Aminopeptidase N fluorescent probe as well as preparation method and application thereof

Abstract

The invention provides a fluorescent probe compound which is novel in structure and shown in a formula (I) and application of the fluorescent probe compound in detection of aminopeptidase N. The fluorescent probe compound can detect the aminopeptidase N in an aqueous solution system, has specificity, excellent selectivity, high sensitivity and the like on detection of the aminopeptidase N and can be used for quantitative detection of the aminopeptidase N. The preparation method of the fluorescent probe compound as shown in the formula (I) is simple in synthesis step, good in biocompatibility, short in reaction time, convenient to purify and simple in process.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical detection, and specifically discloses an aminopeptidase N fluorescent probe, a preparation method thereof, and an application thereof. Background Art

[0002] Aminopeptidase N, also known as alanine aminopeptidase or CD13, is a zinc ion-dependent exopeptidase that can hydrolyze neutral or basic amino acids at the N-terminus of protein polypeptide chains, activating a series of important in vivo biochemical reactions. Aminopeptidase N is widely present in mammals, exists in the cell membrane in the form of a homodimer, and has a variety of important physiological functions in the human body. When other urinary proteins are at normal levels, the presence of aminopeptidase N in urine indicates early kidney injury. Drug-induced liver injury is considered to be the main cause of acute liver injury, and abnormal expression of some enzymes such as aminopeptidase and oxidoreductase may be a precursor of drug-induced liver injury. In addition, aminopeptidase N is also a promising cancer biomarker because it exhibits enhanced enzyme activity in cancer cells. Therefore, the development of a highly sensitive and highly selective real-time detection method for aminopeptidase N will promote the diagnosis and pathophysiological research of aminopeptidase N-related diseases.

[0003] Currently, methods for detecting aminopeptidase N include colorimetric method, high performance liquid chromatography, nuclear magnetic resonance method, etc. However, these analytical methods often have disadvantages such as poor sensitivity, long response time, large amount of sample required, and destructive detection. The fluorescent probe method has attracted much attention due to its advantages such as high sensitivity, rapid response, and suitability for detecting trace samples. Currently, the number of fluorescent probes for detecting aminopeptidase N is limited, and they often have disadvantages such as cumbersome synthesis and poor biocompatibility. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a fluorescent probe compound shown in the following formula (I):

[0005]

[0006] Wherein, R1 and R2 are the same or different, and are independently selected from the following groups: H, halogen (such as F, Cl, Br, I), unsubstituted or optionally substituted by one, two or more R s substituted C 1-10 alkyl, C 1-10 alkoxy;

[0007] R s is selected from H, C 1-10 alkyl, C 1-10 alkoxy, halogen;

[0008] R3 is selected from H, halogen, C 1-6 alkyl, C 1-6 alkoxy;

[0009] R4 is selected from H, halogen, C 1-6 alkyl, C 1-6 alkoxy;

[0010] m and n are the same or different and are each independently selected from 0, 1, 2, 3, 4.

[0011] According to an embodiment of the present invention, R1 and R2 are the same or different and are each independently selected from the following groups: H, halogen, unsubstituted or optionally substituted by one, two or more R s substituted C 1-6 alkyl, C 1-6 alkoxy; preferably, R1 and R2 are the same or different and are each independently selected from the following groups: H, halogen, unsubstituted or optionally substituted by one, two or more R s substituted C 1-3 alkyl, C 1-3 alkoxy.

[0012] According to an embodiment of the present invention, R s is selected from H, C 1-6 alkyl, C 1-6 alkoxy, halogen; preferably, R s is selected from the following groups: H, halogen, C 1-3 alkyl, C 1-3 alkoxy; more preferably, R s is selected from the following groups: H, halogen.

[0013] According to an embodiment of the present invention, R1 is selected from the following groups: H, halogen, C 1-6 alkyl, C 1-6 alkoxy; preferably, R1 is selected from the following groups: H, halogen, C 1-3 alkyl, C 1-3 alkoxy; exemplified by H, halogen, methyl, ethyl, propyl, methoxy, ethoxy, propoxy.

[0014] According to an embodiment of the present invention, R2 is selected from the following groups: H, halogen, C 1-6 alkyl, C 1-6 alkoxy; preferably, R2 is selected from the following groups: H, halogen, C 1-3 alkyl, C 1-3 alkoxy; exemplified by H, halogen.

[0015] According to an embodiment of the present invention, R3 is selected from H, halogen, C 1-4 alkyl, C 1-4 alkoxy; exemplified by H, halogen, methyl, ethyl, propyl, butyl, isobutyl, methoxy, ethoxy, propoxy, butoxy.

[0016] According to an embodiment of the present invention, R4 is selected from H, halogen, C 1-4 alkyl, C 1-4 alkoxy; exemplary are H, halogen, methyl, ethyl, propyl, butyl, isobutyl, methoxy, ethoxy, propoxy, butoxy.

[0017] According to an embodiment of the present invention, the compound of formula (I) is selected from the following compounds:

[0018]

[0019] The present invention also provides a preparation method of the above-mentioned fluorescent probe compound of formula (I), and the preparation method includes the following steps:

[0020] (a) React the compound shown in formula (II) with the compound shown in formula (III) to obtain the compound shown in formula (IV);

[0021] (b) React the compound shown in formula (IV) with R5-NH-CH(R4)-COOH to obtain the compound shown in formula (V);

[0022] (c) React the compound shown in formula (V) with an acid to obtain the compound shown in formula (I);

[0023] The reaction route is as follows:

[0024]

[0025] wherein, R1, R2, R3, R4, m, n have the definitions as described above;

[0026] R5 is an amino protecting group, such as a Boc protecting group.

[0027] According to an embodiment of the present invention, in step (a), the reaction is carried out in the presence of a solvent; preferably, the solvent is an organic solvent, such as ethanol.

[0028] According to an embodiment of the present invention, in step (a), the reaction is carried out in the presence of a base; preferably, the base is an inorganic base; the inorganic base can be selected from one or more of sodium hydroxide and potassium hydroxide, for example.

[0029] According to an embodiment of the present invention, in step (a), the molar ratio of the compound shown in formula (II) to the compound shown in formula (III) is (1 to 5):1, preferably 2:1.

[0030] According to an embodiment of the present invention, in step (a), the reaction temperature is 20°C - 80°C, such as 80°C.

[0031] According to an embodiment of the present invention, in step (a), the reaction time is 2 h - 24 h, for example, 12 h.

[0032] According to an embodiment of the present invention, in step (b), the reaction is carried out in a solvent; preferably, the solvent is an organic solvent; for example, dichloromethane.

[0033] According to an embodiment of the present invention, in step (b), the reaction is carried out in the presence of a catalyst; the catalyst can be, for example, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU).

[0034] According to an embodiment of the present invention, in step (b), the reaction is carried out in the presence of a base; preferably, the base is an organic base; the organic base can be, for example, N,N-diisopropylethylamine (DIEA).

[0035] According to an embodiment of the present invention, in step (b), the reaction temperature is 10 °C - 40 °C, for example, room temperature.

[0036] According to an embodiment of the present invention, in step (b), the reaction time is 1 h to 12 h, for example, 8 h.

[0037] According to an embodiment of the present invention, in step (b), the molar ratio of the compound represented by formula (IV) to R5-NH-CH(R4)-COOH is 1:(1 - 5), preferably 1:2.

[0038] According to an embodiment of the present invention, in step (b), the molar ratio of the compound represented by formula (IV) to the catalyst is 1:(1 - 5), preferably 1:2.

[0039] According to an embodiment of the present invention, in step (b), the molar ratio of the compound represented by formula (IV) to the organic base is 1:(2 - 10), preferably 1:3.

[0040] According to an embodiment of the present invention, in step (c), the reaction is carried out in the presence of a solvent; preferably, the solvent is an organic solvent; more preferably, the reaction is carried out in a mixed solvent of dichloromethane and trifluoroacetic acid; in the mixed solvent, the volume ratio of the two is preferably 1:(0.2 - 5), for example, 1:1.

[0041] According to an embodiment of the present invention, in step (c), the reaction temperature of the reaction is 10 °C - 40 °C, for example, room temperature.

[0042] According to an embodiment of the present invention, in step (c), the reaction time is 0.1 h to 12 h, for example, 2 h.

[0043] The present invention also provides the use of the fluorescent probe compound represented by the above formula (I) in detecting the content of aminopeptidase N in a sample.

[0044] The present invention also provides the use of the fluorescent probe compound represented by the above formula (I) in preparing a reagent or kit for detecting the content of aminopeptidase N in a sample.

[0045] According to an embodiment of the present invention, the sample is, for example, urine, water sample, cell, tissue, etc.

[0046] According to an embodiment of the present invention, the detection includes visual qualitative detection, fluorescence detection, and cell imaging detection.

[0047] According to an embodiment of the present invention, the detection includes qualitative detection and quantitative detection.

[0048] According to an embodiment of the present invention, the detection can be carried out in a variety of buffer solutions, such as PBS buffer solution, Tris-HCl buffer solution, etc. According to an embodiment of the present invention, the buffer solution contains bovine serum albumin (BSA); preferably, the concentration of bovine serum albumin is 0.5 mg / mL - 5.0 mg / mL, such as 1 mg / mL. According to an embodiment of the present invention, the pH of the buffer solution is 7.4.

[0049] The present invention also provides the use of the fluorescent probe compound represented by the above formula (I) in preparing an aminopeptidase N imaging agent. Preferably, it is used in preparing an imaging agent for aminopeptidase N in cells and / or bacteria.

[0050] According to an embodiment of the present invention, the above application is for non-disease diagnosis or treatment purposes.

[0051] The present invention also provides the use of the compound represented by formula (IV) in preparing the fluorescent probe compound represented by the above formula (I),

[0052]

[0053] wherein, R1, R2, R3, m, and n have the definitions described above.

[0054] The present invention also provides the use of the compound represented by formula (V) in preparing the fluorescent probe compound represented by the above formula (I),

[0055]

[0056] wherein, R1, R2, R3, R4, R5, m, and n have the definitions described above.

[0057] Beneficial effects

[0058] The present invention provides a fluorescent probe compound of formula (I) with a novel structure and its application in detecting aminopeptidase N. The fluorescent probe compound can detect aminopeptidase N in an aqueous solution system, and has specificity, excellent selectivity, high sensitivity, etc. for the detection of aminopeptidase N, and can be used for the quantitative detection of aminopeptidase N.

[0059] The preparation method of the fluorescent probe compound of formula (I) in the present invention has simple synthesis steps, good biocompatibility, short reaction time, convenient purification, and simple process.

[0060] Term Definition and Explanation

[0061] Unless otherwise defined, all scientific and technical terms herein have the same meaning as commonly understood by those skilled in the art to which the claimed subject matter belongs. It should be understood that the above summary and the following detailed description are exemplary and explanatory only, and do not limit the subject matter of the present application in any way. In the present application, unless otherwise specified, the term "comprising" and other forms, such as "including", "containing", and "having", are not restrictive.

[0062] The term "C 1-10 alkyl" should be understood to represent a straight-chain or branched-chain saturated monovalent hydrocarbon group having 1 to 10 carbon atoms, preferably C 1-6 alkyl. "C 1-6 alkyl" should be understood to preferably represent a straight-chain or branched-chain saturated monovalent hydrocarbon group having 1, 2, 3, 4, 5, or 6 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, etc. or their isomers. In particular, the group has 1, 2, 3, 4, 5, 6 carbon atoms ("C 1-6 alkyl"), such as methyl, ethyl, propyl, butyl, isopropyl, isobutyl, sec-butyl, tert-butyl, and more particularly, the group has 1, 2, or 3 carbon atoms ("C 1-3 alkyl"), such as methyl, ethyl, n-propyl, or isopropyl.

[0063] The above definition of the term "C 1-10 alkyl" also applies to other terms containing "C 1-10 alkyl", such as the term "C 1-10 alkoxy". Brief Description of the Drawings

[0064] Figure 1 The fluorescence intensity diagram of compound (1) in PBS buffer solution containing 1 g / mL bovine serum albumin with the change of aminopeptidase N concentration, and the linear fitting of fluorescence intensity at 512 nm with the change of aminopeptidase N concentration.

[0065] Figure 2 The fluorescence response diagram of compound (1) to different analytes at 512 nm. Among them, 0 is the control group, 1 is sodium ion, 2 is potassium ion, 3 is iron ion, 4 is magnesium ion, 5 is chloride ion, 6 is bromide ion, 7 is iodide ion, 8 is sulfate ion, 9 is cysteine, 10 is homocysteine, 11 is glutathione, 12 is glucose, 13 is glycine, 14 is alanine, 15 is hypochlorous acid, 16 is γ-glutamyltransferase, 17 is alkaline phosphatase, 18 is carboxylesterase 2, 19 is acetylcholinesterase, 20 is aminopeptidase N. Detailed implementation manners

[0066] The technical solutions of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are only for illustrative explanation of the present invention and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0067] Unless otherwise specified, the raw materials and reagents used in the following embodiments are all commercially available products or can be prepared by known methods.

[0068] Preparation Example 1:

[0069]

[0070] Add p-methoxyacetophenone (3.01 g, 20 mmol) (2) and p-aminobenzaldehyde (1.21 g, 10 mmol) (3) to a three-necked flask, and then add a small amount of solid sodium hydroxide. Under nitrogen protection, add anhydrous ethanol solvent (20 mL) to the flask with a disposable syringe. Under vigorous stirring, the reaction system is refluxed at 80 °C for 12 h. After the reaction is completed, it is cooled to room temperature, and 50 mL of dichloromethane and 50 mL of brine are added to the system. After extracting 3 times with 50 mL of dichloromethane, the combined organic phases are dried with anhydrous calcium chloride, the organic solvent is removed under vacuum, and the crude product is purified by a silica gel column for chromatography, where the eluent is petroleum ether / ethyl acetate (3:1, v / v) to obtain 1.45 g of yellow solid (yield 57%) (4).

[0071] For compound 4 1 1H NMR, 13 13C NMR, and mass spectrometry data are as follows:

[0072] 1 1H NMR (300 MHz, DMSO-d6) δ (ppm): 8.10 (d, J = 6 Hz, 2H), 7.58 - 7.54 (m, 4H), 7.05 (d, J = 6 Hz, 2H), 6.60 (d, J = 6 Hz, 2H), 5.86 (s, 2H), 3.86 (s, 3H); 13 13C NMR (100 MHz, DMSO-d6) δ (ppm): 187.50, 163.20, 152.27, 145.11, 131.69, 131.35, 130.91, 122.59, 115.86, 114.32, 114.07, 55.97; ESI-MS: m / z calcd for: [C 16 H 15 NO2 + H] + 254.118, found: 254.1176.

[0073] Preparation Example 2

[0074]

[0075] After adding Boc-L-alanine (1.89 g, 10 mmol), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (3.80 g, 10 mmol) and N,N-diisopropylethylamine (DIEA) (1.93 g, 15 mmol) into a flask, 20 mL of anhydrous dichloromethane was added. Stir vigorously at room temperature for 1 hour. After all the solids were dissolved, compound (4) (1.27 g, 5 mmol) was added thereto. After continuing to stir vigorously at room temperature for 8 hours, the reaction was stopped. 30 mL of dichloromethane and 50 mL of brine were added to the system. After extracting 3 times with 50 mL of dichloromethane, the combined organic phases were dried over anhydrous calcium chloride, and the organic solvents were removed under vacuum. The crude product was purified by a silica gel column for chromatography, wherein the eluent was petroleum ether / ethyl acetate (3.5:1, v / v), and 1.42 g of a white solid (yield 67%) (5) was obtained.

[0076] For compound 5 1 1H NMR, 13 13C NMR, and mass spectrometry data are as follows:

[0077] 1 1H NMR (300 MHz, CDCl3) δ (ppm): 8.60 (s, 1H) 8.07 (d, J = 6 Hz, 2H) 7.75 - 7.50 (m, 7H) 7.03 (d, J = 6 Hz, 2H) 4.30 (s, 1H) 3.92 (s, 3H) 1.51 - 1.43 (m, 12H);13 13C NMR (100 MHz, CDCl3) δ (ppm): 188.67, 171.13, 163.38, 143.37, 139.93, 131.27, 130.75, 129.30, 129.26, 113.83, 77.34, 76.71, 55.47, 28.33, 17.37; ESI-MS: m / z calcd for: [C 24 H 28 N2O5 + Na] + , 447.189, found: 447.1890。

[0078] Preparation Example 3:

[0079]

[0080] Add 424 mg of compound (5) to a flask, and then add 10 mL of a mixed solvent of dichloromethane and trifluoroacetic acid (v:v = 1:1). After vigorously stirring at room temperature for 1 hour, add 20 mL of dichloromethane and 30 mL of water to the reaction system. After extraction and separation, collect the aqueous phase, and add triethylamine until the pH is neutral. Filter to collect the resulting pale yellow precipitate, and wash the solid successively with water and dichloromethane, and finally dry to obtain 103 mg of pale yellow solid (1) (yield 78%).

[0081] For compound 1 1 1H NMR, 13 13C NMR, and mass spectrometry data are as follows:

[0082] 1 1H NMR (300 MHz, DMSO-d6) δ (ppm): 10.66 (s, 1H) 8.23 - 8.13 (m, 5H), 7.92 - 7.84 (m, 3H), 7.12 - 7.07 (d, J = 6 Hz, 2H), 4.04 (d, J = 4 Hz, 1H) 3.88 (s 3H) 1.48 (d J = 4 Hz 3H); 13 13C NMR (100 MHz, DMSO-d6) δ (ppm): 187.74, 172.69, 163.30, 155.70, 143.34, 141.64, 131.26, 131.12, 130.14, 120.79, 119.57, 114.46, 78.57, 56.03, 28.68, 18.38; ESI-MS: m / z calcdfor: [C 19 H 20 N2O3 + H] + 325.155, found: 325.1548。

[0083] The reaction route is as follows:

[0084]

[0085] Example 1

[0086] The synthesized compound (1) above was used to detect aminopeptidase N at different concentrations.

[0087] The detection method is as follows: Weigh 3.24 mg of compound (1) and dissolve it in 10 mL of dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 1 mM. Dilute the stock solution with PBS buffer solution (pH = 7.4) containing 1 mg / mL bovine serum albumin (BSA), and simultaneously add 0 - 50 ng / mL of aminopeptidase N to obtain a sample with a concentration of 10 μM of compound (1). Incubate the sample at a constant temperature of 37 °C for 1 hour, and then use a fluorescence spectrometer to detect its fluorescence spectrum with an excitation light wavelength of 350 nm (see Figure 1 ).

[0088] As can be seen from Figure 1 , when there is no aminopeptidase N in the solution, the luminescence of the sample is negligible. As the concentration of aminopeptidase N increases, the luminescence of the sample at 512 nm gradually increases. Moreover, the luminescence of the sample at 512 nm shows a good linear relationship with the concentration of aminopeptidase N. According to the formula LOD = 3σ / k, the detection limit of the probe for APN is calculated to be 0.058 ng / mL, indicating that the probe has excellent sensitivity for the detection of APN (where σ is the standard deviation of 9 blank experiments, and k is the Figure 1 slope of the linear fitting in

[0089] Example 2

[0090] The synthesized compound (1) above was used for the selective detection of aminopeptidase N.

[0091] Take 3.24 mg of compound (1) and dissolve it in 10 mL of dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 1 mM. Dilute the stock solution with PBS buffer solution (pH = 7.4) containing 1 mg / mL bovine serum albumin (BSA), and add or not add 20 different analytical substances according to different experimental groups. Among them, 0 is the control group, 1 is sodium ion, 2 is potassium ion, 3 is iron ion, 4 is magnesium ion, 5 is chloride ion, 6 is bromide ion, 7 is iodide ion, 8 is sulfate ion, 9 is cysteine, 10 is homocysteine, 11 is glutathione, 12 is glucose, 13 is glycine, 14 is alanine, 15 is hypochlorous acid, 16 is γ-glutamyltransferase, 17 is alkaline phosphatase, 18 is carboxylesterase 2, 19 is acetylcholinesterase, 20 is aminopeptidase N.

[0092] Incubate the samples at a constant temperature of 37 °C for 1 hour, then use a fluorescence spectrometer to detect their fluorescence spectra with an excitation light wavelength of 350 nm, and count the fluorescence intensity of each group of fluorescence spectra at 512 nm. The results are as Figure 2 shown. Only when aminopeptidase N ( Figure 2 sample 20 in) is added, the fluorescence intensity of the sample shows a significant increase; and this fluorescence intensity is more than 17 times that of other samples. The above results indicate that compound (1) has excellent selectivity for the detection of aminopeptidase N.

[0093] Example 3

[0094] Use the synthesized compound (1) above to detect aminopeptidase N in urine.

[0095] Take 3.24 mg of compound (1) and dissolve it in 10 mL of dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 1 mM. Dilute human urine 10-fold with PBS buffer solution first, and then add 0 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL of aminopeptidase N respectively. Next, dilute the above samples with PBS buffer solution (pH = 7.4) containing 1 mg / mL bovine serum albumin (BSA). Dilute the compound (1) sample 100-fold and the urine sample 10-fold respectively, and react the compound (1) sample with the urine sample in a water bath at 37 °C for 2 hours. After the reaction, use a fluorescence spectrometer to detect its fluorescence spectrum (λ ex = 420 nm), record the fluorescence intensity at 512 nm, subtract the background fluorescence, and calculate the concentration and standard deviation of aminopeptidase N in the diluted sample according to the Figure 1 standard curve in, and multiply it by 100 times to get the concentration of aminopeptidase N in the original sample.

[0096] Table 1 shows the detection results of compound (1) for APN in human urine and urine supplemented with 500 - 1500 ng / mL aminopeptidase N (APN), and the comparison with the test results of a commercial kit. The standard deviation was calculated based on the results of 5 parallel experiments.

[0097] The concentration of aminopeptidase N measured in the above experiment showed good agreement with the value measured by a commercial kit (Biolab Technology BL10715 - A). The above experimental results indicate that compound (1) can be used to detect the APN content in different urine samples.

[0098] Table 1 Detection results of compound (1) for aminopeptidase N in urine

[0099]

[0100] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A fluorescent probe compound represented by the following formula (I): Among them, R1 and R2 are the same or different and are independently selected from the following groups: H, halogen, unsubstituted or optionally substituted by one, two or more R s substituted C 1-10 alkyl, C 1-10 alkoxy; R s selected from H, C 1-10 alkyl, C 1-10 alkoxy, halogen; R3 is selected from H, halogen, C 1-6 alkyl, C 1-6 alkoxy; R4 is selected from H, halogen, C 1-6 alkyl, C 1-6 alkoxy; m and n are the same or different and each independently selected from 0, 1, 2, 3, 4.

2. The compound according to claim 1, wherein R1 and R2 are the same or different and are each independently selected from the following groups: H, halogen, unsubstituted or optionally substituted by one, two or more R s substituted C 1-6 alkyl, C 1-6 alkoxy; preferably, R1 and R2 are the same or different and are each independently selected from the following groups: H, halogen, unsubstituted or optionally substituted by one, two or more R s substituted C 1-3 alkyl, C 1-3 alkoxy.

3. The compound according to claim 1 or 2, characterized in that, R s selected from H, C 1-6 alkyl, C 1-6 alkoxy, halogen; preferably, R s is selected from the following groups: H, halogen, C 1-3 alkyl, C 1-3 alkoxy; Preferably, R1 is selected from the following groups: H, halogen, C 1-6 alkyl, C 1-6 alkoxy; Preferably, R2 is selected from the following groups: H, halogen, C 1-6 alkyl, C 1-6 alkoxy; Preferably, R3 is selected from H, halogen, C 1-4 alkyl, C 1-4 alkoxy; Preferably, R4 is selected from H, halogen, C 1-4 alkyl, C 1-4 alkoxy.

4. The compound according to any one of claims 1 - 3, characterized in that, The compound of formula (I) is selected from the following compounds:

5. A method for preparing the fluorescent probe compound of formula (I) according to any one of claims 1-4, the preparation method comprising the following steps: (a) Reacting the compound represented by formula (II) with the compound represented by formula (III) to obtain the compound represented by formula (IV); (b) Reacting the compound represented by formula (IV) with R5-NH-CH(R4)-COOH to obtain the compound represented by formula (V); (c) Reacting the compound represented by formula (V) with an acid to obtain the compound represented by formula (I); The reaction route is as follows: Among them, R1, R2, R3, R4, m, n have the definitions as described in any one of claims 1-4; R5 is an amino protecting group.

6. Use of the fluorescent probe compound of formula (I) according to any one of claims 1-4 in detecting the content of aminopeptidase N in a sample.

7. Use of the fluorescent probe compound of formula (I) according to any one of claims 1-4 in preparing a reagent or kit for detecting the content of aminopeptidase N in a sample.

8. Use of the fluorescent probe compound of formula (I) according to any one of claims 1-4 in preparing an aminopeptidase N imaging agent.

9. Use of the compound represented by the following formula (IV) in preparing the fluorescent probe compound of formula (I) according to any one of claims 1-4, Among them, R1, R2, R3, m, n have the definitions as described in any one of claims 1-4.

10. Use of the compound represented by the following formula (V) in preparing the fluorescent probe compound of formula (I) according to any one of claims 1-4, Among them, R1, R2, R3, R4, R5, m, n have the definitions as described in any one of claims 1-4.

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