A zwitterionic fluorescent compound and its preparation method and application

Abstract

The present invention relates to an amphoteric ion fluorescent compound, a preparation method thereof, and an application thereof. The amphoteric ion fluorescent compound uses a derivative of a semi-cyanine dye structure as a fluorophore, and is respectively connected with an R2 group (—OH, —NH2 or a fragment containing a biomarker response group) and a fragment containing a quaternary ammonium at both ends. It not only has high sensitivity, good biocompatibility and optical stability, but also has good water solubility and renal clearance efficiency through the cooperation with a substitution group or a fragment. As a fluorescent probe, it can more accurately perform early diagnosis of diseases, intraoperative navigation treatment, tissue and organ function evaluation, etc.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and more specifically, to a zwitterionic fluorescent compound and a preparation method and application thereof. Background Art

[0002] The urinary system is composed of kidneys, ureters, bladder, and urethra, and is an important excretion pathway for human metabolic products. During the excretion process, abnormal metabolic waste, foreign microorganisms, drugs, etc. in the body can easily cause infection and damage to surrounding tissues and cells, leading to a series of diseases. The current clinical diagnosis of urinary system diseases often relies on traditional imaging methods. However, these methods have defects such as high ionizing radiation, low sensitivity, invasiveness, and high cost, making it difficult to diagnose early and implement intervention. Compared with traditional imaging technology, optical imaging / detection technology has the advantages of high sensitivity, strong specificity, high safety, and convenience and popularity. Therefore, the use of non-invasive, non-ionizing radiation, highly specific and sensitive fluorescence imaging technology to detect urinary system diseases has excellent application prospects.

[0003] At present, the probes used for kidney disease detection mainly include inorganic nanoprobes and organic molecular probes. The metabolism of inorganic nanoprobes is limited by the pore size of the glomerular basement membrane, and has strict selectivity for the size of nanoprobes. Only inorganic nanoparticles with a hydrated diameter of less than 6nm and a low protein binding rate can be effectively excreted through the kidneys. Secondly, inorganic nanomaterials have a slow metabolism rate and can be captured and accumulated by the endothelial reticular system of the liver when circulating in the body, resulting in long-term, potential biological toxicity. In addition, the preparation scale of inorganic nanoprobes is limited, the price is high, and there are difficulties in the reproducibility and quantification of synthetic production. In contrast, organic molecular probes have the advantages of fast metabolism, high biocompatibility, and modifiable structure. They have a wide range of applications in the biomedical field, including cell imaging, tumor diagnosis and treatment, and clinical intraoperative navigation. The use of organic molecules to construct renal clearance fluorescent probes is mainly achieved by coupling hydrophilic macromolecules (such as polyethylene glycol, cyclodextrin, polysaccharide compounds, etc.) to achieve good water solubility and renal metabolism effects. For example, the patent with publication number CN115947946A provides a hydrophilic organic macromolecule renal clearance type fluorescent probe. However, due to the presence of a large number of complex glycoprotein enzymes in the body, such hydrophilic organic macromolecule labeled molecular probes are easily degraded in the body; in addition, fluorescent coupling labeled hydrophilic macromolecules are usually polymers, so there are often problems such as unstable synthesis process and large differences between different batches, which affect subsequent biological applications.

[0004] Therefore, the development of single-molecule, label-free, renal-cleared fluorescent probes is of great significance for the diagnosis and treatment of urinary system diseases. Summary of the invention

[0005] The primary purpose of the present invention is to overcome the problems of the existing organic macromolecular renal clearance fluorescent probe being easily degraded in vivo and the unstable product batches, and to provide a zwitterionic fluorescent compound.

[0006] A further object of the present invention is to provide a method for preparing the above zwitterionic fluorescent compound.

[0007] A further object of the present invention is to provide the use of the above zwitterionic fluorescent compound or its pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, tautomer in the preparation of fluorescent probes.

[0008] The above-mentioned object of the present invention is achieved by the following technical solutions:

[0009] A zwitterionic fluorescent compound having a structure shown in any one of formulas (I) to (V):

[0010]

[0011]

[0012] Among them, R 1 for -COO - , -SCN, -N 3 or R 2 -OH, -NH 2 , A is a self-eliminating group, B is a biomarker response group; n is an integer of 0 to 50, n1 is an integer of 0 to 20, and n2 is an integer of 0 to 20.

[0013] The present invention uses a specific main structure as a fluorophore (chromophore) and connects a specific R 2 Groups (-OH, -NH 2 or containing a biomarker response group), and a sulfonic acid group and a fragment containing a quaternary ammonium are respectively connected to a specific substitution position at the other end of the main structure to obtain a zwitterionic fluorescent compound. Among them, the fluorophore has a good quantum yield, and the biomarker response group can be activated by the corresponding biomarker, thereby causing changes in the molecular structure and optical properties, generating a stronger fluorescent signal, and can be monitored in real time using fluorescent imaging technology; the zwitterionic fluorescent compound not only has high sensitivity, good biocompatibility and optical stability, but also has good water solubility and renal clearance efficiency through coordination with the substitution group or fragment.

[0014] It should be noted that in formula (I) to formula (V) All of them belong to the structure of indole compounds and can be equivalently replaced in the present invention; Comparison With one more benzene ring, the conjugation effect is enhanced. Both have conjugation effect. As the number of phenyl groups increases, the ultraviolet absorption wavelength gradually increases. Both can also emit fluorescence, and the fluorescence wavelength also gradually increases.

[0015] The zwitterionic fluorescent compounds provided by the present invention can be used as fluorescent probes and prepared into preparations for various modes of administration (intravenous injection, intraperitoneal injection or spraying). Taking the intravenous injection mode as an example, after the zwitterionic fluorescent compounds of the present invention are intravenously injected, since the expression content of biomarkers in healthy tissues is very low, the biomarker response group of the fluorescent probe is not activated, and no change in the probe electron cloud density occurs, so no obvious fluorescent optical signal is detected in healthy tissues. When the urinary system is lesioned, biomarkers are expressed in large quantities, and the zwitterionic fluorescent compounds metabolized by the kidneys are activated, releasing strong fluorescent signals, which can be monitored in real time by fluorescent imaging, and dynamic monitoring of lesions in the urinary system and visual identification of lesion areas can be achieved, providing more accurate guidance for precision surgical treatment, so as to improve surgical efficacy and patient prognosis, and is expected to provide a new auxiliary method for human disease diagnosis and treatment.

[0016] Preferably, the self-eliminating group A is

[0017] It should be understood that the self-eliminating group A is When , the left connection site is connected to the biomarker response group B; the self-eliminating group A is When the left connection site is connected to B; the self-eliminating group A is When , the right joining site is connected to B.

[0018] The biomarker response group B can be selected according to different target biomarkers. The present invention provides a series of different biomarker response groups.

[0019] Preferably, the biomarker response group B is

[0020]

[0021] After research, the relationship between the above biomarker response groups and the specific excitation between biomarkers is as follows:

[0022]

[0023]

[0024]

[0025] Preferably, n is an integer of 1-20.

[0026] Preferably, n1 is an integer of 1-10.

[0027] Preferably, n2 is an integer of 1-10.

[0028] Preferably, it has the following structure:

[0029]

[0030]

[0031] The preparation method of the above zwitterionic fluorescent compound comprises the following steps:

[0032] S1. Compound 1 represented by formula (1) and compound 6 represented by formula (6) undergo condensation reaction to obtain compound 8 represented by formula (8);

[0033] or the compound 1 represented by formula (1) and the compound 7 represented by formula (7) undergo a condensation reaction to obtain the compound 9 represented by formula (9);

[0034] Or the compound 2 represented by formula (2) and the compound 6 represented by formula (6) undergo a condensation reaction to obtain the compound 12 represented by formula (12);

[0035] Or the compound 2 represented by formula (2) and the compound 7 represented by formula (7) undergo a condensation reaction to obtain the compound 13 represented by formula (13);

[0036] Or the compound 3 represented by formula (3) and the compound 6 represented by formula (6) undergo condensation reaction to obtain the compound 16 represented by formula (16);

[0037] Or the compound 3 represented by formula (3) and the compound 7 represented by formula (7) undergo condensation reaction to obtain the compound 17 represented by formula (17);

[0038] Or the compound 4 represented by formula (4) and the compound 6 represented by formula (6) undergo condensation reaction to obtain the compound 20 represented by formula (20);

[0039] Or the compound 4 represented by formula (4) and the compound 7 represented by formula (7) undergo condensation reaction to obtain the compound 21 represented by formula (21);

[0040] Or the compound 5 represented by formula (5) and the compound 6 represented by formula (6) undergo condensation reaction to obtain the compound 24 represented by formula (24);

[0041] Or the compound 5 represented by formula (5) and the compound 7 represented by formula (7) undergo condensation reaction to obtain the compound 25 represented by formula (25);

[0042] S2. Compound 8 undergoes a demethylation protection reaction to obtain compound 10 represented by formula (10);

[0043] Or compound 12 undergoes a demethylation protection reaction to obtain compound 14 represented by formula (14);

[0044] Or compound 16 undergoes a demethylation protection reaction to obtain compound 18 represented by formula (18);

[0045] Or compound 20 undergoes a demethylation protection reaction to obtain compound 22 represented by formula (22);

[0046] Or compound 24 undergoes a demethylation protection reaction to obtain compound 26 represented by formula (26);

[0047] Or compound 9 is subjected to reduction reaction to obtain compound 11 represented by formula (11);

[0048] Or compound 13 is subjected to reduction reaction to obtain compound 15 represented by formula (15);

[0049] Or compound 17 is subjected to reduction reaction to obtain compound 19 represented by formula (19);

[0050] Or compound 21 is subjected to reduction reaction to obtain compound 23 represented by formula (23);

[0051] Or compound 25 is subjected to reduction reaction to obtain compound 27 represented by formula (27);

[0052] S3. Compound 10 and R 2 -H undergoes a substitution reaction to obtain a zwitterionic fluorescent compound as shown in formula (I);

[0053] or compound 14 with R 2 -H undergoes a substitution reaction to obtain a zwitterionic fluorescent compound as shown in formula (II);

[0054] or compound 18 with R 2 -H undergoes a substitution reaction to obtain a zwitterionic fluorescent compound as shown in formula (III);

[0055] or compound 22 with R 2 -H undergoes a substitution reaction to obtain a zwitterionic fluorescent compound as shown in formula (IV);

[0056] or compound 26 with R 2 -H undergoes a substitution reaction to obtain a zwitterionic fluorescent compound as shown in formula (V);

[0057] or compound 11 and R 2 -H undergoes a condensation reaction to obtain a zwitterionic fluorescent compound as shown in formula (I);

[0058] or compound 15 with R 2 -H undergoes a condensation reaction to obtain a zwitterionic fluorescent compound as shown in formula (II);

[0059] or compound 19 with R 2 -H undergoes a condensation reaction to obtain a zwitterionic fluorescent compound as shown in formula (III)

[0060] or compound 23 with R 2 -H undergoes a condensation reaction to obtain a zwitterionic fluorescent compound as shown in formula (IV);

[0061] or compound 27 with R 2 -H undergoes a condensation reaction to obtain a zwitterionic fluorescent compound as shown in formula (V);

[0062]

[0063]

[0064] Preferably, the condensation reaction in step S1 is carried out at a temperature of 45 to 100° C. and for a time of 5 to 12 hours.

[0065] Preferably, the solvent used in the condensation reaction in step S1 is one or more of anhydrous ethanol, anhydrous methanol, N,N-dimethylformamide or acetic anhydride, and the activating agent used is one or more of potassium carbonate, cesium carbonate, sodium acetate or potassium acetate.

[0066] Preferably, the temperature of the demethylation protection reaction in step S2 is 0-30° C., and the time is 5-12 h; the temperature of the reduction reaction in step S2 is 45-100° C., and the time is 5-12 h.

[0067] Preferably, the solvent used in the demethylation protection reaction in step S2 is one or more of ethanol, dichloromethane, methanol or N,N-dimethylformamide, and the activating agent used is one or more of boron tribromide, aluminum chloride, sodium ethanethiolate or trimethylsilane iodide; the solvent used in the reduction reaction in step S2 is one or more of ethanol, methanol, N,N-dimethylformamide or water, and the reducing agent used is one or more of palladium carbon, hydrogen, stannous chloride or iron powder.

[0068] Preferably, the temperature of the substitution reaction in step S3 is 45-100° C., and the time is 5-12 h; the temperature of the condensation reaction in step S3 is 0-45° C., and the time is 8-24 h.

[0069] Preferably, the solvent selected for the substitution reaction in step S3 is one or more of anhydrous methanol, anhydrous N,N-dimethylformamide, anhydrous acetonitrile or anhydrous tetrahydrofuran; the solvent selected for the condensation reaction in step S3 is one or more of anhydrous methanol, anhydrous N,N-dimethylformamide, anhydrous dichloromethane or anhydrous acetonitrile, the condensation reagent selected is one or more of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), dicyclohexylcarbodiimide (DCC) or benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (PyBop), and the activator selected is one or more of potassium carbonate, N,N-diisopropylethylamine, triethylamine, pyridine or trimethoprim.

[0070] Preferably, the compound 2 in step S1 is prepared by the following steps:

[0071] S11. Compound 28 represented by formula (28) undergoes substitution reaction with compound 29 represented by formula (29) to obtain compound 2;

[0072]

[0073] More preferably, the substitution reaction in step S11 is carried out at 80-150° C. for 24-72 hours, and the solvent is one or more of N,N-dimethylformamide, toluene or o-dichlorobenzene.

[0074] Preferably, the compound 3 in step S1 is prepared by the following steps:

[0075] S12. Compound 30 represented by formula (30) undergoes substitution reaction with compound 31 represented by formula (31) to obtain compound 3;

[0076]

[0077] More preferably, the substitution reaction temperature in step S12 is 80-150° C., the time is 24-72 h, and the solvent is one or more of N,N-dimethylformamide, toluene or o-dichlorobenzene;

[0078] Preferably, the compound 7 in step S1 is prepared by the following steps:

[0079] S13. Compound 32 represented by formula (32) undergoes bromination reaction to obtain compound 33 represented by formula (33);

[0080] S14. Compound 33 undergoes a cyclization reaction with compound 34 represented by formula (34) to obtain compound 7;

[0081]

[0082] More preferably, the bromination reaction temperature in step S13 is 0-30° C., the time is 4-12 h, and the solvent is one or more of N,N-dimethylformamide, dichloromethane, tetrahydrofuran or acetonitrile;

[0083] More preferably, the cyclization reaction temperature in step S14 is 0-30° C., the time is 4-12 h, the solvent is one or more of N,N-dimethylformamide, dichloromethane, tetrahydrofuran or acetonitrile, and the activator is one or more of potassium carbonate, cesium carbonate, sodium acetate or potassium acetate.

[0084] The use of the above zwitterionic fluorescent compound or its pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, tautomer in the preparation of fluorescent probes also falls within the protection scope of the present invention.

[0085] Preferably, the fluorescent probe is a renal-cleared fluorescent probe.

[0086] Preferably, the fluorescent probe is a constantly-on fluorescent probe or an excitation fluorescent probe.

[0087] Preferably, the pharmaceutically acceptable salt is hydrochloride, hydrobromide, nitrate, methylnitrate, sulfate, bisulfate, aminosulfate, phosphate, acetate, glycolate, phenylacetate, propionate, butyrate, isobutyrate, valerate, maleate, hydroxymaleate, acrylate, fumarate, malate, tartrate, citrate, salicylate, para-aminosalicylate, glycolate, lactate, enanthate, phthalate, oxalate, succinate, benzoate, o-acetyl At least one of oxybenzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, mandelate, tannate, formate, stearate, ascorbate, palmitate, oleate, pyruvate, bishydroxynaphthoate, malonate, laurate, glutarate, glutamate, propionate lauryl sulfate, methanesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-aminobenzenesulfonate, p-toluenesulfonate (toluenesulfonate) or naphthalene-2-sulfonate.

[0088] Compared with the prior art, the present invention has the following beneficial effects:

[0089] The zwitterionic fluorescent compound provided by the present invention uses a derivative of a hemicyanine dye structure as a fluorophore, modifies a sulfonic acid group on the indole ring, and connects R 2 Groups (-OH, -NH 2Or containing biomarker response groups) and fragments containing quaternary ammonium, not only have high sensitivity, good biocompatibility and optical stability, but also have good water solubility and renal clearance efficiency through coordination with substituted groups or fragments. As fluorescent probes, they can more accurately diagnose diseases at an early stage, navigate treatment during surgery, evaluate tissue and organ functions, etc., and can play an important role in future medical optical examinations, with excellent application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1 Schematic diagram of the preparation method of the zwitterionic fluorescent compound ZWCYOH1 of Example 1;

[0091] Figure 2 Schematic diagram of the preparation method of the zwitterionic fluorescent compound ZWCYOH2 of Example 2;

[0092] Figure 3 Schematic diagram of the preparation method of the zwitterionic fluorescent compound ZWCYOH3 of Example 3;

[0093] Figure 4 The zwitterionic fluorescent compound ZWCYNH of Example 4 2 A schematic diagram of a preparation method of ;

[0094] Figure 5 The zwitterionic fluorescent compound ZWCYNH of Example 5 2 Schematic diagram of the preparation method of P;

[0095] Figure 6 The zwitterionic fluorescent compounds ZWCYOH1, ZWCYOH2, ZWCYOH3, ZWCYNH 2 Ultraviolet absorption spectrum of

[0096] Figure 7 The zwitterionic fluorescent compounds ZWCYOH1, ZWCYOH2, ZWCYOH3, ZWCYNH 2 Fluorescence spectrum of

[0097] Figure 8 The zwitterionic fluorescent compounds ZWCYOH1, ZWCYOH2, ZWCYOH3, ZWCYNH 2 Urine recovery rate within 24 hours after injection;

[0098] Fig. 9 The zwitterionic fluorescent compound ZWCYNH of Example 5 2 P fluorescence spectrum changes in response to in vitro proteases;

[0099] Fig.10The zwitterionic fluorescent compound ZWCYNH of Example 5 2 In vivo imaging of P in a mouse model of acute kidney injury;

[0100] Fig.11 The zwitterionic fluorescent compound ZWCYNH of Example 5 2 Ex vivo organ imaging of P in a mouse model of acute kidney injury. DETAILED DESCRIPTION

[0101] In order to more clearly and completely describe the technical solution of the present invention, the present invention is further described in detail through specific embodiments below. It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention. Various changes can be made within the scope of the rights of the present invention.

[0102] Example 1

[0103] This embodiment provides a zwitterionic fluorescent compound, and the schematic diagram of the preparation method thereof is shown in Figure 1 , specifically including the following steps:

[0104] 1. Take a 100mL double-necked round-bottom flask, under an argon atmosphere, add anhydrous dichloromethane (30mL), N,N-dimethylformamide (6mL, 77.5mmol), phosphorus tribromide (2mL, 21.4mmol) in sequence, stir at room temperature for 30min, add compound A1 (2mL, 19.3mmol), and stir at room temperature overnight. After the reaction is completed, slowly add the reaction solution dropwise to an ice-cold saturated sodium bicarbonate solution and spin dry. Extract with ethyl acetate three times, dry with anhydrous sodium sulfate, and spin dry to obtain a light yellow to brown oily liquid compound B1. No further purification is required, and it can be directly put into the next step of reaction.

[0105] 2. Take a 100mL double-necked round-bottom flask, under an argon atmosphere, add compound C1 (741mg, 4.9mmol), cesium carbonate (3.9g, 12.1mmol), compound B1 (764mg, 4.1mmol), N,N-dimethylformamide (20mL) in sequence, and stir at room temperature overnight. After the reaction is completed, spin dry, extract with ethyl acetate three times, dry with anhydrous sodium sulfate, and spin dry. The crude product is separated and purified by column chromatography, and the elution system is petroleum ether: ethyl acetate = 15:1. Collect the purified product and spin dry to obtain a light yellow to brown solid compound D1 (560mg, yield 58%). 1 H NMR (400 MHz, CDCl 3): δ10.32(s,1H),7.08(d,J=9.1Hz,1H),6.69–6.63(m,3H),3.84(s,2H),2. 60–2.54(m,1H),2.47–2.41(m,1H),1.74–1.68(m,1H).LRMS(ESI)m / z:[M+H] + Calcd for C 15 H 15 O 3 243.10;Found 243.10.

[0106] 3. Take a 100mL round-bottom flask, add compound E1 (3.76g, 20mmol), acetic acid (30mL), compound F1 (3.2mL, 30mmol), anhydrous sodium acetate (3.2g, 38mmol) in sequence, heat to 80°C in an oil bath, and react overnight. After the reaction is completed, no treatment is required, and the sample is directly loaded on the column chromatography, and the target product is eluted with dichloromethane: methanol = 15:1. Spin dry to obtain pink powder compound G1 (4.0g, yield 84%). 1 HNMR (400 MHz, DMSO-d 6 ): δ7.63(s,1H),7.55(d,J=7.9Hz,1H),7.34(d,J=7.9Hz,1H),2.21(s,3H),1.25(s,6H).LRMS(ESI)m / z:[M+H] + Calcd for C 11 H 14 NO 3 S240.07; Found 240.19.

[0107] 4. Take a 100mL round-bottom flask, add compound G1 (1.5g, 6.0mmol), compound H1 (3.1g, 12.0mmol), and toluene (35mL) in sequence, heat to 100°C in an oil bath, and react for 24h. After the reaction is completed, spin dry and wash with dichloromethane to obtain pink powder compound I1. No further purification is required and it can be directly used in the next step.

[0108] 5. Take a 50mL round-bottom flask, under an argon atmosphere, add compound I1 (170mg, 0.5mmol), compound D1 (137mg, 0.6mmol), anhydrous sodium acetate (246mg, 3.0mmol), and anhydrous ethanol (15mL) in sequence, heat the oil bath to 55°C, and react overnight. After the reaction is completed, spin dry, add boron tribromide (1.5mL, 15.6mmol) under an ice bath, and continue to stir overnight. After the reaction is completed, slowly add the reaction solution dropwise to an ice-cold saturated sodium bicarbonate solution, spin dry, and separate by high performance liquid chromatography to obtain a green powder compound J1 (90mg, yield 33%), which is a zwitterionic fluorescent compound, denoted as ZWCYOH1. 1 H NMR (400 MHz, DMSO-d 6 ): δ11.02(s,1H),8.62(d,J=14.6Hz,1H),7.91(s,1H),7.73(d,J=8.2Hz,1H ),7.68(s,1H),7.56(dd,J=16.6,8.5Hz,2H),7.02(s,1H),6.90(dd,J=8.5, 2.2Hz,1H),6.45(d,J=14.6Hz,1H),4.37(t,J=7.3Hz,2H),3.08(s,9H),2.8 0–2.67(m,4H),2.21(s,2H),1.78(s,6H),1.23(s,4H).LRMS(ESI)m / z:[M+H] + Calcd for C 31 H 38 N 2 O 5 S550.25; Found 549.92.

[0109] The structural formula of the zwitterionic fluorescent compound provided in this embodiment is as follows:

[0110] It can be used as a always-on fluorescent probe.

[0111] Example 2

[0112] This embodiment provides a zwitterionic fluorescent compound, and the schematic diagram of the preparation method thereof is shown in Figure 2 , specifically including the following steps:

[0113] 1. Take a 50mL round-bottom flask and add compound A2 (576mg, 4.0mmol), tert-butanol (15mL), potassium tert-butoxide (538mg, 4.8mmol), and compound B2 (0.4mL, 4.0mmol) in sequence. Stir at 70℃ overnight. After the reaction is completed, spin dry, extract three times with n-hexane, take the aqueous phase, adjust the solution pH to 1, spin dry, and dry. Add methanol (5mL) to dissolve, filter, take the filtrate, and directly put it into the next step.

[0114] 2. Take a 50mL round-bottom flask, add the methanol solution of compound C2 (i.e., the filtrate obtained in step 1), and then add 1mol / L sodium hydroxide solution (14mL, 14.0mmol). Heat the oil bath to 70°C and react overnight. After the reaction is completed, spin dry and adjust the solution pH to 1, spin dry, and dry. Add 20mL acetone to re-dissolve, filter, and spin dry the filtrate to obtain compound D2 (300mg, yield 36%). LRMS (ESI) m / z: [M+H] + Calcd for C 8 H 17 O 4 S209.08; Found 209.12.

[0115] 3. Take a 50mL round-bottom flask and add compound D2 (300mg, 1.4mmol), compound E2 (188mg, 1.0mol), and acetic acid (10mL) in sequence. Heat the oil bath to 95°C and reflux overnight. After the reaction is completed, wash with isopropanol solution, filter, and take the filter residue as compound F2 (176mg, yield 48%). LRMS (ESI) m / z: [M+H] + Calcd for C 14 H 19 NO 6 S 2 362.07; Found 362.23.

[0116] 4. Take a 100mL round-bottom flask, add compound F2 (1.3g, 3.0mmol), o-dichlorobenzene (30mL), compound H1 (3.2g, 12.0mmol), sodium iodide (900mg, 6mmol) in sequence, heat to 100℃ in an oil bath, and react for 24h. After the reaction is completed, spin dry, add ethyl acetate to wash, and obtain compound H2. No further purification is required, and it can be directly used in the next step.

[0117] 5. Take a 100mL double-necked round-bottom flask, add compound H2 (567mg, 1.2mmol), compound D1 (410mg, 1.8mmol, prepared in Example 1), anhydrous sodium acetate (590mg, 2.4mmol), and anhydrous ethanol (30mL) in sequence, heat to 55°C in an oil bath, and react overnight. After the reaction is completed, spin dry, add boron tribromide (1.5mL, 15.6mmol) in an ice bath, and continue stirring overnight. After the reaction is completed, slowly add the reaction solution dropwise to an ice-cold saturated sodium bicarbonate solution, spin dry, and separate by high performance liquid chromatography to obtain green powder compound I2 (249mg, yield 31%), which is a zwitterionic fluorescent compound, recorded as ZWCYOH2. 1 HNMR (500MHz, Methanol-d 4 ): δ8.55(s,1H),7.91(d,J=10.4Hz,1H),7.80(s,2H),7.53(d,J=9.0Hz,1H),7.41(d,J=8.6Hz,1H),6.86 (d,J=9.0Hz,2H),6.77(s,2H),6.61(d,J=8.1Hz,2H),6.29(d,J=13.5Hz,3H),4.57(s,1H),4.27–4.20(m, 1H),3.85(s,1H),3.67(s,1H),3.19(dd,J=26.2,16.2Hz,1H),2.82(s,1H),2.66(d,J=23.7Hz,1H),2.33( s,1H),1.95(s,1H),1.81(s,1H),1.60(s,1H),1.31(d,J=14.9Hz,1H),1.14(s,1H).LRMS(ESI)m / z:[M+H] + Calcd for C 34 H 43 N 2 O 8 S 2 671.25; Found 671.77.

[0118] The structural formula of the zwitterionic fluorescent compound provided in this embodiment is as follows:

[0119] It can be used as a always-on fluorescent probe.

[0120] Example 3

[0121] This embodiment provides a zwitterionic fluorescent compound, and the schematic diagram of the preparation method thereof is shown in Figure 3 , specifically including the following steps:

[0122] 1. Take a 100mL round-bottom flask, add compound A3 (10.3g, 65.0mmol), water (16mL), sodium hydroxide (2.6g, 65.0mmol) in sequence, and stir at room temperature for 5h. After the reaction is completed, stand and separate. Collect the organic phase in a new 100mL round-bottom flask, add 1,2-dichloroethane (20mL) and compound B3 (8.5mL, 97.5mmol), heat the oil bath to 50℃, and react overnight. After the reaction is completed, add ethyl acetate for washing to obtain compound C3 (13.7g, yield 87%). 1 HNMR (400MHz, D 2 O): δ3.64(s,2H),3.44(dd,J=8.4,5.6Hz,4H),3.07(s,6H),2.91(s,2H),2.19(dd,J=11.9,7.1Hz,4H).

[0123] 2. Take a 100 mL round-bottom flask, add compound G1 (4.0 g, 16.0 mmol, prepared in Example 1), o-dichlorobenzene (30 mL), compound C3 (4.0 g, 12.0 mmol), sodium iodide (1.8 g, 12 mmol) in sequence, heat the oil bath to 100 ° C, and react for 24 hours. After the reaction is completed, spin dry, separate by high performance liquid chromatography, and obtain pink solid compound D3 (3.0 g, yield 42%). 1 HNMR (500MHz, Methanol-d 4 ): δ8.23–8.09(m,2H),8.06(s,1H),4.84–4.61(m,1H),3.88–3.76(m,2H),3.72–3.55(m,1H),3.46–3.37(m,1H), 3.27–3.21(m,1H),2.92(s,1H),2.60(d,J=47.6Hz,1H),2.32(d,J=6.4Hz,1H),1.74(s,4H).LRMS(ESI)m / z:[MH] - Calcd for C 19 H 29 N 2 O 6 S 2 445.15; Found 445.22.

[0124] 3. Take a 100mL double-necked round-bottom flask, add compound D3 (760mg, 1.7mmol), compound D1 (470mg, 2.1mmol, prepared in Example 1), anhydrous sodium acetate (845mg, 10mmol), N,N-dimethylformamide (5mL) in sequence, and heat to 55°C in an oil bath to react overnight. After the reaction is completed, spin dry, add boron tribromide (1.5mL, 15.6mmol) in an ice bath, and continue stirring overnight. After the reaction is completed, slowly add the reaction solution dropwise to an ice-cold saturated sodium bicarbonate solution, spin dry, and separate by high performance liquid chromatography to obtain green powder compound E3 (379mg, yield 34%), which is a zwitterionic fluorescent compound, recorded as ZWCYOH3. 1 H NMR (500MHz, Methanol-d 4 ): δ8.29(d,J=13.7Hz,1H),7.86(s,1H),7.82(d,J=7.8Hz,1H),7.78(d,J=6.7 Hz,1H),7.45(d,J=9.0Hz,1H),7.22(d,J=8.3Hz,1H),6.76(dd,J=9.1,1.6Hz,1 H),6.56(s,1H),6.04(d,J=13.5Hz,1H),3.63–3.54(m,6H),3.16(s,6H),2.83 (dd,J=23.2,16.7Hz,12H),2.26(dd,J=20.9,7.4Hz,6H).LRMS(ESI)m / z:[M+H] + Calcd for C 33 H 41 N 2 O 8 S 2 657.23;Found 657.68.

[0125] The structural formula of the zwitterionic fluorescent compound provided in this embodiment is as follows:

[0126] It can be used as a always-on fluorescent probe.

[0127] Example 4

[0128] This embodiment provides a zwitterionic fluorescent compound, and the schematic diagram of the preparation method thereof is shown in Figure 4 , specifically including the following steps:

[0129] 1. Take a 100mL double-necked round-bottom flask, and under an argon atmosphere, add compound A4 (500mg, 3.0mmol), cesium carbonate (3.6g, 10.8mmol), N,N-dimethylformamide (20mL), and compound B1 (1.4g, 5.0mmol, prepared in Example 1) in sequence. The reaction was stirred at room temperature overnight. Extracted with ethyl acetate three times, dried over anhydrous sodium sulfate, and then spin-dried. The crude product was separated and purified by column chromatography, and the elution system was petroleum ether: ethyl acetate = 10:1. The eluted and purified product was collected and spin-dried to obtain a light yellow to brown solid compound B4 (629mg, yield 82%). 1 H NMR (400 MHz, CDCl 3 ): δ10.38(s,1H),7.93(d,J=10.2Hz,2H),7.28(d,J=8.2Hz,1H),6.70(s,1H),2. 68–2.62(m,2H),2.47(t,J=5.7Hz,2H),1.80–1.73(m,2H).LRMS(ESI)m / z:[M+H] + Calcd for C 14 H 11 NO 4 258.08;Found 258.26.

[0130] 2. Take a 100mL double-necked round-bottom flask, under an argon atmosphere, add compound D3 (1.0g, 2.2mmol, prepared in Example 3), compound B4 (310mg, 1.2mmol), anhydrous sodium acetate (590mg, 7.2mmol), N,N-dimethylformamide (15mL), and acid anhydride (15mL) in sequence, and heat the oil bath to 55°C to react overnight. After the reaction is completed, spin dry. Add methanol (30mL) and stannous chloride (4.7g, 25.0mmol) in concentrated hydrochloric acid solution, heat the oil bath to 55°C to react overnight. After the reaction is completed, add saturated sodium bicarbonate solution dropwise until the pH reaches 8. Spin dry, separate by high performance liquid chromatography, and obtain green powder compound C4 (366mg, yield 56%), which is a zwitterionic fluorescent compound, denoted as ZWCYNH 2 . 1 H NMR (500MHz, Methanol-d 4): δ8.50(d,J=13.5Hz,1H),7.78(dd,J=30.2,13.5Hz,2H),7.43(d,J=8.7Hz,1H),7.28(d,J=8.1Hz,1H ),7.16(s,1H),7.01(s,1H),6.83(d,J=8.5Hz,1H),6.14(d,J=13.7Hz,1H),3.60–3.46(m,4H),3.07(s, 3H), 2.79 (d, J = 12.3Hz, 2H), 2.72 (d, J = 22.0Hz, 2H), 2.11 (t, J = 7.2Hz, 3H), 1.86 (s, 2H), 1.74 (d, J = 8. 9Hz,2H),1.51(dd,J=14.1,7.1Hz,3H),1.47–1.37(m,3H),1.21(d,J=34.8Hz,6H).LRMS(ESI)m / z:[MH] - Calcd for C 33 H 40 N 3 O 7 S 2 654.24;Found 654.20.

[0131] The structural formula of the zwitterionic fluorescent compound provided in this embodiment is as follows:

[0132] It can be used as a always-on fluorescent probe.

[0133] Example 5

[0134] This embodiment provides a zwitterionic fluorescent compound, which is in the ZWCYNH of embodiment 4. 2 The biomarker response group is connected to the above to obtain a schematic diagram of its preparation method. Figure 5 , specifically including the following steps:

[0135] 1. Take a 50mL round-bottom flask and add compound A5 (130mg, 0.2mmol), O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (140mg, 0.4mmol), anhydrous N,N-dimethylformamide (15mL) and N,N-diisopropylethylamine (30μL, 0.2mmol) in sequence. After stirring at room temperature for 20 minutes, add compound C4 (50mg, 0.08mmol) obtained in Example 4, continue stirring at room temperature, and react overnight. After the reaction is completed, spin dry, no other treatment is required, and it is put into the next step.

[0136] 5. Take a 25mL round-bottom flask, add compound B5 and trifluoroacetic acid (1mL, 13mmol). Stir for 8h. After the reaction is completed, spin dry and separate by high performance liquid chromatography to obtain blue powder compound C5 (26mg, yield 29%), which is a zwitterionic fluorescent compound, denoted as ZWCYNH 2 P. 1 H NMR (500 MHz, D 2 O): δ8.58(d,J=14.3Hz,1H),8.44(s,1H),7.95(s,1H),7.89(d,J=8.3Hz,1H),7.81(s,1H),7.38(d,J=8.3Hz,1H),7.33–7.25(m,2H) ,6.32(d,J=14.5Hz,1H),4.71(dd,J=9.7,6.1Hz,363H),4.64–4.56(m,5H),4.42–4.32(m,3H),4.22(dd,J=13.7,7.5Hz,4H),4.02(d, J=7.5Hz,1H),3.69(d,J=7.2Hz,2H),3.66–3.59(m,2H),3.56–3.43(m,4H),3.10(s,2H),2.92(dd,J=18.2,11.0Hz,2H),2.71–2.58(m ,4H),2.36–2.13(m,6H),1.99(s,1H),1.72(s,2H),1.57(d,J=4.4Hz,2H),1.40–1.20(m,6H),0.85–0.71(m,4H).LRMS(ESI)m / z:[MH] - Calcd for C 54 H 72 N 7 O 17 S 2 1154.45;Found1154.45.

[0137] The zwitterionic fluorescent compound provided in this embodiment has the following structural formula:

[0138] It can be used as an activated fluorescent probe.

[0139] Performance Testing

[0140] 1. Spectral test: prepare 0.05 mg / mL constant-bright fluorescent probes ZWCYOH1, ZWCYOH2, ZWCYOH3, and ZWCYNH 2PBS solution and methanol solution. The absorption spectrum of each sample at 400-900nm was measured by UV spectrometer, and the fluorescence spectrum of each sample was measured by fluorescence spectrometer (excitation wavelength: 660nm). The test results are as follows Figure 6 and Figure 7 As shown, since the fluorophores of each probe are all hemicyanine structures, there is no obvious difference in the maximum absorption and maximum emission wavelengths of the spectrum.

[0141] 2. Renal clearance efficiency test: prepare 0.05 mg / mL constant-bright fluorescent probes ZWCYOH1, ZWCYOH2, ZWCYOH3, and ZWCYNH 2 PBS solution. Twelve Balb / c mice were randomly divided into three groups, with three mice in each group. 75 μL of probe solution was injected into the tail vein of the mice. After injection, the mice were placed individually in a clean metabolic cage, and urine was collected within 24 hours and the volume was recorded. A standard curve of probe concentration-chromatographic peak area was prepared by high performance liquid chromatography to calculate the recovery rate of the probe in mouse urine.

[0142] Test results such as Figure 8 As shown. ZWCYOH1, ZWCYOH2, ZWCYOH3, ZWCYNH 2 All of them can be metabolized through the urinary system and exist in the urine. Among them, the constant-bright probes ZWCYOH2, ZWCYOH3, and ZWCYNH 2 The charge distribution of the three is more balanced, and the urine recovery rates of the three are higher, which are 88%, 91% and 93% respectively, which proves that the zwitterionic fluorescent probe provided by the present invention has good renal clearance efficiency and can be used to detect urinary system diseases.

[0143] ZWZ 2 The results of the renal clearance test of P were similar, with a recovery rate of 91%.

[0144] 3. Test of the responsiveness of the activation probe: 10 μM of the activation probe ZWCYNH 2 The P solution was incubated with the apoptotic protease caspase-8 in a buffer solution at 37°C. The fluorescence spectrum of the solution was measured. The test results are shown in Fig. 9 As shown, the polypeptide sequence of the probe is recognized and cleaved by caspase-8 protease, the fluorescence emission wavelength of the solution is red-shifted, and the fluorescence intensity increases.

[0145] 4. Establishment of acute kidney injury mouse model: 18 Balb / c mice were randomly divided into 6 groups, namely blank control group (control group), NAC control group, model group (12h group, 24h group, 48h group, 72h group), three mice in each group. (1) Control group: intraperitoneal injection of 0.4mL normal saline; (2) AKI (acute kidney injury) model group: intraperitoneal injection of 20mg / kg cisplatin solution, and imaging examination was performed at 12h, 24h, 48h, and 72h after injection; (3) NAC group: tail vein injection of 400mg / kg NAC solution, 30min later, intraperitoneal injection of 20mg / kg cisplatin solution, and imaging examination was performed at 48h after cisplatin injection. Finally, the mice were euthanized and imaging of each organ was performed.

[0146] The test results are as follows Fig.10 As shown, the probe ZWCYNH 2 After P was injected into the body, the expression of caspase-8 was relatively low in the control group and the 12h group because the kidneys were normal and without lesions. The fluorescence intensity of the probe was also low. As the time after the injection of cisplatin increased, cisplatin gradually damaged the kidney tissue, induced acute kidney injury, and caused the expression of caspase-8 protein in the kidney to gradually increase. When the probe was injected into the body, its polypeptide sequence was recognized and cut by the caspase-8 protease, exposing the free amino group, restoring the "D-π-A" structure of the probe, and showing extremely strong fluorescence.

[0147] The test results are as follows Fig.11 As shown, the signal of the liver is weaker than that of the kidney, proving that the probe is mainly metabolized by the kidney rather than the liver. At the same time, the kidney signal of each group increases with the extension of cisplatin injection time, that is, the expression of capase-8 protein gradually increases, which can achieve early diagnosis of the disease.

[0148] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. An amphoteric ionic fluorescent compound, characterized in that, it has the structure shown in formula (Ⅲ): Formula (Ⅲ), Among them, R 1 for ; R 2 for , B is the biomarker response group; n is an integer from 1 to 20, n 2 is an integer from 1 to 10; The biomarker response group B is .

2. The amphoteric ionic fluorescent compound according to claim 1, characterized in that, it has the structure shown as follows: 。 3. A preparation method of the amphoteric ionic fluorescent compound according to claim 2, characterized in that, it comprises the following steps: S1. Take a 100 mL two-necked round-bottom flask. Under an argon atmosphere, sequentially add 2.2 mmol of compound D3, 1.2 mmol of compound B4, 7.2 mmol of anhydrous sodium acetate, 15 mL of N,N-dimethylformamide, and 15 mL of acid anhydride. Then, heat in an oil bath to 55 °C and react overnight; after the reaction is completed, spin-dry; add 30 mL of methanol and a stannous chloride concentrated hydrochloric acid solution containing 25.0 mmol of stannous chloride, heat in an oil bath to 55 °C and react overnight; after the reaction is completed, dropwise add saturated sodium bicarbonate solution until the pH reaches 8; spin-dry and separate by high performance liquid chromatography to obtain the green powder compound C4; S2. Take a 50 mL round-bottom flask and sequentially add 0.2 mmol of compound A5, 0.4 mmol of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 15 mL of anhydrous N,N-dimethylformamide, and 0.2 mmol of N,N-diisopropylethylamine; stir at room temperature for 20 minutes, then add 0.08 mmol of compound C4, continue to stir at room temperature, and react overnight; after the reaction ends, spin-dry to obtain compound B5, without other treatment, and directly put it into the next step; S3. Take a 25 mL round-bottom flask, add compound B5 and 13 mmol of trifluoroacetic acid; stir for 8 h; after the reaction ends, spin-dry and separate by high performance liquid chromatography to obtain the amphoteric ionic fluorescent compound according to claim 2; 。 4. Use of the amphoteric ionic fluorescent compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof in the preparation of a fluorescent probe.

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