Matrine fluorescent probe, preparation method and application

Abstract

The invention relates to the technical field of chemical synthesis and fluorescence imaging, in particular to a matrine fluorescent probe, a preparation method and application. The method comprises the following steps: dissolving 2, 3, 3-trimethylindole S-1 in acetonitrile, reacting with methyl iodide, and washing the product to obtain light pink solid S-2; the preparation method comprises the following steps: dissolving 2, 3, 3-trimethylindole S-1 in methylbenzene to react with 6-bromohexanoic acid, filtering and drying to obtain a purple red solid S-3; weighing the S-2 and the S-4 to react to generate a compound S-5, and continuously adding the S-3 to react to obtain a dark purple solid S-6 (namely cyano dye CY7-COOH); the preparation method comprises the following steps: weighing Me3SiN3, and reacting the Me3SiN3 with HOAc, sophocarpine (S-7) and DBU in sequence to obtain a light yellow viscous oily substance S-8; dissolving the S-8 in absolute methanol, and reacting with Pd / C to obtain a faint yellow oily substance S-9; dissolving S-9 and S-6 in dichloromethane, adding HATU and DIEA, reacting and purifying to obtain a target product dark purple solid TZ-1, namely the matrine fluorescent probe. The fluorescent probe is based on the fluorescence characteristic of CY7 dye, a conventional medicine matrine has fluorescence performance through multi-step chemical synthesis, and the fluorescent probe can be applied to the field of fluorescence imaging of biological cells, tissues and experimental animals. The fluorescent probe disclosed by the invention is good in chemical stability and light stability and strong in fluorescence signal, and has profound biomedical research significance.

Description

Technical Field

[0001] The present invention relates to the technical fields of chemical synthesis and fluorescence imaging, and particularly relates to a matrine fluorescent probe, a preparation method and an application thereof. Background Art

[0002] As an alkaloid with significant potential for drug development, matrine has a variety of different pharmacological activities and has far-reaching significance in aspects such as anti-tumor, anti-virus, anti-inflammatory, anti-myocardial fibrosis, antibacterial, anti-parasitic, etc.; however, at the same time, matrine is accompanied by characteristics such as low bioavailability, liver and kidney damage caused by long-term use, central nerve paralysis and disorder, poor liposolubility, and short in vivo half-life, which add challenges to its clinical application. Understanding the target organs of drugs can optimize dosage forms, enhance the utilization rate and efficacy of drugs, and avoid drug toxicity. This requires researchers to determine the main target organs of matrine in vivo through pharmaceutical research. Conventional in vivo metabolism detection methods of drugs (such as high performance liquid chromatography) require a large amount of samples, a long test cycle, and low instrument sensitivity. In vivo imaging technology allows repeated evaluation in the same group of animals, reducing the minimum number of animals required for each group; it has sufficient sensitivity and dynamic range to continuously track the entire kinetic process of drugs for a long time without damaging the animal body, and can be used as a substitute or supplement for traditional preclinical efficacy research.

[0003] Therefore, providing a synthesized matrine fluorescent probe to provide technical support for subsequent distribution research of matrine in mice is a technical problem that needs to be solved by those skilled in the art. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to endow matrine with fluorescence properties, and to develop a stable and strong-signal matrine fluorescent probe and its preparation method.

[0005] In order to achieve the above purpose, the technical solution adopted in this patent is as follows:

[0006] In the first aspect, the present invention provides a matrine fluorescent probe, the structure of which is shown in Figure (Ⅰ); the structure of matrine is shown in Figure (Ⅱ); the structure of CY7-COOH cyanine dye is shown in Figure (Ⅲ):

[0007]

[0008] It should be noted that the molecular formula of the fluorescent probe of the present invention is C 49 H 64 N5O2 +, with a relative molecular mass of 755.0835, the probe is a dark purple solid powder, insoluble in water and easily soluble in dimethyl sulfoxide. After testing, the probe of the present invention has the strongest fluorescence signal in the wavelength range of 710 - 780, belonging to a near-infrared fluorescence probe, with less background interference, can penetrate tissues for deep imaging, and is suitable for small animal in vivo imaging. The probe of the present invention has good stability, and the fluorescence quenching is not obvious within 48 hours, and can be used for long-term monitoring.

[0009] In a second aspect, the present invention provides a method for preparing the above-mentioned matrine fluorescence probe, comprising the following steps:

[0010] Dissolve 2,3,3-trimethylindole S-1 in acetonitrile and react with methyl iodide, wash the product to obtain a light pink solid S-2; dissolve 2,3,3-trimethylindole S-1 in toluene and react with 6-bromohexanoic acid, filter and dry to obtain a purplish red solid S-3; weigh S-2 and react with S-4 to form compound S-5, then continue to add S-3 to react to obtain a dark purple solid S-6, namely the cyanine dye CY7-COOH; weigh Me3SiN3 and react with HOAc, sophocarpine (S-7), and DBU in sequence to obtain a light yellow viscous oil S-8; dissolve S-8 in anhydrous methanol and react with Pd / C to obtain a light yellow oil S-9; dissolve S-9 and S-6 in dichloromethane, add HATU and DIEA to react and purify to obtain the target product, a dark purple solid TZ-1, namely the matrine fluorescence probe.

[0011] Specifically, the method for preparing the matrine fluorescence probe is as follows:

[0012] (1) Dissolve 2,3,3-trimethylindole S-1 in acetonitrile, add methyl iodide, and heat the reaction mixture under reflux conditions; cool the reaction solution to room temperature, let it stand for several hours to fully precipitate the solid, then filter to collect the filter cake, wash the filter cake with petroleum ether, and then wash it again with a petroleum ether / acetonitrile mixed solvent, and then drain the solvent and dry it to obtain a light pink solid S-2, and its synthesis route is:

[0013]

[0014] (2) Dissolve 2,3,3-trimethylindole S-1 in toluene, then add 6-bromohexanoic acid, and heat the reaction mixture under reflux conditions for reaction; cool the reaction solution to room temperature, let it stand for several hours, and the reaction solution in the reaction flask will be stratified up and down, remove the upper layer of liquid, and retain the lower layer of viscous solid; add ethyl acetate to the reaction flask, heat and stir, then let it stand, and remove the liquid; add ethyl acetate again, heat and stir and then let it stand, filter, wash the filter cake with petroleum ether, filter again, collect the solid, and dry it to obtain a purplish red solid S-3, and its synthesis route is:

[0015]

[0016] (3) At room temperature, weigh S-2 into a round-bottom flask, add Ac2O and stir to dissolve, then add S-4. The system starts to heat up, and continue stirring the reaction. At this time, S-5 is generated in the reaction, and no treatment is required. Then add S-3, and then drip anhydrous pyridine. After adding, continue stirring the reaction; turn off the heating and cool the reaction solution to room temperature;

[0017] Take a beaker, add deionized water, and while stirring with a glass rod, add the cooled reaction solution. A viscous dark blue solid precipitates in the system, showing a purple luster; after the reaction flask is rinsed with a small amount of pyridine, the liquid is also added to the water; then adjust the pH of the system with dilute hydrochloric acid, let it stand, discard the liquid in the beaker, and retain the solid in the beaker; add deionized water to the beaker, ultrasonic and discard the aqueous phase, and then add deionized water again, ultrasonic and discard the aqueous phase;

[0018] Collect the solid in the beaker, perform silica gel column chromatography, first use pure ethyl acetate, and then use a gradient elution system of dichloromethane / methanol to obtain a dark purple solid S-6, that is, the cyanine dye CY7-COOH. Its synthetic route is:

[0019]

[0020] (4) Weigh Me3SiN3 into a round-bottom flask, add toluene and stir to dissolve in the system, then add HOAc and stir at room temperature, then add sophocarpine S-7, and then add DBU. The system has a slight exothermic temperature rise, and continue stirring the reaction at room temperature; first use a water jet vacuum pump to distill off most of the solvents and reagents under reduced pressure, and then replace it with a rotary vane vacuum oil pump to continue pumping to obtain a light yellow viscous oil S-8. Its synthetic route is:

[0021]

[0022] (5) At room temperature, add anhydrous methanol to the round-bottom flask of the previous step S-8, stir to dissolve, then displace with N2 protection, add 10% content specification Pd / C, and then displace the system with H2, and continue stirring the reaction; remove H2, displace the system with N2 atmosphere, filter with a Buchner funnel, wash the filter cake with anhydrous methanol, combine the filtrates, and concentrate the filtrates under reduced pressure to dryness to obtain a light yellow oil S-9. Its synthetic route is:

[0023]

[0024] (6) Dissolve the prepared S-9 uniformly in 10 mL of dichloromethane for standby. Weigh S-6, namely CY7-COOH, put it in a round-bottom flask, add dichloromethane and stir to dissolve. Then displace with N2 for protection, cool down in an ice bath, add HATU, stir, add DIEA, continue to stir, and then add the dichloromethane solution of S-9. Remove the ice bath and continue to stir at room temperature for 3 h. Transfer the reaction solution into a separatory funnel, add dichloromethane, and then wash it once with dilute hydrochloric acid, once with deionized water, once with potassium carbonate solution, once with deionized water, and once with saturated brine. Then collect the dichloromethane phase, dry it with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to dryness to obtain the crude product. The crude product is eluted by gradient elution on a silica gel column to obtain a dark purple solid TZ-1. Its synthetic route is as follows:

[0025]

[0026] Optionally, in step (1), the molar ratio of 2,3,3-trimethylindole to methyl iodide is 1:1.1; the mass-volume ratio of 2,3,3-trimethylindole to acetonitrile is 1 g:5 mL; the volume ratio of petroleum ether to acetonitrile is 1:1; the reflux heating time is 15 h.

[0027] Optionally, in step (2), the molar ratio of 2,3,3-trimethylindole to 6-bromohexanoic acid is 1:1; the reflux heating time is 24 h; add ethyl acetate equal to the volume of toluene solvent to the viscous solid, heat up to 60 °C and stir for 30 min, repeat this step 2 times and then filter.

[0028] Optionally, in step (3), the molar ratio of S-2, S-4 to S-3 is 1:1.1:1.1; the volume ratio of Ac2O to anhydrous pyridine is 2:1; when S-2 and S-4 react, heat up to an internal temperature of 120 °C and continue to stir and react for 30 min, add S-3 and continue to react at 120 °C for 3 h; adjust the pH of the reactant to 2-3 with dilute hydrochloric acid, let it stand for two hours, and wash ultrasonically with deionized water 2 times; the solid product is eluted first with ethyl acetate and then with a gradient of dichloromethane / methanol.

[0029] Optionally, in step (4), the molar ratio of Me3SiN3, HOAc, sophocarpine to DBU is 6.4:6.2:1:0.25; the reaction solvent is toluene; the product is evacuated with a vacuum water pump and then with a vacuum oil pump at 40 °C for 1 h.

[0030] Optionally, in step (5), the mass of Pd / C is 130 mg; the reactant and Pd / C are stirred and reacted under H2 conditions for 24 h.

[0031] Optionally, in step (6), the molar ratio of S-6, HATU, and DIEA is 1:3:6; the end product is washed successively with 50 mL of 0.5 N dilute hydrochloric acid, deionized water, 5% potassium carbonate solution, 50 mL of deionized water, and saturated brine, and eluted using silica gel column chromatography.

[0032] In a third aspect, the present invention provides the use of the above-mentioned matrine fluorescent probe in the fields of biomolecule detection and medical diagnosis.

[0033] Specifically, the present invention provides the use of the above-mentioned matrine fluorescent probe in small animal in vivo imaging technology and drug in vivo distribution research.

[0034] Compared with the prior art, the beneficial technical effects brought by the present invention are as follows:

[0035] Fluorescence imaging technology has been widely used in the fields of biomolecule detection, medical diagnosis, etc. due to its many advantages. In the present invention, matrine is linked with CY7-COOH through a chemical reaction, making the matrine drug have fluorescence properties. This probe belongs to a near-infrared fluorescent probe and has more obvious advantages; it has strong penetration into biological tissues, is less affected by light scattering of biological tissues in the body, has higher near-infrared imaging brightness and imaging efficiency; has high resolution, and near-infrared imaging has almost no interference from fluorescence background, thus providing real-time imaging information with higher signal-to-noise ratio and high spatio-temporal resolution, making this technology more suitable for in vivo research. Description of the Drawings

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0037] Figure 1 CNMR spectrum of S-6 (CY7-COOH cyanine dye) prepared in Example 1 of the present invention;

[0038] Figure 2 HNMR spectrum of S-6 (CY7-COOH cyanine dye) prepared in Example 1 of the present invention;

[0039] Figure 3 Mass spectrum of S-6 (CY7-COOH cyanine dye) prepared in Example 1 of the present invention;

[0040] Figure 4 HNMR spectrum of TZ-1 (matrine fluorescent probe) prepared in Example 1 of the present invention;

[0041] Figure 5 Mass spectrum of TZ-1 (matrine fluorescent probe) prepared in Example 1 of the present invention;

[0042] Figure 6 Fluorescence image of S-6 (CY7-COOH cyanine dye) prepared in Example 1 of the present invention;

[0043] Figure 7 Fluorescence image of TZ-1 (matrine fluorescent probe) prepared in Example 1 of the present invention;

[0044] Figure 8 Synthesis route diagram of TZ-1 (matrine fluorescent probe);

[0045] Figure 9 Fluorescence imaging atlas of TZ-1 (A) and CY7-COOH (B) in mice;

[0046] Figure 10 Effects of CY7-COOH and TZ-1 on the PRRSV N gene of PAMs cells. Different lowercase letters (a, b, c, d, e) indicate significant differences between groups (p < 0.05). Detailed implementation manners

[0047] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0048] The special term "embodiment" here, any embodiment described as "exemplary" does not have to be construed as superior to or better than other embodiments. For the performance index tests in the embodiments of this application, unless otherwise specified, the conventional test methods in the art are adopted. It should be understood that the terms described in this application are only used to describe specific embodiments and are not intended to limit the content disclosed in this application.

[0049] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the technical field to which this application belongs; the test methods and technical means not otherwise specifically noted in this application refer to the experimental methods and technical means commonly adopted by those of ordinary skill in the art.

[0050] To better illustrate the content of this application, numerous specific details are given in the following detailed embodiments. Those skilled in the art should understand that this application can still be implemented without some specific details. In the embodiments, some methods, means, instruments, equipment, etc. well known to those skilled in the art are not described in detail in order to highlight the main idea of this application.

[0051] Under the premise of no conflict, the technical features disclosed in the embodiments of the present application can be combined arbitrarily, and the resulting technical solutions belong to the contents disclosed in the embodiments of the present application.

[0052] The invention discloses a matrine fluorescent probe, a preparation method and an application thereof.

[0053] In order to better understand the present invention, the present invention is further specifically described below through the following examples, but it should not be understood as a limitation of the present invention. Some non-essential improvements and adjustments made by technicians in this field based on the above invention content are also considered to fall within the protection scope of the present invention.

[0054] Example 1

[0055] In an embodiment of the present invention:

[0056] Required experimental equipment: electronic balance, RE-52AA rotary evaporator, DF-101S thermal collector constant temperature heating magnetic stirrer, electric constant temperature blast drying oven, etc.

[0057] Required experimental reagents: 2,3,3-trimethylindole (CAS: 1640-39-7), iodomethane (CAS: 74-88-4), 6-bromohexanoic acid (CAS: 4224-70-8), pentadienal diphenylamine hydrochloride (CAS: 1497-49-0), sophorafoetida (CAS: 145572-44-7), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, CAS: 6674-22-2 ), trimethylsilyl azide (CAS: 4648-54-8), palladium on carbon (CAS: 7440-05-3), 2-(7-azobenzotriazole)-tetramethyluronium hexafluorophosphate (HATU, CAS: 148893-10-1), pyridine, HOAc, DIEA, acetonitrile, dichloromethane, methanol, sodium chloride, petroleum ether, ethyl acetate, etc. The main raw materials were purchased from Leyan, Anage, Bid Pharmaceutical, McLean, Aladdin, Xiens and other reagent platforms.

[0058] 1. Synthesis of S-2

[0059] Procedure: 2,3,3-Trimethylindole S-1 (5 g, 31.4 mmol, 1 eq.) was dissolved in acetonitrile (25 mL), and then iodomethane (5 g, 35.23 mmol, 1.1 eq.) was added and the reaction mixture was heated under reflux for 15 hours.

[0060] Workup: Cool the reaction solution to room temperature, let it stand for several hours to fully precipitate the solid, then filter to collect the filter cake. Wash the filter cake once with 10 mL of petroleum ether, wash it again with 10 mL of a mixed solvent of petroleum ether / acetonitrile (1:1), then drain the solvent and dry to obtain a light pink solid S-2 (9.0 g), with a yield of 95.2%.

[0061] 1 H NMR (400 MHz, DMSO) δ 7.93 - 7.90 (m, 1H), 7.84 - 7.82 (m, 1H), 7.65 - 7.59 (m, 2H), 3.98 (s, 3H), 2.78 (s, 3H), 1.53 (s, 6H).

[0062] 2. Synthesis of S-3

[0063] Procedure: Dissolve 2,3,3-trimethylindole S-1 (6.37 g, 40.0 mmol, 1 eq.) in toluene (20 mL), then add 6-bromohexanoic acid (7.8 g, 40.0 mmol, 1 eq.). Heat the reaction mixture under reflux for 24 hours.

[0064] Workup: (1) Cool the reaction solution to room temperature. After standing for several hours, the reaction solution in the reaction flask is layered. Remove the upper layer of liquid and retain the lower viscous solid. (2) Add 20 mL of ethyl acetate to the reaction flask, heat to 60 °C, stir for 30 min, then let it stand for 15 min and remove the liquid; add 20 mL of ethyl acetate again, heat to 60 °C, stir for 30 min, then let it stand for 15 min, filter. Pulp the filter cake with petroleum ether, filter again, collect the solid, and dry to obtain a purple-red solid S-3 (11.2 g), with a yield of 79.0%.

[0065] 1 H NMR (400 MHz, DMSO) δ 8.00 - 7.97 (m, 1H), 7.86 - 7.84 (m, 1H), 7.63 - 7.61 (m, 2H), 4.47 (t, J = 7.8 Hz, 2H), 2.86 (s, 3H), 2.23 (t, J = 7.3 Hz, 2H), 1.89 - 1.81 (m, 2H), 1.60 - 1.51 (m, 8H), 1.47 - 1.41 (m, 2H).

[0066] 3. Synthesis of S-6

[0067] Operation: At room temperature, weigh S-2 (3010 mg, 10 mmol, 1 eq.) into a 100 mL round-bottom flask, add 30 mL of Ac2O and stir to dissolve. After 10 min, add S-4 (3130 mg, 11 mmol, 1.1 eq.). The system starts to heat up. After the internal temperature reaches 120 °C, continue stirring and reacting for 30 min. At this time, S-5 is generated in the reaction and does not need to be treated. Then add S-3 (3900 mg, 11 mmol, 1.1 eq.), and then dropwise add 15 mL of anhydrous pyridine. After adding, the system continues to stir and react at 120 °C for 3 h.

[0068] Work-up: Turn off the heating and cool the reaction solution to room temperature. Take a 500 mL beaker, add 300 mL of deionized water, and add the cooled reaction solution while stirring with a glass rod. A viscous dark blue solid precipitates in the system, showing a purple luster. After rinsing the reaction flask with a small amount of pyridine, the liquid is also added to the water. Then adjust the pH of the system to 2 - 3 with dilute hydrochloric acid, let it stand for 2 h, discard the liquid in the beaker, and retain the solid in the beaker; add 100 mL of deionized water to the beaker, ultrasonic for 15 min and then discard the aqueous phase. Then add 100 mL of deionized water again, ultrasonic for 15 min and then discard the aqueous phase. Collect the solid in the beaker, perform silica gel column chromatography, first use pure ethyl acetate, and then use a gradient elution system of dichloromethane / methanol to collect the target product to obtain a dark purple solid S-6 (1.3 g), with a yield of 20.4%. S-6 is the cyanine dye CY7-COOH.

[0069] 1 H NMR (400 MHz, DMSO) δ 7.87 (td, J = 13.1, 6.0 Hz, 2H), 7.76 (t, J = 12.8 Hz, 1H), 7.57 (dd, J = 7.4, 3.4 Hz, 2H), 7.42 - 7.34 (m, 4H), 7.25 - 7.19 (m, 2H), 6.58 - 6.49 (m, 2H), 6.34 (dd, J = 13.8, 4.3 Hz, 2H), 4.05 (t, J = 7.5 Hz, 2H), 3.59 (s, 3H), 2.18 (t, J = 7.3 Hz, 2H), 1.70 - 1.63 (m, 13H), 1.58 - 1.50 (m, 3H), 1.42 - 1.33 (m, 2H).

[0070] 1313C NMR (100 MHz, DMSO-d6) δ 174.83, 172.47, 171.38, 151.28, 143.35, 142.66, 141.40, 128.92, 128.87, 125.72, 125.13, 124.92, 122.85, 122.75, 111.45, 111.36, 104.56, 103.98, 49.11, 49.06, 43.81, 34.05, 31.63, 27.69, 27.49, 27.18, 26.17, 24.72.

[0071] MS (ESI) m / z (M)+ (C34H41N2O2+) The calculated value by ChemDraw is 509.3163, and the measured value is 509.3167.

[0072] 4. Synthesis of S-8

[0073] Procedure: Weigh Me3SiN3 (3000 mg, 26.04 mmol, 6.4 eq.) into a 25 mL round-bottom flask, add 10 mL of toluene to the system and stir to dissolve. Then add HOAc (1500 mg, 25 mmol, 6.2 eq.). After stirring at room temperature for 30 min, add sophocarpine S-7 (1000 mg, 4.06 mmol, 1 eq.), and then DBU (150 mg, 1 mmol, 0.25 eq.). The system has a slight exothermic temperature rise. Continue to stir and react at room temperature for 24 h.

[0074] Workup: Remove most of the solvents and reagents by rotary evaporation under reduced pressure with a vacuum water pump. Then replace the rotary vane vacuum oil pump and continue to pump under vacuum at 40 °C for 1 h to obtain a light yellow viscous oil S-8 (crude product 1.27 g), which is directly used for the next step.

[0075] 5. Synthesis of S-9

[0076] Procedure: At room temperature, add 15 mL of anhydrous methanol to the round-bottom flask of the above-mentioned S-8 (crude product 1.27 g). After stirring to dissolve, replace with N2 protection, add 10% Pd / C (130 mg, 10% w / w.), and then replace the system with H2. Continue to stir and react for 24 h.

[0077] Workup: Remove H2, replace the system with N2 atmosphere, filter with a Buchner funnel, wash the filter cake with 10 mL of anhydrous methanol, combine the filtrates, and concentrate the filtrates under reduced pressure to dryness to obtain a light yellow oil S-9 (crude product, 1.07 g), which is directly used for the next step.

[0078] 11H NMR (600 MHz, DMSO-d6, TMS): δ 8.50 - 7.80 (m, 2H), 4.11 (dd, 1H, J = 12.0 Hz, 4.2 Hz), 3.96 - 3.89 (m, 1H), 3.53 - 3.45 (m, 1H), 3.01 (t, 1H, J = 12.0 Hz), 2.80 - 2.70 (m, 2H), 2.62 - 2.57 (m, 1H), 2.40 - 2.35 (m, 1H), 2.15 - 1.96 (m, 3H), 1.95 - 1.82 (m, 3H), 1.66 - 1.52 (m, 2H), 1.50 - 1.26 (m, 5H);

[0079] MS (ESI) m / z (M)+ (C15H26N3O+) calculated by ChemDraw is 264.21, found 264.20.

[0080] 6. Synthesis of TZ-1

[0081] Procedure: The crude product S-9 (1.07 g) from the previous step was dissolved uniformly in 10 mL of dichloromethane for standby; 948 mg (1.49 mmol, 1 eq.) of S-6 was weighed into a 100 mL round-bottom flask, dissolved by stirring in 35 mL of dichloromethane, then replaced with N2 for protection, cooled in an ice bath, HATU (1700 mg, 4.47 mmol, 3 eq.) was added, and after stirring for 15 min, DIEA (1155 mg, 8.94 mmol, 6 eq.) was added. After continuing to stir for 20 min, the dichloromethane solution of S-9 was added, the ice bath was removed, and stirring was continued at room temperature for 3 h.

[0082] Workup: The reaction solution was transferred to a separatory funnel, 200 mL of dichloromethane was added, and then it was washed successively with 50 mL of 0.5 N dilute hydrochloric acid once, 50 mL of deionized water once, 50 mL of 5% potassium carbonate solution once, 50 mL of deionized water once, and 50 mL of saturated brine once; then the dichloromethane phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to dryness to obtain the crude product. The crude product was purified by gradient elution on a silica gel column to obtain a dark purple solid TZ-1 (246 mg), with a yield of 18.7%.

[0083] 11H NMR (400 MHz, DMSO) δ 7.91 - 7.83 (m, 3H), 7.78 - 7.72 (m, 1H), 7.58 - 7.56 (m, 2H), 7.42 - 7.33 (m, 4H), 7.25 - 7.20 (m, 2H), 6.56 - 6.48 (m, 2H), 6.36 - 6.31 (m, 2H), 4.16 - 4.13 (m, 1H), 4.04 - 3.93 (m, 3H), 3.91 - 3.80 (m, 1H), 3.58 - 3.56 (m, 4H), 2.98 - 2.86 (m, 2H), 2.78 - 2.63 (m, 3H), 2.44 - 2.27 (m, 2H), 2.20 - 2.12 (m, 1H), 2.12 - 1.96 (m, 4H), 1.87 - 1.77 (m, 4H), 1.68 - 1.59 (m, 13H), 1.59 - 1.19 (m, 17H);

[0084] MS (ESI) m / z (M)+ (C49H64N5O2+) The calculated value by ChemDraw is 754.5055, and the measured value is 754.5063. Test example

[0085] Required experimental materials: The PAMs used in this experiment were isolated and preserved by our laboratory. Matrine was purchased from the National Institutes for Food and Drug Control, China. PRRSV was derived from the highly pathogenic PRRSV live vaccine (JXA1 strain), which was amplified and preserved by our laboratory. After determination, its TCID 50 was 10 -5.69 / mL. 1640 medium (Gibco, USA), fetal bovine serum (CellMax, China), penicillin-streptomycin mixture (Solarbio, China), CCK8 (Boster, China), PBS (Solarbio, China), reverse transcription kit (Tolo, China), SYBR (polymerase, China);

[0086] Required experimental equipment: Fluorescence inverted microscope (Olympus, Japan), CO2 incubator (Heal force, China), pipettor (Eppendorf, Germany), laminar flow hood (Shanghai Boxun, China), micro nucleic acid and protein concentration detector (Thermo Scientific, USA), biological safety cabinet (Likon, China), high-speed centrifuge, PCR instrument (Bori Technology, China), ABI 7500 quantitative PCR instrument (Applied Biosystems, USA), etc.;

[0087] Matrine was used as a positive control in this experiment at a concentration of 0.4 mg / kg (preliminary laboratory verification showed that this concentration was safe and effective against PRRSV), TZ-1 concentrations were 1.67 μg / mL, 0.835 μg / mL, and 0.4175 μg / mL, and CY7-COOH concentrations were 2.22 μg / mL, 1.11 μg / mL, and 0.55 μg / mL.

[0088] 1. Efficacy test of TZ-1 and CY7-COOH

[0089] Use culture medium to adjust PAMs to an appropriate concentration. After 12 hours, change the medium and add culture medium of different treatment groups: blank control group (Cell control), virus control group (Virus control), matrine positive control group (0.4 mg / mL, MT), high (1.67 μg / mL, TVH), medium (0.835 μg / mL, TVM), and low (0.4175 μg / mL, TVL) doses of TZ-1 drug groups, as well as high (2.22 μg / mL, CVH), medium (1.11 μg / mL, CVM), and low (0.55 μg / mL, CVL) doses of CY7-COOH drug groups. After 12 hours of culture, cell samples were collected.

[0090] After extracting total RNA from cells in each treatment group according to the TRlzol instructions, the RNA was reverse transcribed into cDNA according to the Tolo Harbor kit instructions and then used for qRT-PCR detection. The reaction system was established according to the instructions of the Polymer fluorescent quantitative reagent, and the expression of PRRSVN mRNA was detected by qRT-PCR. The results are shown in Figure 10 The primer sequences corresponding to each gene in the reaction are shown in Table 1.

[0091] Table 1 Summary of primer sequence information

[0092]

[0093]

[0094] All data are expressed as mean ± standard deviation (Mean ± SD), and the differences among groups were analyzed using one-way analysis of variance in GraphPad Prism 8.0.2 software. Different lowercase letters (a, b, c) indicate significant differences (p < 0.05). Figure 9 .

[0095] The results showed that the high (1.67 μg / mL, TVH) and medium (0.835 μg / mL, TVM) doses of matrine fluorescent probe (TZ-1) had therapeutic effects on PRRS virus infection, and the therapeutic effect of the high-dose matrine fluorescent probe was equivalent to that of matrine used as a positive control drug, proving that the matrine fluorescent probe had higher activity. High (2.22 μg / mL, CVH), medium (1.11 μg / mL, CVM), and low (0.55 μg / mL, CVL) doses of CY7-COOH had no therapeutic effects, and the medium and high doses promoted virus infection. Therefore, it can be inferred that the therapeutic effect of the matrine fluorescent probe does not come from CY7-COOH.

[0096] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A matrine fluorescent probe, characterized in that, The structure of the probe is shown in Figure (Ⅰ); the structure of matrine is shown in Figure (Ⅱ); the structure of CY7-COOH cyanine dye is shown in Figure (Ⅲ):

2. A preparation method of the matrine fluorescence probe as described in claim 1, characterized in that, The method includes the following steps: Dissolve 2,3,3-trimethylindole S-1 in acetonitrile and react it with methyl iodide, wash the product to obtain a light pink solid S-2; dissolve 2,3,3-trimethylindole S-1 in toluene and react it with 6-bromohexanoic acid, filter and dry to obtain a purple-red solid S-3; weigh S-2 and react it with S-4 to form compound S-5, then continue to add S-3 and react to obtain a deep purple solid S-6, namely the cyanine dye CY7-COOH; weigh Me3SiN3 and react it with HOAc, sophocarpine (S-7), and DBU in sequence to obtain a light yellow viscous oil S-8; dissolve S-8 in anhydrous methanol and react it with Pd / C to obtain a light yellow oil S-9; dissolve S-9 and S-6 in dichloromethane, add HATU and DIEA, react and purify to obtain the target product, a deep purple solid TZ-1, namely the matrine fluorescent probe described above.

3. The preparation method of the matrine fluorescent probe according to claim 2, characterized in that, The specific operation of the method is as follows: (1) Dissolve 2,3,3-trimethylindole S-1 in acetonitrile, add methyl iodide, and heat the reaction mixture under reflux conditions; cool the reaction solution to room temperature, let it stand for several hours to fully precipitate solids, then filter to collect the filter cake. Wash the filter cake with petroleum ether, wash it again with a mixed solvent of petroleum ether / acetonitrile, and then drain the solvent and dry it to obtain a light pink solid S-2. The synthesis route is as follows: (2) Dissolve 2,3,3-trimethylindole S-1 in toluene, then add 6-bromohexanoic acid, and heat the mixture under reflux conditions; cool the reaction solution to room temperature, let it stand for several hours, and the reaction solution in the reaction flask will be layered up and down, remove the upper layer of liquid, and retain the lower viscous solid; add ethyl acetate to the reaction flask, heat and stir, then let it stand, remove the liquid; add ethyl acetate again, heat and stir and then let it stand, filter, wash the filter cake with petroleum ether, filter again, collect the solid, and dry to obtain a purple-red solid S-3. Its synthesis route is: (3) At room temperature, weigh S-2 in a round-bottom flask, add Ac2O and stir to dissolve, then add S-4, the system starts to heat up, continue to stir and react. At this time, S-5 is formed and does not need to be treated. Continue to add S-3, and then dropwise add anhydrous pyridine. After adding, continue to stir and react; turn off the heating, and cool the reaction solution to room temperature; Take a beaker, add deionized water, and while stirring with a glass rod, add the cooled reaction solution. A viscous dark blue solid will precipitate from the system, showing a purple luster; after the reaction flask is rinsed with a small amount of pyridine, the liquid is also added to the water; then adjust the pH of the system with dilute hydrochloric acid, let it stand, discard the liquid in the beaker, and retain the solid in the beaker; add deionized water to the beaker, ultrasonically discard the aqueous phase, and then add deionized water again, ultrasonically discard the aqueous phase; Collect the solid in the beaker, perform silica gel column chromatography, first use pure ethyl acetate, and then use a gradient elution system of dichloromethane / methanol to obtain a deep purple solid S-6, namely the cyanine dye CY7-COOH. Its synthesis route is: (4) Weigh Me3SiN3 in a round-bottom flask, add toluene to the system and stir to dissolve, then add HOAc and stir at room temperature, then add sophocarpine S-7, and then add DBU. The system has a slight exothermic temperature rise, and continue to stir and react at room temperature; first use a water aspirator to reduce the pressure and rotary evaporate to remove most of the solvents and reagents, and then replace it with a rotary vane vacuum oil pump to continue pumping to obtain a light yellow viscous oil S-8. Its synthesis route is: (5) At room temperature, anhydrous methanol was added to the round-bottom flask in the previous step S-8. After stirring and dissolving, N2 was replaced for protection. 10% Pd / C was added, and then the system was replaced with H2, and stirring reaction was continued; H2 was removed, the system was replaced with N2 atmosphere, filtered with a Buchner funnel, the filter cake was washed with anhydrous methanol, the filtrates were combined, and the filtrate was concentrated under reduced pressure to dryness to obtain a pale yellow oily substance S-9. Its synthetic route is as follows: (6) Dissolve the previous step S-9 uniformly with 10 mL of dichloromethane for standby. Weigh S-6, that is, CY7-COOH, into a round-bottom flask, add dichloromethane and stir to dissolve, then replace N2 for protection, cool down in an ice bath, add HATU, stir and then add DIEA, continue stirring and then add the dichloromethane solution of S-9, remove the ice bath, and continue stirring at room temperature for 3 h; transfer the reaction solution into a separatory funnel, add dichloromethane, and then wash once with dilute hydrochloric acid, once with deionized water, once with potassium carbonate solution, once with deionized water, and once with saturated brine; then collect the dichloromethane phase, dry it with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure to dryness to obtain the crude product, and the crude product was eluted by gradient elution on a silica gel column to obtain a dark purple solid TZ-1. Its synthetic route is as follows:

4. The preparation method of the matrine fluorescence probe according to claim 3, characterized in that, In step (1), the molar ratio of 2,3,3-trimethylindole to methyl iodide is 1:1.1; the mass-volume ratio of 2,3,3-trimethylindole to acetonitrile is 1 g:5 mL; the volume ratio of petroleum ether to acetonitrile is 1:1; the reflux heating time is 15 h.

5. The preparation method of the matrine fluorescent probe according to claim 3, wherein, In step (2), the molar ratio of 2,3,3-trimethylindole to 6-bromohexanoic acid is 1:1; the reflux heating time is 24 h; add ethyl acetate equal to the volume of toluene solvent to the viscous solid, heat to 60 °C and stir for 30 min, and repeat this step 2 times and then filter.

6. The preparation method of the matrine fluorescent probe according to claim 3, wherein In step (3), the molar ratio of S-2, S-4 to S-3 is 1:1.1:1.1; the volume ratio of Ac2O to anhydrous pyridine is 2:1; When S-2 reacts with S-4, the internal temperature is raised to 120 °C and stirring reaction is continued for 30 min. After adding S-3, the reaction is continued at 120 °C for 3 h; The reactant is adjusted to pH 2-3 with dilute hydrochloric acid, left standing for two hours, and ultrasonically washed 2 times with deionized water; the solid product is eluted by gradient elution with ethyl acetate first and then dichloromethane / methanol.

7. The preparation method of the matrine fluorescent probe according to claim 3, characterized in that, In step (4), the molar ratio of Me3SiN3, HOAc, sophocarpine to DBU is 6.4:6.2:1:0.25; the reaction solvent is toluene; the product is evacuated at 40 °C for 1 h with a vacuum water pump first and then a vacuum oil pump.

8. The preparation method of the matrine fluorescence probe according to claim 3, characterized in that, In step (5), the mass of Pd / C is 130 mg; the reactant and Pd / C are stirred and reacted under H2 conditions for 24 h.

9. The preparation method of the matrine fluorescent probe according to claim 3, wherein, In step (6), the molar ratio of S-6, HATU to DIEA is 1:3:6; the final product is washed successively with 50 mL of 0.5 N dilute hydrochloric acid, deionized water, 5% potassium carbonate solution, 50 mL of deionized water, and saturated brine, and eluted with a silica gel column chromatography.

10. Use of the matrine fluorescence probe as described in claim 1 or the matrine fluorescence probe prepared by the method as described in any one of claims 2-3 in the fields of biomolecule detection and medical diagnosis.

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