Acid-induced in situ generation of luminescent cationic radical probes and their preparation and application

Abstract

The present invention belongs to the technical field of medical materials, and discloses an acid-induced in-situ generated luminescent cationic free radical probe and its preparation and application. The luminescent cationic free radical probe is obtained by oxidizing a compound of formula I under acidic conditions, and its structure is formula II. The present invention also discloses a method for preparing the luminescent cationic free radical probe. The cationic free radical probe of the present invention is generated by an in-situ reaction in an acidic environment, does not require separation, has the advantages of red to near-infrared luminescence, high generation efficiency, and good stability, and can be used for fluorescence imaging. The cationic free radical probe of the present invention can realize in-situ imaging and gastrointestinal imaging in an acidic environment of the stomach. The probe of the present invention is also used to prepare a probe for monitoring the digestion process of food in the stomach or to prepare a reagent for monitoring treatment with gastric acid-resistant drugs that neutralize gastric acid.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical materials and relates to a red to near-infrared luminescent cationic free radical generated in situ under acidic conditions, and specifically relates to an acid-induced luminescent pyrrole cationic free radical probe, its preparation, and application in gastrointestinal imaging and detecting the effects of gastric acid-resistant drugs. Background Art

[0002] Gastrointestinal imaging and sensing technologies with high spatiotemporal resolution are crucial for the diagnosis and drug development of gastrointestinal diseases. Common clinical conditions, such as abnormal gastric emptying, require analysis of gastrointestinal structure and physiological function for accurate diagnosis. However, current clinical imaging technologies, such as ultrasound, computed tomography, and magnetic resonance imaging, are typically only used to assess the structural status of the gastrointestinal tract, while gastrointestinal function analysis requires invasive and uncomfortable endoscopic examinations or extraction of gastric fluid. To facilitate the diagnosis and drug development of gastrointestinal diseases, probes with non-invasive gastrointestinal imaging and in situ sensing capabilities are highly desirable.

[0003] Near-infrared fluorescence imaging has significant advantages such as strong penetration and low autofluorescence interference, and is widely used in imaging-mediated surgery. In recent years, a series of red-near-infrared emitting organic fluorophores have been developed for gastrointestinal imaging and sensing, which have the advantages of high spatiotemporal resolution and convenient operation, such as cyanine and benzodibenzodiazole derivatives. However, the preparation of these fluorophores with closed-shell electron structures is cumbersome, which seriously limits their application. Compared with traditional saturated electron fluorophores, the energy gap between the excited state and the ground state of radical cations is much smaller, which provides the possibility of developing small-sized near-infrared fluorophores. However, the radical cations reported in existing studies are almost all non-luminescent, which seriously limits their biological applications.

[0004] Therefore, the development of a cation radical fluorescent probe with simple synthesis and the ability to image the stomach in situ and monitor the gastrointestinal tract is highly desirable. The pyrrole derivative precursor of the present invention can be converted in situ into a pyrrole cation radical with red to near-infrared fluorescence in the acidic stomach environment, successfully enabling gastrointestinal imaging. It also has potential applications in monitoring gastric digestion and the effectiveness of gastric acid-resistant drugs. Summary of the Invention

[0005] To overcome the shortcomings and defects of the prior art, the primary objective of the present invention is to provide a luminescent cationic radical probe (i.e., an acid-induced in situ generated luminescent cationic radical probe) and its preparation method. The infrared to near-infrared luminescent cationic radical probe of the present invention is obtained by in situ generation of a dimethylpyrrole derivative precursor under acidic conditions (e.g., in the acidic stomach). The probe of the present invention enables fluorescence imaging of the gastrointestinal tract with high temporal and spatial resolution.

[0006] Another object of the present invention is to provide applications of the aforementioned luminescent cationic radical probe. The cationic radical probe is used to prepare a gastrointestinal fluorescent imaging agent. The probe of the present invention is also used to monitor gastric emptying. Furthermore, the cationic radical probe of the present invention can also be used to monitor the effectiveness of gastric acid-resistant drugs.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A luminescent cationic radical probe is obtained by oxidizing a compound of formula I under acidic conditions, and its structure is formula II;

[0009] The structure of the probe:

[0010]

[0011] Compounds of formula I:

[0012] In Formula I and Formula II, R 1 is hydrogen, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

[0013] R 2 、R 5 Each is independently halogen, substituted or unsubstituted alkyl, alkyloxy, alkylamino, aryl, heteroaryl, aryloxy (Ar-O-), arylamino (Ar-NH-), arylthio (Ar-S-), heteroaryloxy, heteroarylamino, heteroarylthio;

[0014] R 3 、R 4 Each is independently hydrogen, halogen, substituted or unsubstituted alkyl, alkyloxy, alkylamino, aryl, heteroaryl, aryloxy (Ar-O-), arylamino (Ar-NH-), arylthio (Ar-S-), heteroaryloxy, heteroarylamino, heteroarylthio.

[0015] R 1 In the above, the aryl group refers to a monocyclic or polycyclic aromatic group having 6 to 20 carbon atoms. Representative aryl groups include phenyl, naphthyl, anthracenyl, and pyrenyl.

[0016] The substituted aryl group refers to a hydrogen atom on the aryl ring being replaced by one or more of an alkoxy group, an amino group, or a carboxyl group;

[0017] The heteroaryl group refers to a monocyclic or polycyclic heteroaromatic group having 1 to 20 carbon atoms and 1 to 4 heteroatoms selected from N, S, and O. Representative heteroaryl groups include: pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, thiazolyl, indolyl, azanaphthyl, azaanthryl, and azapyrenyl;

[0018] R 2 、R3 、R 4 、R 5 In which the alkyl group is C 1-30 Alkyl, alkyloxy is C 1-30 Alkyloxy, alkylamino is C 1-30 Alkylamino, alkylthio is C 1-30 alkylthio;

[0019] R 3 、R 3 、R 4 、R 5 In the substituted alkyl group, the hydrogen on the alkyl group is replaced by a hydroxyl group, a methoxy group, a carboxyl group, or a halogen group;

[0020] R 2 、R 3 、R 4 、R 5 In the above, the aryl group refers to a monocyclic or polycyclic aromatic group having 6 to 20 carbon atoms, and representative aryl groups include: phenyl, naphthyl, anthracenyl, pyrenyl; the aryl groups in the aryloxy, arylamino and arylthio groups are each independently a monocyclic or polycyclic aromatic group having 6 to 20 carbon atoms, and representative aryl groups include: phenyl, naphthyl, anthracenyl, pyrenyl;

[0021] The heteroaryl group refers to a monocyclic or polycyclic heteroaromatic group having 1-20 carbon atoms and 1-4 heteroatoms selected from N, S, and O. Representative heteroaryl groups include: pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, thiazolyl, indolyl, azanaphthyl, azaanthryl, and azapyrenyl; the heteroaryl groups in the heteroaryloxy, heteroarylamino, and heteroarylthio groups are each independently a monocyclic or polycyclic heteroaromatic group having 1-20 carbon atoms and 1-4 heteroatoms selected from N, S, and O. Representative heteroaryl groups include: pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, thiazolyl, indolyl, azanaphthyl, azaanthryl, and azapyrenyl.

[0022] The luminescent cationic radical probe is stable in an acidic environment.

[0023] R 1 is phenyl or methoxyphenyl, R 2 and R 5 Each is a methyl group, R 3 is -CH2-OH, R 4 is hydrogen or piperidinyl-1-methylene

[0024] The acidic condition refers to pH ≤ 3 (eg pH = 1 to 3). The acidic condition can be an acidic solution or an acidic buffer solution.

[0025] The oxidation is oxidation with an oxidant, wherein the oxidant is oxygen, a single-electron oxidant, or an oxygen-containing atmosphere.

[0026] The preparation method of the near-infrared luminescent cationic radical probe (Formula II) comprises the following steps: subjecting the compound of Formula I to an oxidation reaction under acidic conditions to obtain the near-infrared luminescent cationic radical probe (Formula II).

[0027] Compound of formula II: R 1 、R 2 、R 3 、R 4 、R 5 As defined above in Formula I.

[0028] Reaction equation:

[0029]

[0030] The reaction is carried out under acidic conditions (pH ≤ 3, such as pH = 1 to 3);

[0031] The acidic conditions refer to acidic solutions, which refer to all acidic aqueous solutions with a pH of ≤ 3 (pH = 1 to 3), including Britton-Robinson buffer and simulated gastric acid buffer (HCl);

[0032] The oxidation refers to oxidation under the action of an oxidant; the oxidant is oxygen or an atmosphere containing oxygen (such as air).

[0033] The compound of formula I can be prepared into a solution in a solvent and then mixed with an acidic solution; or it can be directly mixed with an acidic solution; the concentration of the compound of formula I in the solvent (the solvent can be dimethyl sulfoxide (DMSO), acetonitrile (MeCN), ethanol (CH3CH2OH), methanol (CH3OH), etc.) is 1 μM to 1000 mM; the volume ratio of the acidic aqueous solution (pH = 2 to 3) to the solution of the compound of formula I is 99:1.

[0034] When the compound of formula I is directly mixed with the acidic solution, the concentration of the compound of formula I in the acidic solution is 1 μM to 1000 mM.

[0035] The reaction time is 5 to 120 minutes.

[0036] The reaction temperature is room temperature.

[0037] The cationic free radical probe is used to prepare a fluorescent probe for gastrointestinal imaging.

[0038] The cationic free radical-based near-infrared luminescent probe is used for in situ detection of the gastric emptying process (ie, the probe of the present invention is used as a probe for in situ detection of the gastric emptying process). The gastric emptying process refers to the process of food digestion in the gastrointestinal tract.

[0039] In addition, the cation radical probe of the present invention is used in monitoring the effects of gastric acid resistance drugs.

[0040] The food refers to rice paste or water.

[0041] The gastric acid resistant drug is a drug administered to the stomach where gastric acid secretion is excessive to neutralize gastric acid.

[0042] The gastric acid neutralizing drug is preferably a sodium bicarbonate tablet.

[0043] The cationic radical probe of the present invention is generated by in-situ reaction in an acidic environment without separation, has the advantages of red to near-infrared luminescence, high generation efficiency and good stability, and can be used for fluorescence imaging.

[0044] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0045] 1. The cationic free radical fluorescent probe of the present invention can be generated in situ in the stomach and used for gastrointestinal fluorescence imaging with high temporal and spatial resolution.

[0046] 2. The cationic free radical probe of the present invention has near-infrared fluorescence and can be used to monitor the gastric emptying process and gastric acid resistance therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The structural formulas of different pyrrole derivatives and their photos under fluorescent light in buffer solutions with different pH values; (A) Structural formulas of pyrrole derivatives; (B) Photos of different pyrrole derivatives under fluorescent light in buffer solutions with different pH values;

[0048] Figure 2 P3 (2mM) is in situ oxidized into a cationic free radical compound P3 in an acidic buffer solution (pH = 2.0-2.6) and air. ·+ Absorption spectrum and fluorescence emission spectrum of: (A) Absorption spectrum; (B) Fluorescence emission spectrum, excitation wavelength is 600nm; (C) Curves formed by absorption value and fluorescence intensity at different pH values;

[0049] Figure 3 P6 (2mM) is in situ oxidized into a cationic free radical compound P6 in an acidic buffer solution (pH = 2.0-3.0) and air. ·+Absorption spectrum and fluorescence emission spectrum of: (A) Absorption spectrum; (B) Fluorescence emission spectrum, excitation wavelength is 600nm; (C) Curves formed by absorption value and fluorescence intensity at different pH values;

[0050] Figure 4 P7 (2mM) is in situ oxidized into a cationic free radical compound P7 in an acidic buffer solution (pH = 2.0-3.0) and air. ·+ Absorption spectrum and fluorescence emission spectrum of: (A) Absorption spectrum; (B) Fluorescence emission spectrum, excitation wavelength is 600nm; (C) Curves formed by absorption value and fluorescence intensity at different pH values;

[0051] Figure 5 For P3 ·+ Electron spin resonance (EPR) spectrum of solid powder;

[0052] Figure 6 For P3 ·+ H NMR spectra of P3 in deuterated water (pH = 2.0) and deuterated acetonitrile;

[0053] Figure 7 P3 (5 mM) was converted to P3 in acidic buffer (pH = 2.0) ·+ And the absorption spectrum, emission spectrum and intensity change diagram over time: (A) P3 ·+ Time-dependent absorption spectrum of (B) P3 ·+ Time-dependent fluorescence emission spectra of (C) and (D) P3 ·+ The relationship between the absorption intensity at 600nm and the fluorescence intensity at 680nm and time; the excitation wavelength is 600nm;

[0054] Figure 8 For P3 ·+ Emission spectra and intensity changes of the fluorescent dye Amplex Red in working solution with changes in P3 concentration; (A) Fluorescence emission spectra of Amplex Red with changes in P3 concentration; (B) Relationship between the fluorescence intensity of Amplex Red at 590 nm and P3 concentration;

[0055] Figure 9 P3 contains different metal ions (Na + , K + Mg 2+ , Ca 2+ ), antioxidants (reduced glutathione, ascorbic acid), sugars (glucose, soluble starch) and proteins (pepsin, hemoglobin, papain, ovalbumin, lactoferrin, lactoglobulin, transferrin and bovine serum albumin) in an acidic buffer (pH = 2.0)·+ Absorption spectrum, emission spectrum and intensity change value: (A) Absorption spectrum (B) Fluorescence emission spectrum, excitation wavelength is 600nm; (C) P3 ·+ The ratio of fluorescence intensity at 680 nm in the presence of different relevant interferents to that in pure acidic buffer (pH = 2.0) (I / I0);

[0056] Figure 10 For P3 ·+ Fluorescence spectra and intensity changes of P3 (2mM) in a mixed solution of glycerol and water with increasing glycerol content: (A) Fluorescence spectrum, excitation wavelength is 600nm; (B) ·+ The ratio of the emission intensity at 680 nm in mixed solutions of glycerol and water with different contents to the emission intensity at 680 nm in aqueous solution (I / I0);

[0057] Figure 11 For P3 ·+ (2mM) Fluorescence lifetime spectra and fluorescence lifetime change diagram of P3 in a mixed solution of glycerol and water with increasing glycerol content: (A) Fluorescence lifetime spectrum, emission wavelength is 680nm; (B) ·+ Fluorescence lifetime change value of mixed solution of glycerol and water with different contents;

[0058] Figure 12 Figure 3 shows in vivo fluorescence imaging and changes in gastric fluorescence intensity over time after oral gavage of nude mice with 70 μL of an aqueous solution containing 1.5 mg of P3: (A) In vivo imaging of mice after oral gavage; (B) Changes in the ratio of fluorescence intensity at different times to the fluorescence intensity at 0 minute (I / I0) in the corresponding gastric region;

[0059] Figure 13 The images show the time-dependent fluorescence imaging of the laparotomy and the corresponding anatomical organs after the nude mice were gavaged with 70 μL of an aqueous solution containing 1.5 mg of P3.

[0060] Figure 14 Fluorescent imaging of the gastrointestinal anatomy of nude mice at different time points after oral administration of 150 μL rice paste containing 1.5 mg P3 or 70 μL aqueous solution containing 1.5 mg P3;

[0061] Figure 15 This is the electron spin resonance (EPR) spectrum of the blue contents of the stomach collected after nude mice were gavage-fed with 150 μL rice paste containing 1.5 mg P3;

[0062] Figure 16Preliminary experiments and modeling experiments for monitoring gastric acid resistance in mice; (A) In vivo imaging of two groups of normal nude mice after oral administration of 200 μL of acidic buffer (pH 2.0) and neutral buffer (pH 7.0) containing 1.5 mg of P3; (B) In vivo fluorescence imaging of normal nude mice and gastric acid resistance model mice after oral administration of 70 μL of aqueous solution containing 1.5 mg of P3;

[0063] Figure 17 Microscopic images of tissue sections and liver and kidney function analysis of nude mice and normal nude mice after oral gavage with 70 μL aqueous solution containing 1.5 mg P3; (A) Microscopic photos of tissue sections; (B) Liver and kidney function indicators. DETAILED DESCRIPTION

[0064] The present invention is further described in detail below with reference to the examples, the purpose of which is to provide a better understanding of the research content of the present invention, but the embodiments of the present invention are not limited thereto.

[0065] Example 1: Compound P3 in acidic buffer (pH = 2.0-2.6) ·+ In situ generation

[0066]

[0067] 30 μL of DMSO mother solution (200 mM) of compound P3 (2,5-dimethyl-3-hydroxymethylene-1-phenyl-1H-pyrrole) was added to 2970 μL of acidic buffer (pH = 2.0-2.6) (P3 can be added directly to the acidic buffer and then sonicated for 5 minutes; the acidic buffer here specifically refers to a buffer with a pH of 2.0-2.6 prepared by glacial acetic acid, boric acid, phosphoric acid and sodium hydroxide). After mixing, the protonated pyrrole compound P3 was rapidly converted into the corresponding cationic free radical compound P3 ·+ (carried out in air atmosphere), P3 can be proved by bright field photos, UV-visible absorption spectrum and fluorescence spectrum test ·+ The result is as follows. Figure 1 and Figure 2 shown.

[0068] Example 2: P6 in acidic buffer (pH = 2.0-3.0) ·+ In situ generation

[0069]

[0070] 30 μL of DMSO stock solution (200 mM) of compound P6 (4-(piperidin-1-methylene)-2,5-dimethyl-3-hydroxymethylene-1H-pyrrole) was added to 2970 μL of acidic buffer (pH = 2.0-3.0). After mixing, the pyrrole compound P6 was converted into the corresponding cationic free radical compound P6 within 30 minutes. ·+ (carried out in air atmosphere), the bright field photograph, UV-visible absorption spectrum and fluorescence spectrum test can prove P6 ·+ The result is as follows. Figure 1 and Figure 3 shown.

[0071] Example 3: P7 in acidic buffer (pH = 2.0-3.0) ·+ In situ generation

[0072]

[0073] 30 μL of compound P7 (1-(4-methoxyphenyl)-2,5-dimethyl-3-hydroxymethylene-1H-pyrrole) DMSO stock solution (200 mM) was added to 2970 μL of acidic buffer (pH = 2.0-3.0). After mixing, the protonated pyrrole compound P7 was converted into the corresponding cationic free radical compound P7 within 30 minutes. ·+ (carried out in air atmosphere), the bright field photographs, UV-visible absorption spectra and fluorescence spectra can be used to prove that P7 ·+ The result is as follows. Figure 1 and Figure 4 shown.

[0074] Example 4:

[0075] Verify the generation of pyrrole cation radical:

[0076] (1) Electron paramagnetic resonance (EPR) test

[0077] 10 mg of compound P3 was added to 1 mL of BR buffer solution (pH 2.0) and sonicated (480W, 10 minutes) to obtain a blue solution, which was freeze-dried to obtain a blue powder. The blue solid powder was then placed in an EPR tube and the EPR spectrum was recorded on a Bruker E500-10 / 12 electron paramagnetic resonance CW (20 mW) for 5 minutes. Figure 5 As shown in the figure, an obvious free radical signal peak with a g value of 2.0031 was collected, proving that the pyrrole cation radical P3 ·+ Generation.

[0078] (5) Nuclear magnetic resonance characterization

[0079] 5.5 mg of compound P3 was added to 550 μL of deuterated water (pH 2.0) and 550 μL of deuterated acetonitrile, respectively, and sonicated (480W, 10 minutes). The deuterated solution was then recorded on a Bruker AV 400 NMR spectrometer, and the spectra were processed for comparison. Figure 6 It can be seen that there is no NMR signal of compound P3 in acidic deuterated water, which indirectly indicates that P3 generates cationic free radical P3 in deuterated water (pH 2.0). ·+ .

[0080] Example 5

[0081] P3 ·+ Formation process and mechanism verification:

[0082] (1)P3 ·+ Absorption spectra, fluorescence emission spectra, and corresponding intensity changes over time

[0083] 30 μL of DMSO stock solution of compound P3 (200 mM) was added to 2970 μL of acidic buffer (the acidic buffer here specifically refers to a pH 2.0 buffer prepared from glacial acetic acid, boric acid, phosphoric acid, and sodium hydroxide), mixed, and then time-dependent UV-visible absorption and fluorescence spectra were measured (2 h). Figure 7 In acidic buffer (pH 2.0), compound P3 can be observed to have a stable absorption peak of 600 nm and a fluorescence emission peak of 680 nm. From the absorption peak, it can be seen that P3 can be converted into a cationic radical P3 with red to near-infrared emission within 5 min. ·+ (570nm to 600nm), where Figure 7 The inset in A refers to the photographs of compound P3 under fluorescent light at different reaction time points (0.1, 10, 30, and 120 minutes) after the addition of acidic buffer (pH 2.0).

[0084] (2) Verify P3 ·+ Trace amounts of H2O2 are produced during the generation process

[0085] 2 μL of different concentrations of P3 ·+(P3: 0, 25, 50, 100, 150, 200 μM) blue solution was added to 48 μL of PBS solution, and then 50 μL of Amplex Red working solution (4.85 mL PBS, 100 μL 10 U / mL HRP enzyme and 50 μL 10 mM Amplex Red (McLean) DMSO stock solution) was added. Three replicate wells were set up for each concentration and incubated at 30°C for 30 minutes before fluorescence emission spectrum testing. The excitation wavelength was 530 nm and the emission wavelength was 560-700 nm. Figure 8 It can be seen that with P3 ·+ As the concentration of (P3) increases, the fluorescence of Amplex Red working solution (H2O2 probe) at 590 nm increases, which shows that P3 ·+ The production process will produce trace amounts of H2O2.

[0086] Example 6

[0087] P3 ·+ Anti-interference experiment:

[0088] Pre-formulated with different metal ions (Na + , K + Mg 2+ , Ca 2+ ), antioxidants (reduced glutathione, ascorbic acid), sugars (glucose, soluble starch) and proteins (pepsin, hemoglobin, papain, ovalbumin, lactoferrin, lactoglobulin, transferrin and bovine serum albumin) in an acidic buffer (the acidic buffer here specifically refers to a pH 2.0 buffer prepared from glacial acetic acid, boric acid, phosphoric acid and sodium hydroxide) (pH 2.0). 30 μL of the DMSO mother solution of compound P3 (200 mM) was added to 2970 μL of the above acidic buffer, mixed and incubated for 2 hours, and the blue P3 ·+ The solution was tested for UV-visible absorption and fluorescence spectra, with an excitation wavelength of 600 nm. The corresponding samples were imaged with a small animal imaging device to obtain fluorescence images. Figure 9 It can be seen that different metal ions, common antioxidants and sugars will not affect P3 ·+ The fluorescence of P3 ·+ fluorescence.

[0089] Example 7

[0090] Verification of protein enhanced fluorescence (viscosity experiment):

[0091] Glycerol and water were mixed in different volume ratios (water / glycerol = 100 / 0, 90 / 10, 80 / 20, 60 / 40, 40 / 60, 20 / 80, 10 / 90) to form mixed solutions with different glycerol contents. P3 prepared in acidic buffer (pH 2.0) was added. ·+ Compound P3 was dissolved in these mixed solutions to a final concentration of 2 mM, and then the fluorescence emission spectrum (excitation wavelength is 600 nm) and fluorescence lifetime curve (emission wavelength is 680 nm) were tested. The fluorescence lifetime was obtained by origin double exponential fitting. Figures 11-12 As shown in Figure 2, with the continuous increase of glycerol content (from 0% to 90%), the cationic radical P3 ·+ The relative fluorescence intensity ratio (I / I0, I and I0 refer to P3 ·+ The fluorescence intensity and fluorescence lifetime (fitted value) of the compound in aqueous solution with different glycerol contents gradually increased, which indicates that the fluorescence intensity and photophysical properties of the compound will be further enhanced when the molecular motion is restricted.

[0092] Example 8

[0093] Animal experimentation: Male 8-week-old Balb / c nude mice were purchased from Guangzhou Yancheng Biotechnology Co., Ltd. The mice were acclimated for 1 week and fasted for 24 hours before oral gavage.

[0094] (1) In vivo imaging

[0095] Mice were anesthetized beforehand, and 1.5 mg of compound P3 (DMSO) was dispersed in 70 μL of ultrapure water. The molecule was then administered orally to the mouse's stomach. In vivo fluorescence imaging was then performed at room temperature using a Bruker MI SE 721 small animal imager. Imaging was performed every 10 minutes until 60 minutes, every 30 minutes until 120 minutes, and then every 60 minutes until 180 minutes. Two imaging steps were performed sequentially: 590 nm / 700 nm, exposure time 60 seconds, and white light exposure time 0.175 seconds. Figure 12 After 10 minutes of imaging, obvious near-infrared fluorescence signals can be seen in the mouse stomach. Until 180 minutes of imaging, obvious near-infrared fluorescence signals appear in the intestinal area. Under the same conditions, fluorescence imaging quantitative analysis was performed using Bruker MI SE 721. Fluorescence quantitative analysis was performed on the mouse stomach signals at all the above time points. The fluorescence average of three points in the stomach area at 0 min was taken as I0, and the fluorescence average of three points in the stomach area at other time points was taken as I. The fluorescence ratio (I / I0) was used to semi-quantitatively analyze the signal changes in the mouse stomach. At different time points, the mice were euthanized and the gastrointestinal tract of the mice was dissected out. White light photography was performed, and the blue contents of the stomach could be seen.

[0096] (2) In vivo imaging by laparotomy

[0097] The mice were anesthetized in advance, and then an incision was made in the abdomen to expose the gastrointestinal tract. 1.5 mg of compound P3 (DMSO) was dispersed in 70 μL of ultrapure water and then gavage was administered to the stomach of the anesthetized mice. In vivo fluorescence imaging was then performed at 37°C using a small animal imager Bruker MI SE 721. Imaging was performed every 20 minutes until 120 minutes. Two imaging steps were performed in sequence: 590 nm / 700 nm, exposure time 60 seconds, and white light exposure time 0.175 seconds. Figure 13 After 20 minutes of imaging, the fluorescent signal in the stomach was strongest, while the intestinal fluorescence signal increased over time. Finally, the mice were euthanized and all organs except the gastrointestinal tract were dissected, and no fluorescent signal was found.

[0098] (3) Monitoring of gastric digestion process

[0099] Mice were anesthetized and then gavaged with either a P3 / rice paste mixture (1.5 mg of P3 (DMSO) in 150 μL of commercially prepared rice paste) or a P3 aqueous solution (1.5 mg of P3 (DMSO) in 70 μL of ultrapure water). The mice were euthanized and dissected at various time points (10, 60, and 180 minutes) for ex vivo fluorescence imaging of the isolated gastrointestinal tract. Two imaging steps were performed: 590 nm / 700 nm exposure time of 60 seconds, followed by a white light exposure time of 0.175 seconds. Figure 14 In the experiment, the rice paste group containing chemical P3 was compared with the water group. It can be seen that the fluorescence signal in the stomach of the rice paste group did not weaken significantly within 180 minutes, indicating that it takes longer time for food to be empty (>180 minutes). However, the stomach of the water group was empty within 180 minutes, and there was only a weak fluorescence signal in the intestine, indicating that pure water is digested quickly in the gastrointestinal tract.

[0100] (4) Verify P3 ·+ Generated in situ in the stomach

[0101] The P3 / rice paste mixture (same as above) was poured into the stomach of anesthetized mice and incubated at room temperature for 2 hours. The mice were euthanized and the stomachs were dissected out. The blue stomach contents of mice in each parallel group were collected and dehydrated using a freeze dryer to obtain a blue solid. The blue solid powder was then placed in an EPR tube, and the EPR spectrum was recorded on a Bruker E500-10 / 12 electron paramagnetic resonance CW (20 mW) within 5 minutes. Figure 15 Medium EPR spectrum Figure 1 The signal peak with g value = 2.0031 indicated the generation of pyrrole cation radicals in the stomach.

[0102] (5) Monitoring of gastric acid resistance therapy

[0103] Before modeling, a preliminary experiment was conducted: 1.5 mg of P3 (DMSO) was dispersed in 200 μL of pH 2.0 and pH 7.0 buffer solutions. The two solutions were then injected into the stomachs of two groups of mice, respectively. The mice were incubated for 2 hours for in vivo fluorescence imaging. Two imaging steps were performed in sequence: 590 nm / 700 nm, exposure time 60 s, and white light exposure time 0.175 s. Figure 16 As shown in A, only the acidic group can produce fluorescent signals when injected into the mouse stomach, while the neutral group has no signals.

[0104] Mice were randomly divided into a normal group and an acid-resistant group. The acid-resistant group was treated with sodium bicarbonate tablets (250 mg / kg -1 The normal group was induced by oral administration (0.2 mL of normal saline 1 hour before administration). A P3 aqueous solution (1.5 mg of P3 (DMSO) dispersed in 70 μL of ultrapure water) was then instilled into the stomachs of both groups of mice. In vivo fluorescence imaging was performed for 2 hours. Two imaging steps were performed sequentially: 590 nm / 700 nm, exposure time 60 s, and white light exposure time 0.175 s. Figure 16 As shown in B, compared with the strong fluorescence signal of the normal group, the anti-gastric acid treatment group did not have any fluorescence signal.

[0105] (6) H&E staining and liver and kidney index analysis ( Figure 17 )

[0106] Randomly selected mice were gavaged with P3 solution (1.5 mg P3 dispersed in 70 μL water). Two hours after gavage, blood (approximately 500 μL) was collected from the fundus venous plexus and stored on ice before centrifugation at 3500 rpm for 20 minutes to prevent clotting. Serum samples (200 μL) were analyzed for total protein (TP), albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), carbon dioxide (CO2), uric acid (UA), and urea (UREA) using a Roche Cobas E602 electrochemiluminescence immunoassay analyzer. Stomach, liver, spleen, kidney, and heart of the mice were removed and placed in 4% paraformaldehyde (PFA) for histological examination 2 hours after gavage with P3 solution.

[0107] All tissues were fixed with 4% PFA, dehydrated with ethanol, embedded in paraffin, and cut into 20 mm coronal cryostat sections for hematoxylin and eosin (H&E) staining. Paraffin was removed by washing with xylene, and sections were then incubated with hematoxylin and eosin for 4 minutes and washed with distilled water. Stained sections were examined using a Nikon ECLIPSE 80i microscope.

[0108] like Figure 17A, Compared with the control group, the main organs (stomach, liver, spleen, kidney, lung, heart) of mice treated with P3 showed no obvious damage or inflammatory lesions. Figure 17 B, The expression levels of biochemical indices of liver and kidney function in P3-treated mice were also similar to those in the control group.

[0109] In summary, these results confirm that the pyrrole radical cation P3 generated in situ in the stomach ·+ It has good biocompatibility and is suitable for in vivo gastrointestinal imaging.

[0110] P6 of the present invention ·+ and P7 ·+ It can also perform gastrointestinal imaging, but the P6 molecule conversion efficiency is lower and the luminescence is weaker, and the imaging effect is not as good as P3. ·+ .

[0111] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for preparing a luminescent cationic radical probe, characterized in that: The following steps are involved: The compound of formula I is subjected to an oxidation reaction under acidic conditions of pH ≤ 3 to obtain a cationic radical probe having red to near-infrared luminescence; Compounds of formula I: ; The structure of the cationic radical fluorescent probe is Formula II: Formula II In Formula I and Formula II, R 1 is phenyl or methoxyphenyl, R 2 and R 5 Each is a methyl group, R 3 is -CH2-OH, R 4 is hydrogen or piperidinyl-1-methylene; The reaction is carried out in an atmosphere containing oxygen; the acidic condition is an acidic solution with a pH value of ≤ 3.

2. The method for preparing the luminescent cationic radical probe according to claim 1, wherein: The concentration of the compound of formula I in the acidic solution is 1 μM to 1000 mM.

3. The method for preparing the luminescent cationic radical probe according to claim 1, wherein: The reaction time is 5~120min.

4. The use of the luminescent cationic radical probe obtained by the preparation method according to any one of claims 1 to 3, characterized in that: The fluorescent probe of the luminescent cationic free radical is used to prepare a fluorescent probe or imaging agent for in situ imaging in an acidic environment of the stomach and high temporal and spatial resolution imaging of the gastrointestinal tract.

5. The use of the luminescent cationic radical probe obtained by the preparation method according to any one of claims 1 to 3, characterized in that: The fluorescent probe of the luminescent cationic free radical is used to prepare a probe for monitoring the food digestion process in the stomach, or the fluorescent probe of the luminescent cationic free radical is used to prepare a reagent for monitoring gastric acid resistance therapy.

Images