-2；
Figure PCTCN2021137625-appb-000012
步骤A4，将化合物Ⅶ-1和甲基格氏试剂(CH 3MgBr)混合，反应完毕后将得到的混合液倒入高氯酸(HClO 4)水溶液中，得到化合物Ⅷ-1；将化合物Ⅶ-2和甲基格氏试剂混合，反应完毕后将得到的混合液倒入高氯酸(HClO 4)水溶液中，得到化合物Ⅷ-2；
Figure PCTCN2021137625-appb-000013
步骤A5，将化合物Ⅷ-1、化合物Ⅷ-2、提供或制备化合物Ⅸ混合，反应制得所述荧光化合物Ⅰ，
Figure PCTCN2021137625-appb-000014
在一种可能的实现方式中，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均为氢，R 12和R 22均为N(CH 2CH 3) 2，R 33、R 43均为N(CH 3) 2，X、Y均选自硫原子，该荧光化合物的结构如式Ⅺ所示：
Figure PCTCN2021137625-appb-000015
其中，荧光化合物Ⅺ的制备方法，包括步骤A1-1、步骤A2-1、步骤A3-1和步骤A4-1，详细步骤如下。
步骤A1-1，如下所示，在氮气氛围下，依次将25mmol氢化钠(NaH)、10mL甲苯和18mmol化合物Ⅳ加入至100mL圆底烧瓶中，搅拌过程中，将10mL浓度为0.9mol/L的化合物Ⅲ-3的甲苯溶液滴加进圆底烧瓶中，回流反应2小时，冷却至室温。将反应体系倒入冰水中，加入10mL浓度为1mol/L的盐酸中和强碱，调节pH至中性，然后加入乙酸乙酯萃取，收集有机相，用无水硫酸钠干燥后，减压蒸馏浓缩有机相，最后使用硅胶柱层析纯化得白色固体产物Ⅴ-3，产率78％。
Figure PCTCN2021137625-appb-000016
步骤A2-1，如下所示，将20mL多聚磷酸(PPA)加入圆底烧瓶中，搅拌并加热至95℃，随后依次加入20mmol化合物Ⅴ-3和20mmol化合物Ⅵ-3，反应3小时，缓慢加入冰水，再加入二氯甲烷萃取，收集有机相，用无水硫酸钠干燥，减压蒸馏浓缩有机相，最后使用硅胶柱层析纯化得黄绿色固体产物Ⅶ-3。
Figure PCTCN2021137625-appb-000017
在一实施方式中，Ⅶ-3还可以通过如下方法制备，第一步，将20mL多聚磷酸(PPA)加入圆底烧瓶中，搅拌并加热至95℃，随后依次加入20mmol化合物Ⅴ-3和20mmol化合物Ⅵ-4，反应3小时，缓慢加入冰水，再加入二氯甲烷萃取，收集有机相，用无水硫酸钠干燥，减压旋转蒸发浓缩，硅胶柱层析纯化得黄色固体产物Ⅶ-4，产率38％；
第二步，在氮气氛围下，将8mmol化合物Ⅶ-4、0.2mmol三(二亚苄基丙酮)二钯(Pd 2(dba) 3)、0.2mmol 2-二环己膦基-2'-(N,N-二甲胺)-联苯(Davephos)、20mmol碳酸铯(Cs 2CO 3)、40mL无水二氧六环(dioxane)和4mL二乙胺(HNEt 2)，搅拌并加热至回流，反应18小时，待温度降至室温，过滤并收集滤液，减压蒸馏浓缩滤液，最后使用硅胶柱层析纯化得黄绿色固体产物Ⅶ-3，产率73％。
Figure PCTCN2021137625-appb-000018
步骤A3-1，氮气氛围下，将2.9mmol化合物Ⅶ-3、30mL无水四氢呋喃(THF)加入圆底烧瓶，将反应体系置于冰水浴中，搅拌中滴加8.7mL浓度为1mol/L的甲基格氏试剂(CH 3MgBr)，室温搅拌2小时后，将反应体系倒入10％HClO 4水溶液中，加入二氯甲烷萃取，收集有机相，加入无水硫酸钠干燥，减压蒸馏浓缩有机相，得到蓝色固体产物Ⅷ-3，产率96％。
Figure PCTCN2021137625-appb-000019
步骤A4-1，氮气氛围下，依次将0.2mmol化合物Ⅷ-3、0.1mmol化合物Ⅸ、8mL甲苯和2mL正丁醇。加热至回流搅拌22小时。减压旋转蒸发浓缩，加入5mL丙酮，搅拌5分钟，过滤，收集黑色固体产物Ⅺ,产率61％。
Figure PCTCN2021137625-appb-000020
请参阅图1，将荧光化合物Ⅺ溶于二氯甲烷，测量其吸收光谱，测试所用的吸收光谱仪的型号为岛津UV-3600Plus。荧光化合物Ⅺ的吸收波段在900-1200纳米，最大吸收波长为1082纳米，荧光化合物Ⅺ的吸收光谱半峰宽比较窄，对光的选择性高。在900-1200纳米波段内，生物组织吸收值低，荧光和光声成像背景低，具有更高的灵敏度
请参阅图2，将荧光化合物Ⅺ溶于二氯甲烷，用980纳米的激光激发，测量其荧光光谱，测试所用的荧光光谱仪的型号为爱丁堡FLS920。荧光化合物Ⅺ的发射波段在1000-1400纳米，最大发射波长为1116纳米，落在近红外二区，生物组织的自荧光较弱，且荧光化合物Ⅺ荧光成像具有较深的穿透深度，使荧光化合物Ⅺ具有更卓越的成像优势。
在一实施方式中，在近红外二区激光照射下，荧光化合物Ⅺ由基态跃迁到激发态，然后以光和热的形式释放能量回到基态，其中以光的形式释放能量的部分，产生荧光信号；以热的形式释放能量的部分，产生光声信号。
本申请一实施方式提供一种探针，该探针包括上述任意一项的荧光化合物。该探针受激光激发后，可产生荧光和光声信号，用于荧光成像和光声成像。
在一种可能的实现方式中，探针还包括双亲性辅料，所述双亲性辅料的一端为亲水基团，所述双亲性辅料的另一端为疏水基团。双亲性辅料包括马来酸酐-1-十八碳烯交替共聚物(PMAO)、泊洛沙姆(聚氧乙烯聚氧丙烯醚嵌段共聚物)、二硬脂酰基磷脂酰乙醇胺-聚乙二 醇(DSPE-mPEG)、聚丙二醇-聚乙二醇-聚丙二醇三嵌段共聚物(PEG-b-PPG-b-PEG)、二油酰基磷脂酰乙醇胺-聚乙二醇(DOPE-mPEG)、苄泽(Brij)类表面活性、牛血清白蛋白(BSA)。双亲性辅料可增加探针在水中的溶解度，使探针更好地应用在生物技术及生命科学领域。
在一实施方式中，双亲性辅料为马来酸酐-1-十八碳烯交替共聚物(PMAO)。PMAO的结构如式Ⅻ所示，其中n为正整数。荧光化合物Ⅰ的水溶性较差，与生物体相容性差，加入PMAO后，一方面，PMAO结构中的烷基长链对荧光化合物Ⅰ进行缠绕包裹，在荧光化合物Ⅰ分子外围形成疏水环境；另一方面，PMAO的酸酐结构在碱性条件下水解后得到如式XIII所示的羧酸盐，从而获得亲水端，实现水溶性。水解后的PMAO的烷基链形成疏水环境，增强了荧光化合物Ⅰ的稳定性和维持了荧光化合物Ⅰ良好的光学性质；亲水性的羧酸盐结构增加了探针的水溶性，从而提高了探针生物相容性。在一实施方式中，PMAO重均分子量(M w)为30000-50000。
Figure PCTCN2021137625-appb-000021
在一种可能的实现方式中，荧光化合物Ⅰ和PMAO的质量比为1∶(5-1000)。在一实施方式中，荧光化合物Ⅰ和PMAO的质量比为1∶(20-1000)。在一实施方式中，荧光化合物Ⅰ和PMAO的质量比为1∶(100-1000)。在一实施方式中，荧光化合物Ⅰ和PMAO的质量比为1∶(100-600)。在一实施方式中，荧光化合物Ⅰ和PMAO的质量比为1∶(200-800)。在一实施方式中，荧光化合物Ⅰ和PMAO的质量比为1∶(400-1000)。在一实施方式中，荧光化合物Ⅰ和PMAO的质量比可以为1∶20、1∶50、1∶100、1∶200、1∶400、1∶600、1∶800、1∶1000。
在一种可能的实现方式中，探针的制备方法包括步骤B1、步骤B2、步骤B3、步骤B4，具体步骤如下。
步骤B1，将荧光化合物Ⅰ和双亲性辅料溶于有机溶剂中得到澄清溶液；在一实施方式中，有机溶剂选自二氯甲烷或三氯甲烷中至少一种；该步骤使荧光化合物Ⅰ和双亲性辅料混合均匀；
步骤B2，除去步骤B1澄清溶液中的有机溶剂得到膜状混合物；
步骤B3，向步骤B2中的膜状混合物加入水，超声后得到探针水溶液；在一实施方式中，该探针水溶液可直接用于荧光和光声成像；在水中PMAO结构中的烷基长链对荧光化合物Ⅰ进行缠绕包裹，得到纳米粒子水溶液；
步骤B4，除去步骤B3中的水，得到探针；该探针为固体纳米粒子，便于保存，将其溶于水后可用于荧光和光声成像。
在一种可能的实现方式中，探针包括荧光化合物Ⅺ和PMAO(M w＝30000-50000)，荧光化合物Ⅺ和PMAO的质量比为1∶100，该探针的制备方法包括步骤B1-1、步骤B2-1、步骤B3-1、步骤B4-1，具体步骤如下所示。
步骤B1-1，将1毫克的荧光化合物Ⅺ和100毫克PMAO，溶于10-50毫升的二氯甲烷或三氯甲烷中得到澄清溶液；
步骤B2-1，减压蒸馏除去步骤B1-1溶液中的有机溶剂得到膜状混合物；
步骤B3-1，向步骤B2-1中膜状混合物加入10-50毫升水，超声后得到探针水溶液；或向步骤B2-1中膜状混合物加入0.1摩尔每升的氢氧化钠水溶液或0.1摩尔每升的4-二甲胺基吡啶水溶液，超声后得到澄清溶液，超滤或透析除去溶液中的氢氧化钠或4-二甲胺基吡啶，得到探针水溶液；
步骤B4-1，减压蒸馏或冷冻干燥除去步骤B3-1中的水，得到探针。
在一实施方式中，在近红外二区激光照射下，探针中的荧光化合物Ⅺ由基态跃迁到激发态，然后以光和热的形式释放能量回到基态，其中以光的形式释放能量的部分，产生荧光信号；以热的形式释放能量的部分，产生光声信号。
在一实施方式中，探针的响应波长处于近红外二区(1000-1700nm)，该波长激光组织穿透能力强，该波段下，血液和组织自发荧光和光散射较弱，因此具有高灵敏度和大成像深度。
在一种可能的实现方式中，如上任意一项所述的探针可用于荧光成像。
在一种可能的实现方式中，如上任意一项所述的探针可用于光声成像。
在一种可能的实现方式中，如上任意一项所述的探针可用于荧光和光声成像。
为了说明本申请荧光化合物的成像效果，本申请还做了探针中荧光化合物Ⅺ和PMAO不同质量比的效果实施例。具体的，控制荧光化合物Ⅺ的浓度不变，按照荧光化合物Ⅺ和PMAO的质量比为1∶100、1∶200、1∶400、1∶600、1∶800、1∶1000的比例分别制得探针P1、探针P2、探针P3、探针P4、探针P5、探针P6。探针P1-P6在水中的吸收光谱图如图3所示，测试所用的吸收光谱仪的型号为INESAL3S。961纳米处的吸收峰为荧光化合物Ⅺ的聚集峰，随着PMAO质量比例的增加，探针在961纳米处的吸收值逐渐降低，说明PMAO的加入使荧光化合物Ⅺ在溶液中更加分散，随着PMAO质量比例的增加，荧光化合物Ⅺ的聚集程度降低，分散程度越高。
控制探针P1-P6在980nm处具有相同的吸收值，以980纳米为激发波长，对探针P1-P6进行荧光光谱测试，探针P1-P6在水中的荧光光谱图如图4所示，探针P1的荧光强度最小，探针P6的荧光强度最大，探针P1、探针P2、探针P3、探针P4、探针P5、探针P6的荧光强依次增加。
控制荧光化合物Ⅺ在水中的浓度不变(50ug/ml)，制备不同比例的探针的水溶液，在1064纳米激光激发下进行荧光成像(如图5所示)，荧光成像所用激光器型号为北京镭志威LWIRPD-1F。荧光化合物Ⅺ和PMAO的质量比在1∶(100-600)范围内，荧光强度随PMAO使用比例的增加而增强；在1∶(600-1000)范围内，荧光强度无明显增强。PMAO的加入，使探针成像时的荧光强度增强，当PMAO和荧光化合物Ⅺ的质量比超过1∶600时，PMAO对探针成像的荧光强度的增强效果不明显。
控制荧光化合物Ⅺ在水中的浓度不变(50ug/ml)，制备不同比例探针的水溶液，在1064纳米脉冲激光激发下进行光声成像，光声成像所用激光器型号为Innolas EVOs OPO-532。荧光化合物Ⅺ和PMAO的质量比在1∶100时光声信号强度最强(如图6所示)，荧光化合物Ⅺ和PMAO的质量比在1∶(200-1000)荧光强度无明显区别。
以荧光化合物Ⅺ和PMAO质量比为1∶1000所制备的探针为例，相同激发条件下，探针荧光信号强度随探针浓度具有线性关系(如图7a和图7b所示)，探针的浓度增大，探针 的荧光信号越强。
以荧光化合物Ⅺ和PMAO质量比为1∶1000所制备的探针为例，相同激发条件下，探针光声信号强度随探针浓度具有线性关系(如图8a和图8b所示)，探针的浓度增大，探针的光声信号越强。
探针具有良好稳定性，以荧光化合物Ⅺ和PMAO质量比为1∶1000所制备的探针为例(如图9所示)，探针的水溶液在4℃避光条件下，第3天的吸收光谱与第一天的吸收光谱基本重叠；与第一天的吸收光谱相比，第8天的吸收光谱无明显变化，最大吸收峰峰值变化小于5％，说明探针具有良好的稳定性。
以上对本申请实施例所提供的荧光化合物和探针进行了详细介绍，本文中应用了具体个例对本申请的原理及实施例进行了阐述，以上实施例的说明只是用于帮助理解本申请的方法及其核心思想；同时，对于本领域的一般技术人员，依据本申请的思想，在具体实施例及应用范围上均会有改变之处，综上所述，本说明书内容不应理解为对本申请的限制。

Claims (10)

  1. 一种荧光化合物，其特征在于，所述荧光化合物的结构通式如式Ⅰ所示：
    Figure PCTCN2021137625-appb-100001
    其中，所述X、Y独立地选自硫原子或氧原子；
    所述R 11-R 14、R 21-R 24、R 31-R 35、R 41-R 45独立地选自氢、取代或未取代的第一烷基、第一取代基和第二取代基，所述第一取代基为OR a或SR b，所述R a、R b独立地选自氢、取代或未取代的第二烷基和取代或未取代的第一酰基中的一种；
    所述第二取代基为N(R c)R d，所述R c、R d独立地选自氢、取代或未取代的第三烷基和取代或未取代的第二酰基中的一种。
  2. 根据权利要求1所述的荧光化合物，其特征在于，所述取代或未取代的第一烷基、所述取代或未取代的第二烷基、所述取代或未取代的第一酰基、所述取代或未取代的第三烷基和所述取代或未取代的第二酰基的碳原子数为1-18。
  3. 根据权利要求1所述的荧光化合物，其特征在于，所述取代或未取代的第一烷基、所述取代或未取代的第二烷基、所述取代或未取代的第三烷基包括被羧酸基、磺酸基、硫酸基、磷酸基、氨基、季铵基中的至少一种取代的烷基；所述取代或未取代的第一酰基、所述取代或未取代的第二酰基包括被羧酸基、磺酸基、硫酸基、磷酸基、氨基、季铵基中的至少一种取代的酰基。
  4. 根据权利要求1所述的荧光化合物，其特征在于，所述荧光化合物的结构通式如式Ⅱ所示：
    Figure PCTCN2021137625-appb-100002
    所述R 1、R 2、R 3、R 4独立地选自氢、所述取代或未取代的第一烷基、所述第一取代基和所述第二取代基。
  5. 根据权利要求4所述的荧光化合物，其特征在于，所述R 1、R 2、R 3、R 4独立地选自所述第一取代基或所述第二取代基，所述第一取代基中，所述R a、R b独立地表示氢、碳原子数为1-4的烷基、碳原子数为1-4的酰基中的一种；所述第二取代基中，所述R c、R d独立地表示氢、碳原子数为1-4的烷基，或所述R c和所述R d其中一个为氢，所述R c和所述R d中另一个为碳原子数为1-4的酰基。
  6. 根据权利要求1所述的荧光化合物，其特征在于，所述荧光化合物Ⅰ的制备方法，包括以下步骤：
    (1)提供或制备如下所示的化合物Ⅲ-1、化合物Ⅲ-2，以及提供或制备化合物Ⅳ；
    Figure PCTCN2021137625-appb-100003
    (2)将所述化合物Ⅲ-1、所述化合物Ⅳ和强碱催化剂混合，反应得到如下所示的化合物Ⅴ-1；将所述化合物Ⅲ-2、所述化合物Ⅳ和强碱催化剂混合，反应得到如下所示的化合物Ⅴ-2；
    Figure PCTCN2021137625-appb-100004
    (3)将所述化合物Ⅴ-1、提供或制备如下所示的化合物Ⅵ-1、多聚磷酸混合反应得到如下所示的化合物Ⅶ-1；将所述化合物Ⅴ-2、提供或制备如下所示的化合物Ⅵ-2、多聚磷酸混合反应得到如下所示的化合物Ⅶ-2；
    Figure PCTCN2021137625-appb-100005
    (4)将所述化合物Ⅶ-1和甲基格氏试剂混合，反应完毕后将得到的混合液倒入高氯酸水溶液中，得到如下所示的化合物Ⅷ-1；将所述化合物Ⅶ-2和甲基格氏试剂混合，反应完毕后将得到的混合液倒入高氯酸水溶液中，得到如下所示的化合物Ⅷ-2；
    Figure PCTCN2021137625-appb-100006
    (5)将所述化合物Ⅷ-1、所述化合物Ⅷ-2、提供或制备化合物如下所示的Ⅸ混合，反应制得所述荧光化合物Ⅰ，
    Figure PCTCN2021137625-appb-100007
  7. 一种探针，其特征在于，所述探针包括如权利要求1-6任意一项所述的荧光化合物。
  8. 根据权利要求7所述的探针，其特征在于，所述探针还包括双亲性辅料，所述双亲性辅料或所述双亲性辅料的水解产物的一端为亲水基团，所述双亲性辅料或所述双亲性辅料的水解产物的另一端为疏水基团。
  9. 根据权利要求8所述的探针，其特征在于，所述双亲性辅料为马来酸酐-1-十八碳烯交替共聚物，所述马来酸酐-1-十八碳烯交替共聚物的重均分子量为30000-50000，所述荧光化合物和所述马来酸酐-1-十八碳烯交替共聚物的质量比为1∶(5-1000)。
  10. 根据权利要求7所述的探针，其特征在于，所述探针的制备方法包括以下步骤：
    (1)将所述荧光化合物和所述双亲性辅料溶于有机溶剂中得到澄清溶液；
    (2)除去步骤(1)所述澄清溶液中的有机溶剂得到膜状混合物；
    (3)向步骤(2)中所述膜状混合物加入水，超声后得到探针水溶液；
    (4)除去步骤(3)中的水，得到探针。
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