A Bioluminescent Probe for Detecting Norepinephrine, Its Preparation Method and Use

Abstract

The present invention provides a bioluminescence probe for detecting norepinephrine, its preparation method and use, belonging to the field of biological detection. The bioluminescence probe of the present invention has high selectivity and high responsiveness to norepinephrine, can specifically recognize norepinephrine, has high sensitivity and low detection limit, and can be used for the precise detection of norepinephrine. In the range of pH 1.0 to 7.4, the bioluminescence probe of the present invention has excellent stability and can meet the application requirements under different physiological environments in vivo. At the same time, the norepinephrine bioluminescence probe of the present invention can also visually track exogenous norepinephrine at the in vivo level, and perform dynamic visual analysis on patients with diseases related to abnormal expression of norepinephrine involving nerves, tumors, blood pressure and heart diseases, which is beneficial to the clinical detection of norepinephrine and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and in particular relates to a bioluminescent probe for detecting norepinephrine, a preparation method and application thereof. Background Art

[0002] With the increase of social pressure in recent years, the number of patients with depression has increased significantly, and the number of patients with depression worldwide has exceeded 264 million. Depression is closely related to the decrease of norepinephrine (NE) levels in the brain. NE is the central substance for the synthesis of monoamine catecholamine neurotransmitters, which is synthesized from dopamine and epinephrine under the catalysis of phenylethanolamine N-methyltransferase (PMNT). However, the structure and properties of norepinephrine are very similar to those of the other two catecholamine neurotransmitters, epinephrine (EP) and dopamine (DA), so it is challenging to specifically capture and detect norepinephrine.

[0003] Methods for detecting norepinephrine in vivo are constantly evolving. Traditional methods such as colorimetry and radioimmunoassay have many interferences and cannot meet the analysis of biological samples. With the development of analytical instruments, more sensitive and specific methods have emerged, such as fluorescence spectroscopy, high performance liquid chromatography and capillary electrophoresis. However, these methods have many problems, such as: the detection limit does not meet the requirements, the detection sensitivity is low, the reaction time is long, the anti-interference ability is poor, and it cannot be used for dynamic visualization detection of living bodies.

[0004]

[0005] The Yin Caixia research group at the School of Chemistry and Chemical Engineering, Shanxi University (Na Z, Fangjun H, Yongkang Y, and Caixia Y. Specific Fluorescent Probe Based on “Protect-Deprotect” To Visualize the Norepinephrine Signaling Pathway and Drug Intervention Tracers. J. Am. Chem. Soc. 2020, 142, 17751-17755.) reported a fluorescent probe for detecting norepinephrine, which can specifically detect norepinephrine and image it in vivo. This report adopted a “protect-deprotect” strategy: a cyanine dye with a longer emission wavelength was modified with water-soluble sulfonate and protected by a carbonate connected to a leaving group thiol; the special β-hydroxyethylamine moiety in norepinephrine can cause the protecting group to leave through nucleophilic substitution and intramolecular nucleophilic reaction, releasing the fluorophore, thus realizing the specific red fluorescence detection of norepinephrine. This report also achieved the imaging of norepinephrine neurotransmission stimulated by potassium ions and for the first time performed real-time fluorescence imaging of the norepinephrine level in the rat brain after antidepressant stimulation. However, on the one hand, this fluorescent probe requires incident light of a certain wavelength for excitation, resulting in photobleaching, photo-stimulation, and background interference of the excitation light, and at the same time causing the excitation wavelength to be relatively short, making it difficult to penetrate deep tissues and unable to achieve non-invasive in vivo detection. On the other hand, in order to meet the application requirements in different physiological environments in vivo, it is necessary to develop a probe with excellent luminescence stability in physiological environments within a wide pH range.

[0006] Therefore, it is of great significance to develop a bioluminescent probe that can not only achieve non-invasive in vivo detection of norepinephrine but also has excellent luminescence stability. Summary of the Invention

[0007] The purpose of the present invention is to provide a bioluminescent probe that can not only achieve non-invasive in vivo detection of norepinephrine but also has excellent luminescence stability, and its preparation method and uses.

[0008] The present invention provides a compound represented by Formula I, or a salt thereof, or a stereoisomer thereof:

[0009]

[0010] Wherein, R 1 、R 2 、R 3 、R 4 、R 5 are each independently selected from hydrogen, C1 ~C 6 alkyl, C 1 ~C 6 alkoxy, halogen, hydroxyl, carboxyl, amino, nitro.

[0011] Furthermore, the compound is as shown in Formula II:

[0012]

[0013] Wherein, R 1 , R 3 , R 4 , R 5 are independently selected from hydrogen, carboxyl, C 1 ~C 3 alkyl.

[0014] Furthermore, the structural formula of the compound is as shown in Formula III:

[0015]

[0016] The present invention also provides a preparation method of the compound shown in Formula III, which comprises the following steps:

[0017]

[0018] Step 1: React raw material I, raw material II and raw material III in an organic solvent to obtain intermediate IV;

[0019] Step 2: React intermediate IV with D-cysteine in a solvent to obtain the compound shown in Formula III.

[0020] Furthermore, in Step 1, the organic solvent is toluene, the reaction is carried out in the presence of a base, and the molar ratio of raw material I, raw material II, raw material III and the base is (1~4):(0.5~1):(0.5~1):(2~4); the molar volume ratio of raw material I to the organic solvent is (2~4):(20~40) mmol / mL; the reaction is carried out in an inert gas atmosphere, the temperature of the reaction is 0~120 °C, and the reaction time is 1~7 h;

[0021] And / or, in step 2, the reaction comprises the following steps: dissolving intermediate IV in an organic solvent, adding an aqueous methanol solution containing D-cysteine, and reacting in an inert gas atmosphere; the molar ratio of intermediate IV to D-cysteine is (0.5 - 1):(1 - 2); the organic solvent is a mixed solution of methanol and dichloromethane, and the molar volume ratio of intermediate IV to the organic solvent is (0.5 - 1):(10 - 20) mmol / mL; the molar volume ratio of intermediate IV to the aqueous methanol solution containing D-cysteine is (0.5 - 1):(3 - 6) mmol / mL; the reaction temperature is 0 - 100 °C, and the reaction time is 0.5 - 4 h.

[0022] Further, in step 1, the base is an organic base; the molar ratio of raw material I, raw material II, raw material III, and the base is 1.2:1:1:2; the molar volume ratio of raw material I to the organic solvent is 1.2:20 mmol / mL; the reaction temperature is 50 - 90 °C, and the reaction time is 3 - 5 h;

[0023] And / or, in step 2, the molar ratio of intermediate IV to D-cysteine is 1:1; the molar volume ratio of intermediate IV to the organic solvent is 0.5:5 mmol / mL; the molar volume ratio of intermediate IV to the aqueous methanol solution containing D-cysteine is 1:4 mmol / mL; in the mixed solution of methanol and dichloromethane, the volume ratio of methanol to dichloromethane is 1:(1 - 3); in the aqueous methanol solution containing D-cysteine, the volume ratio of methanol to water is (1 - 2):1; the reaction temperature is 25 °C, and the reaction time is 0.5 h.

[0024] Further, the base is triethylamine.

[0025] The present invention also provides a norepinephrine-specific detection kit, which contains the above-mentioned compound, or its salt, or its stereoisomer.

[0026] The present invention also provides the use of the above-mentioned compound, or its salt, or its stereoisomer in the preparation of a bioluminescence probe or kit for detecting norepinephrine.

[0027] Further, the bioluminescence probe or kit can detect the norepinephrine level in vitro or in vivo.

[0028] The bioluminescent probe of the present invention has excellent stability. The test results show that within the pH range of 1.0 to 7.4, the bioluminescent probe of the present invention has excellent stability and can meet the application requirements in different physiological environments in vivo. In the exploration and attempt of the specific synthesis methodology of this probe, during the post-treatment process of the second-step synthesis, the present invention adopts the methods of low-temperature extraction and low-temperature rotary evaporation, which can greatly improve the stability of the probe in the aqueous phase and different organic phases. Finally, the probe is stored in a 4°C refrigerator, enabling the probe to remain stable for a long time.

[0029] The bioluminescent probe of the present invention adopts a "Turn-on" strategy: the D-luciferin (D-fluorescein) substrate leaves the carbonate linking group thiol; in norepinephrine, the β-hydroxyethylamine moiety causes the protecting group to leave through nucleophilic substitution and intramolecular nucleophilic reaction, releasing D-luciferin. In the presence of luciferase, ATP, O 2 and Mg 2+ , through an enzyme-catalyzed chemical reaction, the chemical energy in the living body is converted into light energy.

[0030] The bioluminescent probe of the present invention has high selectivity and high responsiveness to norepinephrine, can specifically recognize norepinephrine, has high sensitivity and low detection limit, and can be used for the precise detection of norepinephrine.

[0031] The probe of the present invention does not require external excitation light, avoiding photobleaching, light stimulation, and background interference of the excitation light, and has higher sensitivity. At the same time, since the probe achieves the detection purpose by converting chemical energy into light energy, non-invasive in vivo detection can be realized.

[0032] The present invention has realized for the first time the real-time in vivo non-invasive detection of the norepinephrine level in the mouse brain after antidepressant stimulation, and for the first time adopted a method with high sensitivity, real-time monitoring, and non-invasiveness to detect norepinephrine. The norepinephrine bioluminescent probe of the present invention can visually track exogenous norepinephrine at the in vivo level, and perform dynamic visual analysis on patients with diseases related to abnormal norepinephrine expression involving nerves, tumors, blood pressure, and heart diseases, which is beneficial to the clinical detection of norepinephrine and has broad application prospects.

[0033] The preparation method of the bioluminescent probe of the present invention is simple, the raw materials are easily available, and it is suitable for large-scale production.

[0034] Obviously, based on the above content of the present invention, according to the common general technical knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modification, substitution, or change can also be made.

[0035] The following is a further detailed description of the above content of the present invention in the form of specific embodiments. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. Description of the Drawings

[0036] Figure 1 1H NMR spectrum of the bioluminescent probe prepared for Example 1 1 1H NMR spectrum.

[0037] Figure 2 13C NMR spectrum of the bioluminescent probe prepared for Example 1 13 13C NMR spectrum.

[0038] Figure 3 ESI-HRMS spectrum of the bioluminescent probe prepared for Example 1

[0039] Figure 4 HPLC spectrum of the bioluminescent probe prepared for Example 1

[0040] Figure 5 Selection of different neurotransmitters by the bioluminescent probe of the present invention

[0041] Figure 6 Response of the bioluminescent probe of the present invention to different concentrations of norepinephrine solution. Among them, Figure A shows the bioluminescence intensity of the probe solution in different concentrations of norepinephrine solution, and Figure B shows the response regression curve of the probe in the norepinephrine solution of 0 - 250 μM.

[0042] Figure 7 Bioluminescence signal after injecting exogenous norepinephrine or fluoxetine solution into mice. Among them, Figure A shows the influence of the fluoxetine administration group and the blank control group on the bioluminescence system, Figure B shows the quantification of the bioluminescence intensity of the fluoxetine administration group and the blank control group, Figure C shows the influence of the norepinephrine administration group and the blank control group on the bioluminescence system, and Figure D shows the quantification of the bioluminescence intensity of the norepinephrine administration group and the blank control group.

[0043] Figure 8 Change of bioluminescence signal intensity in mice over time after injecting 0.2 mL of 100 μM norepinephrine solution and 100 μM bioluminescent probe solution via the tail vein. Among them, Figure A shows the change of bioluminescence intensity over time in the experimental group and the control group after administering the bioluminescent probe, Figure B shows the quantification of the intensity change of norepinephrine over time in the experimental group and the control group, and Figure C shows the quantitative comparison of the bioluminescence intensity between the experimental group and the control group at the 18th minute after administering the bioluminescent probe.

[0044] Figure 9The change of bioluminescence signal intensity in mice with the variation of exogenous norepinephrine. Among them, Figure A shows the change of bioluminescence intensity at the 18th minute after the bioluminescence probe is given in the experimental group and the control group injected with different concentrations of norepinephrine, and Figure B shows the quantification of the bioluminescence intensity at the 18th minute after the bioluminescence probe is given in the experimental group and the control group injected with different concentrations of norepinephrine.

[0045] Figure 10 The imaging of the bioluminescence probe of the present invention in mice with endogenous norepinephrine production stimulated by drugs. Among them, Figure A shows the change of bioluminescence intensity at the 18th minute after the bioluminescence probe is given in the experimental group and the control group injected with different concentrations of fluoxetine, and Figure B shows the quantification of the bioluminescence intensity at the 18th minute after the bioluminescence probe is given in the experimental group and the control group injected with different concentrations of fluoxetine. Detailed implementation mode

[0046] The raw materials and equipment used in the present invention are all known products, obtained by purchasing commercially available products.

[0047] Example 1. Preparation of the bioluminescence probe of the present invention

[0048] The synthesis route is as follows:

[0049]

[0050] Step 1: Mix and dissolve 1.2 mmol of raw material I and 1 mmol of raw material III in 20 mL of toluene, add 2 mmol of organic base triethylamine (TEA), react at 120 °C for 3 h under nitrogen protection, then cool the reaction system to 50 °C, add 1 mmol of raw material II, and react at 50 °C for 2 h to obtain 157 mg of intermediate IV, with a yield of 62.3%.

[0051] Intermediate IV: mp: 91.0 - 91.4 °C. 11 H NMR (400 MHz, DMSO-d 6 ) δ 8.33 (dd, J = 5.6, 3.2 Hz, 2H), 7.67 (dd, J = 9.0, 2.4 Hz, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.4 Hz, 2H), 2.36 (s, 3H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 206.75, 171.22, 150.27, 149.34, 137.48, 136.26, 125.22, 123.12, 115.82, 113.24, 37.48, 29.45, 27.83. 13 C NMR (101 MHz, DMSO-d6 ) δ 177.56, 145.32, 135.15, 133.77, 133.24, 131.76, 129.12, 127.43, 126.14, 125.65, 116.32, 116.08, 14.61.

[0052] Step 2: Dissolve 1 mmol of Intermediate Ⅳ in a mixed solution of 10 mL of methanol and dichloromethane (the volume ratio of methanol to dichloromethane is 1:1.5), and then slowly add dropwise 4 mL of an aqueous methanol solution containing 1 mmol of D-cysteine (the volume ratio of methanol to water is 1:1) at 0 °C. React at 25 °C for 0.5 h under nitrogen protection to obtain a reaction solution containing the crude probe. Adjust the pH of the reaction solution to 5 with hydrochloric acid, filter and dry after precipitation of solids to obtain 49 mg of the bioluminescent probe of the present invention, with a yield of 41.7%.

[0053] The bioluminescent probe of the present invention: 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.90 (d, J = 8.6 Hz, 1H), 7.49–7.41 (m, 3H), 7.31 (d, J = 7.8 Hz, 2H), 7.04 (d, J = 8.6 Hz, 1H), 3.77–3.72 (m, 1H), 3.53 (dd, J = 9.6, 8.6 Hz, 2H), 2.35 (s, 3H). 113 C NMR (101 MHz, DMSO-d 6 ) δ 168.79, 166.89, 151.24, 149.65, 140.75, 135.56, 135.16, 130.68, 125.12, 123.94, 122.17, 121.77, 117.45, 116.03, 107.16, 82.23, 21.26. ESI-MS calcd. for C 19 H 14 N 2 O 4 S 3 (M + H) + 431.0116, found 431.0184.

[0054] The characterization spectra of the bioluminescent probe of the present invention are shown in Figures 1 to 4 .

[0055] The beneficial effects of the present invention are demonstrated by the following experimental examples.

[0056] Test Example 1, Selectivity of the Bioluminescent Probe of the Present Invention for Norepinephrine

[0057] 1. Test method

[0058] (1) Preparation of solutions

[0059] Solvent: 50 mM Tris-HCl buffer solution (pH 7.4, containing 10 mM MgCl 2 ).

[0060] Preparation of the bioluminescent probe solution: First, dissolve the bioluminescent probe prepared in Example 1 with DMSO to obtain a probe stock solution with a concentration of 10 mM; then dilute the probe stock solution with the solvent Tris-HCl buffer solution to a probe solution with a concentration of 25 μM.

[0061] Preparation of the enzyme working solution: Dissolve 60 μg of commercial luciferase on ice in 3 mL of Tris-HCl buffer solution, and add 2 mM ATP to obtain it (prepare and use immediately).

[0062] (2) Steps for the selectivity test of the bioluminescent probe of the present invention for norepinephrine

[0063] Prepare neurotransmitter solutions containing norepinephrine (5 mM), epinephrine (5 mM), dopamine (5 mM), 5-hydroxytryptamine (5 mM), γ-aminobutyric acid (5 mM), L-cysteine (500 μM), homocysteine (100 μM), reduced glutathione (10 μM), lysine (500 μM), serine (500 μM), and threonine (500 μM) respectively. The aforementioned neurotransmitter solutions are obtained by separately dissolving the corresponding neurotransmitter solids in Tris-HCl buffer solution, and the concentration of each is 25 μM, which is 10 times the probe concentration. Add 50 μL of the aforementioned neurotransmitter solutions to 50 μL of the probe solution with a concentration of 25 μM in a 96-well plate and incubate at 37 °C for 30 min. Then, add 50 μL of the enzyme working solution to each experimental well, and immediately use the CCD detector (Charge-coupled Device, CCD) of the small animal in vivo imaging system to record the bioluminescence signals of each well within 1 min after adding the enzyme working solution, and measure the bioluminescence phenomenon and the change in the number of photons in the well plate.

[0064] 2. Test results

[0065] The selectivity of the bioluminescent probe of the present invention for different neurotransmitters is as Figure 5 shown. It can be Figure 5 seen that the bioluminescent probe of the present invention has high selectivity for norepinephrine, and the bioluminescence signal is more than 9 times that of other neurotransmitters.

[0066] The above experimental results show that the bioluminescent probe of the present invention has excellent specificity and selectivity for norepinephrine and can be used to detect norepinephrine.

[0067] Experimental Example 2, Responsiveness of the Bioluminescent Probe of the Present Invention to Norepinephrine

[0068] 1. Test Method

[0069] (1) Preparation of Solutions

[0070] Solvent: 50 mM Tris-HCl buffer solution (pH 7.4, containing 10 mM MgCl 2 )

[0071] Preparation of norepinephrine solution: Norepinephrine was prepared using the above solvent to obtain norepinephrine solutions with different concentrations. The norepinephrine concentrations were 0 μM, 1 μM, 2 μM, 5 μM, 10 μM, 25 μM, 50 μM, 100 μM, 250 μM, 500 μM, and 1000 μM respectively

[0072] Preparation of bioluminescent probe solution: The probe solution with a concentration of 25 μM was prepared according to the method described in Experimental Example 1. First, the bioluminescent probe prepared in Example 1 was dissolved with DMSO to obtain a probe stock solution with a concentration of 10 mM; then the probe stock solution was diluted with Tris-HCl buffer solution to a bioluminescent probe solution with a concentration of 25 μM

[0073] Preparation of enzyme working solution: The same as Experimental Example 1

[0074] (2) Test Steps for the Responsiveness of the Bioluminescent Probe of the Present Invention to Norepinephrine

[0075] Mix 50 μL of bioluminescent probe solution and 50 μL of norepinephrine solutions with different concentrations, incubate at 37 °C for 60 min in a 96-well plate. Subsequently, add 50 μL of enzyme working solution to each experimental well, and immediately use the CCD detector of the small animal in vivo imaging system to photograph and record the bioluminescence signals of each well within 1 min after adding the enzyme working solution, and measure the bioluminescence phenomenon and the change in the number of photons in the well plate

[0076] 2. Test Results

[0077] The response of the bioluminescent probe of the present invention to norepinephrine solutions with different concentrations is as Figure 6 shown. It can be seen from Figure 6 that: The bioluminescent probe of the present invention has good responsiveness to norepinephrine. The response regression curve shows that within the range of norepinephrine concentration from 0 to 250 μM, after the substrate norepinephrine interacts with the probe, the detected bioluminescence signal shows a good linear relationship with the norepinephrine concentration, R 2=0.991 indicates a strong correlation between the two, and the detection limit of the probe for norepinephrine can reach 1 μM.

[0078] The above experimental results show that the bioluminescent probe of the present invention has high sensitivity in detecting norepinephrine, can detect very low concentrations of norepinephrine, and can be used for the precise detection of norepinephrine.

[0079] Test Example 3. Effects of Injecting Exogenous Norepinephrine or Fluoxetine into Mice on the Bioluminescent System

[0080] 1. Test Method

[0081] (1) Test animals: Transgenic FVB-luc+ mice (provided by the School of Pharmacy, Shandong University, transgenic mice stably expressing luciferase, male, 6 - 8 weeks old, 22 - 26 g).

[0082] (2) Test grouping:

[0083] Experimental group (norepinephrine or fluoxetine): First, inject 0.2 mL of norepinephrine solution (norepinephrine solution (10 mM) or fluoxetine solution (50 mg / kg) prepared with physiological saline) via the tail vein, and then inject 0.2 mL of commercially available hydroxyfluorescein (50 μM, first prepared as a 10 mM stock solution with DMSO and then diluted to a concentration of 50 μM with physiological saline) via the tail vein, and incubate in vivo for 10 min;

[0084] Blank control group (Vehicle): First, inject 0.2 mL of physiological saline intraperitoneally, and then inject 0.2 mL of commercially available hydroxyfluorescein (100 μM, first prepared as a 10 mM stock solution with DMSO and then diluted to a concentration of 100 μM with physiological saline) intraperitoneally, and incubate in vivo for 10 min.

[0085] (3) Imaging conditions: The CCD detector (Charge-coupled Device, CCD) of the small animal in vivo imaging system takes pictures every 3 min for 30 min, with an exposure time of 1 s each time, and records the luminescence of each time period and the total number of photons during this time.

[0086] 2. Test Results

[0087] The bioluminescence signals of mice after injecting exogenous norepinephrine or fluoxetine solution are as Figure 7 shown. It can be Figure 7 seen that the changes in bioluminescence signals of mice in the experimental group and the blank control group are similar, and there is no significant difference between groups.

[0088] The above experimental results indicate that the injection of exogenous norepinephrine or fluoxetine has no effect on the bioluminescence imaging of living animals.

[0089] Test Example 4: Changes in Bioluminescence Signal Intensity in Mice with Exogenous Norepinephrine Concentration and Time

[0090] 1. Test Method

[0091] (1) Test animals: Transgenic FVB-luc+ mice (provided by the School of Pharmacy, Shandong University, transgenic mice stably expressing luciferase, male, 6 - 8 weeks old, 22 - 26 g).

[0092] (2) Test groups:

[0093] Experimental group: Inject 0.2 mL of norepinephrine solution with different concentrations (norepinephrine solution prepared with physiological saline, concentrations are 100 μM, 200 μM, and 500 μM respectively) via the tail vein, and then inject an equal volume of the bioluminescence probe solution prepared in Example 1 (100 μM, first prepared as a 10 mM stock solution with DMSO and then diluted to a concentration of 10 μM with physiological saline) via the tail vein;

[0094] Control group (Vehicle): Inject 0.2 mL of physiological saline via the tail vein, and then inject an equal volume of the bioluminescence probe solution prepared in Example 1 (100 μM, first prepared as a 10 mM stock solution with DMSO and then diluted to a concentration of 100 μM with physiological saline) via the tail vein.

[0095] (3) Imaging conditions: The CCD detector (Charge-coupled Device, CCD) of the small animal in vivo imaging system takes pictures every 3 minutes for 60 minutes, with an exposure time of 1 s each time. The luminescence situation in each time period is photographed and the total number of photons in this period is recorded.

[0096] 2. Test Results

[0097] The changes in bioluminescence signal intensity in mice with exogenous norepinephrine concentration are as Figure 8 and 9 shown. It can be seen from the figure that: there is only a weak bioluminescence signal in the control group; while there is a significantly enhanced bioluminescence signal in the experimental group, and the signal intensity shows a positive correlation with the exogenous norepinephrine concentration, and the bioluminescence intensity of the mice injected with exogenous norepinephrine reaches the maximum at 18 minutes.

[0098] The above experimental results indicate that the bioluminescence probe of the present invention can be used for visual analysis of the dynamic changes of exogenous norepinephrine levels in vivo.

[0099] Experimental Example 5. Imaging Application of the Bioluminescent Probe of the Present Invention in Mice with Drug-Stimulated Endogenous Norepinephrine Production

[0100] 1. Experimental Method

[0101] (1) Experimental animals: Transgenic FVB-luc+ mice (provided by the School of Pharmacy, Shandong University, transgenic mice stably expressing luciferase, male, 6 - 8 weeks old, 22 - 26 g).

[0102] (2) Experimental groups:

[0103] Experimental group: According to the reference (Na Z, Fangjun H, Yongkang Y, and Caixia Y. Specific Fluorescent Probe Based on “Protect-Deprotect” To Visualize the Norepinephrine Signaling Pathway and Drug Intervention Tracers. J. Am. Chem. Soc. 2020, 142, 17751 - 17755.), a mouse model with high expression of norepinephrine was established using fluoxetine. The experimental group was divided into: 1 mg / kg group, 50 mg / kg group, and 100 mg / kg group according to different fluoxetine administration doses. Then, 0.2 mL of the bioluminescent probe solution prepared in Example 1 (200 μM, first prepared as a 10 mM stock solution with DMSO and then diluted to a concentration of 200 μM with physiological saline) was injected via the tail vein;

[0104] Control group: 0.2 mL of the bioluminescent probe solution prepared in Example 1 (100 μM, first prepared as a 10 mM stock solution with DMSO and then diluted to a concentration of 100 μM with physiological saline) was injected via the tail vein into normal untreated mice.

[0105] (3) Imaging conditions: The CCD detector (Charge-coupled Device, CCD) of the small animal in vivo imaging system took pictures every 3 min for 30 min, with an exposure time of 1 s each time. The luminescence situation in each time period was photographed and the total number of photons in this period was recorded.

[0106] 2. Experimental Results

[0107] The imaging situation of the bioluminescent probe of the present invention in mice with drug-stimulated endogenous norepinephrine production is as Figure 10 shown. From Figure 10It can be seen that the bioluminescence signal intensity in the control group of mice was relatively stable. Compared with the control group, the bioluminescence signal intensity in the experimental group of mice was significantly enhanced, and it became stronger and stronger with the increase of the concentration of fluoxetine.

[0108] Fluoxetine is a drug clinically used to treat depression, and it can stimulate the endogenous production of norepinephrine. The above experimental results show that the bioluminescence probe of the present invention can not only be used to visually analyze the dynamic changes of exogenous norepinephrine levels in vivo, but also be used to visually analyze the dynamic production levels of endogenous norepinephrine in vivo, and can be used to assist in screening antidepressant drugs.

[0109] Test Example 6: Stability test of the bioluminescence probe of the present invention

[0110] 1. Test method

[0111] Take a 10 mM stock solution of the probe (prepared in Example 1), and dilute the probe to a final concentration of 100 μM with solutions of pH = 1.0, 4.5, 6.6, and 7.4 respectively. The samples are placed in an incubator at 37 °C, and samples are taken at different time points of t = 0, 15, 30, 60, 90, and 120 min for detection by HPLC. The normalization method is used to calculate the relative content of the probe. The probe detected at t = 0 is 100%, and the relative ratios at other time points are calculated (compared with t = 0).

[0112] 2. Test results

[0113] Table 1. Changes in the content of the bioluminescence probe in buffer solutions with different pH values under physiological conditions (unit: %)

[0114]

[0115]

[0116] As shown in Table 1, it can be seen that the bioluminescence probe of the present invention has excellent stability in solvents with pH values ranging from 1.0 to 7.4 within 0 - 120 min, and can meet the application requirements under different physiological environments in vivo.

[0117] In summary, the present invention provides a bioluminescence probe for detecting norepinephrine, its preparation method and uses. The bioluminescence probe of the present invention has high selectivity and high responsiveness to norepinephrine, can specifically recognize norepinephrine, has high sensitivity and low detection limit, and can be used for the precise detection of norepinephrine. In the range of pH from 1.0 to 7.4, the bioluminescence probe of the present invention has excellent stability and can meet the application requirements under different physiological environments in vivo. At the same time, the norepinephrine bioluminescence probe of the present invention can also visually track exogenous norepinephrine at the in vivo level, and perform dynamic visual analysis on patients with diseases related to abnormal expression of norepinephrine involving nerves, tumors, blood pressure and heart diseases, which is beneficial to the detection of norepinephrine clinically and has broad application prospects.

Claims

1. A compound represented by Formula III:

2. A method for preparing the compound according to Claim 1, characterized in that: it comprises the following steps: Step 1: React raw material I, raw material II and raw material III in an organic solvent to obtain intermediate IV; Step 2: React intermediate IV with D-cysteine in a solvent to obtain the compound represented by Formula III.

3. According to the preparation method described in Claim 2, characterized in that: In Step 1, the organic solvent is toluene, the reaction is carried out in the presence of a base, and the molar ratio of raw material I, raw material II, raw material III and the base is (1-4):(0.5-1):(0.5-1):(2-4); the millimole volume ratio of raw material I to the organic solvent is (2-4):(20-40) mmol / mL; the reaction is carried out in an inert gas atmosphere, the temperature of the reaction is 0-120 °C, and the reaction time is 1-7 h; and / or, in Step 2, the reaction comprises the following steps: Dissolve intermediate IV in an organic solvent, add a methanol aqueous solution containing D-cysteine, and react in an inert gas atmosphere; the molar ratio of intermediate IV to D-cysteine is (0.5-1):(1-2); the organic solvent is a mixed solution of methanol and dichloromethane, and the millimole volume ratio of intermediate IV to the organic solvent is (0.5-1):(10-20) mmol / mL; the millimole volume ratio of intermediate IV to the methanol aqueous solution containing D-cysteine is (0.5-1):(3-6) mmol / mL; the temperature of the reaction is 0-100 °C, and the reaction time is 0.5-4 h.

4. According to the preparation method described in Claim 2, characterized in that: In Step 1, the reaction is carried out in the presence of a base, and the base is an organic base; the molar ratio of raw material I, raw material II, raw material III and the base is 1.2:1:1:2; the millimole volume ratio of raw material I to the organic solvent is 1.2:20 mmol / mL; the temperature of the reaction is 50-90 °C, and the reaction time is 3-5 h; and / or, in Step 2, the reaction comprises the following steps: Dissolve intermediate IV in an organic solvent, add a methanol aqueous solution containing D-cysteine, and react in an inert gas atmosphere; the molar ratio of intermediate IV to D-cysteine is 1:1; the organic solvent is a mixed solution of methanol and dichloromethane, and the millimole volume ratio of intermediate IV to the organic solvent is 0.5:5 mmol / mL; the millimole volume ratio of intermediate IV to the methanol aqueous solution containing D-cysteine is 1:4 mmol / mL; in the mixed solution of methanol and dichloromethane, the volume ratio of methanol to dichloromethane is 1:(1-3); in the methanol aqueous solution containing D-cysteine, the volume ratio of methanol to water is (1-2):1; the temperature of the reaction is 25 °C, and the reaction time is 0.5 h.

5. According to the preparation method described in Claim 4, characterized in that: The base is triethylamine.

6. A noradrenaline specific detection kit, characterized in that: It comprises the compound as described in claim 1.

7. Use of the compound as described in claim 1 in the preparation of a bioluminescence probe or kit for detecting noradrenaline.

8. According to the use as claimed in claim 7, wherein: the bioluminescence probe or kit is capable of detecting the level of noradrenaline in vitro or in vivo.

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