A benzothiazole-chalcone structural compound and a preparation method and application thereof

Abstract

This invention discloses a compound with a benzothiazole-chalcone structure, its preparation method, and its applications. The compound is prepared by synthesizing the target compound K3 from 5-methylsalicylaldehyde and 2-aminothiophenol via a multi-step reaction. This compound exhibits a specific "fluorescence-on" response to carboxylesterase in a DMSO-PBS buffer system, showing significant fluorescence enhancement only in the presence of carboxylesterase at 596 nm, and demonstrates good anti-interference performance with a detection limit of 95 mU / mL. Utilizing the principle that organophosphorus pesticides specifically inhibit carboxylesterase, this compound / carboxylesterase system has been successfully applied to the quantitative detection of organophosphorus pesticides, achieving a detection limit at the 1 μg / L level. This compound also exhibits good biocompatibility and can be used for fluorescence imaging of carboxylesterase activity in human gastric cancer cells. Furthermore, test strips prepared based on this compound can achieve rapid and visual on-site screening of carboxylesterase and organophosphorus pesticides under ultraviolet light through fluorescence color changes.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence analysis technology, and relates to a benzothiazole-chalcone compound, its preparation method, and its application in the rapid detection of carboxylesterases and organophosphorus pesticides. Background Technology

[0002] Carboxylesterases (CEs) are a class of serine hydrolases widely found in animals, plants, and humans, participating in the metabolism of various endogenous ester substrates and exogenous drugs and toxins. Abnormal expression of CEs is closely related to liver disease, metabolic disorders, and cancer, and their activity is also an important biomarker for evaluating the toxicity of environmental pollutants (especially organophosphorus pesticides). Therefore, developing highly sensitive and selective methods for detecting CE activity is of great significance for basic biological research, early disease diagnosis, and food safety and environmental monitoring.

[0003] Organophosphorus pesticides (OPs), as a class of highly effective and broad-spectrum insecticides, are widely used in the cultivation and storage of Chinese medicinal herbs. However, their residue problems seriously threaten the safety of Chinese medicinal herbs and public health. Currently, the legal detection of OPs in Chinese medicinal herbs mainly relies on gas chromatography (GC) and chromatography-mass spectrometry (GC-MS) techniques. Although these methods have high accuracy and sensitivity, they generally suffer from drawbacks such as cumbersome sample pretreatment, expensive equipment, high operational expertise, and long detection cycles, making it difficult to meet the needs of rapid on-site screening in planting bases, agricultural markets, and other similar locations.

[0004] In recent years, biosensing technology based on the principle of enzyme inhibition has provided a new direction for rapid detection, especially fluorescent probe methods using carboxylesterases (CEs) as recognition elements, which have attracted widespread attention due to their high sensitivity and fast response. However, existing CE fluorescent probe technologies still face challenges when applied to the complex matrix of traditional Chinese medicine: on the one hand, complex endogenous substances in the extract of medicinal materials (such as pigments, polyphenols, metal ions, etc.) can easily interfere with the probe signal, resulting in insufficient anti-interference ability and specificity of existing probes; on the other hand, most probe research is limited to solution phase analysis and has failed to develop portable, visual detection forms (such as test strips) suitable for rapid on-site screening. Therefore, there is an urgent need in this field for a novel fluorescent probe and detection system that can specifically overcome the interference of complex matrices in traditional Chinese medicine, achieve highly sensitive and selective detection of trace organophosphorus pesticides, and can be transformed into a simple-to-use rapid on-site screening tool. This invention aims to solve the above-mentioned technical problems. Summary of the Invention

[0005] The purpose of this invention is to provide a novel benzothiazole-chalcone compound and its preparation method; Another object of the present invention is to provide the application of the benzothiazole-chalcone compound in the detection of carboxylesterases; Another object of the present invention is to provide the application of the benzothiazole-chalcone compound in the detection of organophosphorus pesticides.

[0006] I. Compounds with benzothiazole-chalcone structure This invention provides a benzothiazole-chalcone compound with the following structural formula: This compound uses benzothiazole and chalcone as a bichromophore skeleton, and connects benzoyl recognition groups through ester bonds to form a fluorescent probe molecule with intramolecular charge transfer properties.

[0007] II. Preparation methods of benzothiazole-chalcone compounds The method for preparing the compound of the present invention includes the following steps: (1) Synthesis of intermediate c: 5-methylsalicylaldehyde, 2-aminothiophenol and sodium metabisulfite were dissolved in DMF, heated to 110±5 °C and refluxed for 4~6 h. After cooling, distilled water was added to precipitate the solid, and after purification, intermediate c was obtained. (2) Synthesis of intermediate d: Intermediate c and hexamethylenetetramine were added to trifluoroacetic acid under nitrogen protection and the mixture was heated to 65~75 °C and refluxed. After the reaction was completed, the mixture was neutralized to pH=6, and a precipitate was formed. The intermediate d was obtained by column chromatography. (3) Synthesis of compound K1: intermediate d and 1-acetylnaphthalene were dissolved in anhydrous ethanol and refluxed at 100±5 °C with piperidine as catalyst. After the reaction was completed, compound K1 was obtained by extraction, drying, concentration and column chromatography. (4) Synthesis of target compound K3: At 0 °C, compound K1 was dissolved in a mixed solvent of pyridine and anhydrous dichloromethane, benzoyl chloride was added, and after the reaction, the target compound K3 was obtained by extraction, drying, concentration and column chromatography purification.

[0008] In the above preparation method, preferred process conditions include: In step (1), the molar ratio of 5-methylsalicylaldehyde to 2-aminothiophenol is 1:1.1 to 1:1.2; the mass ratio of 2-aminothiophenol to sodium metabisulfite is 1:1.26 to 1:1.3; and the volume ratio of distilled water to DMF added after the reaction is 6 to 7:1. In step (2), the mass ratio of intermediate c to hexamethylenetetramine is 1:1.1 to 1:1.2; In step (3), the mass ratio of 1-acetylnaphthalene to intermediate d is 1:1.4 to 1:1.6; In step (4), the molar ratio of compound K1 to benzoyl chloride is 1:2 to 1:4.

[0009] III. Applications of benzothiazole-chalcone structural compounds 1. Application in the detection of carboxylesterase The benzothiazole-chalcone compound of this invention can be used to prepare reagents for detecting carboxylesterase. The specific detection principle is as follows: the compound itself exhibits weak fluorescence. When it reacts with carboxylesterase, the enzyme catalyzes the hydrolysis of the ester bond, releasing the phenolic hydroxyl product, restoring the intramolecular charge transfer effect, and producing a significant "fluorescence-on" response. This detection is based on the fluorescence "on" response. After the compound reacts with carboxylesterase in a DMSO-PBS buffer, the fluorescence intensity at a wavelength of 596 nm is significantly enhanced. Preferably, the volume ratio of DMSO to PBS in the buffer is 1:9, the pH is 7.4, and the reaction temperature is 37°C.

[0010] 2. Application in the detection of organophosphorus pesticides The benzothiazole-chalcone compound of this invention can also be used to prepare products for detecting organophosphorus pesticides. The detection principle is as follows: organophosphorus pesticides can specifically inhibit the activity of carboxylesterases. Quantitative detection of organophosphorus pesticides is achieved by detecting changes in the fluorescence intensity of the compound / carboxylesterase system. When organophosphorus pesticides are present, carboxylesterase activity is inhibited, preventing effective hydrolysis of the probe molecule, weakening or eliminating the fluorescence enhancement effect, and the fluorescence intensity is negatively correlated with the pesticide concentration. Preferably, the organophosphorus pesticide is dichlorvos or phoxim.

[0011] 3. Detection Method Based on the above applications, the present invention also provides a method for detecting carboxylesterases or organophosphorus pesticides using the compound, comprising: incubating the sample to be tested with the compound in a suitable buffer system, measuring the fluorescence intensity at 596 nm, and calculating the concentration of the analyte according to a standard curve.

[0012] Compared with the prior art, the present invention has the following beneficial effects: This invention, for the first time, integrates benzothiazole and chalcone chromophores through rational molecular design, constructing a novel fluorescent probe backbone with push-pull electron effects. This results in a larger Stokes shift and higher fluorescence quantum yield, effectively reducing background interference. Experiments show that the detection limit of this probe for carboxylesterases reaches 95 mU / mL, and it exhibits a specific fluorescent response only to carboxylesterases in the presence of various amino acids, metal ions, biothiols, and potential interfering enzymes (such as acetylcholinesterase, butyrylcholinesterase, and trypsin), demonstrating excellent anti-interference ability. The probe stabilizes within 40 min when reacting with carboxylesterases, and the fluorescence intensity of the enzymatic digest remains stable within 110 min, providing a wide reading time window for detection.

[0013] Cytotoxicity assays showed that the cell viability remained above 60% after 24 hours of incubation with the probe, demonstrating good biocompatibility. Live-cell fluorescence imaging experiments confirmed that the probe could successfully enter human gastric cancer HGC-27 cells and visualize the detection of endogenous carboxylesterases. Test strips prepared based on this probe can visually determine carboxylesterase activity and the presence of organophosphorus pesticides through fluorescence color changes under ultraviolet light irradiation, requiring no large-scale instruments and suitable for rapid on-site screening at the grassroots level.

[0014] Furthermore, the method of this invention has been successfully applied to the quantitative detection of organophosphorus pesticide residues in traditional Chinese medicinal materials (Plantago asiatica, Houttuynia cordata, and Taraxacum mongolicum), with recovery rates ranging from 97.90% to 105.06%. The precision, repeatability, and stability are good (RSDs are all less than 2%), demonstrating that the probe can effectively overcome interference from the complex matrix of traditional Chinese medicinal materials and has practical application value. In summary, this invention provides a benzothiazole-chalcone fluorescent probe that integrates high sensitivity, high selectivity, good biocompatibility, rapid response, and visual detection. It provides a high-performance tool for carboxylesterase activity analysis, organophosphorus pesticide residue detection, and live-cell imaging, and has broad application prospects in bioanalysis, environmental monitoring, food safety, and traditional Chinese medicine quality control. Attached Figure Description

[0015] Figure 1 This is the 1H NMR spectrum (CDCl3-d6) of the probe compound K3 of this invention.

[0016] Figure 2 This is the carbon NMR spectrum (CDCl3-d6) of the probe compound K3 of this invention.

[0017] Figure 3 This is a high-resolution mass spectrum of the probe compound K3 of this invention.

[0018] Figure 4This is the infrared spectrum of the probe compound K3 of this invention.

[0019] Figure 5 The fluorescence spectra of the probe compound K3 (20 μM) of this invention after adding different analytes to DMSO-PBS buffer (DMSO:PBS=1:9, v / v, pH=7.4).

[0020] Figure 6 These are fluorescence images of the probe compound K3 of this invention after the addition of different analytes under 365 nm ultraviolet light irradiation.

[0021] Figure 7 This figure shows the anti-interference experimental results of the probe compound K3 of this invention in DMSO-PBS buffer for the detection of carboxylesterase.

[0022] Figure 8 The image shows the fluorescence titration spectra of the probe compound K3 of this invention against different concentrations of carboxylesterase in DMSO-PBS buffer.

[0023] Figure 9 This is a fitted curve of the fluorescence intensity of the probe compound K3 at 596 nm as a function of carboxylesterase concentration.

[0024] Figure 10 The graph shows the variation of the autofluorescence intensity of the probe compound K3 under different pH conditions.

[0025] Figure 11 The graph shows the changes in fluorescence intensity after the probe compound K3 reacts with carboxylesterase under different pH conditions.

[0026] Figure 12 This is a line graph comparing the effects of different pH values ​​on the fluorescence intensity of probe K3 and the K3+CEs system.

[0027] Figure 13 The graph shows the variation of the autofluorescence intensity of the probe compound K3 under different temperature conditions.

[0028] Figure 14 The graph shows the changes in fluorescence intensity after the probe compound K3 reacts with carboxylesterase under different temperature conditions.

[0029] Figure 15 This is a curve comparison showing the effect of different temperatures on the fluorescence intensity of probe K3 and the K3+CEs system.

[0030] Figure 16 This is a time-kinetic curve of the reaction between the probe compound K3 of this invention and carboxylesterase.

[0031] Figure 17This is a fluorescence stability graph of the probe compound K3 and the K3+CEs system of the present invention within 110 min.

[0032] Figure 18 Bar chart showing cell viability of HGC-27 cells after incubation with different concentrations of probe K3 for 24 h.

[0033] Figure 19 The images show fluorescence imaging of HGC-27 cells after incubation of the probes K3 and K3+CEs of this invention (Figure a1 is the dark field image of K3; Figure a2 is the bright field image of K3; Figure a3 is the superimposed image of dark and bright fields of K3; Figure b1 is the dark field image of K3-CEs; Figure b2 is the bright field image of K3-CEs; Figure b3 is the superimposed image of dark and bright fields of K3-CEs).

[0034] Figure 20 This is the NMR titration spectrum of the probe compound K3 of this invention before and after the reaction with carboxylesterase.

[0035] Figure 21 This is a high-resolution mass spectrum of the probe compound K3 of this invention before and after its reaction with carboxylesterase.

[0036] Figure 22 The infrared spectra of the probe compound K3 of this invention before and after its reaction with carboxylesterase are shown.

[0037] Figure 23 This is a comparison of the fluorescence spectra of the probe compound K3 of this invention before and after its reaction with carboxylesterase.

[0038] Figure 24 This is a frontier molecular orbital distribution diagram (DFT calculation) of the enzyme digestion products of probe K3 and K3+CEs of this invention.

[0039] Figure 25 The fluorescence titration spectra of the probe K3+CEs system of this invention after adding different concentrations of dichlorvos are shown.

[0040] Figure 26 This is a linear fitting curve of fluorescence inhibition rate versus the logarithm of dichlorvos concentration in the K3+CEs probe system of this invention.

[0041] Figure 27 The images show the fluorescence titration spectra of the K3+CEs probe system of this invention after adding different concentrations of phoxim.

[0042] Figure 28 This is a linear fitting curve of fluorescence inhibition rate versus the logarithm of phoxim concentration in the K3+CEs probe system of this invention.

[0043] Figure 29The fluorescence response of the K3 probe test strip of this invention under a 365nm ultraviolet lamp after being coated with CEs and a CEs + dichlorvos mixed solution, respectively.

[0044] Figure 30 The fluorescence response of the K3 probe test strip of this invention under a 365nm UV lamp after being coated with CEs and a CEs + phoxim mixed solution, respectively. Detailed Implementation

[0045] The preparation of the benzothiazole-chalcone compound K3 described in this invention and its application in carboxylesterase detection and organophosphorus pesticide residue analysis are further described in detail below through specific embodiments, but the scope of protection of this invention is not limited thereto.

[0046] Example 1: Synthesis of probe compound K3 The synthetic route for the benzothiazole-chalcone compound K3 described in this invention is as follows: The specific preparation steps are as follows: (1) 5-Methylsalicylaldehyde (0.68 g, 5 mmol), 2-aminothiophenol (751.14 mg, 6 mmol), and sodium metabisulfite (950.5 mg, 5 mmol) were added sequentially to the reactor and dissolved in 12 mL of DMF. The mixture was heated to 110 °C and refluxed for 5 h. The reaction was monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature, and 70 mL of distilled water was slowly added. The mixture was stirred until a large amount of white solid precipitated. The solid was collected by suction filtration, washed three times with distilled water, and finally dried to obtain white crystalline product c, with a yield of 85%. mp 131-133 °C; Mass spectrometry (ESI-MS): m / z = 241.1 [M+H]⁺ (calculated value C) 14 H 11 NOS: 241.1).

[0047] (2) Compound c (1.45 g, 6 mmol) and hexamethylenetetramine (1.68 g, 12 mmol) were added to a 100 mL Schlenk reaction flask. The flask was evacuated and purged with nitrogen. After adding trifluoroacetic acid, the mixture was heated to 72 °C and refluxed overnight. The reaction progress was monitored by TLC. After the reaction was complete, the mixture was cooled to room temperature and slowly poured into ice water. The mixture was neutralized to pH 6 with saturated NaHCO3 solution, resulting in a pale yellow precipitate d. The solid was collected by filtration and washed three times with distilled water. The crude product was purified by column chromatography (silica gel 100-200 mesh, eluent: petroleum ether / ethyl acetate = 5:1, v / v), and concentrated by rotary evaporation to obtain a pale yellow solid with a yield of 85%. mp174-175℃. ¹H NMR (400 MHz, CDCl3) δ: 12.99 (s, 1H), 10.47 (s,1H), 8.00 (dt, J =0.9, 8.2 Hz, 1H), 7.91 (m, 2H), 7.68 (d, J =2.9 Hz, 1H), 7.43(ddd, J =1.2, 7.2, 8.2 Hz, 1H), 2.39 (s, 3H), mass spectrometry (ESI-MS): m / z = 269 [M+H]⁺ (calculated C) 15 H 11 NO2S: 269.1).

[0048] (3) Compound d (269 mg, 1.0 mmol) and 1-acetylnaphthalene (170 mg, 1.0 mmol) were dissolved in anhydrous ethanol (10 mL), and about 3 drops of piperidine were added as a catalyst. The reaction mixture was refluxed at 100 °C for 24 h. After the reaction was complete, it was cooled to room temperature and extracted with dichloromethane (3 × 20 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (silica gel 100-200 mesh, eluent: petroleum ether / ethyl acetate = 5:1, v / v) to give the target compound K1 (332 mg, yield 75%). Molecular formula: C 27 H 19 NO2S. mp 138-140 ℃. ¹H NMR (400 MHz, CDCl3) δ 13.18 (s, 1H), 8.40 (m, 1H), 8.00 (m, 2H), 7.93 (m, 3H), 7.85 (m, 2H), 7.56 (m, 4H), 7.47 (m, 3H), 7.39 (ddd, J=1.2, 7.2,8.3 Hz, 1H), 2.35 (s, 3H).

[0049] (4) Compound K1 (50.52 mg, 0.12 mmol) was dissolved in a mixed solvent of pyridine and anhydrous dichloromethane (pyridine / dichloromethane = 1:1, total volume 4 mL) at 0 °C, and acyl chloride (0.36 mmol, 3.0 equiv) was added sequentially. The reaction solution was stirred at 0 °C for 30 min, then slowly heated to room temperature and stirred overnight. The reaction progress was monitored by TLC. After the reaction was completed, dichloromethane (5 mL) was added for dilution, the solution was transferred to a separatory funnel, washed with water (10 mL), and extracted three times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 50:1, v / v) to obtain the target compound K3 in 55% yield. mp201–203 ℃, FT-IR (KBr, cm⁻¹): 1740.27 (C=O), 1638.60 (C=C), 1280-1250 (COC); 1 H NMR (400 MHz, CDCl3): δ 8.24 (m, 2H), 8.15 (m, 2H), 7.81 (m, 4H), 7.70(d, J =7.4 Hz, 2H), 7.62 (s, 1H), 7.49 (m, 6H), 7.32 (m, 2H), 7.13 (t, J =7.7 Hz, 1H), 2.52 (s, 3H); 13 C NMR (101 MHz, CDCl3): δ 195.71, 165.05, 162.07, 152.90,145.58, 139.56, 136.93, 136.34, 135.42, 134.10, 133.75, 132.94, 131.61,130.70, 130.48, 130.02, 129.34,128.90, 128.87, 128.45, 127.54, 127.35,126.52, 126.44, 125.62, 125.51, 124.25, 123.43, 121.49, 21.15; HRMS(ESI)m / z:[M+H]⁺.525.14716 (calcd. for C 34 H 23 NO3S, 525.6).

[0050] Example 2: Fluorescence response test of probe compound K3 to carboxylesterase Transfer 5 mL of probe compound K3 in DMSO-PBS buffer solution (DMSO:PBS = 1:9, v / v, pH = 7.4, probe concentration 2.0 × 10⁻⁶). -5 M) was added to a series of colorimetric tubes, and the following analytes were added respectively: 1. K3 (20 μM), 2. CEs (3.0 U / mL), 3. Ala (0.01 mM), 4. Met (0.01 mM), 5. Asp (0.01 mM), 6. Pro (0.01 mM), 7. Leu (0.01 mM), 8. Glu (0.01 mM), 9. His (0.01 mM), 10. Ser (0.01 mM), 11. Val (0.01 mM), 12. Phe (0.01 mM), 13. Lys (0.01 mM), 14. Arg (0.01 mM), 15. Thr (0.01 mM), 16. GSH (0.01 mM), 17. Hcy (0.01 mM), 18. Cys (0.01 mM), 19. Na + (0.01 mM), 20 K + (0.01 mM), 21. Ca 2+ (0.01 mM), 22.Mg 2+ (0.01 mM), 23. Zn 2+ (0.01 mM), 24. Fe 3+ (0.01 mM), 25. Cu 2+ (0.01 mM), 26. Co 2+ (0.01 mM), 27. Cl - (0.01 mM), 28. NO3 - (0.01 mM), 29. CO3 2- (0.01mM), 30. SO4 2-31. EDTA (0.01 mM), 32. Sucrose (0.01 mg / mL), 33. Glucose (0.01 mg / mL), 34. Maltose (0.01 mg / mL), 35. LZ (3.0 U / mL), 36. AChE (3.0 U / mL), 37. BChE (3.0 U / mL), 38. D-biotin (0.01 mg / mL), 39. Trypsin (3.0 U / mL). After thorough mixing, the mixture was incubated at 37°C for 60 min, and then the fluorescence spectra of each system were measured (excitation wavelength 390 nm, emission wavelength scanning range 450–750 nm, slit width 10 nm). The results are as follows: Figure 5 and Figure 6 As shown, the system with only CEs added exhibited significant fluorescence enhancement at 596 nm, with the solution changing from pale yellow to bright yellow; other analytes did not cause significant fluorescence changes. Anti-interference experiment ( Figure 7 This indicates that the addition of the other analytes in the presence of CEs has no significant effect on the fluorescence intensity, proving that the probe has high selectivity for CEs.

[0051] Example 3: Sensitivity and detection limit of probe compound K3 for detecting carboxylesterases In the test system of Example 2, the concentration of probe K3 was fixed at 20 μM, and different concentrations of CEs (0, 0.01, 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 3.0 U / mL) were added. After incubation at 37 °C for 60 min, the fluorescence intensity at 596 nm was measured. The results are as follows. Figure 8 As shown, the fluorescence intensity gradually increases with increasing CE concentration. Within the range of 0-3.0 U / mL, the fluorescence intensity exhibits a good linear relationship with the CE concentration. Figure 9 The linear equation is: F 596 = 285.26576 X + 94.35879 (R) 2 =0.99389), where X is the concentration of CEs (U / mL). The detection limit is calculated to be 95 mU / mL using the 3σ / s method.

[0052] Example 4: Effects of pH and temperature on the detection system K3 solution and K3+CEs (3.0 U / mL) system were prepared according to the method in Example 2. The pH was adjusted to 3.0, 4.0, 5.0, 6.0, 7.0, 7.4, 8.0, and 9.0, respectively, and the fluorescence intensity was measured. The results are as follows: Figure 10-12As shown, K3 itself exhibits weak and stable fluorescence within the pH range of 3.0–9.0; the K3+CEs system shows significantly enhanced fluorescence within the pH range of 6.0–9.0, and this fluorescence increases with increasing pH. Considering the physiological conditions of biological samples, pH 7.4 is preferred.

[0053] Separately, K3 solution and K3+CEs system were incubated at different temperatures (0, 10, 25, 37, 55 ℃) for 60 min, and the fluorescence intensity was measured. The results are as follows: Figure 13-15 As shown, K3 itself has good thermal stability; the K3+CEs system exhibits the strongest fluorescence at 37 ℃, and the fluorescence intensity decreases at temperatures above or below this point, therefore the preferred reaction temperature is 37 ℃.

[0054] Example 5: Reaction Kinetics and Stability CEs (0–0.6 U / mL) were added to a K3 solution (20 μM), and the fluorescence intensity at 596 nm was continuously measured over time at 37 °C. The results are as follows: Figure 16 As shown, the fluorescence intensity rapidly increased within 15 min and tended to stabilize after 40 min; therefore, a reaction time of 60 min was chosen to ensure complete reaction. Stability experiment ( Figure 17 This indicates that the fluorescence intensity of K3 itself and the K3+CEs system changes only slightly within 110 min, demonstrating good time stability.

[0055] Example 6: Cytotoxicity and Live Cell Imaging Experiments Human gastric cancer HGC-27 cells were co-incubated with different concentrations of K3 (0, 5, 10, 15, 20, 25 μM) for 24 h, and cell viability was determined by the MTT assay. The results are as follows: Figure 18 As shown, the cell viability remained above 60% after incubation with 20 μM K3, indicating that the probe had low cytotoxicity. Cell imaging experiment: HGC-27 cells were divided into two groups: one group was treated with only K3 (20 μM), and the other group was treated with K3 (20 μM) followed by CEs (0.6 U / mL). After incubation for 30 min, the cells were observed using a fluorescence microscope (excitation wavelength 390 nm, emission channel 500-600 nm). The results are shown below. Figure 19 As shown, cells with only K3 added showed no fluorescence (a2), while cells with K3+CEs added showed significantly enhanced orange fluorescence (b2), demonstrating that the probe can enter the cell and respond to endogenous carboxylesterases.

[0056] Example 7: Mechanism analysis of probe compound K3 recognizing CEs Through high-resolution mass spectrometry, 1 The response mechanism of the fluorescent probe K3 to carboxylesterases (CEs) was investigated by 1H NMR titration, FT-IR, fluorescence spectroscopy, and DFT calculation. High-resolution mass spectrometry (such as...) Figure 21 As shown in the figure, a new peak appears at m / z 422.12280 after K3 reacts with CEs, confirming that the ester bond is hydrolyzed to generate a phenolic hydroxyl product. 1 H NMR titration showed a phenolic hydroxyl signal at δ≈13 ppm (e.g. Figure 20 (As shown). FT-IR spectrum (e.g. Figure 22 As shown in the figure, the characteristic peak of the ester group of K3 is weakened, and at 3448 cm⁻¹ -1 An OH absorption peak appears at [location missing]. Fluorescence spectroscopy indicates that the emission peaks of K3+CEs coincide with those of compound K1 (e.g., [missing information]). Figure 23 As shown). Combined with DFT calculation (such as... Figure 24 As shown in the figure, the response mechanism is as follows: benzoyl group inhibits ICT, leading to fluorescence quenching; CEs catalyze the hydrolysis of ester bonds to release phenolic hydroxyl groups, and restore ICT to enhance fluorescence.

[0057] Example 8: Practical application of probe compound K3 in the detection of CEs 1. To verify the practicality of probe K3, this experiment will use a 2.0 × 10⁻⁶ probe. -5 10 mL each of mmol / L K3 solution, K3-CEs solution, K3-CEs-dichlorvos solution, and K3-CEs-phoxim solution were poured into filter paper petri dishes, incubated for at least 24 h, and the color changes were observed under UV light. Simultaneously, visual detection was performed using test strips. The test strips were immersed in DMSO / PBS solution containing the above solutions, incubated for 24 h, dried, and observed under UV light. The results showed that the system containing only K3 (test strips and filter paper) was dark gray; after the addition of CEs, the fluorescence significantly increased, turning bright orange; while in the system containing dichlorvos or phoxim, the fluorescence was significantly quenched. These results indicate that the probe K3 can achieve rapid and visual detection of CEs and their inhibitors (dichlorvos, phoxim) on filter paper and test strips, without the need for large instruments, and has the potential to be developed into a portable detection tool (e.g., Figure 29 and Figure 30 (As shown).

[0058] 2. Application in rapid detection of pesticide residues in Chinese medicinal herbs 2.1 Preparation of probe K3 stock solution: Accurately weigh 52.6 mg of probe K3 solid and place it in a 50 mL volumetric flask. Add a mixture of DMSO and PBS (V:V = 1:9) to the mark, shake well, and let stand. The final concentration is 2.0 × 10⁻⁶. -3 mol / L.

[0059] Preparation of CEs working solution: Accurately weigh 0.02 mg of CEs solid and dilute to 2 mL with PBS buffer to obtain a CEs working solution with a concentration of 10 U / mL. This solution should be prepared and used immediately.

[0060] 2.2 Preparation of pesticide residue samples from fresh Chinese medicinal herbs 2.2.1 Preparation of Fresh Chinese Medicinal Herbs to Simulate Pesticide Contamination Using fresh plantain, houttuynia cordata, and dandelion as research subjects, the herbs were first washed and dried with gauze, then cut into small cubes and mixed thoroughly. 5.0 g of each herb sample was accurately weighed and evenly spread on a clean tray. Using a sprayer, an acetone solution of the target organophosphorus pesticide (such as dichlorvos or phoxim) was evenly sprayed onto the surface of the herbs, with different pesticide contamination gradients (0, 200, 500, and 1500 mg / kg). The samples were air-dried in a fume hood in the dark for 2-4 hours, while parallel samples without pesticide application were prepared as blank controls.

[0061] 2.2.2 Preparation of pesticide extract Sample extraction: Weigh 5.0 g of each contaminated sample and blank control sample for each concentration gradient, add 10 mL of PBS buffer (pH=7.4), shake for 3 min, let stand, and filter to obtain the pesticide extract.

[0062] Pesticide detection system: In PBS buffer (pH=7.4), pesticide extract, CEs working solution and DMSO were added sequentially, mixed and incubated at 37℃ for 20 min. Then, probe K3 stock solution (K3:DMSO and PBS 1:9) was added, and incubation continued for 40 min. Finally, the fluorescence spectrum was measured.

[0063] Data analysis: Using the fluorescence intensity of the blank control group as a benchmark, the enzyme activity inhibition rate of the samples was calculated. The pesticide concentration was inversely estimated using the standard curve of "inhibition rate - logarithm of pesticide concentration" and converted into the residue in the original medicinal material (mg / kg). Finally, the recovery rate was calculated.

[0064] 2.3 Establishment of the Standard Curve Preparation of standard solutions: Add 3 mg / mL dichlorvos and phoxim standard solutions dropwise to the probe working solution containing CEs (final probe concentration 2.0 × 10⁻⁶). -5 Fluorescent titration was performed using pesticide concentration (mol / L). Fluorescence measurement: The fluorescence intensity change of probe K3 was monitored at 596 nm (λex = 390 nm, slit width 10 nm, voltage 680 V). Standard curve plotting: A standard curve was plotted with the logarithm of pesticide concentration on the x-axis and the fluorescence intensity change on the y-axis.

[0065] 2.4 Detection of pesticide content in Chinese medicinal herbs 2.4.1 Determination of probe K3: Take 50 μL of 2.0 × 10 -3Add the mol / L probe K3 stock solution to the fresh Chinese medicinal herb pesticide extract prepared in step “4.2.2”, and adjust the volume to 5 mL with PBS buffer. Mix well and incubate at 37 °C for 60 min.

[0066] Fluorescence intensity measurement: The fluorescence intensity of probe K3 (λex / λem=390 / 596 nm) in the above system was measured.

[0067] Calculate the inhibition rate (IR): The inhibition rate is calculated using formula (1): (1) In the formula, F0 is the fluorescence intensity of the blank control (without pesticide extract), and F is the fluorescence intensity of the system after adding pesticide extract.

[0068] 3. Establishment of a method for detecting pesticides in Chinese medicinal herbs using probe K3. Based on the principle of enzyme inhibition, this invention constructs a fluorescence detection system based on probe K3, and evaluates its detection performance using dichlorvos and phoxim as examples. The specific method is as follows: In a 5 mL colorimetric tube, CEs solution (3.0 U / mL), PBS buffer (pH 7.4, 100 mM), DMSO, and different concentrations of dichlorvos (0-1400 μg / L) or phoxim solution (0-1500 μg / L) are added sequentially. After mixing, the mixture is incubated at 37℃ for 20 min; then, probe K3 stock solution (2 mM) is added, and incubation continues for 40 min, followed by immediate measurement of the fluorescence spectrum. In the final reaction system, the concentration of CEs is 3.0 U / mL, the concentration of K3 is 20 μM, the concentration of PBS is 50 mM, and the volume fraction of DMSO is 10%. The results show that as the concentration of dichlorvos or phoxim increases (e.g., ...), the fluorescence spectrum is significantly reduced. Figure 25 and Figure 27 As shown in the figure, the fluorescence intensity of the K3-CEs system decreased in a concentration-dependent manner. When the concentration of dichlorvos reached 1400 μg / L or the concentration of phoxim reached 1500 μg / L, the fluorescence signal decreased to a level similar to that of the K3 blank group. In the range of 0-1400 μg / L, the inhibition rate showed a good linear relationship with the logarithm of the dichlorvos concentration, and the fitting equation was: Inhibition (%) = 86.07671 × log[Dichlorvos] - 181.47101 (R²) 2 = 0.99461), IC50 was 489 μg / L, and detection limit was 1.149 μg / L; in the range of 0-1500 μg / L, the inhibition rate also showed a good linear relationship with the logarithm of phoxim concentration, and the fitting equation was: Inhibition (%) = 96.16083 × log[Phoxim] - 216.73058 (R ...). 2= 0.99775), IC50 was 593 μg / L, and detection limit was 1.049 μg / L. These results indicate that this probe system can sensitively and quantitatively detect the inhibitory effect of organophosphorus pesticides on CEs (e.g., α = 0.99775), Figure 26 and Figure 28 (As shown).

[0069] 3.1 Methodological Examination To verify the reliability of the method for detecting pesticide residues in Chinese medicinal herbs based on the fluorescent probe K3, this study systematically investigated its precision, repeatability, stability, blank interference, and recovery rate. All sample pretreatment was performed according to method "2.2". Fluorescence detection was performed according to method "2.3" to determine the fluorescence intensity of probe K3 (λex / λem = 390 / 596 nm), and the content of the corresponding pesticides was calculated using standard curves.

[0070] 3.1.1 Precision Examination To evaluate the precision of the instrument and detection system, six aliquots of Plantago asiatica (5 g each) were weighed and measured six times consecutively under the same conditions. The fluorescence intensity of probe K3 was recorded, and the concentrations of dichlorvos and phoxim were calculated. The relative RSD of the six measurements was used as the precision index (as shown in Tables 1 and 2). The mean values ​​of probe K3 were 1038.42 ± 2.92 μg / L and 1003.52 ± 8.48 μg / L, with RSDs of 0.28% and 0.84%, respectively. The results indicate that both probes have good precision, verifying the stability and reliability of the method.

[0071] Table 1. Precision assessment of dichlorvos determination based on probe K3. Table 2. Precision evaluation of phoxim determination based on probe K3 3.1.2 Repeatability Test Fluorescence intensity was measured at 596 nm for extracts from six plantain samples (5 g each), and the residues of dichlorvos and phoxim were calculated using a standard curve. Six parallel determinations were performed to assess the repeatability of the method; the results are shown in Tables 3 and 4.

[0072] Data showed that the mean values ​​for probe K3 were 1041.70 ± 12.30 μg / L and 999.70 ± 7.10 μg / L, with RSDs of 1.18% and 0.71%, respectively. Both probes demonstrated good repeatability, indicating that the method is stable and reliable.

[0073] Table 3 Repeatability test of dichlorvos determination based on probe K3 Table 4. Repeatability test of phoxim determination based on probe K3 3.1.3 Stability Test The fluorescence intensity of six plantain extracts (5 g each) was measured at 20, 40, 60, 80, 100, and 120 min at the emission wavelength of their respective fluorescent probes. The results are shown in Tables 5 and 6.

[0074] The fluorescence response of probe K3 also stabilized after 20 min. The average values ​​of dichlorvos and phoxim residues determined by probe K3 were 1045.39 ± 10.04 μg / L and 989.85 ± 9.68 μg / L, respectively, with RSDs of 0.96% and 0.98%. These results indicate that the detection methods of the two probes have good repeatability in terms of time stability.

[0075] Table 5. Stability assessment of dichlorvos determination based on probe K3. Table 6. Stability Study of Phosphate Determination Based on Probe K3 3.1.4 Recovery rate assessment To verify the applicability of the K3 probe in actual samples, three representative samples—Plantago asiatica, Houttuynia cordata, and Taraxacum mongolicum—were selected for recovery experiments. Different concentrations of dichlorvos (500, 1000, 1400 μg / L) and phoxim (500, 1000, 1500 μg / L) standard solutions were added to the sample extracts, and the fluorescence intensity was measured at an emission wavelength of 596 nm. The results showed that the recoveries of dichlorvos in the three samples were 99.65%–105.06%, with standard deviations of 0.17%–2.45%; the recoveries of phoxim were 98.14%–103.94%, with standard deviations of 0.24%–2.45%. All recoveries were within the specified range of 80%–110%, and the standard deviations were small, indicating that the K3 probe has high accuracy and good applicability in various plant samples and can be used for the actual detection of pesticide residues in traditional Chinese medicinal materials.

[0076] Table 7. Results of the recovery rate of dichlorvos determined by probe K3 (n=3) Table 8. Results of recovery of phoxim by probe K3 (n=3) 3.2 Blank control The established extraction method was used to obtain an extract of plantago asiatica without pesticides. The fluorescence intensity of the plantago asiatica without pesticides was detected by the probe compound K3. The results showed that the fluorescence intensity of the plantago asiatica in 6 tests was the same as that of the plantago asiatica without pesticides.

[0077] In summary, this invention provides a benzothiazole-chalcone structured fluorescent probe compound for carboxylesterase and establishes a method for detecting carboxylesterase in solution under fluorescent conditions. In a solution of (DMSO:PBS = 1:9, V / V), various analytes were added, and only when the carboxylesterase changed did it enhance the fluorescence of the solution, exhibiting good anti-interference performance. Using pesticide residues in organophosphorus medicinal herbs as the detection target, a standard curve method was used to achieve ultrasensitive and rapid detection of organophosphorus medicinal herbs. This benzothiazole-chalcone probe compound can effectively determine the content of organophosphorus residues in medicinal herbs, with advantages such as fast analysis speed, stable fluorescence performance, and real-time detection. It is particularly suitable for the detection of organophosphorus pesticide residues in traditional Chinese medicine, food, and other fields, and has broad application value.

Claims

1. A benzothiazole-chalcone compound with the following structural formula: 。 2. The method for preparing the benzothiazole-chalcone compound according to claim 1, characterized in that, Includes the following steps: (1) Dissolve 5-methylsalicylaldehyde, 2-aminobenzylthiophenol and sodium metabisulfite in DMF, heat to 110±5 °C and reflux for 4~6 h, cool and add distilled water to precipitate solid, and purify to obtain intermediate c; (2) Intermediate c and hexamethylenetetramine were added to trifluoroacetic acid under nitrogen protection, and the mixture was heated to 65-75 °C and refluxed. After the reaction was completed, the mixture was neutralized to pH=6, and a precipitate was formed. Intermediate d was obtained by column chromatography. (3) Intermediate d and 1-acetylnaphthalene were dissolved in anhydrous ethanol and refluxed at 100±5 °C with piperidine as catalyst. After the reaction was completed, the mixture was extracted, dried, concentrated and purified by column chromatography to obtain compound K1. (4) At 0 °C, compound K1 was dissolved in a mixed solvent of pyridine and anhydrous dichloromethane, benzoyl chloride was added, and after the reaction, the compound was extracted, dried, concentrated and purified by column chromatography to obtain the target compound K3.

3. The method of claim 2, wherein: In step (1), the molar ratio of 5-methylsalicylaldehyde to 2-aminothiophenol is 1:1.1 to 1:1.2; the mass ratio of 2-aminothiophenol to sodium metabisulfite is 1:1.26 to 1:1.3; and the volume ratio of distilled water to DMF added after the reaction is 6 to 7:

1.

4. The method of claim 2, wherein: In step (2), the mass ratio of intermediate c to hexamethylenetetramine is 1:1.1 to 1:1.

2.

5. The method of claim 2, wherein: In step (3), the mass ratio of 1-acetylnaphthalene to intermediate d is 1:1.4 to 1:1.

6.

6. The method of claim 2, wherein: In step (4), the molar ratio of compound K1 to benzoyl chloride is 1:2 to 1:

4.

7. The use of the benzothiazole-chalcone compound according to claim 1 in the preparation of reagents for detecting carboxylesterases.

8. Use according to claim 7, characterized in that: The detection is based on a fluorescence "on" response. After the compound reacts with carboxylesterase in a DMSO-PBS buffer, the fluorescence intensity at a wavelength of 596 nm is significantly enhanced. The volume ratio of DMSO to PBS in the buffer is 1:9, the pH is 7.4, and the reaction temperature is 37°C.

9. The application of the benzothiazole-chalcone compound according to claim 1 in the preparation of products for detecting organophosphorus pesticides, characterized in that: By utilizing the inhibitory effect of organophosphorus pesticides on carboxylesterases, quantitative detection of organophosphorus pesticides can be achieved by detecting changes in fluorescence intensity of the compound / carboxylesterase system.

10. The application according to claim 9, characterized in that: The organophosphorus pesticide mentioned is dichlorvos or phoxim.

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