Organic fluorescent probe for detecting oxygen deficit as well as preparation method and application of organic fluorescent probe

Abstract

The invention relates to the technical field of organic small-molecule fluorescent probes, in particular to an organic fluorescent probe for detecting oxygen deficit, a preparation method and application, the organic fluorescent probe is a compound DCM-Azo-pNO2, and the structure of the organic fluorescent probe is shown as a formula (I). The core of the method is that strong electron-withdrawing substituent-NO2 is introduced to azo aromatic hydrocarbon, so that the electrophilicity and bond dissociation energy of an azo bond are remarkably enhanced, and the azo bond can be quickly and efficiently reduced and broken by azo reductase. According to the organic fluorescent probe, in an anoxic environment, azo bonds are specifically reduced, an intramolecular charge transfer effect is recovered, and remarkable change of fluorescence from off to on is realized. The fluorescence enhancement multiple of the organic fluorescent probe is as high as 690 times, and compared with 3-6 times of improvement of a traditional probe, a leap in order of magnitude is generated, and the quenching efficiency is greatly improved; and the detection limit of the mimic enzyme SDT is as low as 0.12 M, so that the detection sensitivity is greatly improved.

Description

Technical Field

[0001] This invention relates to the field of organic small molecule fluorescent probe technology, specifically to an organic fluorescent probe for detecting hypoxia, its preparation method, and its application. Background Technology

[0002] The hypoxic microenvironment of solid tumors is a key indicator for assessing their malignancy, predicting radiotherapy and chemotherapy resistance, and guiding individualized treatment. Therefore, developing technologies that can accurately and rapidly detect hypoxia has significant clinical implications.

[0003] Currently, various techniques are used for hypoxia detection. Optical imaging has become a research hotspot due to its radiation-free nature, low cost, and direct coupling with commercial handheld / endoscopic devices. Previous studies have constructed fluorescent probes based on the cleavage of –N=N– bonds by azo reductase (AzoR). However, these probes all employ azo backbones with symmetrical or weak electron-donating / withdrawing substitutions, resulting in low quenching efficiency, with fluorescence enhancement only 3–6 times before and after hypoxia.

[0004] Therefore, there is an urgent need to develop an organic fluorescent probe with high quenching efficiency for detecting hypoxia. Summary of the Invention

[0005] To address the above problems, this invention provides an organic fluorescent probe for detecting hypoxia, its preparation method, and its application.

[0006] This invention is achieved through the following technical solution: An organic fluorescent probe for detecting hypoxia, wherein the organic fluorescent probe is the compound DCM-Azo-pNO2, and its structure is shown in formula (I): Formula (I).

[0007] The method for preparing the organic fluorescent probe includes the following steps: (1) Using tetrahydrofuran as a solvent, 2'-hydroxyacetophenone and ethyl acetate were condensed in the presence of sodium hydride to obtain compound 1.

[0008] (2) Using acetic acid as a solvent, compound 1 was reacted with sulfuric acid to undergo a catalytic ring-closure reaction to obtain compound 2.

[0009] (3) Using acetic anhydride as a solvent, compound 2 was reacted with malononitrile by Knoevenagel condensation to obtain compound 3.

[0010] (4) Under a nitrogen atmosphere, using toluene as a solvent, compound 3 and p-acetaminobenzaldehyde were condensed in the presence of piperidine and acetic acid to obtain the fluorophore DCM-NH2.

[0011] (5) Using dichloromethane and water as solvents, 4-nitroaniline and potassium peroxymonosulfate were oxidized under a nitrogen atmosphere to obtain compound 4; wherein the mass ratio of 4-nitroaniline to potassium peroxymonosulfate was 0.2:1.74.

[0012] (6) Using acetic acid as a solvent, compound 4 was subjected to a diazotization coupling reaction with the fluorophore DCM-NH2 to obtain DCM-Azo-pNO2; wherein the mass ratio of compound 4 to fluorophore DCM-NH2 was 60:63.8.

[0013] .

[0014] Preferably, in step (6), the temperature of the diazotization coupling reaction is 40°C and the time is 12 hours.

[0015] Application of the organic fluorescent probe in the preparation of agents for detecting hypoxia.

[0016] Preferably, the formulation is used to detect hypoxia at the cellular level.

[0017] Preferably, the cells are HeLa cells.

[0018] Preferably, the formulation is used to detect hypoxia at the tissue level.

[0019] Preferably, the tissue is tumor tissue.

[0020] Compared with the prior art, the present invention has the following beneficial effects: This invention provides an organic fluorescent probe for detecting hypoxia, wherein the organic fluorescent probe is the compound DCM-Azo-pNO2, the structure of which is shown in formula (I). The core of this invention lies in introducing a strong electron-withdrawing substituent –NO2 – onto an azo aromatic hydrocarbon, significantly enhancing the electrophilicity and bond dissociation energy of the azo bond, thereby enabling it to be rapidly and efficiently reduced and broken by azo reductase. Under hypoxic conditions, the azo bond of this organic fluorescent probe is specifically reduced, restoring the intramolecular charge transfer effect and achieving a significant change in fluorescence from "off" to "on". Experiments of this invention demonstrate that the fluorescence enhancement factor of this organic fluorescent probe is as high as 690 times, a leap of orders of magnitude compared to the mere 3-6 times improvement of traditional probes, greatly improving quenching efficiency; and the detection limit for the enzyme SDT is as low as 0.12 µM, greatly improving detection sensitivity. Furthermore, it exhibits a highly specific response to azo reductase, while showing almost no response to other common ions and reducing substances in the body, ensuring accurate and reliable detection results. This invention has demonstrated in both cell models and mouse ischemia models that the probe can achieve rapid and significant fluorescence "on" imaging with consistent and reliable results. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is the synthetic route for the DCM-Azo series of organic fluorescent probes of this invention.

[0023] Figure 2 The organic fluorescent probe DCM-Azo-pNO2 of this invention 1 H NMR spectrum.

[0024] Figure 3 This is the 13C NMR spectrum of the organic fluorescent probe DCM-Azo-pNO2 of this invention.

[0025] Figure 4 The response mechanism diagram of the organic fluorescent probe DCM-Azo-pNO2 of this invention is shown below. 1 H NMR spectrum; Figure 4 In the diagram, A represents the response mechanism of DCM-Azo-pNO2; B represents the state before the DCM-Azo-pNO2 response. 1 1H NMR spectrum; C represents DCM-Azo-pNO2 after NMR. 1 H NMR spectrum.

[0026] Figure 5 The images show the fluorescence titration spectra of the organic fluorescent probe of this invention at different SDT concentrations. Figure 5 In the figure, A represents DCM-Azo-pNO2; B represents DCM-Azo-pCN; C represents DCM-Azo-BOC; D represents DCM-Azo-POC; E represents DCM-Azo-pBr; F represents DCM-Azo-EOC; G represents DCM-Azo-pH; and H represents DCM-Azo-pOCH3. Note: The concentrations of SDT in the curves from bottom to top in the figure are 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, 1.0 mM, 1.2 mM, 1.4 mM, 1.6 mM, and 1.8 mM, respectively.

[0027] Figure 6 This is a titration curve of the fluorescence intensity response of the organic fluorescent probe DCM-Azo-NO2 of this invention to different concentrations of SDT. Detailed Implementation

[0028] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0029] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention and in its specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0030] The beneficial effects of the present invention will be illustrated below through specific embodiments.

[0031] The inventive concept of this invention is as follows: The present invention provides an organic fluorescent probe for detecting hypoxia, the core structure of which includes a near-infrared fluorophore DCM-NH2 and an azobenzene recognition unit regulated by a substituent R and linked to it by a linking group.

[0032] The substituent R is used to adjust the electron cloud density and electrophilicity of the azo bond (–N=N–).

[0033] R is a strong electron-withdrawing substituent nitro (–NO2) with a Hammitt constant σ greater than 0.7.

[0034] The organic fluorescent probe of this invention works based on an intramolecular charge transfer mechanism: under normoxic conditions, the strongly electron-withdrawing azo bond acts as a highly efficient electron / energy acceptor, quenching the fluorescence of the fluorophore DCM-NH2, and the probe is in an "off" state; under hypoxic conditions, the azo reductase highly expressed in the organism specifically recognizes and catalyzes the reduction and cleavage of the azo bond, releasing the original DCM-NH2 fluorophore, restoring the ICT effect, and thus generating a strong fluorescence "on" signal in the near-infrared region (λem=641nm).

[0035] 1. Substituent effect: By introducing substituents with different electron-withdrawing abilities, the electrophilicity of the –N=N– bond in azo aromatic hydrocarbons can be regulated, and a series of Azo-Rs recognition sites responsive to azo reductase (AzoR) can be constructed.

[0036] 2. Intramolecular charge transfer (ICT) mechanism: Due to the strong quenching effect of the intramolecular –N=N– bond, the probe is initially in the “off” state; in the presence of azo reductase (AzoR), the enzyme catalyzes the reduction and cleavage of the –N=N– bond, the ICT effect is restored, and the fluorescence emission is significantly enhanced at λem=641nm.

[0037] Example 1 The preparation method of the DCM-Azo-pNO2 probe is as follows: (1) Synthesis of Compound 1: Sodium hydride (3.2 g, 133 mmol) was dissolved in tetrahydrofuran (40 mL). Then, a mixture of 2'-hydroxyacetophenone (2.7 mL, 20.0 mmol), ethyl acetate (5.9 mL, 60.0 mmol), and tetrahydrofuran (10 mL) was added dropwise to the stirred solution. The reaction mixture was refluxed at 60 °C for 6 hours. After the reaction was confirmed to be complete by thin-layer chromatography (TLC), the reaction mixture was poured into ice water (50 mL). The pH of the solution was adjusted to 7 with an appropriate amount of hydrochloric acid. Subsequently, the solution was extracted with ethyl acetate (3 × 100 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. A light brown solid (3.12 g, yield 87%) was given, which was Compound 1, and was used directly in subsequent reactions without further purification.

[0038] .

[0039] (2) Synthesis of Compound 2: Compound 1 (1.78 g, 10 mmol) was dissolved in acetic acid (20 mL), followed by the addition of sulfuric acid (98%, 1.0 mL) with continuous stirring. The reaction mixture was refluxed at 120 °C for 30 min. After the reaction was complete, the resulting solution was transferred to 20 mL of ice water, and the pH was adjusted to approximately 8 with saturated sodium carbonate solution. The mixture was extracted three times with ethyl acetate (50 mL each time), and the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The crude product was purified by column chromatography using a mixture of petroleum ether and ethyl acetate (2:1, v / v) as the eluent to give 1.40 g of a dark brown product, which was Compound 2, with an overall yield of 83%.

[0040] The 1H and 1C NMR spectra of compound 2 are shown below: 1 H NMR (500 MHz, Methanol - d4) δ 8.00 (s, 1H), 7.67 (s, 1H), 7.42 (s,1H), 7.37 (s, 1H), 6.15 (s, 1H), 2.37 (s, 4H).

[0041] 13C NMR (126 MHz, Methanol - d4) δ 180.15, 169.29, 157.77, 135.16, 126.28, 125.98, 123.99, 119.06, 110.70, 49.00, 20.48.

[0042] .

[0043] (3) Synthesis of Compound 3: Compound 2 (0.83 g, 5.0 mmol) and malononitrile (0.47 g, 7.5 mmol) were dissolved in 10 mL of acetic anhydride. The reaction mixture was heated to 140 °C and refluxed for 12 hours. Then, 10 mL of deionized water was added to remove excess acetic anhydride. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was complete, 20 mL of ice water was added to quench the reaction mixture, followed by extraction with dichloromethane (3 × 50 mL). The crude product was purified by column chromatography using a solvent system of petroleum ether and ethyl acetate (v / v 2:1). An orange flocculent solid was given, which was compound 3, in a yield of 50% (0.39 g).

[0044] The 1H and 1C NMR spectra of compound 3 are shown below: 1H NMR (600 MHz, Chloroform - d) δ 8.88 (s, 1H), 7.71 (d, J = 15.6Hz, 1H), 7.47 - 7.41 (m, 2H), 6.69 (s, 1H), 2.43 (s, 3H).

[0045] 13C NMR (150 MHz, Methanol - d4) δ 168.77, 157.30, 134.79, 25.97, 125.70, 123.66, 118.74, 111.94, 110.52, 49.00, 20.39.

[0046] .

[0047] (3) Synthesis of the fluorophore DCM-NH2: Compound 3 (63.0 mg, 0.30 mmol) and p-acetaminobenzaldehyde (45.0 mg, 0.28 mmol) were dissolved in toluene (10 mL) at room temperature under a nitrogen atmosphere, followed by the addition of piperidine (0.15 mL) and acetic acid (0.15 mL). The mixture was refluxed at 115 °C for 3 hours to obtain an orange precipitate. After filtering the precipitate, the orange solid was refluxed in a solution of concentrated hydrochloric acid and ethanol (2:1, 30 mL) for 2 hours. The pH of the solution was adjusted to neutral. The aqueous solution was extracted three times with ethyl acetate (50 mL each time). After drying with anhydrous sodium sulfate, the organic layer was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using a mixture of dichloromethane and methanol (20:1, v / v) as the eluent. A red solid (33 mg) was obtained, which was the fluorophore DCM-NH2, with a yield of 43%.

[0048] The 1H and 1C NMR spectra of the fluorophore DCM-NH2 are shown below: 1 H NMR (600 MHz, DMSO - d6) δ 8.73 (s, 1H), 7.89 (s, 1H), 7.77 (s,1H), 7.63 (s, 1H), 7.58 (s, 1H), 7.49 (d, J = 8.4 Hz, 2H), 7.10 (s, 1H), 6.86(s, 1H), 6.62 (d, J = 8.3 Hz, 2H), 6.03 (s, 2H).

[0049] 13C NMR (150 MHz, DMSO - d6) δ 169.19, 161.50, 150.08, 144.44,140.11, 133.99, 131.88, 128.33, 126.66, 123.35, 121.92, 114.15, 49.00.

[0050] .

[0051] (5) Dissolve 4-nitroaniline (1.5 mmol, 200 mg) in dichloromethane (2 mL). Dissolve potassium persulfate (Oxone, 1.95 equivalents, 5.64 mmol, 1.74 g) in water (8 mL) and add it to the above reaction mixture. Stir the reaction mixture vigorously overnight at room temperature under a nitrogen atmosphere. After the reaction is complete, extract the mixture with dichloromethane. The organic layer is then... 1 The mixture was washed with M HCl solution, dried with anhydrous sodium sulfate, and finally the solvent was removed under reduced pressure to give a brown solid intermediate, namely compound 4, with a yield of 98%, corresponding to 240 mg.

[0052] The 1H and 1C NMR spectra of compound 4 are shown below: 1 H NMR (600 MHz, DMSO-d6) δ 8.53 (d, J = 8.6 Hz, 2H), 8.15 (d, J = 8.6Hz, 2H).

[0053] 13C NMR (150 MHz, DMSO-d6) δ 163.21, 150.56, 125.76, 121.62, 39.52.

[0054] .

[0055] (6) Compound 4 (60 mg, 0.4 mmol) was added to a 100 mL round-bottom flask, followed by a mixture containing the fluorophore DCM-NH2 (63.8 mg, 0.5 equivalent) and acetic acid. The resulting solution was stirred at 40 °C for approximately 12 hours. The reaction was confirmed to be complete by thin-layer chromatography (TLC), indicated by the disappearance of the starting spot. The reaction was then terminated, and the pH of the reaction mixture was adjusted to approximately 7. Dichloromethane was extracted with the aqueous phase (3 × 20 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent. Purification was performed by column chromatography using a mixture of petroleum ether and ethyl acetate (2:1, v / v) as eluent to give an orange-red solid target organic fluorescent probe DCM-Azo-pNO2 in 50% yield, corresponding to 44 mg.

[0056] Its 1H and 1C NMR spectra are shown below: 1 H NMR (500 MHz, DMSO-d6) δ 8.52 (d, J = 9.2 Hz, 2H), 8.47 (d, J = 9.2Hz, 2H), 8.41 (d, J = 9.0 Hz, 2H), 8.22 (d, J = 9.0 Hz, 2H), 8.09 (s, 1H),8.01 - 7.98 (m, 1H), 7.76 (d, J = 8.5 Hz, 2H), 7.60 (s, 1H), 7.45 (s, 1H),6.25 (s, 1H).

[0057] 13C NMR (150 MHz, DMSO-d6) δ 156.70, 156.46, 151.67, 138.63, 130.97,127.54, 124.79, 123.47, 123.17, 122.90, 122.43, 118.85, 116.43, 113.33,78.40, 78.35.

[0058] The synthetic route of the DCM-Azo probe is as follows: Figure 1 As shown; DCM-Azo-pNO2 1 H NMR image as follows Figure 2 As shown; the 13C NMR spectrum of DCM-Azo-pNO2 is as follows. Figure 3 As shown.

[0059] To directly verify the response mechanism of the probe DCM-Azo-pNO2 at the molecular structure level, proton NMR spectra before and after the response were performed. 1 A comparative experiment using ¹H NMR was conducted. The specific method was as follows: The probe DCM-Azo-pNO2 was dissolved in PBS buffer (20 mM, pH 7.4, PBS / DMSO = 9:1, volume ratio, containing 0.2% Tween 80) to prepare a 10 µM solution. Subsequently, sodium dithionite (SDT) was added to this solution to achieve a final concentration of 1.8 mM, and the solution was incubated at room temperature in the dark. Samples of the solution before and after the reaction were taken for further analysis. 1 H NMR test.

[0060] Response mechanism diagram of DCM-Azo-pNO2 and before and after response 1 H NMR spectrum as shown Figure 4As shown, DCM-Azo-pNO2 can undergo the expected specific shearing reaction under simulated reducing conditions, thus verifying its design principle and responsiveness to hypoxic microenvironments.

[0061] Comparative Example 1 The fluorophore DCM-NH2 was synthesized using the exact same method as in Example 1.

[0062] Preparation process of DCM-Azo-pCN: Compound 5 (54 mg, 0.4 mmol) was synthesized from 4-cyanoaniline using the exact same synthetic method as compound 4 in Example 1, and added to a 100 mL round-bottom flask, followed by the addition of a mixture containing the fluorophore DCM-NH2 (63.8 mg, 0.5 equivalent) and acetic acid. The resulting solution was stirred at 40 °C for approximately 12 hours. The reaction was confirmed by thin-layer chromatography (TLC) as the starting spot disappeared. The reaction was then terminated, and the pH of the reaction mixture was adjusted to approximately 7. Dichloromethane was extracted with the aqueous phase (3 × 20 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent. Purification was performed by column chromatography using a mixture of petroleum ether and ethyl acetate (2:1, v / v) as eluent to give an orange-red solid target organic fluorescent probe DCM-Azo-pCN in 47% yield, corresponding to 47 mg.

[0063] The chemical structure of compound 5 is shown below: .

[0064] Comparative Example 2 The fluorophore DCM-NH2 was synthesized using the exact same method as in Example 1.

[0065] Preparation process of DCM-Azo-BOC: Compound 6 was synthesized using the same synthetic method as compound 4 in Example 1, with the fluorophore DCM-NH2. Compound 6 (135 mg, 0.42 mmol) and tert-butyl carbamate (49 mg, 0.42 mmol) were placed in a 100 mL round-bottom flask. A mixed solvent of acetic acid and ethanol (4:1, 12 mL) was then added, and the mixture was stirred overnight at 40 °C. The reaction was confirmed by thin-layer chromatography (TLC) as the starting spot disappeared. The reaction was then terminated, and the pH of the reaction mixture was adjusted to 7. Dichloromethane was extracted with the aqueous phase (3 × 20 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent. Purification was performed by column chromatography using a mixture of petroleum ether and ethyl acetate (2:1, v / v) as eluent to give a yellow solid target organic fluorescent probe DCM-Azo-BOC in 46% yield, corresponding to 60 mg.

[0066] The chemical structure of compound 6 is shown below: .

[0067] Comparative Example 3 The fluorophore DCM-NH2 was synthesized using the exact same method as in Example 1.

[0068] Preparation process of DCM-Azo-POC: Compound 6 was synthesized using the same synthetic method as compound 4 in Example 1, with the fluorophore DCM-NH2. Compound 6 (40 mg, 0.12 mmol) and isopropyl carbamate (13 mg, 0.12 mmol) were placed in a 100 mL round-bottom flask. A mixed solvent of acetic acid and ethanol (4:1, 12 mL) was then added, and the mixture was stirred overnight at 40 °C. The reaction was confirmed by thin-layer chromatography (TLC) as the starting spot disappeared. The reaction was then terminated, and the pH of the reaction mixture was adjusted to approximately 7. Dichloromethane was extracted with the aqueous phase (3 × 20 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent. Purification was performed by column chromatography using a mixture of petroleum ether and ethyl acetate (2:1, v / v) as eluent to give a yellow solid target organic fluorescent probe DCM-Azo-POC in 55% yield, corresponding to 22 mg.

[0069] Comparative Example 4 The fluorophore DCM-NH2 was synthesized using the exact same method as in Example 1.

[0070] Preparation process of DCM-Azo-pBr: Compound 7 (70 mg, 0.4 mmol) was synthesized from 4-bromoaniline using the exact same synthetic method as compound 4 in Example 1, and added to a 100 mL round-bottom flask, followed by the addition of a mixture containing the fluorophore DCM-NH2 (63.8 mg, 0.5 equivalent) and acetic acid. The resulting solution was stirred at 40 °C for approximately 12 hours. The reaction was confirmed by thin-layer chromatography (TLC) as the starting spot disappeared. The reaction was then terminated, and the pH of the reaction mixture was adjusted to approximately 7. Dichloromethane was extracted with the aqueous phase (3 × 20 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent. Purification was performed by column chromatography using a mixture of petroleum ether and ethyl acetate (2:1, v / v) as eluent to give an orange-red solid target organic fluorescent probe DCM-Azo-pBr in 40% yield, corresponding to 42.3 mg.

[0071] The chemical structural formula of compound 7 is shown below: .

[0072] Comparative Example 5 The fluorophore DCM-NH2 was synthesized using the exact same method as in Example 1.

[0073] Preparation process of DCM-Azo-EOC: Compound 6 was synthesized using the same synthetic method as compound 4 in Example 1, with the fluorophore DCM-NH2. Compound 6 (130 mg, 0.40 mmol) and tert-butyl carbamate (35.62 mg, 0.40 mmol) were placed in a 100 mL round-bottom flask. A mixed solvent of acetic acid and ethanol (4:1, 12 mL) was then added, and the mixture was stirred overnight at 40 °C. The reaction was confirmed by thin-layer chromatography (TLC) as the starting spot disappeared. The reaction was then terminated, and the pH of the reaction mixture was adjusted to approximately 7. Dichloromethane was extracted with the aqueous phase (3 × 20 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent. Purification was performed by column chromatography using a mixture of petroleum ether and ethyl acetate (2:1, v / v) as eluent to give a yellow solid target organic fluorescent probe DCM-Azo-EOC in 50% yield, corresponding to 66 mg.

[0074] Comparative Example 6 The fluorophore DCM-NH2 was synthesized using the exact same method as in Example 1.

[0075] Preparation process of DCM-Azo-pH: Compound 8 (47 mg, 0.4 mmol) was synthesized from aniline using the same synthetic method as Compound 4 in Example 1 (Example 5). The compound was added to a 100 mL round-bottom flask, followed by the addition of a mixture containing the fluorophore DCM-NH2 (63.8 mg, 0.5 equivalent) and acetic acid. The resulting solution was stirred at 40 °C for approximately 12 hours. The reaction was confirmed by thin-layer chromatography (TLC) as the starting material spot disappeared. The reaction was then terminated, and the pH of the reaction mixture was adjusted to approximately 7. Dichloromethane was extracted with the aqueous phase (3 × 20 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent. Purification was performed by column chromatography using a mixture of petroleum ether and ethyl acetate (2:1, v / v) as eluent to give an orange-red solid target organic fluorescent probe DCM-Azo-pH in 38% yield, corresponding to 35 mg.

[0076] The chemical structure of compound 8 is shown below: .

[0077] Comparative Example 7 The fluorophore DCM-NH2 was synthesized using the exact same method as in Example 1.

[0078] Preparation process of DCM-Azo-pOCH3: Compound 6 was synthesized from the fluorophore DCM-NH2 using the exact same synthetic method as compound 4 in Example 1. Compound 6 (100 mg, 0.31 mmol) and 4-methoxyaniline (35.62 mg, 0.40 mmol) were placed in a 100 mL round-bottom flask. A mixed solvent of acetic acid and ethanol (4:1, 12 mL) was then added, and the mixture was stirred overnight at 40 °C. The reaction was confirmed by thin-layer chromatography (TLC) as the starting spot disappeared. The reaction was then terminated, and the pH of the reaction mixture was adjusted to approximately 7. Dichloromethane was extracted with the aqueous phase (3 × 20 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent. Purification was performed by column chromatography using a mixture of petroleum ether and ethyl acetate (2:1, v / v) as eluent to give a yellow solid target organic fluorescent probe DCM-Azo-pOCH3 in 52% yield, corresponding to 52 mg.

[0079] Experimental Example 1 To quantitatively evaluate the in vitro response performance of a series of probes, fluorescence titration spectroscopy experiments were performed. The specific methods are as follows:

[0080] The DCM-Azo-pNO2, DCM-Azo-pCN, DCM-Azo-BOC, DCM-Azo-POC, DCM-Azo-pBr, DCM-Azo-EOC, DCM-Azo-pH, and DCM-Azo-pOCH3 solutions prepared in Examples 1 and Comparative Examples 1-7 were mixed with a PBS:DMSO mixed solution to prepare 20 mM solutions of DCM-Azo-pNO2, DCM-Azo-pCN, DCM-Azo-BOC, DCM-Azo-POC, DCM-Azo-pBr, DCM-Azo-EOC, DCM-Azo-pH, and DCM-Azo-pOCH3, respectively. The PBS:DMSO mixed solution was prepared by mixing 20 mM PBS buffer with a certain concentration of DMSO at a volume ratio of 9:1.

[0081] Sodium dithionite (SDT), the reducing agent used in simulating a reduction environment, was weighed at concentrations of 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, 1.0 mM, 1.2 mM, 1.4 mM, 1.6 mM, and 1.8 mM. Each SDT was dissolved in 1 mL of 20 mM PBS buffer. The different concentrations of SDT were then mixed with 1 mL of 20 mM organic fluorescent probe solution, followed by fluorescence detection. The fluorescence intensity of the probe before the addition of SDT was defined as F0, and the corresponding fluorescence intensity after reaction with different concentrations of SDT was defined as F. Excitation was performed at a wavelength of 490 nm, with a spectral range of 520 nm to 800 nm and a slit width of 2 nm.

[0082] The fluorescence emission spectra of each probe at different SDT concentrations are plotted as follows: Figure 5 As shown (the horizontal axis represents the emission wavelength, and the vertical axis represents the fluorescence intensity), the "fluorescence enhancement factor" of each probe is obtained by calculating the F / F0 ratio before and after adding the highest concentration of 1.8 mM SDT to each probe.

[0083] like Figure 5 As shown, the fluorescence enhancement folds of each substituent are as follows: -pNO2 (690x) > -pCN (315x) > -BOC (304x) > -POC (175x) > -pBr (164x) > -EOC (118x) > -pH (65x) > -pOCH3 (45x). It is evident that the fluorescence enhancement fold and response speed of DCM-Azo-pNO2 are significantly superior to other probes in the same series.

[0084] A linear fit was performed between the fluorescence intensity measured for NO2 and the SDT concentration to obtain... Figure 6 The fitted curve was y = 478.03x - 93.83. The fluorescence intensity of the probe before the addition of SDT was defined as F0, and the corresponding fluorescence intensity after reacting with different concentrations of SDT was defined as F. A straight line was fitted with SDT concentration as the x-axis and F / F0 as the y-axis, showing a good linear relationship within the SDT concentration range of 0 mM to 1.8 mM. The detection limit of the probe was calculated, and according to the 3σ / slope rule, the detection limit was 0.12 µM.

[0085] Experiment Example 2 Rapid hypoxia imaging at the cellular level Reagents: The DCM-Azo-pNO2, DCM-Azo-pCN, DCM-Azo-BOC, DCM-Azo-POC, DCM-Azo-pBr, DCM-Azo-EOC, DCM-Azo-pH, and DCM-Azo-pOCH3 solutions prepared in Examples 1 and Comparative Examples 1-7 were mixed with a PBS:DMSO mixed solution to prepare 5µM solutions of DCM-Azo-pNO2, DCM-Azo-pCN, DCM-Azo-BOC, DCM-Azo-POC, DCM-Azo-pBr, DCM-Azo-EOC, DCM-Azo-pH, and DCM-Azo-pOCH3, respectively. The PBS:DMSO mixed solution was prepared by mixing 20µM PBS buffer with a specific concentration of DMSO at a volume ratio of 9:1.

[0086] Target: HeLa cells Hypoxia Model: Coverslip-Restricted Oxygen Diffusion Method Experimental Procedure: HeLa cells were incubated for 2 hours in DMEM medium with 5 μM DCM-Azo-pNO2, 5 μM DCM-Azo-pCN, 5 μM DCM-Azo-BOC, 5 μM DCM-Azo-POC, 5 μM DCM-Azo-pBr, 5 μM DCM-Azo-EOC, 5 μM DCM-Azo-pH, and 5 μM DCM-Azo-pOCH3 solutions, respectively. Each solution was added in 100 μL. Subsequently, each group of cells was seeded onto glass slides and covered with coverslips to establish a hypoxic environment. Under hypoxic conditions, fluorescence imaging was performed at 0, 15, 30, 60, and 120 minutes. Fluorescence signals were detected in the red channel (600 nm–650 nm) using an excitation wavelength of 493 nm.

[0087] There is almost no fluorescence at 0 min; a bright red signal (600nm~650nm) appears at 15 min.

[0088] The signal intensity at 120 min increased by about 15 times compared to 0 min, and DCM-Azo-pNO2 showed a significantly faster increase than other substituent probes (weak signals only appeared at 30 min to 60 min).

[0089] Experimental Example 3 In vivo hypoxia imaging in a mouse ischemia model Animals: Eight groups of C57BL / 6 mice were used. Local ischemia was established by ligating the left hind limb for 20 minutes. Dosage: The DCM-Azo-pNO2, DCM-Azo-pCN, DCM-Azo-BOC, DCM-Azo-POC, DCM-Azo-pBr, DCM-Azo-EOC, DCM-Azo-pH, and DCM-Azo-pOCH3 prepared in Examples 1 and Comparative Examples 1-7 were mixed with PBS:DMSO to prepare 50µM solutions of DCM-Azo-pNO2, DCM-Azo-pCN, DCM-Azo-BOC, DCM-Azo-POC, DCM-Azo-pBr, DCM-Azo-EOC, DCM-Azo-pH, and DCM-Azo-pOCH3, respectively. Specifically, 5 mmol of each of the above-mentioned organic fluorescent probes was weighed on a balance and added to 10 mL of PBS:DMSO to prepare a 500µM stock solution. Dilute 100 μL of the stock solution to 1 mL with a PBS:DMSO mixture to obtain a 50 µM solution. Administer 50 μL of each organic fluorescent probe intramuscularly. The PBS:DMSO mixture is prepared by mixing 20 M PBS buffer with a specific concentration of DMSO at a volume ratio of 9:1.

[0090] Equipment: Small animal imaging system, excitation 493nm, reception 600nm~650nm Experimental Procedure: Mice were randomly divided into eight groups. To simulate a hypoxic environment in vivo, a tourniquet was applied to the left hind limb of each mouse for 20 minutes. Subsequently, different probes were administered intramuscularly: 50 μM DCM-Azo-pNO2 solution, 50 μM DCM-Azo-pCN solution, 50 μM DCM-Azo-BOC solution, 50 μM DCM-Azo-POC solution, 50 μM DCM-Azo-pBr solution, 50 μM DCM-Azo-EOC solution, 50 μM DCM-Azo-pH solution, and 50 μM DCM-Azo-pOCH3 solution. In vivo fluorescence imaging was performed using a small animal imaging system at 5, 30, 60, and 120 minutes post-injection. Mice whose left hind limb received an equal volume of saline injection served as a control group. Fluorescence images were captured in the red channel from 600 nm to 650 nm.

[0091] The results showed that fluorescence was visible 5 minutes after injection of DCM-Azo-pNO2; the signal intensity reached its peak at 30 minutes; and it maintained high brightness at 120 minutes. The effect was significantly better than other substituent probes, verifying its ability to visualize hypoxia in vivo in "seconds".

[0092] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0093] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this invention should be determined by the appended claims.

Claims

1. An organic fluorescent probe for detecting hypoxia, characterized in that, The organic fluorescent probe is the compound DCM-Azo-pNO2, whose structure is shown in formula (I): Formula (I).

2. The method for preparing the organic fluorescent probe as described in claim 1, characterized in that, Includes the following steps: (1) Using tetrahydrofuran as a solvent, 2'-hydroxyacetophenone and ethyl acetate were condensed in the presence of sodium hydride to obtain compound 1; (2) Using acetic acid as a solvent, compound 1 was reacted with sulfuric acid to undergo a catalytic ring-closure reaction to obtain compound 2; (3) Using acetic anhydride as a solvent, compound 2 was reacted with malononitrile by Knoevenagel condensation to obtain compound 3; (4) Under a nitrogen atmosphere, using toluene as a solvent, compound 3 was condensed with p-acetaminobenzaldehyde in the presence of piperidine and acetic acid to obtain the fluorophore DCM-NH2; (5) Using dichloromethane and water as solvents, 4-nitroaniline was oxidized with potassium peroxymonosulfate under a nitrogen atmosphere to obtain compound 4; wherein the mass ratio of 4-nitroaniline to potassium peroxymonosulfate was 0.2:1.

74. (6) Using acetic acid as a solvent, compound 4 was subjected to a diazotization coupling reaction with the fluorophore DCM-NH2 to obtain DCM-Azo-pNO2; wherein the mass ratio of compound 4 to fluorophore DCM-NH2 was 60:63.

8. 。 3. The preparation method according to claim 2, characterized in that, In step (6), the diazotization coupling reaction is carried out at a temperature of 40°C for 12 hours.

4. The application of the organic fluorescent probe as described in claim 1 in the preparation of agents for detecting hypoxia.

5. The application as described in claim 4, characterized in that, The formulation is used to detect hypoxia at the cellular level.

6. The application as described in claim 5, characterized in that, The cells in question are HeLa cells.

7. The application as described in claim 4, characterized in that, The formulation is used to detect hypoxia at the tissue level.

8. The application as described in claim 7, characterized in that, The tissue in question is tumor tissue.

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