A fluorescent probe compound, a preparation method thereof and an application thereof

Abstract

The present invention relates to the field of applications of fluorescent probes, and discloses a fluorescent probe compound, a preparation method thereof and an application. The compound has a structure shown in formula (I). The compound provided by the present invention can be used as a fluorescent probe, has the advantages of high spatio-temporal resolution, visualization, non-invasiveness, etc., and can be used for the activity test and tracking of related targets in vivo.

Description

Technical Field

[0001] The present invention relates to the field of applications of fluorescent probes, and specifically relates to a fluorescent probe compound, a preparation method thereof, and an application thereof. Background Art

[0002] As a typical representative of ferrooxidoreductase, 4-hydroxyphenylpyruvate dioxygenase (HPPD) is a key enzyme in the tyrosine metabolic pathway in organisms, widely present in aerobic organisms, and is also an important target for developing drugs and pesticides.

[0003] HPPD can catalyze the oxidation of 4-hydroxyphenylpyruvic acid (HPPA) to produce homogentisic acid (HGA). In plants, homogentisic acid is an important precursor for the synthesis of plastoquinone and tocopherol, which is crucial for plant photosynthesis. Therefore, if HPPD is inhibited, plants will turn white and die due to the lack of photosynthetic pigments. Therefore, HPPD can be used as a herbicide target, and since the 1990s, a variety of herbicides targeting HPPD have been on the market.

[0004] In long-term application practice, it has been found that herbicides targeting HPPD have the advantages of being environmentally friendly and not easily generating resistance. Therefore, developing new herbicide varieties targeting HPPD has always been a hot research direction in pesticide research.

[0005] In mammalian bodies, homogentisic acid needs to be further converted into maleylacetoacetate (MAA) under the catalysis of homogentisic acid dioxygenase, and then MAA successively generates acetoacetate and fumarate in maleylacetoacetate isomerase (MAAI) and fumarylacetoacetase (FAH) in mammalian bodies, and finally participates in the tricarboxylic acid cycle in the body. In the field of medical discovery, HPPD inhibitors can be used to treat various diseases caused by abnormal tyrosine metabolism.

[0006] Currently, the methods for evaluating the activity of HPPD inhibitors can be divided into two categories: in vitro methods and in vivo methods.

[0007] In vitro methods mainly evaluate the activity of inhibitors by analyzing methods such as ultraviolet coupling method, isotope labeling method, chromatography, etc. to detect the inhibitory effect of candidate compounds on HPPD in vitro.

[0008] In vivo methods involve directly applying the HPPD inhibitor to living plants and evaluating its activity by observing its killing effect on the plants.

[0009] However, both of these methods currently have some deficiencies. In vitro methods can only provide information on the interaction between the compound to be selected and HPPD. However, the in vivo environment is complex and variable, and the absorption, conduction, and metabolism of the inhibitor by the plant will all affect its effect.

[0010] Therefore, in vitro methods often cannot reflect the effect of the inhibitor in actual applications. Although in vivo methods can reflect the true effect, on the one hand, they are time-consuming (at least more than one week) and not easy to quantify (only relying on visual evaluation of biological activity), and on the other hand, they cannot obtain the interaction between the inhibitor and HPPD in vivo, and off-target effects may occur.

[0011] In summary, it is necessary to provide a new method that can be used for in vivo activity testing and tracking of related targets. Summary of the Invention

[0012] The object of the present invention is to provide a new class of fluorescent probes and methods that can be used for in vivo activity testing and tracking of related targets.

[0013] To achieve the above object, the first aspect of the present invention provides a fluorescent probe compound, which has the structure shown in formula (I):

[0014]

[0015]

[0016] Wherein, in formula (I), n is an integer from 1 to 6;

[0017] Q is the structure shown in formula (Q1) or the structure shown in formula (Q2);

[0018] In formula (Q1) and formula (Q2), R is selected from H, C 1-6 alkyl, -NO2, and halogen.

[0019] The second aspect of the present invention provides a method for preparing the compound described in the first aspect. The method includes: contacting and reacting the compound shown in formula (II) with the compound shown in formula (III) in the presence of a solvent and a basic substance,

[0020]

[0021] Wherein, in formula (II) and formula (III), the definitions of Q, R, and n are the same as those described in the first aspect.

[0022] The third aspect of the present invention provides the application of the fluorescent probe compound described in the first aspect above in constructing a visual in vivo screening method for HPPD inhibitors.

[0023] The compound provided by the present invention can be used as a fluorescent probe, has the advantages of high spatio-temporal resolution, visualization, non-invasiveness, etc., and can be used for activity testing and tracking of related targets in vivo.

[0024] The compound provided by the present invention can be used as a fluorescent probe targeting HPPD and can be made into a fluorescence visualization instrument. Moreover, the present invention has developed a visual in vivo screening method for inhibitors, which can visually evaluate the inhibition status of HPPD in vivo quickly (within 48 hours). BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the visual result of the detection method of the present invention described in Example 3 of the present invention for the inhibition effect of HPPD in rapeseed in vivo. Among them, a represents the visual result of the detection method of the present invention for the inhibition effect of mesotrione on HPPD in rapeseed in vivo; b represents the quantitative data of the fluorescence value in a; c represents the visual result of the detection method of the present invention for the inhibition effect of methyl quizalofop on HPPD in rapeseed in vivo; d represents the quantitative data of the fluorescence value in c.

[0026] Figure 2 It is the visual result of the detection method of the present invention described in Example 4 of the present invention for the inhibition effect of HPPD in zebrafish in vivo. Among them, a represents the visual result of the detection method of the present invention for the inhibition effect of nitisinone on HPPD in zebrafish in vivo; b represents the quantitative data of the fluorescence value in a. DETAILED DESCRIPTION OF THE INVENTION

[0027] The endpoints and any values disclosed in this text are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this text.

[0028] As described above, the first aspect of the present invention provides a fluorescent probe compound, and this compound has the structure shown in formula (I):

[0029]

[0030] Wherein, in formula (I), n is an integer from 1 to 6;

[0031] Q is a structure represented by formula (Q1) or a structure represented by formula (Q2);

[0032] In formula (Q1) and formula (Q2), R is selected from H, C 1-6 alkyl, -NO2 and halogen.

[0033] The C 1-6 alkyl in the present invention includes straight-chain alkyl and branched-chain alkyl with a total number of carbon atoms of 1-6, including but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl.

[0034] Preferably, in formula (I),

[0035] n is 1, 2, 3, 4, 5 or 6;

[0036] Q is a structure represented by formula (Q1) or a structure represented by formula (Q2);

[0037] R is selected from H, C 1-3 alkyl, -NO2, fluorine, chlorine, bromine.

[0038] More preferably, in formula (I),

[0039] n is 1, 2, 3, 4, 5 or 6;

[0040] Q is a structure represented by formula (Q1) or a structure represented by formula (Q2);

[0041] R is selected from H, methyl, -NO2, fluorine, chlorine, bromine.

[0042] Preferably, in formula (I),

[0043] n is 4;

[0044] Q is a structure represented by formula (Q2);

[0045] R is selected from H, methyl, -NO2, fluorine, chlorine, bromine.

[0046] Particularly preferably, in formula (I),

[0047] n is 4;

[0048] Q is a structure represented by formula (Q1) or a structure represented by formula (Q2);

[0049] R is selected from H, -NO2.

[0050] The present invention has no particular requirements for the specific method of preparing the aforementioned compound. Those skilled in the art can determine a suitable synthetic route in combination with the structural formula characteristics of the present invention and the known knowledge in the art to obtain the aforementioned compound. However, in order to obtain the compound of the present invention with higher purity and yield, the method described in the second aspect of the present invention is provided to prepare the compound of the present invention.

[0051] As described above, the second aspect of the present invention provides a method for preparing the compound described in the first aspect, the method comprising: contacting the compound represented by formula (II) with the compound represented by formula (III) in the presence of a solvent and a basic substance,

[0052]

[0053] wherein, in formula (II) and formula (III), the definitions of Q, R and n are the same as those described in the first aspect.

[0054] Preferably, the conditions of the contacting reaction include: the temperature is 0 - 50 °C and the time is 12 - 48 h.

[0055] Preferably, the contacting reaction is carried out in the presence of a catalyst.

[0056] Preferably, the catalyst is 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N-ethyldiisopropylamine with a molar ratio of 1:1 - 3.

[0057] Preferably, the solvent is selected from at least one of dichloromethane, acetonitrile, and DMF.

[0058] Preferably, the basic substance is at least one organic base selected from triethylamine, p-dimethylaminopyridine, pyridine, and N,N-diisopropylethylamine.

[0059] Preferably, the molar ratio of the amount of the compound represented by formula (II) to the amount of the compound represented by formula (III) is 1 - 3:1.

[0060] The present invention has no particular limitation on the sources of the compound represented by formula (II) and the compound represented by formula (III). Those skilled in the art can determine a suitable synthetic route by using the methods known in the art or can obtain them through commercial purchase. An exemplary obtaining method is provided in the following text of the present invention, and those skilled in the art should not understand it as a limitation to the present invention.

[0061] Preferably, in formula (II), Q is a structure represented by formula (Q1), and the compound represented by formula (II) is prepared by a method comprising the following steps: in the presence of a solvent, reacting a compound represented by formula (II-1), a compound represented by formula (II-2), oxalyl chloride (CO(Cl)2) and triethylamine in a molar ratio of 1:1 - 1.2:1 - 2:1.5 - 3 in a first reaction.

[0062]

[0063] Preferably, the conditions of the first reaction include: the temperature is -20°C to 20°C, and the time is 12 - 48 h.

[0064] Preferably, in the first reaction, the solvent is selected from at least one of dichloromethane, acetonitrile, and tetrahydrofuran.

[0065] Preferably, the first reaction is carried out in the presence of a first catalyst, and the first catalyst is acetone cyanohydrin.

[0066] Preferably, in formula (II), Q is a structure represented by formula (Q2), and the compound represented by formula (II) is prepared by a method comprising the following steps:

[0067] S1: In the presence of a solvent, reacting a compound represented by formula (II-3), a compound represented by formula (II-4), triphosgene in a molar ratio of 1.5 - 3:1.5 - 3:1 in a second reaction to obtain a compound represented by formula (II-5);

[0068] S2: Reacting the compound represented by formula (II-5), DCC and HCOOH in a molar ratio of 1:1.5 - 3:5 - 9 in a third reaction to obtain a compound represented by formula (II-6);

[0069] S3: Reacting the compound represented by formula (II-6), the compound represented by formula (II-2), oxalyl chloride (CO(Cl)2) and triethylamine in a molar ratio of 1:1 - 1.2:1 - 2:1.5 - 3 in a fourth reaction to obtain a compound represented by formula (II-7);

[0070] S4: In the presence of ethyl acetate, hydrolyzing the compound represented by formula (II-7) in an aqueous lithium hydroxide solution to obtain a compound represented by formula (II-8).

[0071]

[0072] Preferably, the conditions of the second reaction include: the temperature is 0 - 100°C, and the time is 12 - 48 h.

[0073] Preferably, in the second reaction, the solvent is selected from at least one of 1,4-dioxane, pyridine, and DMF.

[0074] Preferably, the second reaction is carried out in the presence of an inorganic base cesium carbonate.

[0075] Preferably, the conditions of the third reaction include: the temperature is 0 - 100 °C, and the time is 12 - 48 h.

[0076] Preferably, in the third reaction, the solvent is selected from at least one of 1,4-dioxane, pyridine, and DMF.

[0077] Preferably, the third reaction is carried out in the presence of an organic base.

[0078] Preferably, the third reaction is carried out in the presence of 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene and Pd(OAc)₂ as catalysts with a molar ratio of 1:0.001 - 0.005.

[0079] Preferably, the conditions of the fourth reaction include: the temperature is -20 °C to 20 °C, and the time is 12 - 48 h.

[0080] Preferably, in the fourth reaction, the solvent is selected from at least one of dichloromethane, acetonitrile, acetone, and tetrahydrofuran.

[0081] Preferably, the fourth reaction is carried out in the presence of a second catalyst, and the second catalyst is acetone cyanohydrin.

[0082] Preferably, the conditions of the hydrolysis include: the temperature is -20 °C to 20 °C, and the time is 2 - 12 h.

[0083] Preferably, the compound shown in formula (III) is prepared by the method disclosed in CN103820104A.

[0084] Without special instructions, the present invention has no special requirements and limitations on the amounts of the solvent and the catalyst. Those skilled in the art can determine the appropriate amounts according to the characteristics of the reaction and the known knowledge in the art. The present invention will not elaborate herein, and those skilled in the art should not consider it as a limitation to the present invention.

[0085] As described above, the third aspect of the present invention provides the application of the fluorescent probe compound described in the first aspect in constructing a visual in vivo screening method for HPPD inhibitors.

[0086] Preferably, applying the fluorescent probe of the present invention to construct a visual in vivo screening method for HPPD inhibitors includes at least the following preferred embodiments.

[0087] Preferred specific implementation 1, in-vivo screening steps based on plants:

[0088] (1) Apply HPPD inhibitor to the cultivated plant seedlings, or soak the plant seedlings in an aqueous solution of HPPD inhibitor at different concentrations, and let stand for 8 to 48 hours;

[0089] (2) Transfer the above plants and soak them in an aqueous solution containing the compound of the present invention (10 - 50 μM), and let stand for 1 to 3 hours; at the same time, prepare some untreated plants and soak them in the same concentration of probe solution for the same time as a control;

[0090] (3) Place the above plants soaked with the probe in a multi-functional laser imager for imaging, and evaluate the activity of the HPPD inhibitor by comparing the differences in fluorescence intensities between the control group and the experimental group.

[0091] Preferred specific implementation 2, in-vivo screening steps based on zebrafish:

[0092] (1) Add a certain concentration of HPPD inhibitor to the culture medium of zebrafish larvae 3 days after hatching, and let stand for 30 min;

[0093] (2) Transfer the zebrafish to fresh culture medium, and microinject 20 pmol of the compound of the present invention, and incubate for 1 hour; at the same time, prepare some untreated zebrafish and microinject the same dose of the probe as a control;

[0094] (3) Place the above-treated zebrafish under a fluorescence inverted microscope for imaging, and evaluate the activity of the HPPD inhibitor by comparing the differences in fluorescence intensities between the control group and the experimental group.

[0095] The visualization in-vivo screening method for 4-hydroxyphenylpyruvate dioxygenase provided by the present invention is a fluorescence detection method developed by combining a class of fluorescence probes targeting 4-hydroxyphenylpyruvate dioxygenase with a fluorescence visualization instrument. The activity of 4-hydroxyphenylpyruvate dioxygenase in vivo (such as plants and zebrafish, etc.) is quantified through the change in fluorescence intensity, and then the biological activity of the inhibitor at the in-vivo level is evaluated.

[0096] The method of the present invention can quickly and accurately visualize the measurement of the activity of 4-hydroxydioxygenase at the in-vivo level and evaluate the inhibitory effect of the inhibitor.

[0097] Compared with the traditional in-vitro inhibitor screening method or greenhouse herbicidal activity evaluation method, the method of the present invention mainly has the following prominent advantages:

[0098] (1) Compared with traditional in vitro screening methods for 4-hydroxyphenylpyruvate dioxygenase inhibitors (such as oxygen consumption method, high performance liquid chromatography method, isotope method, etc.), this method can effectively and reliably evaluate the absorption, conduction, metabolism and other abilities of inhibitors in vivo, while in vitro bioactivity methods cannot;

[0099] (2) Compared with traditional in vivo screening methods, this method can visually evaluate the effect of inhibitors in vivo in real time and in situ;

[0100] (3) This method can quickly observe whether the inhibitor to be tested is off-target.

[0101] The present invention will be described in detail below by way of examples. In the following examples, unless otherwise specified, the raw materials used are all ordinary commercially available products.

[0102] Herein, room temperature or normal temperature both refer to 25±2°C.

[0103] Example 1

[0104] Synthesis of the fluorescent probe compound HPD-1, and the synthesis route is as follows:

[0105]

[0106] (1) Synthesis of Intermediate 1:

[0107] 2-Nitro-4-methoxycarbonylbenzoic acid (5.6 mmol) and dichloromethane (50 mL) were added to a reaction flask and cooled to 0 °C. Oxalyl chloride (7 mmol) was slowly added dropwise to the reaction system with stirring. After the addition was complete, a drop of dry DMF was added. The system was stirred at 0 °C for 2 h, then slowly warmed to room temperature and reacted overnight. The reaction solvent was removed under reduced pressure, and 50 mL of dry dichloromethane was added to the system. Under an ice-water bath, 1,3-cyclohexanedione (5.6 mmol) and triethylamine (11.2 mmol) were added to the system and stirred for 2 h. After the reaction was completed, saturated sodium carbonate (50 mL) was added to the system and stirred vigorously for 15 min. The aqueous layer was separated, and the organic layer was washed once with 1 M HCl solution (50 mL) and saturated brine (50 mL) respectively. After drying over anhydrous sodium sulfate, the solvent was removed, and the obtained residue was directly used in the next reaction without separation. 50 mL of acetonitrile, triethylamine (11.2 mmol), and a drop of acetone cyanohydrin were added to the obtained residue. The obtained solution was stirred at room temperature for 12 h. After the reaction was completed, the reaction solvent was removed under reduced pressure. 50 mL of dichloromethane and 50 mL of 1 M HCl solution were added to the reaction flask and stirred vigorously for 30 min. The organic layer was separated, and the aqueous layer was extracted once with 20 mL of dichloromethane. The organic layers were combined, washed once with saturated brine (50 mL), dried over anhydrous sodium sulfate, and the solvent was removed. The obtained residue was purified by column chromatography to obtain the product. Yield: 25%, 1 H NMR (400 MHz, CDCl3) δ 16.33 (s, 1H), 8.91 (d, J = 1.6 Hz, 1H), 8.40 (dd, J = 8.0, 1.2 Hz, 1H), 7.36 (d, J = 7.6 Hz, 1H), 4.11 (brs, 1H), 2.83 (t, J = 6.0 Hz, 2H), 2.40 (t, J = 6.4 Hz, 2H), 2.14–2.02 (m, 2H).

[0108] (2) Synthesis of Intermediate 2

[0109] Intermediate 1 (1 mmol) was added to 5 mL of acetone. After the solid of Intermediate 1 was dissolved, 5 mL of 10 wt% lithium hydroxide aqueous solution was added, and then stirred for 4 h. Then 2 M hydrochloric acid was added to the reaction system to neutralize to pH = 7, and a solid precipitated. Filtration gave 252 mg of Intermediate 2.

[0110] (3) Synthesis of Probe Compound HPD-1:

[0111] Dissolve intermediate 2 (0.5 mmol) in 10 mL of dichloromethane, and then successively add N,N-diisopropylethylamine (DIPEA, 1 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 1 mmol) and intermediate 3 (0.5 mmol), and stir for 16 h. After monitoring the reaction by TLC and completion of the reaction, add 10 mL of water, extract three times with 10 mL of dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, remove the solvent, and the resulting residue can be obtained the target HPD-1 after column chromatography. 1 H NMR (400 MHz, DMSO-d6) δ 8.66 (d, J = 12.0 Hz, 2H), 8.51 (d, J = 4.0 Hz, 2H), 8.34 (s, 1H), 8.09 (d, J = 36.0 Hz, 3H), 7.58 (s, 1H), 7.43 (d, J = 8.0 Hz, 2H), 7.22 (s, 1H), 6.61 (s, 1H), 4.94 (s, 1H), 3.30 (s, 4H), 3.05 (s, 6H), 2.75 (s, 4H), 1.95 (s, 2H), 1.57 (d, J = 8.0 Hz, 4H), 1.38 (s, 2H), 1.29 (s, 2H). HRMS calculated value for 38 H 38 N5O7 + [C

[0112] Example 2

[0113] Synthesis of the fluorescent probe compound HPD-2, and the synthetic route is as follows:

[0114]

[0115] Substance A: CAS No. 1345444-50-9, English name 6-Amino-3-iodo-2-methyl-benzoic acid, and the structural formula is

[0116] (1) Synthesis of intermediate 4:

[0117] Add triphosgene (3.6 mmol) and 1,4-dioxane (20 mL) to a reaction flask and cool to 0 °C. Slowly add dropwise a solution composed of methyl 4-aminobenzoate (8 mmol) and 20 mL of 1,4-dioxane with stirring. After stirring the system at this temperature for 30 min, continue to stir the reaction at room temperature for 3 h, and then heat to reflux for 2 h. Cool the system to room temperature and remove the reaction solvent. Add substance A (7.2 mmol) and pyridine 60 mL to the system with stirring and heat to reflux overnight. After the reaction is completed, remove pyridine under reduced pressure. Add DMF (30 mL) and cesium carbonate (10 mmol) to the system and stir at room temperature for 12 h. After the reaction is completed, pour the system into 200 mL of water, add 100 mL of ethyl acetate for extraction, separate the organic layer, and wash the organic layer with water (50 mL) and saturated brine (50 mL) respectively once. After drying over anhydrous sodium sulfate, remove the solvent under reduced pressure. The obtained residue is subjected to column chromatography to obtain intermediate 4. Yield: 47%, 1 H NMR (400 MHz, DMSO-d6) δ 8.22 (d, J = 8.8 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.54 - 7.46 (m, 2H), 7.45 - 7.33 (m, 3H), 7.32–7.24 (m, 2H), 7.20 (d, J = 8.8 Hz, 1H), 5.37 (s, 2H), 3.49 (s, 3H), 2.85 (s, 3H), 2.54 (s, 3H).

[0118] (2) Synthesis of intermediate 5:

[0119] Add intermediate 4 (2.2 mmol), DCC (4.4 mmol), Xanphos (0.0069 mmol), Pd(OAc)2 (0.0069 mmol) to a reaction flask, add DMF (10 mL) under nitrogen protection, and slowly add dropwise HCOOH (15.4 mmol) to the reaction system. After the addition, add triethylamine (4.4 mmol) and heat to 80 °C for 2 h. After the reaction is completed, filter off the insoluble matter. Pour the filtrate into 100 mL of water and stir vigorously for 20 min. The obtained solid is filtered by suction, washed with water to obtain intermediate 5. Yield: 87%, 1 H NMR (400 MHz, DMSO-D6) δ 13.20 (brs, 1H), 8.03 (d, J = 8.8 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.53 - 7.47 (m, 2H), 7.46–7.35 (m, 4H), 7.33–7.26 (m, 2H), 5.37 (s, 2H), 3.53 (s, 3H), 2.83 (s, 3H), 2.54 (s, 3H).

[0120] (3) Synthesis of Intermediate 6:

[0121] The synthesis method of Intermediate 6 is similar to that of Intermediate 1. Yield: 46%, 1 H NMR (400 MHz, CDCl3) δ 8.14 (d, J = 8.8 Hz, 1H), 7.47 - 7.32 (m, 6H), 7.22–7.13 (m, 3H), 5.37 (s, 2H), 3.63 (s, 3H), 2.81 (t, J = 6.4 Hz, 2H), 2.65 (s, 6H), 2.48–2.39 (m, 2H), 2.12–2.02 (m, 2H).

[0122] (4) Synthesis of Intermediate 7:

[0123] Add Intermediate 6 (1 mmol) to 5 ml of acetone. After the solid of Intermediate 1 is dissolved, add 5 ml of 10 wt% lithium hydroxide aqueous solution, and then stir for 4 hours. Then add 2M hydrochloric acid to the reaction system to neutralize to pH = 7, and a solid precipitates. Filter to obtain 374 mg of Intermediate 7.

[0124] (5) Synthesis of Probe Compound HPD-2:

[0125] Dissolve Intermediate 7 (0.5 mmol) in 10 mL of dichloromethane, and then successively add N,N-diisopropylethylamine (1 mmol), 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1 mmol) and Intermediate 3 (0.5 mmol), and stir for 16 h. After monitoring the reaction by TLC and completion, add 10 mL of water, extract three times with 10 mL of dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, remove the solvent, and subject the obtained residue to column chromatography to obtain HPD-2. 1 H NMR (400 MHz, DMSO-d6) δ 8.64 (s, 1H), 8.51 (s, 1H), 8.15–7.94 (m, 5H), 7.98 (d, J = 29.2 Hz, 1H), 7.92 (s, 1H), 7.79 (s, 2H), 7.43 (d, J = 8.0 Hz, 2H), 7.26 (d, J = 68.9 Hz, 4H), 6.80 (d, J = 87.2 Hz, 2H), 3.89 (s, 4H), 3.30 (s, 2H), 3.16 (s, 1H), 3.02 (s, 7H), 2.59 (s, 1H), 2.33 (s, 3H), 1.62–1.51 (m, 6H), 1.33 (d, J = 36.0 Hz, 4H). HRMS calculated value [C 48 H 47 NO7 +, 819.3501; Measured value, 819.3510.

[0126] Example 3

[0127] Construction and Application of a Visual HPPD Inhibitor-Based In Vivo Activity Screening Method

[0128] (1) The rape plants cultivated for 5 days were incubated with clear water, clear water containing different concentrations of mesotrione (commercial HPPD inhibitor, 10 μM, 50 μM, 100 μM, and 200 μM), and clear water containing different concentrations of quizalofop-methyl (commercial HPPD inhibitor, 10 μM, 50 μM, 100 μM, and 200 μM) for 24 hours.

[0129] (2) The above-treated rape samples were incubated in a PBS solution containing 10 μM of HPD-1 for 2 hours.

[0130] (3) The rape samples treated with the probe were placed in a multifunctional laser imaging system (Amersham Typhoon 5) and imaged using the Cy5 channel, and the fluorescence values were calculated to evaluate the inhibitory ability of the inhibitor.

[0131] The imaging results are shown as Figure 1 shown in, in which, as can be seen in a, compared with the normal group without inhibitor treatment, as the concentration of mesotrione increased, the fluorescence intensity of the plants in the inhibitor treatment group decreased significantly.

[0132] Figure 1 In, as can be seen in b, after quantitative calculation, compared with the normal group, the relative fluorescence intensity of the mesotrione-treated inhibitor group decreased significantly, and the greater the concentration of mesotrione, the more obvious the decrease.

[0133] Figure 1 In, as can be seen in c, compared with the normal group without inhibitor treatment, as the concentration of quizalofop-methyl increased, the fluorescence intensity of the plants in the inhibitor treatment group decreased significantly.

[0134] Figure 1 In, as can be seen in d, after calculation, compared with the normal group, the relative fluorescence intensity of the quizalofop-methyl-treated inhibitor group decreased significantly, and the greater the concentration of quizalofop-methyl, the more obvious the decrease. Thus, as can be seen from Figure 1 it can be seen that under the action of the two commercial inhibitors, the fluorescence decreased significantly, showed an obvious concentration dependence, and was accurately quantified and consistent with the actual in vitro test results.

[0135] When the present invention uses HPD-2 to replace HPD-1 for the same experiment, similar effects can be obtained.

[0136] Example 4

[0137] Construction and Application of a Visual HPPD Inhibitor-Based In Vivo Activity Screening Method for Zebrafish

[0138] (1) Zebrafish larvae hatched for 3 days were placed in a culture medium containing different concentrations of nitisinone (a commercial HPPD inhibitor, 25 μg / mL, 50 μg / mL, 100 μg / mL). The control group was without nitisinone, and they were incubated in the culture medium for 30 min.

[0139] (2) The above-treated zebrafish were microinjected with 20 pmol of HPD-1 and incubated in fresh culture medium for 1 hour.

[0140] (3) The zebrafish treated with the probe were placed in an inverted fluorescence microscope (IX71) and imaged using the NG channel, and the fluorescence values were calculated to evaluate the inhibitory ability of the inhibitor.

[0141] The imaging results are shown as Figure 2 shown in. As can be seen in a, after microinjecting HPD-1, significant fluorescence appeared in the abdomens of zebrafish in different groups. And compared with the normal group without inhibitor treatment, after the zebrafish were treated with the HPPD inhibitor nitisinone, the abdominal fluorescence decreased significantly.

[0142] Figure 2 As can be seen in b, through quantitative calculation, compared with the normal group, the relative fluorescence intensity of the nitisinone treatment group of the inhibitor decreased significantly, and the greater the concentration of nitisinone, the more obvious the degree of decrease.

[0143] Thus, as can be seen from Figure 2 , under the action of nitisinone at different concentrations, the fluorescence decreased significantly and showed an obvious concentration dependence.

[0144] When the present invention uses HPD-2 to replace HPD-1 for the same experiment, similar effects can be obtained.

[0145] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A fluorescent probe compound, characterized in that, The compound has the structure shown in formula (I): Wherein, in formula (I), n is an integer from 1 to 6; Q is the structure shown in formula (Q1) or the structure shown in formula (Q2); In formulas (Q1) and (Q2), R is selected from H, C 1-6 alkyl, -NO2 and halogen.

2. The compound according to claim 1, wherein In formula (I), n is 1, 2, 3, 4, 5 or 6; Q is the structure shown in formula (Q1) or the structure shown in formula (Q2); R is selected from H, C 1-3 alkyl, -NO2, fluorine, chlorine, bromine.

3. The compound according to claim 2, wherein In formula (I), n is 1, 2, 3, 4, 5 or 6; Q is the structure shown in formula (Q1) or the structure shown in formula (Q2); R is selected from H, methyl, -NO2, fluorine, chlorine, bromine.

4. The compound according to claim 3, wherein, In formula (I), n is 4; Q is the structure shown in formula (Q1) or the structure shown in formula (Q2); R is selected from H, -NO2.

5. A method for preparing the compound according to any one of claims 1-4, characterized in that, The method includes: in the presence of a solvent and a basic substance, contacting the compound shown in formula (II) with the compound shown in formula (III) for a reaction, Wherein, in formula (II) and formula (III), the definitions of Q, R and n are the same as those described in any one of claims 1-4.

6. The method according to claim 5, wherein, The conditions of the contacting reaction include: the temperature is 0-50 °C and the time is 12-48 h.

7. The method according to claim 5 or 6, wherein The contacting reaction is carried out in the presence of a catalyst.

8. The method according to claim 7, wherein The catalyst is 2-(7-azabenzotriazol)-N,N,N',N'-tetramethylurea hexafluorophosphate and N-ethyldiisopropylamine with a molar ratio of 1:1-3.

9. The method according to claim 5 or 6, wherein The solvent is selected from at least one of dichloromethane, acetonitrile, DMF.

10. The method according to claim 5 or 6, wherein, The basic substance is at least one organic base selected from triethylamine, p-dimethylaminopyridine, pyridine and N,N-diisopropylethylamine.

11. Use of the fluorescent probe compound according to any one of claims 1-4 in constructing a visual in-vivo screening method for HPPD inhibitors.

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