A near-infrared fluorescent probe for detecting carboxylesterase and having antibacterial activity, and its preparation method and application

Abstract

The present invention provides a near-infrared fluorescent probe for detecting carboxylesterase and exhibiting antibacterial activity, as well as its preparation method and application. The probe has the following structural formula: #imgabs0#, wherein R is selected from #imgabs1#, #imgabs2#, #imgabs3#, #imgabs4#, #imgabs5#, or #imgabs6#. The probe of the present invention utilizes a hemicyanine derivative as a parent nucleus, introduces p-aminobenzenesulfonamide as an antibacterial active group, and utilizes acid esters containing different R groups as recognition groups for detecting carboxylesterase. Carboxylesterase-induced ester cleavage is accompanied by a significant enhancement in the fluorescence signal. The probe can effectively detect carboxylesterase from different proteases, amino acids, and yin and yang samples, exhibiting good selectivity and high sensitivity. The probe's action on carboxylesterase is accompanied by a distinct color change visible to the naked eye, and the probe exhibits a certain degree of antibacterial activity, resulting in successful applications for the detection and imaging of carboxylesterase in biological systems.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic synthesis, and in particular relates to a near-infrared fluorescent probe for detecting carboxylesterase and having antibacterial activity, and a preparation method and application thereof. Background Art

[0002] Carboxylesterase (CEs) is a member of the serine protease family and is widely present in animals, plants and microorganisms. It can catalyze the transesterification and hydrolysis of esters, thioesters, amides and carbamates, regulate various metabolic functions, including gene expression, ester metabolism, substance transport and substrate-specific detoxification, and play an important role in regulating various biological activities of organisms.

[0003] To date, several methods for detecting carboxylesterases have been reported, including immunology, chromatography, chemiluminescence, mass spectrometry, and fluorescence. However, most of these methods are complex, require expensive equipment, and lack real-time detection. Compared with traditional methods, fluorescent probes offer advantages such as low cost, ease of use, high sensitivity, and short response time. They can also enable real-time, non-destructive imaging of CEs and are rapidly developing. Sulfonamides, one of the earliest chemically synthesized antimicrobial drugs, play a vital role in the treatment of infectious diseases. Furthermore, sulfonamide derivatives have been reported to interact with a variety of enzymes and receptors and exhibit diverse biological activities, including antibacterial, antifungal, antiparasitic, anticancer, anti-inflammatory, and antiviral properties. Structural modifications of sulfonamides have revealed a wide range of bioactivities, demonstrating significant pharmaceutical potential and development value. Probes are important tools in analytical chemistry. In recent years, small molecule probes, including those with photoacoustic imaging and photothermal therapy, have emerged. However, few reports have reported the integration of drug intermediates with fluorophores to enable probes with both detection and pharmacological activity.

[0004] Fluorescent probes have become powerful tools for observing small molecules within cells and are of great value in biomedical research. Combining fluorescent probes with drug intermediates offers new possibilities and directions for disease diagnosis and drug development. Therefore, developing a fluorescent probe that combines detection capabilities with pharmacological activity is of great significance for drug development and clinical diagnosis. Summary of the Invention

[0005] To overcome the challenges of the prior art, the present invention provides a fluorescent probe for detecting carboxylesterase with antibacterial activity. This fluorescent probe has a rapid response to carboxylesterase and exhibits good selectivity and linearity. It has been successfully used for imaging carboxylesterase in living cells and in zebrafish, enabling imaging of endogenous carboxylesterase from Staphylococcus aureus and demonstrating moderate antibacterial activity.

[0006] The present invention also provides a preparation method and application of the fluorescent probe.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] A near-infrared fluorescent probe for detecting carboxylesterase and having antibacterial activity, the structural formula of which is as follows:

[0009] Where R is selected from And R is When the compound is recorded as CYB-E1, R is When the compound is recorded as CYB-E2, R is When the compound is recorded as CYB-E3, R is When the compound is recorded as CYB-E4, R is When the compound is recorded as CYB-E5, R is When , the compound is recorded as CYB-E6.

[0010] The preparation method of the fluorescent probe for detecting carboxylesterase and having antibacterial activity is as follows: Figure 1 As shown, the following steps are included:

[0011] (1) Cyclohexanone, PBr3 and N,N-dimethylformamide react in dichloromethane to obtain compound I;

[0012] (2) 2,4-dihydroxybenzaldehyde, 3,4-dihydro-2H-pyran and pyridinium p-toluenesulfonate react in dichloromethane to obtain compound II;

[0013] (3) Compound I, Compound II and potassium carbonate react in N,N-dimethylformamide to obtain Compound III;

[0014] (4) Compound III is reacted with trifluoroacetic acid in dichloromethane with stirring to obtain compound IV;

[0015] (5) p-Aminobenzenesulfonamide reacts with chloroacetyl chloride in acetone to obtain compound V;

[0016] (6) Compound V reacts with 2,3,3-trimethyl-3H-indole in acetonitrile to obtain compound VI;

[0017] (7) Compound IV and triethylamine react with acetyl chloride, butyryl chloride, 2-thenoyl chloride, 2-furoyl chloride, benzoyl chloride or N,N-dimethylformyl chloride to obtain compound VII;

[0018] (8) Compound VI reacts with compound VII in ethanol to obtain the fluorescent probes CYB-E1, CYB-E2, CYB-E3, CYB-E4, CYB-E5, and CYB-E6.

[0019] The structures of Compound I, Compound II, Compound III, Compound IV, Compound V, Compound VI and Compound VII are as follows: Figure 1 shown.

[0020] Preferably, the preparation process of compound I in step (1) is as follows: under ice bath conditions, PBr3 is dropped into a mixed solution of DMF and dichloromethane, stirred for 0.5h to 1h, and then cyclohexanone is dropped into the aforementioned mixed solution, stirred until the reaction is complete and then neutralized to neutrality, the product is extracted with dichloromethane, and concentrated by rotary evaporation to obtain the target product.

[0021] Preferably, the preparation process of compound II in step (2) is as follows: 2,4-dihydroxybenzaldehyde and pyridinium p-toluenesulfonate are weighed separately and added to dichloromethane, and then 3,4-dihydro-2H-pyran is added under a protective atmosphere. After stirring and refluxing, the reaction is completed, and then rotary evaporation and column chromatography are performed to obtain compound II.

[0022] Preferably, the preparation process of compound III in step (3) is specifically as follows: adding the prepared compound I, compound II and potassium carbonate to DMF, stirring at 35-45° C. until the reaction is complete, cooling to room temperature, filtering and concentrating to obtain a concentrated solution of compound III.

[0023] Preferably, the preparation process of compound IV in step (4) is specifically as follows: dissolving the concentrated solution obtained in step (3) in dichloromethane, adding trifluoroacetic acid to adjust the pH to 6-7, stirring at 35-45° C. after the reaction is complete, cooling to room temperature, filtering, and washing the solid with ethanol to obtain compound IV.

[0024] Preferably, the preparation process of compound V in step (5) is specifically as follows: p-aminobenzenesulfonamide is weighed and added to acetone, chloroacetyl chloride is added dropwise, and after stirring at 70-80° C. for complete reaction, the mixture is cooled to room temperature, ice water is added, and the obtained mixture is stirred and filtered. The collected solid is washed with ice water, and then recrystallized with ethanol, filtered, and the obtained solid is dried to obtain compound V.

[0025] Preferably, the preparation process of compound VI in step (6) is as follows: under a nitrogen atmosphere, compound V obtained in step (5) is dissolved in acetonitrile, 2,3,3-trimethyl-3H-indole is added, and the reaction is refluxed until completion, the reaction solution is vacuum evaporated, dichloromethane is added, and the reaction solution is filtered under reduced pressure to obtain a solid compound VI.

[0026] Preferably, the preparation process of different compounds in step (7) is specifically as follows: under a nitrogen atmosphere, compound IV and triethylamine are added to dichloromethane, acetyl chloride, butyryl chloride, 2-thiophenecarbonyl chloride, 2-furoyl chloride, benzoyl chloride or N,N-dimethylformyl chloride is added under ice bath conditions, and after stirring for complete reaction, rotary evaporation and column chromatography are performed to obtain the corresponding compound VII.

[0027] Preferably, the preparation process of the fluorescent probe in step (8) is specifically as follows: under a nitrogen atmosphere, compound VI is dissolved in ethanol, and the compounds obtained in step (7) are added respectively, and the reaction is refluxed under stirring until a solid precipitate is formed, and the obtained solid is filtered, and the obtained solid is washed with ethyl acetate and then subjected to column chromatography to obtain fluorescent probes CYB-E1, CYB-E2, CYB-E3, CYB-E4, CYB-E5 and CYB-E6 respectively.

[0028] Preferably, in step (1), the molar ratio of PBr3, DMF and cyclohexanone is (2.5-3.0):(2.0-2.5):1. In step (2), the molar ratio of 2,4-dihydroxybenzaldehyde, 3,4-dihydro-2H-pyran and p-toluenesulfonic acid pyridinium salt is 1:(2.0-2.5):0.05. In step (3), the molar ratio of compound I, compound II and potassium carbonate is 1:1:3. In step (5), the molar ratio of p-aminobenzenesulfonamide and chloroacetyl chloride is 1:(1-1.5). In step (6), the molar ratio of compound V and 2,3,3-trimethyl-3H-indole is 1:(1-1.5). In step (7), the molar ratio of compound IV, triethylamine, and acetyl chloride, butyryl chloride, 2-thenoyl chloride, 2-furoyl chloride, benzoyl chloride, or N,N-dimethylformyl chloride is 1:(1-2):(1-2). In step (8), the molar ratio of compound VI to the different compounds obtained in step (7) is 1:(1-1.5).

[0029] The fluorescent probe is used in the detection of carboxylesterase for the purpose of non-disease diagnosis.

[0030] Application of the above fluorescent probes in the preparation of cell fluorescence imaging agents.

[0031] Application of fluorescent probes in preparing zebrafish fluorescence imaging doses.

[0032] Application of fluorescent probes in the preparation of antibacterial activity.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. The ester bond of the fluorescent probe of the present invention can be hydrolyzed and cleaved by carboxylesterase, accompanied by a significant increase in NIR fluorescence emission. Simultaneously, the probe can rapidly distinguish carboxylesterase from other bioactive small molecules such as proteases, amino acids, or anions and cations based on their different kinetic rates. Furthermore, the probe exhibits a significant change in fluorescence intensity accompanied by a distinct color change, visible to the naked eye.

[0035] 2. The fluorescent probe of the present invention is simple to synthesize and has good biocompatibility. The probe successfully performs fluorescence imaging in living cells and zebrafish, and can sensitively and selectively identify changes in the content of carboxylesterase in cells and zebrafish. In addition, the probe can achieve tracking imaging of Staphylococcus aureus carboxylesterase and exhibit antibacterial activity, showing strong application prospects in biomedicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The synthetic route of the near-infrared fluorescent probe prepared in Example 1;

[0037] Figure 2 The H NMR spectrum of the fluorescent probe CYB-E1 prepared in Example 1;

[0038] Figure 3 The NMR carbon spectrum of the fluorescent probe CYB-E1 prepared in Example 1;

[0039] Figure 4 The H NMR spectrum of the fluorescent probe CYB-E2 prepared in Example 1;

[0040] Figure 5 The NMR carbon spectrum of the fluorescent probe CYB-E2 prepared in Example 1;

[0041] Figure 6 The H NMR spectrum of the fluorescent probe CYB-E3 prepared in Example 1;

[0042] Figure 7 The NMR carbon spectrum of the fluorescent probe CYB-E3 prepared in Example 1;

[0043] Figure 8 The H NMR spectrum of the fluorescent probe CYB-E4 prepared in Example 1;

[0044] Figure 9 The NMR carbon spectrum of the fluorescent probe CYB-E4 prepared in Example 1;

[0045] Figure 10 The H NMR spectrum of the fluorescent probe CYB-E5 prepared in Example 1;

[0046] Figure 11 The NMR carbon spectrum of the fluorescent probe CYB-E5 prepared in Example 1;

[0047] Figure 12 The H NMR spectrum of the fluorescent probe CYB-E6 prepared in Example 1;

[0048] Figure 13 The NMR carbon spectrum of the fluorescent probe CYB-E6 prepared in Example 1;

[0049] Figure 14The selectivity test of the near-infrared fluorescent probe prepared in Example 1 for identifying carboxylesterase; a is the initial screening of the selectivity of fluorescent probes CYB-E1, CYB-E2, CYB-E3, CYB-E4, CYB-E5 and CYB-E6, b is the selectivity test result of fluorescent probe CYB-E3, other enzymes, amino acids or ions, and c is the selectivity test result of fluorescent probe CYB-E4 for other enzymes, amino acids or ions; in Figure a, from left to right are blank, carboxylesterase (CEs), acetylcholinesterase (AChE) butyrylcholinesterase (BChE); in Figures b and c: from left to right: (1) blank (2) carboxylesterase (CEs) (3) trypsin (4) lysozyme (5) bovine serum albumin (6) lipase (7) pepsin (8) cellulase (9) peroxidase (10) Ser (11) Glu (12) Met (13) Phe (14) Cys (15) GSH (16) Hys (17) Tyr (18) Lys (19) Leu (20) Mg 2+ (21)Cu 2+ (22)Zn 2+ (23)Fe 3+ (24)Fe 2+ (25)Co 2+ (26)Gd 2+ (27)F - (28)S 2- (29)Br - (30) NO3 - (31)ClO4 - (32)AcO - (33)SCN -

[0050] Figure 15 Figure 1 is a graph showing the spectral properties of the near-infrared fluorescent probe CYB-E4 prepared in Example 1; Figure a is a graph showing the ultraviolet absorption spectrum of the fluorescent probe CYB-E4 in response to different concentrations of carboxylesterase; Figure b is a graph showing the fluorescence spectrum of the fluorescent probe CYB-E4 in response to different concentrations of carboxylesterase; Figure c is a graph showing the relationship between the fluorescence intensity at 710 nm and the concentration of carboxylesterase after the fluorescent probe CYB-E4 responds to different concentrations of carboxylesterase; Figure d is a linear relationship analysis showing the relationship between the fluorescence intensity at 710 nm and the concentration of carboxylesterase after the fluorescent probe CYB-E4 responds to different concentrations of carboxylesterase;

[0051] Figure 16 The fluorescence intensity at 710 nm and time after the near-infrared fluorescent probe CYB-E4 prepared in Example 1 was co-incubated with carboxylesterase (0.15 U / mL)

[0052] Figure 17This is a biological imaging image of the near-infrared fluorescent probe CYB-E4 prepared in Example 1 when detecting carboxylesterase in living cells;

[0053] Figure 18 This is a biological imaging image of the near-infrared fluorescent probe CYB-E4 prepared in Example 1 detecting carboxylesterase in zebrafish;

[0054] Figure 19 This is a biological imaging image of carboxylesterase in Staphylococcus aureus using the near-infrared fluorescent probe CYB-E4 prepared in Example 1. DETAILED DESCRIPTION

[0055] The present invention will be further described below with reference to the embodiments and drawings, but they are not intended to limit the present invention.

[0056] Example 1

[0057] The preparation process of the fluorescent probe in this embodiment is as follows Figure 1 As shown, the specific steps include:

[0058] (1) Preparation of Compound Ⅰ

[0059]

[0060] Under ice-bath conditions, PBr3 (14.3 mL, 153 mmol) was slowly added dropwise to a mixed solution of DMF (10.5 mL, 137 mmol) and CHCl (60 mL) and stirred for 40 min. Cyclohexanone (5 mL, 56.5 mmol) was slowly added, the ice bath removed, and the mixture stirred at room temperature for 30 h. After the reaction, the reaction solution was slowly added to 100 mL of ice-cold water, and the pH was adjusted to neutral with anhydrous sodium carbonate. The mixture was extracted with CHCl (3 × 50 mL). The organic phases were combined, washed with saturated brine, dried over NaSO, and distilled under reduced pressure to obtain a reddish-brown oily liquid. A total of 8.6 g of Compound I was obtained with a yield of 81.1%, which was directly carried to the next step without purification.

[0061] (2) Preparation of Compound II

[0062]

[0063] 2,4-Dihydroxybenzaldehyde (2.76 g, 20 mmol) and PPTS (pyridinium p-toluenesulfonate, 0.25 g, 1 mmol) were added to a 100 mL round-bottom flask containing 25 mL of CH2Cl2. Under nitrogen, 3,4-dihydro-2H-pyran (4.15 mL, 45.5 mmol) was added to the reaction flask and refluxed at 40°C. After completion of the reaction, the mixture was concentrated and purified by silica gel column chromatography (petroleum ether:ethyl acetate 15:1) to afford approximately 3.90 g of Compound II as a white solid in an approximately 88% yield. The H NMR spectrum of Compound II is as follows: 1 H NMR (400MHz, CDCl3) δ11.36(s,1H),9.72(s,1H),7.43(d,J=8.6Hz,1H),6.65(dd,J=8.6,2.3Hz,1H),6.62(d,J=2.2Hz,1H ),5.50(t,J=3.2Hz,1H),3.86–3.78(m,1H),3.66–3.59(m,1H),1.88–1.85(m,2H),1.72–1.66(m,2H),1.63–1.56(m,2H). 13 C NMR (100MHz, CDCl3) δ194.69,164.44,164.26,135.40,115.86,109.50,103.77,96.32,62.29,30.05,25.06,18.52.

[0064] (3) Preparation of Compound III

[0065]

[0066] Under nitrogen, anhydrous potassium carbonate (3.73 g, 27 mmol), compound I (1.7 g, 9 mmol), and compound II (2.00 g, 9 mmol) were added to a round-bottom flask containing 10 mL of DMF and stirred at 40°C. The reaction solution was filtered under reduced pressure to remove the potassium carbonate solid, and then concentrated to obtain compound III, which was directly used in the next step without purification.

[0067] (4) Preparation of Compound IV

[0068]

[0069] 5 mL of CH2Cl2 and compound III were transferred into a 50 mL round-bottom flask. After adjusting the pH to 6 with TFA (trifluoroacetic acid), the mixture was heated in a 40°C constant temperature water bath with stirring. After the reaction was complete, the mixture was filtered under reduced pressure and the solid was washed with ice-cold ethanol to obtain about 1.22 g of compound IV as a yellow solid product with a yield of 59.4%. The H NMR spectrum of compound IV ( 1H NMR) and carbon spectroscopy ( 13 C NMR) data are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.17 (s, 1H), 7.17 (d, J = 8.2Hz, 1H), 6.90 (s, 1H), 6. 62–6.57(m,2H),2.54–2.50(m,2H),2.27(t,J=6.0Hz,2H),1.65–1.56(m,2H). 13 C NMR (100MHz, DMSO-d6) δ186.10,160.33,159.72,152.76,128.13,127.62,124.71,113.06,112.01,111.30,101.88,28.95,21.23,20.01.

[0070] (5) Preparation of Compound V

[0071]

[0072] Under nitrogen, p-aminobenzenesulfonamide (1.72 g, 10 mmol) was suspended in acetone. Chloroacetyl chloride (1.34 g, 12 mmol) was added dropwise at room temperature. The mixture was stirred at 70°C for 1 hour. After the reaction, the mixture was cooled to room temperature. Ice water was added and stirring continued for 10 minutes. The mixture was filtered under reduced pressure and washed with ice water. Multiple recrystallizations from ethanol afforded 2.36 g of a white solid in a 95.1% yield. The H NMR and C NMR data for Compound V are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.62(s,1H),7.76(m,4H),7.28(s,2H),4.30(s,2H). 13 C NMR (100MHz, DMSO-d6) δ165.18,141.34,138.96,126.80,118.99,43.54.

[0073] (6) Preparation of Compound VI

[0074]

[0075] Under nitrogen protection, compound V (1.29 g, 5 mmol) was dissolved in CH3CN, 2,3,3-trimethyl-3H-indole (0.8 g, 6 mmol) was added, and the mixture was stirred under reflux at 85°C for 12 hours. After the reaction, the solvent was removed by vacuum rotary evaporation, CH2Cl2 solvent was added, and the mixture was filtered under reduced pressure to obtain approximately 1.69 g of compound VI with a yield of 91.2%. The H NMR spectrum of compound VI ( 1H NMR) and carbon spectroscopy ( 13 C NMR) data are as follows: 1 H NMR(400MHz,DMSO-d6)δ11.88(s,1H),7.98(s,1H),7.87(s,1H),7.84–7.7 5(m,4H),7.63(s,2H),7.32(s,2H),5.72(s,2H),2.87(s,3H),1.59(s,6H). 13 C NMR (100MHz, DMSO-d6) δ199.75,162.35,141.39,141.08,139.27,129.58,129.14,126.78,123.63,118.96,114.90,54.45,50.27,22.11,14.47.

[0076] (7) Preparation of Compound VII

[0077]

[0078] Under nitrogen, compound IV (228 mg, 0.1 mmol), triethylamine (202 mg, 0.2 mmol), and 5 mL of CHCl were mixed in an ice-water bath for 5 minutes. Acetyl chloride, butyryl chloride, 2-thenoyl chloride, 2-furoyl chloride, benzoyl chloride, or N,N-dimethylformyl chloride (0.1 mmol) was then added and stirred for 5 minutes. The ice bath was removed and the mixture was stirred at room temperature for 1 hour. After the reaction, the solvent was removed and the product was purified by silica gel column chromatography with CHCl:CHOH (60:1) to yield a yellow solid.

[0079] (8) Preparation of fluorescent probes

[0080]

[0081] Under nitrogen protection, compound VI (37.2 mg, 0.01 mmol), compound VII (0.012 mmol), and 5 mL of ethanol were added to a 25 mL round-bottom flask and refluxed at 80°C for 12 h. After the reaction, the mixture was cooled to room temperature, and the precipitated solid was filtered under reduced pressure. The obtained solid was purified by silica gel column chromatography with CH2Cl2:CH3OH (9:1) to obtain the probe;

[0082] H NMR spectrum of fluorescent probe CYB-E1 ( 1 H NMR) and carbon spectroscopy ( 13 C NMR) Figure 2 and 3 The specific data are as follows: 1H NMR (400MHz, DMSO-d6) δ11.79(s,1H),8.65(d,J=15.0Hz,1H),7.87–7.78(m,5H),7.68(d,J=7.9Hz,1H),7.62(d,J=8.5Hz,1H),7.57–7.46(m,4H),7. 31(s,2H),7.15(dd,J=8.4,2.1Hz,1H),6.74(d,J=15.0Hz,1H),5.63(s,2H ),2.73(t,J=5.9Hz,2H),2.65(t,J=6.1Hz,2H),2.34(s,3H),1.81(s,8H). 13 C NMR (100 MHz, DMSO-d6) δ 180.6, 169.4, 164.1, 160.4, 153.1, 153.0, 146.5, 142.3, 142.1, 141.6, 139.6, 132.4, 130.2, 129.5, 128.9, 127.9, 127.3, 120.0, 119.4, 115.3, 114.0, 113.4, 110.5, 107.2, 106.7, 51.3, 49.0, 29.1, 28.3, 28.0, 24.3, 21.4. High resolution mass spectrometry (HR-MS) (ESI): m / z calcd for C 35 H 34 N3O6S + :624.2163,found:624.2175.

[0083] H NMR spectrum of fluorescent probe CYB-E2 ( 1 H NMR) and carbon spectroscopy ( 13 C NMR) Figure 4 and 5 The specific data are as follows: 1 H NMR(400MHz,DMSO-d6)δ11.96(s,1H),8.64(d,J=14.9Hz,1H),7.88–7.77(m,5H ),7.69(d,J=7.9Hz,1H),7.61(d,J=8.5Hz,1H),7.57–7.44(m,4H),7.32(s,2H), 7.14(dd,J=8.4,2.2Hz,1H),6.74(d,J=15.0Hz,1H),5.67(s,2H),2.71(t,J=5.9 Hz, 2H), 2.63 (m, 4H), 1.80 (s, 8H), 1.69 (q, J = 7.4Hz, 2H), 1.00 (t, J = 7.4Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 180.1, 171.4, 163.8, 159.9, 152.7, 152.5, 146.0, 141.9, 141.8, 141.3, 139.1, 131.8, 129.7, 129.0, 128.4, 127.4, 126.8, 122.8, 119.6, 119.5, 118.9, 114.8, 113.0, 110.0, 106.3, 50.8, 48.4, 35.3, 28.6, 27.5, 23.7, 19.7, 17.7, 13.4. High resolution mass spectrometry (HR-MS) (ESI): m / z calculated for C 37 H 38 N3O6S + :652.2476,found:652.2844.

[0084] H NMR spectrum of fluorescent probe CYB-E3 ( 1 H NMR) and carbon spectroscopy ( 13 C NMR) Figure 6 and 7 The specific data are as follows: 1 H NMR (400MHz, DMSO-d6) δ11.66(s,1H),8.65(d,J=14.9Hz,1H),8.16(dd,J=5.0,1.3Hz ,1H),8.09(dd,J=3.8,1.3Hz,1H),7.84–7.73(m,5H),7.69(d,J=2.2Hz,1H),7.66(d, J=8.3Hz,2H),7.55–7.44(m,3H),7.36(dd,J=5.0,3.8Hz,1H),7.30(m,3H),6.72(d,J =15.1Hz,1H),5.60(s,2H),2.73(t,J=5.9Hz,2H),2.64(t,J=6.1Hz,2H),1.79(s,6H). 13C NMR (100 MHz, DMSO) δ 180.2, 163.7, 159.9, 159.8, 152.7, 152.2, 146.2, 141.9, 141.8, 141.2, 139.2, 136.0, 135.8, 131.8, 131.4, 130.0, 129.0, 129.0, 128.5, 127.5, 126.9, 122.8, 119.9, 119.6, 119.0, 114.9, 113.0, 110.3, 106.3, 50.9, 48.4, 28.7, 27.6, 23.7, 19.8. High resolution mass spectrometry (HR-MS) (ESI): m / z calcd for C 38 H 34 N3O6S2 + :692.1884,found:692.1893.

[0085] H NMR spectrum of fluorescent probe CYB-E4 ( 1 H NMR) and carbon spectroscopy ( 13 C NMR) Figure 8 and 9 The specific data are as follows: 1 H NMR (400MHz, DMSO-d6) δ11.81(s,1H),8.65(d,J=15.0Hz,1H),8.17(d,J=1.0 Hz,1H),7.87–7.75(m,5H),7.71–7.64(m,4H),7.57–7.45(m,3H),7.33(s,2H) ,7.30(dd,J=8.4,2.2Hz,1H),6.86(dd,J=3.6,1.7Hz,1H),6.75(d,J=15.0Hz ,1H),5.64(s,2H),2.73(t,J=6.1Hz,2H),2.64(t,J=6.1Hz,2H),1.80(s,8H). 13 C NMR (100 MHz, DMSO) δ 180.2, 163.7, 159.7, 155.9, 152.6, 151.8, 149.1, 146.1, 142.6, 141.9, 141.8, 141.2, 139.1, 131.6, 129.9, 129.0, 128.5, 127.5, 126.8, 122.8, 120.8, 119.8, 119.5, 118.9, 114.9, 113.4, 113.0, 110.2, 106.4, 50.8, 48.4, 27.5, 23.7, 22.1, 19.7. High resolution mass spectrometry (HR-MS) (ESI): m / z calcd for C 38 H34 N3O7S + :676.2112,found:676.2121.

[0086] H NMR spectrum of fluorescent probe CYB-E5 ( 1 H NMR) and carbon spectroscopy ( 13 C NMR) Figure 10 and 11 The specific data are as follows: 1 H NMR (400MHz, DMSO-d6) δ11.78(s,1H),8.66(d,J=14.9Hz,1H),8.19(d,J=8.0Hz,2H),7.79(m,7H),7.68(m,5H),7.59–7.41(m ,4H),7.33(d,J=9.4Hz,3H),6.74(d,J=15.0Hz,1H),5.62(s,2H),2.74(t,J=6.0Hz,2H),2.65(t,J=6.0Hz,2H),1.79(s,8H). 13 CNMR (100 MHz, DMSO) δ 180.2, 164.4, 163.8, 159.9, 152.7, 152.7, 146.1, 141.9, 141.8, 141.3, 139.1, 134.5, 131.8, 130.0, 129.9, 129.1, 128.5, 127.5, 126.8, 122.8, 119.8, 119.0, 115.0, 113.1, 110.3, 106.3, 54.9, 50.8, 48.4, 28.7, 27.6, 23.8, 19.8. High resolution mass spectrometry (HR-MS) (ESI): m / z calcd for C 40 H 36 N3O6S + :686.2319,found:686.2325.

[0087] H NMR spectrum of fluorescent probe CYB-E6 ( 1 H NMR) and carbon spectroscopy ( 13 C NMR) Figure 12 and 13 The specific data are as follows: 1H NMR(400MHz,DMSO-d6)δ11.81(s,1H),8.65(d,J=14.9Hz,1H),7.92–7.75(m, 5H),7.67(d,J=7.9Hz,1H),7.59(d,J=8.5Hz,1H),7.57–7.43(m,3H),7.30(s, 2H),7.14(dd,J=8.4,2.2Hz,1H),6.71(d,J=15.0Hz,1H),5.62(s,2H),3.09(s ,3H),2.96(s,3H),2.72(t,J=5.9Hz,2H),2.64(t,J=6.1Hz,2H),1.81(s,7H). 13 C NMR (100 MHz, DMSO-d6) δ 179.9, 163.7, 160.2, 153.5, 153.3, 152.6, 141.8, 141.2, 139.1, 132.2, 129.4, 128.9, 128.2, 127.3, 126.8, 122.8, 119.7, 118.9, 118.9, 114.8, 112.9, 109.9, 105.9, 54.9, 50.7, 36.4, 36.2, 28.6, 27.5, 23.7, 19.7. High resolution mass spectrometry (HR-MS) (ESI): m / z calcd for C 36 H 37 N4O6S + :653.2428,found:653.2437.

[0088] The application test of the fluorescent probe prepared in this example is as follows:

[0089] 1) Preparation of stock solution for detection

[0090] a. Fluorescent probe sample solution (1.00×10 -3 mol / L) preparation: accurately weigh 0.0062g (M=624) fluorescent probe CYB-E1, 0.0065g (M=652) fluorescent probe CYB-E2, 0.0069g (M=692) fluorescent probe CYB-E3, 0.0067g (M=676) fluorescent probe CYB-E4, 0.0068g (M=686) fluorescent probe CYB-E5 and 0.0065g (M=653) fluorescent probe CYB-E6, and dissolve them in 10mL dimethyl sulfoxide to make a concentration of 1.00×10 -3 mol / L solution.

[0091] b. Trypsin, lysozyme, bovine serum albumin, lipase, pepsin, cellulase, and peroxidase were prepared in deionized water to a concentration of 1 mg / mL. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were prepared in deionized water to a concentration of 5 U / mL. Carboxylesterase was prepared in deionized water to a concentration of 15 U / mL. Various amino acids and ions were prepared in deionized water to a concentration of 1.0 × 10 -2 mol / L solution.

[0092] The buffer solutions used in the following tests were all mixtures of PBS (10 mM, pH = 7.4) and DMSO in a volume ratio of 9:1, and the experimental water was all deionized water.

[0093] 2) Detection and analysis

[0094] 30 μL (1.00×10 -3 mol / L) was added with 3 mL of buffer and mixed evenly. The fluorescence excitation wavelength was 640 nm, the excitation slit width was 5.0 nm, the emission slit width was 5.0 nm, and the scanning range was set to 650-800 nm. The probe was first scanned alone to detect the fluorescence intensity at the maximum emission wavelength of 710 nm. Subsequently, the analytes (30 μL) (CEs, AChE, BChE) were added in sequence to detect the fluorescence intensity at the maximum emission wavelength of 710 nm. Figure 14 (a) shows a comparison of the fluorescence intensity of all probes and carboxylesterases at the maximum emission wavelength of 710 nm, along with acetylcholinesterase and butyrylcholinesterase from the amino acid protease family. CYB-E1 and CYB-E2 showed strong responses to all three proteases, failing to demonstrate specific recognition. CYB-E3 and CYB-E4 showed no response to acetylcholinesterase and butyrylcholinesterase, but showed good response to carboxylesterase. CYB-E5 showed a weaker response to CEs than CYB-E1 to E4. CYB-E6 showed no response to any of the three enzymes in this system. Therefore, CYB-E3 and CYB-E4 were selected for more detailed selectivity testing. Then, CYB-E3 and CYB-E4 were added with the test substances (30 μL) (trypsin, lysozyme, bovine serum albumin, lipase, pepsin, cellulase, peroxidase, Ser, Glu, Met, Phe, Cys, GSH, Hys, Tyr, Lys, Leu, Mg, etc.) in sequence. 2+ 、Cu 2+ 、Zn 2+ 、Fe 3+ 、Fe 2+ 、Co 2+ 、Gd 2+ 、F - 、S 2- Br- 、NO3 - 、ClO4 - 、AcO - 、SCN - , detect the fluorescence intensity at the maximum emission wavelength of 710nm. Figure 14 As shown in (b), after the addition of carboxylesterase, the fluorescence signal of CYB-E3 increased by more than 6 times, and it had a weak response to copper ions, with the fluorescence signal increased by more than 1.5 times; Figure 14 As shown in (c), the fluorescence signal of CYB-E4 increased by more than 6-fold after the addition of carboxylesterase, while the response to other interfering factors, such as other enzymes, amino acids, anions, and cations, was less than 1-fold. This indicates that CYB-E4 has superior selectivity for carboxylesterase, and subsequent performance testing will focus on CYB-E4.

[0095] Use a pipette to transfer 3 mL of buffer and add 30 μL (1.00 × 10 -3 mol / L) probe CYB-E4 stock solution was then added to different concentrations of carboxylesterase aqueous solution to detect the change trend of the absorbance of the system with the concentration of carboxylesterase. The scanning parameters for the detection of ultraviolet absorption spectrum were set to 450nm as the starting point and 800nm ​​as the end point. The results are as follows Figure 15 In (a), after the probe is added to the carboxylesterase aqueous solution, the absorbance of the system increases with time, accompanied by an obvious red shift phenomenon, and a new peak appears at 675nm. At the same time, after the probe solution is added to the carboxylesterase, the solution changes from dark blue to light blue, indicating that the probe has the ability to detect carboxylesterase with the naked eye.

[0096] 30 μL (1.00 × 10 -3 mol / L) probe CYB-E4 stock solution was added to 3 mL of buffer and mixed evenly. The fluorescence excitation wavelength was 640 nm, the excitation slit width was 5.0 nm, the emission slit width was 5.0 nm, and the scanning range was set to 650-800 nm to obtain the fluorescence spectrum of the fluorescent probe. The fluorescence intensity of the system gradually increased with the increase of carboxylesterase. The results are as follows: Figure 15 As shown in (b); the corresponding relationship between the carboxylesterase concentration and the 710nm fluorescence intensity value is made according to the fluorescence intensity value (710nm) detected at different concentrations Figure 15 (c) and the linear analysis diagram of concentration and fluorescence intensity; the probe can be linearly fitted at a carboxylesterase concentration of 0.03-0.105 U / mL, such as Figure 15As shown in (d), the linear correlation for CYB-E4 is 0.997, and the fitting equation is y = 7769.6x + 331.05429. This indicates that within a certain range of carboxylesterase concentrations, the fluorescence intensity at 710.0 nm is positively correlated. The limit of detection (LOD) for the two probes for carboxylesterase was calculated using the formula: LOD = 3σ / k, where σ is the standard deviation of five replicate blank measurements and k is the slope of the linear relationship between different cysteine ​​concentrations and the corresponding fluorescence intensity at 710 nm. The calculated limit of detection for the CYB-E4 probe for carboxylesterase is 0.2 mU / mL.

[0097] 30 μL (1.00×10 -3 mol / L) probe stock solution was added to 3 mL of buffer and mixed evenly. The fluorescence excitation wavelength was 640 nm, the excitation slit width was 5.0 nm, the emission slit width was 5.0 nm, and the scanning range was set to 650-800 nm. The probe was first scanned alone to detect the fluorescence intensity at the maximum emission wavelength of 710 nm. Figure 16 It can be seen that the probe remains stable in the buffer system. After the addition of carboxylesterase, the fluorescence emission of the probe solution at 710 nm is significantly enhanced, and CYB-E4 requires 30 minutes to fully respond.

[0098] HeLa cells were cultured in DMEM medium with 10% FBS at 37°C and 5% CO2. The cultured HeLa cells were inoculated into confocal culture dishes, and confocal imaging experiments were performed after the cells adhered. The confocal culture dishes were numbered and grouped, and four groups of cells were treated and imaged differently. The first group of cells served as the blank group. The second group of cells was incubated with 5.0μM CYB-E4 at 37°C for 30 minutes, washed three times with HBSS buffer solution, and then imaged. The third group of cells was pretreated with 10μM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) at 37°C for 30 minutes, then incubated with 5μM CYB-C4 for 45 minutes, and washed three times with HBSS buffer solution before imaging. The fourth group of cells was incubated with 30 μM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) at 37°C for 30 minutes, and then incubated with 5 μM CYB-E4 probe for 30 minutes. They were washed three times with HBSS buffer solution before imaging. When fluorescence imaging was performed using a laser confocal microscope, the excitation wavelength was 638 nm and the collection range was 690-730 nm. The biological imaging image of the near-infrared fluorescent probe CYB-E4 detecting carboxylesterase in living cells is shown in Figure 2. Figure 17 As shown. Figure 17As can be seen in group a, cells that were not incubated with the probe did not emit fluorescence. When HeLa cells were incubated with 5 μM probe at 37°C for 30 minutes, red fluorescence was observed. This fluorescence change indicates that the probe is sensitive to the natural level of carboxylesterase in living cells and can be used to monitor endogenous carboxylesterase in cells ( Figure 17 To further confirm that the probe can specifically recognize carboxylesterase in living cells, group c was pretreated with AEBSF (carboxylesterase inhibitor, 10 μM) for 30 min, and then incubated with 5 μM probe for 45 min before confocal imaging. Compared with group b treated with the probe alone, the fluorescence was weakened ( Figure 17 Group c). Group d was pretreated with AEBSF (carboxylesterase inhibitor, 30 μM) for 30 min, and then incubated with 5 μM probe for 30 min before confocal imaging. As the inhibitor concentration increased, the fluorescence further weakened ( Figure 17 (d) Figure 17e shows the quantification of the relative fluorescence intensity of each group. These experimental results demonstrate that the near-infrared fluorescent probe CYB-E4 exhibits excellent responsiveness and selectivity to carboxylesterases in cells, suggesting promising applications in biological testing.

[0099] In order to further study the application of the probe, this application conducted an in vivo imaging experiment of carboxylesterase on zebrafish. Zebrafish larvae will be cultured in E3 embryo culture medium (containing 0.15mM KCl, 0.33mM MgSO4, 0.33mM CaCl2 and 5mM NaCl, pH 7.0±1.0). Three-day-old zebrafish are divided into four groups and placed in glass dishes for experiments. The first group is a blank group, the second group is cultured with only 5μM CYB-E4 for 30min; the third group is cultured with 10μM AEBSF for 30min, and then with 5μM CYB-E4 for 30min; the fourth group is cultured with 30μM AEBSF for 30min, and then with 5μM CYB-E4 for 30min. The excitation wavelength is 638nm, and the collection range is 690-730nm. As Figure 18 As shown, the blank group ( Figure 18 No red fluorescence was observed in group a), and the second group treated with CYB-E4 ( Figure 18 The zebrafish in group b) showed obvious red fluorescence, while the zebrafish in group 3 ( Figure 18 Group c) showed weak fluorescence. Group 4 ( Figure 18 In group (d), the AEBSF concentration was increased, and the fluorescence produced was significantly weaker than that in group (e). 18e shows the quantification of the relative fluorescence intensity of some groups, indicating that the probe can be used for the detection of carboxylesterase in vivo.

[0100] Staphylococcus aureus (ATCC 29213) was cultured in fresh LB broth at 37°C, shaking overnight at 200 rpm. The next day, 1 mL of the overnight bacterial culture was transferred to a 1.5 mL centrifuge tube and centrifuged at 10,000 rpm for 1 minute. The culture medium was discarded, and the cells were washed three times with PBS to collect the cells. For the blank control group, the cells were diluted 1000-fold with HBSS solution and then imaged. For the probe group, the washed cells were incubated in HBSS containing 5 μM CYB-E4 for 30 minutes, collected by centrifugation, and diluted 1000-fold with HBSS solution before imaging. The excitation light source was 638 nm, and the fluorescence signal was collected within the 690-730 nm range. The minimum inhibitory concentration (MIC) of the probe was determined using the broth microdilution method according to CLSI standards: S. aureus ATCC 29213 was incubated in 1 mL of MHB medium, shaken at 37°C, 200 rpm for 3 h, and diluted 1000-fold before use. CHY-E4 and vancomycin were prepared as stock solutions at 25600 μg / mL and 3200 μg / mL, respectively, and diluted to 256 μg / mL and 32 μg / mL, respectively, using enzyme-free sterile double-distilled water. Compound solution (200 μL) was added to well 1 of a 96-well plate, followed by MHB solution (100 μL) in wells 2 to 10, followed by a two-fold serial dilution from well 1. Vancomycin was diluted in the same manner as a quality control drug. The diluted bacterial culture (100 μL) was then added to the drug-addition wells. Add MHB (200 μL) medium to the 11th well as a negative control, and add diluted bacterial culture (200 μL) to the 12th well as a positive control. Incubate at 37°C for 16-18 hours and read the results. Result judgment: When vancomycin reaches the quality control range, the positive control well is turbid and the negative control well is clear. The lowest concentration in the clear well at this time is the MIC. Figure 19 As shown, the blank group ( Figure 19 Group a) was not incubated with the probe, and fluorescence imaging was collected in the range of 690-730 nm. No fluorescence was generated. However, after co-incubation with 5 μM probe CYB-E4, Figure 19 In group b, bacteria are illuminated by red fluorescence, indicating the feasibility of tracking, imaging, and detecting bacteria using active small molecules. Currently, no fluorescent probes that track Staphylococcus aureus and have antibacterial activity have been reported. The sulfonamide group is introduced into the CYB-E4 structure, and thus it has potential antibacterial activity. Therefore, this application evaluated the MIC of the probe, and the results showed that the MIC value of the probe CYB-E4 was 16 μg / mL, indicating certain antibacterial activity. In summary, the probe has certain potential in the analysis and detection of Staphylococcus aureus.

Claims

1. A near-infrared fluorescent probe for detecting carboxylesterase and having certain antibacterial activity, wherein the structure of the fluorescent probe is as follows: Where R is selected from 、 、 、 、 or , and R is When the compound is recorded as CYB-E1, R is When the compound is recorded as CYB-E2, R is When the compound is recorded as CYB-E3, R is When the compound is recorded as CYB-E4, R is When the compound is recorded as CYB-E5, R is When , the compound is recorded as CYB-E6.

2. The method for preparing the near-infrared fluorescent probe for detecting carboxylesterase and having antibacterial activity according to claim 1, characterized in that: The following steps are involved: (1) Cyclohexanone, PBr3 and N,N-dimethylformamide react in dichloromethane to obtain compound I; (2) 2,4-dihydroxybenzaldehyde, 3,4-dihydro-2H-pyran and pyridinium p-toluenesulfonate react in dichloromethane to obtain compound II; (3) Compound I, Compound II and potassium carbonate react in N,N-dimethylformamide to obtain Compound III; (4) Compound III is reacted with trifluoroacetic acid in dichloromethane with stirring to obtain compound IV; (5) p-Aminobenzenesulfonamide reacts with chloroacetyl chloride in acetone to obtain compound V; (6) Compound V reacts with 2,3,3-trimethyl-3H-indole in acetonitrile to obtain compound VI; (7) Compound IV and triethylamine react with acetyl chloride, butyryl chloride, 2-thiophenecarbonyl chloride, 2-furoyl chloride, benzoyl chloride or N,N-dimethylformyl chloride to obtain compound VII; (8) Compound VI reacts with compound VII in ethanol to obtain the fluorescent probes CYB-E1, CYB-E2, CYB-E3, CYB-E4, CYB-E5, and CYB-E6. The structures of Compound I, Compound II, Compound III, Compound IV, Compound V, Compound VI and Compound VII are as follows: , R is 、 、 、 、 or .

3. The method for preparing a near-infrared fluorescent probe for detecting carboxylesterase and having antibacterial activity according to claim 2, characterized in that: Specifically include the following steps: (1) The preparation process of compound I is as follows: PBr3 is added dropwise to a mixed solution of N,N-dimethylformamide and dichloromethane under ice bath conditions, stirred for 0.5 h to 1 h, cyclohexanone is added to the mixed solution, stirred until the reaction is complete, and then neutralized to neutrality, the product is extracted with dichloromethane, and concentrated by rotary evaporation to obtain the target product; (2) The specific preparation process of compound II is as follows: 2,4-dihydroxybenzaldehyde and pyridinium p-toluenesulfonate are weighed separately and added to dichloromethane, and then 3,4-dihydro-2H-pyran is added under a protective atmosphere. After stirring and refluxing, the reaction is completed, and then rotary evaporation and column chromatography are performed to obtain compound II; (3) The preparation process of compound III is as follows: Compound I, compound II and potassium carbonate prepared above are added to N,N-dimethylformamide in sequence, stirred at 35-45°C until the reaction is complete, cooled to room temperature, filtered and concentrated to obtain a concentrated solution of compound III; (4) The preparation process of compound IV is as follows: the concentrated solution obtained in step (3) is dissolved in dichloromethane, trifluoroacetic acid is added to adjust the pH to 6-7, and the reaction is stirred at 35-45°C until the reaction is complete. The mixture is cooled to room temperature, filtered, and the solid is washed with ethanol to obtain compound IV; (5) The preparation process of compound V is as follows: p-aminobenzenesulfonamide is weighed and added to acetone, chloroacetyl chloride is added dropwise, and the reaction is completed by stirring at 70-80°C, then cooled to room temperature, ice water is added, the resulting mixture is stirred, filtered, and the collected solid is washed with ice water, then recrystallized with ethanol, and filtered. The resulting solid is compound V; (6) The preparation process of compound VI is as follows: under a nitrogen atmosphere, compound V obtained in step (5) is dissolved in acetonitrile, 2,3,3-trimethyl-3H-indole is added, and the reaction is refluxed until completion, the reaction solution is rotary evaporated, dichloromethane is added, and the reaction solution is filtered under reduced pressure to obtain a solid compound VI; (7) The preparation process of compound VII is as follows: under nitrogen atmosphere, compound IV and triethylamine are added to dichloromethane, and acetyl chloride, butyryl chloride, 2-thiophenecarbonyl chloride, 2-furocarbonyl chloride, benzoyl chloride or N,N-dimethylformyl chloride is added under ice bath condition. After stirring, the reaction is completed, and then rotary evaporation and column chromatography are performed to obtain compound VII. (8) The specific preparation process of the fluorescent probe CYB-E is as follows: under a nitrogen atmosphere, compound VII and compound VI are dissolved in ethanol, stirred and refluxed until a solid precipitate is formed, and then filtered. The obtained solid is washed with ethyl acetate and then subjected to column chromatography to obtain probes CYB-E1, CYB-E2, CYB-E3, CYB-E4, CYB-E5, and CYB-E6.

4. The method for preparing a near-infrared fluorescent probe for detecting carboxylesterase and having antibacterial activity according to claim 2 or 3, characterized in that: In step (1), the molar ratio of PBr3, N,N-dimethylformamide and cyclohexanone is (2.5~3.0):(2.0~2.5):1; in step (2), the molar ratio of 2,4-dihydroxybenzaldehyde, 3,4-dihydro-2H-pyran and pyridinium p-toluenesulfonate is 1:(2.0~2.5):0.05; in step (3), the molar ratio of compound I, compound II and potassium carbonate is 1:1:3; in step (5), the molar ratio of p-aminobenzenesulfonamide and chloroacetyl chloride is 1:(1 ~1.5); in step (6), the molar ratio of compound V to 2,3,3-trimethyl-3H-indole is 1:(1~1.5); in step (7), the molar ratio of compound IV, triethylamine to acetyl chloride, butyryl chloride, 2-thenoyl chloride, 2-furoyl chloride, benzoyl chloride or N,N-dimethylformyl chloride is 1:(1~2):(1~2); in step (8), the molar ratio of compound VI to compound VII is 1:(1~1.5).

5. Use of the fluorescent probe according to claim 1 in the detection of carboxylesterase for the purpose of non-disease diagnosis.

6. Use of the fluorescent probe according to claim 1 in the preparation of a cell fluorescence imaging agent.

7. Use of the fluorescent probe according to claim 1 in the preparation of a zebrafish fluorescent imaging agent.

8. Use of the fluorescent probe according to claim 1 in the preparation of an antibacterial active agent.

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