Dioxetane chemiluminescent probe suitable for biological orthogonal catalytic reaction as well as synthesis and application of dioxetane chemiluminescent probe

Abstract

The invention discloses a dioxetane chemiluminescent probe suitable for a biological orthogonal catalytic reaction as well as synthesis and application of the dioxetane chemiluminescent probe. A phenolic hydroxyl group active site of the probe is protected by an allyl bio-orthogonal cleavable group, and when a ruthenium metal complex catalyst is not added, the probe is in a silence state, and a chemiluminescence signal is weak; and in the presence of the ruthenium catalyst, the protecting group can be specifically catalytically cracked and removed, so that the probe is quickly activated and converted into a luminous'on 'state, and a strong chemiluminescence signal is generated. According to the invention, efficient combination of biological orthogonal cutting reaction and chemiluminescence signal output is realized, and the prepared biological orthogonal chemiluminescence probe has an excellent on-off ratio and is suitable for high-contrast biological imaging of living cell and living body levels.

Description

Technical Field

[0001] This invention relates to the field of biomedical monitoring and molecular imaging technology, specifically to a dioxane chemiluminescent probe suitable for bioorthogonal catalytic reactions and its synthesis and application. Background Technology

[0002] Currently, chemiluminescence imaging has been widely used in immunoassay and biomarker detection due to its significant advantages, including no need for external light excitation, high signal-to-noise ratio, and high sensitivity. Among numerous chemiluminescence systems, probes based on the 1,2-dioxane backbone are particularly prominent. Their mechanism of generating highly efficient chemiluminescence through ring breaking initiation of electron exchange and subsequent photon release makes them ideal biological probes for high-sensitivity in vitro detection and in vivo imaging. Currently, the activation of such probes mainly relies on the catalytic action of specific biological enzymes (e.g., the luminescence of commercially available adamantyl-1,2-dioxane phosphate probes depends on the catalytic activation of alkaline phosphatase). However, this enzyme-dependent activation mechanism has limitations, such as the susceptibility of enzyme activity to physiological environmental interference, the risk of immunogenicity, and the difficulty in achieving modular probe design.

[0003] In recent years, "click" reactions, also known as bioorthogonal reactions, especially bioorthogonal cleavage reactions driven by exogenous synthetic catalysts (such as transition metal complexes and nanocatalysts), have provided new ideas for overcoming the limitations of the aforementioned chemiluminescence systems. The catalysts for these reactions can be modified through chemical synthesis, such as by coupling with specific small molecule groups, peptides, and antibodies to achieve biological targeting and efficiently catalyze specific chemical reactions. Among these, reactions catalyzed by transition metal complexes (such as ruthenium and palladium complexes) to remove allyl groups and their derivatives (such as allyloxycarbonyl groups and carbamates) have become a highly promising bioorthogonal tool due to their advantages of rapid reaction, mild reaction conditions, and good biocompatibility.

[0004] However, a bioorthogonal catalytic chemiluminescence system that efficiently couples targeted transition metal complexes with chemiluminescent probes capable of generating strong signals is currently lacking. In existing technologies, deallylating reactions are typically used to activate small molecule fluorophores or drug precursors; however, these fluorescence-based signal modes often rely on external excitation sources, easily leading to background interference from the autofluorescence of biological tissues. In contrast, chemiluminescent molecules do not require external excitation and exhibit extremely low background signals, making them more suitable for high-contrast imaging in complex in vivo environments. Therefore, developing a bioorthogonal chemiluminescent probe that can efficiently synergize with transition metal complexes and combines low background with strong signal output is of great significance for advancing in vivo imaging technology. Summary of the Invention

[0005] To overcome the limitations of existing enzyme-activated chemiluminescent probes, this invention provides an allyl-adamantyl-1,2-dioxane (AD-Alloc) chemiluminescent probe that can be activated by an exogenous bioorthogonal catalyst, a ruthenium complex ([Ru]). The phenolic hydroxyl active site of this probe is protected by an allyl bioorthogonally cleavable group. In the absence of a catalyst, the protecting group structure is stable, and the probe is in a "silent" state with extremely low chemiluminescent signal. When encountering a specific catalyst, the protecting group can be specifically and rapidly cleaved through a highly efficient bioorthogonal reaction. After the protecting group leaves, the free phenolic hydroxyl group is exposed, which triggers a highly efficient intramolecular chemiluminescence-initiated electron exchange luminescence (CIEEL) process, leading to the cleavage of the dioxane. The probe then transitions from a "silent" state to an "on" state, generating a strong chemiluminescent signal.

[0006] The technical solution adopted to achieve the above-mentioned objectives of this invention is as follows:

[0007] A dioxane chemiluminescent probe, the structural formula of which is as follows:

[0008]

[0009] The method for synthesizing a dioxane chemiluminescent probe described above includes the following steps:

[0010] S1 and compound 1 undergo a substitution reaction with an allylating agent under alkaline and heated conditions to produce compound 1a, as shown in the following reaction formula:

[0011]

[0012] S2. Compound 1a undergoes hydrolysis under alkaline conditions to produce compound 1b, as shown in the following reaction equation:

[0013]

[0014] Compound S3 and Formula 1b undergo a [2+2] cycloaddition reaction in the presence of a photosensitizer, under light and aerobic conditions, to generate compound AD-Alloc, as shown in the following reaction formula:

[0015]

[0016] Furthermore, in step S1:

[0017] The compound of Formula 1 is a known compound that can be prepared according to the methods reported in the literature.

[0018] The alkaline reagent used to provide an alkaline reaction environment is selected from inorganic bases, preferably alkali metal carbonates or bicarbonates, such as lithium carbonate, sodium carbonate, potassium carbonate, or cesium carbonate.

[0019] The allylating agent is selected from allyl halides, such as allyl chloride, allyl bromide, or allyl iodide.

[0020] The reaction is carried out in an organic solvent, preferably an aprotic polar solvent, such as N,N-dimethylformamide, dimethyl sulfoxide, or acetonitrile.

[0021] The reaction temperature range is from room temperature to the reflux temperature of the solvent used, preferably from 25°C to 80°C.

[0022] The reaction time is from 1 hour to 24 hours, and the reaction progress is monitored by thin-layer chromatography.

[0023] The molar ratio of compound 1, base reagent and allylating reagent can be 1.0:(0.5-8.0):(0.5-8.0).

[0024] In one specific embodiment, the base reagent is potassium carbonate, the allylating agent is allyl bromide, the organic solvent is N,N-dimethylformamide, the reaction temperature is 50°C, the reaction time is 2 hours, and the molar ratio of compound (1), potassium carbonate and allyl bromide is (1.0-1.2):(5.8-6.0):(3.8-4.0).

[0025] Furthermore, in step S2:

[0026] The alkali is selected from alkali metal hydroxides or alkaline earth metal hydroxides, preferably lithium hydroxide, sodium hydroxide or potassium hydroxide.

[0027] The reaction can be carried out in a single solvent or a mixture of solvents, preferably water and at least one water-miscible organic solvent, preferably an alcohol solvent, such as methanol or ethanol.

[0028] The reaction temperature range is 0°C to 50°C, preferably carried out at 25°C.

[0029] The reaction time is from 5 minutes to 3 hours, and the reaction progress is monitored by thin-layer chromatography.

[0030] In one specific embodiment, the base is lithium hydroxide, and the reaction solvent is a mixture of lithium hydroxide aqueous solution, tetrahydrofuran and methanol, and the reaction is carried out at room temperature (25°C) for 10 minutes.

[0031] Furthermore, in step S3:

[0032] The reaction is carried out under illumination, with the light source being visible light, preferably light in the wavelength range of 400-700 nm.

[0033] The photosensitizer is selected from organic dyes capable of generating singlet oxygen, such as methylene blue, Bengal rose red, tetraphenylporphyrin or derivatives thereof.

[0034] The reaction is carried out under aerobic conditions, such as in an oxygen atmosphere.

[0035] The reaction is carried out in an organic solvent selected from halogenated hydrocarbons, alcohols, or ethers, such as dichloromethane, methanol, or tetrahydrofuran.

[0036] The reaction temperature range is from 0°C to the reflux temperature of the solvent used, preferably at 25°C.

[0037] The reaction time is 2 to 24 hours, and the reaction progress is monitored by thin-layer chromatography.

[0038] In one specific embodiment, the photosensitizer was methylene blue, the solvent was dichloromethane, and the reaction was carried out at room temperature (25°C) for 3 hours under yellow LED light irradiation and an oxygen atmosphere.

[0039] A transition metal complex catalyst has the following structural formula:

[0040]

[0041] S4 and compound 2 undergo a substitution reaction with an allylating agent under basic conditions to produce compound 2a, as shown in the following reaction equation:

[0042]

[0043] S5. Compound 2a undergoes a substitution reaction with an amine compound to produce compound 2b, as shown in the following reaction equation:

[0044]

[0045] S6. Compound 2b undergoes hydrolysis under acidic conditions to produce compound 2c, as shown in the following reaction equation:

[0046]

[0047] The acylation reagent of compound S7, formula 2c, undergoes a substitution reaction under basic conditions to generate compound 2d, as shown in the following reaction formula:

[0048]

[0049] S8, the 2d compound, undergoes a coordination reaction with the ruthenium precursor under an inert atmosphere to form the [Ru] compound, as shown in the following reaction formula:

[0050]

[0051] Furthermore, in step S4:

[0052] The alkaline reagent used to provide an alkaline reaction environment is selected from inorganic bases, preferably alkali metal carbonates or bicarbonates. The alkali metal bicarbonate is, for example, sodium bicarbonate or potassium bicarbonate.

[0053] The allylating agent is selected from allyl halides, such as allyl chloride, allyl bromide, or allyl iodide.

[0054] The reaction is carried out in an organic solvent, preferably an aprotic polar solvent, such as N,N-dimethylformamide, dimethyl sulfoxide, or acetonitrile.

[0055] The reaction temperature range is from room temperature to the reflux temperature of the solvent used, preferably from 25°C to 80°C.

[0056] The reaction time is from 0.5 hours to 24 hours, and the reaction progress is monitored by thin-layer chromatography.

[0057] The molar ratio of compound 2, base reagent and allyl bromide can be 1.0:(0.5-8.0):(0.5-8.0).

[0058] In one specific embodiment, the base reagent is sodium bicarbonate, the allylating agent is allyl bromide, the organic solvent is N,N-dimethylformamide, the reaction temperature is 50°C, the reaction time is 2 hours, and the molar ratio of compound 2, base reagent and allyl bromide is (1.0-1.2):(3.0-3.2):(2.8-3.0).

[0059] Furthermore, in step S5:

[0060] The amine compound is tert-butyl 2-(methylamino)propylcarbamate or an analogue thereof.

[0061] The reaction is carried out in an organic solvent, preferably an aprotic polar solvent, such as dimethyl sulfoxide or N,N-dimethylformamide.

[0062] The reaction temperature range is from room temperature (50°C to 150°C), preferably from 80°C to 120°C.

[0063] The reaction time is from 1 hour to 48 hours, and the reaction progress is monitored by thin-layer chromatography.

[0064] The molar ratio of compound 2a to amine compound can be 1.0:(0.5-6.0).

[0065] In one specific embodiment, the amine compound is tert-butyl 2-(methylamino)propylcarbamate, the reaction solvent is dimethyl sulfoxide, the reaction temperature is 100°C, the reaction time is 18 hours, and the molar ratio of compound 2a to tert-butyl 2-(methylamino)propylcarbamate is (1.0-1.2):(3.0-3.2).

[0066] Furthermore, in step S6:

[0067] The acid is selected from organic or inorganic acids, preferably strong organic acids, such as trifluoroacetic acid or p-toluenesulfonic acid.

[0068] The reaction is carried out in an organic solvent selected from halogenated hydrocarbons or ether solvents, such as dichloromethane, chloroform, or tetrahydrofuran.

[0069] The reaction temperature range is 0°C to 40°C, preferably carried out at 25°C.

[0070] The reaction time ranges from 5 minutes to 24 hours, and the reaction progress is monitored by thin-layer chromatography.

[0071] In one specific embodiment, the acid is trifluoroacetic acid, the reaction solvent is dichloromethane, and the reaction is carried out at room temperature (25°C) for 1 hour.

[0072] Furthermore, in step S7:

[0073] The alkaline reagent used to provide an alkaline reaction environment is selected from inorganic bases, preferably alkali metal carbonates or bicarbonates. The alkali metal bicarbonate is, for example, sodium bicarbonate or potassium bicarbonate.

[0074] The acylation reagent is an active carboxylic acid derivative capable of reacting with amines to form amide bonds in an aqueous phase or a water-organic phase mixture. Preferably, the acylation reagent is in the form of an active ester of N-(methoxycarbonyl)maleimide or N-hydroxysuccinimide.

[0075] The reaction is carried out in a mixed solution of water and at least one organic solvent mixed with water. The organic solvent is preferably a nitrile, ether, ketone, or sulfoxide solvent, such as acetonitrile, tetrahydrofuran, acetone, or dimethyl sulfoxide.

[0076] The reaction temperature range is 0°C to 35°C, preferably 0°C to 10°C.

[0077] The reaction time is from 5 minutes to 3 hours, and the reaction progress is monitored by thin-layer chromatography.

[0078] The molar ratio of compound 2c to acylation reagent can be 1.0:(0.5-3.0).

[0079] In one specific embodiment, the base reagent is sodium bicarbonate, the acylation reaction reagent is N-(methoxycarbonyl)maleimide, the reaction solvent is acetonitrile, the reaction temperature is 0°C, the reaction time is 30 minutes, and the molar ratio of compound 2c to N-(methoxycarbonyl)maleimide is (1.0-1.2):(1.0-1.2).

[0080] Furthermore, in step S8:

[0081] The ruthenium precursor is tri(acetonitrile)cyclopentadiene ruthenium hexafluorophosphate or an analogue thereof.

[0082] The reaction is carried out in an inert atmosphere, preferably nitrogen or argon.

[0083] The reaction is carried out in an organic solvent selected from halogenated hydrocarbons, ethers, or nitrile solvents, such as dichloromethane, tetrahydrofuran, or acetonitrile.

[0084] The reaction temperature range is 0°C to 35°C, preferably carried out at 25°C.

[0085] The reaction time is from 10 minutes to 12 hours.

[0086] The molar ratio of the 2d compound to the ruthenium precursor can be 1.0:(0.5-3.0).

[0087] In one specific embodiment, the organic solvent is dichloromethane, and the reaction is carried out at room temperature (25°C) for 30 minutes under a nitrogen atmosphere, with the molar ratio of compound 2d to tri(acetonitrile)cyclopentadiene ruthenium hexafluorophosphate being (0.8-1.0):(1.3-1.5).

[0088] The above describes the application of a dioxane chemiluminescent probe in bioimaging.

[0089] Preferably, the dioxane chemiluminescent probe undergoes a specific bioorthogonal cleavage reaction and emits a chemiluminescent signal in the presence of the transition metal complex catalyst described in claim 6.

[0090] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0091] (1) Excellent probe stability. The AD-Alloc designed in this invention exhibits good chemical stability in physiological buffer solutions. High performance liquid chromatography (HPLC) analysis shows that its decomposition rate is less than 5% after incubation in phosphate buffered saline (PBS) at pH 7.4 at 37°C for 24 hours.

[0092] (2) The chemiluminescence signal is specific. Chemiluminescence experiments show that in the absence of a catalyst, the chemiluminescence background signal of the probe is weak and in a "silent" state; however, after the addition of a catalyst, the probe can undergo a highly efficient and specific bioorthogonal cleavage reaction, quickly switch to the luminescence "on" state, and emit a strong chemiluminescence signal with a peak of 510 nm, which is suitable for live cell and live body imaging.

[0093] (3) Good biocompatibility. Cytotoxicity tests showed that even when the probe prepared in this invention was co-incubated with cells at a high concentration for 24 hours, the effect on cell viability was minimal, indicating that the probe has high biocompatibility and meets the basic requirements for in vivo bioimaging.

[0094] (4) Applicable to in vivo biological imaging. Experiments show that, under the condition of introducing a catalyst, the probe of the present invention can be specifically activated in a living model through a bioorthogonal catalytic reaction, thereby achieving precise chemiluminescence imaging of the target lesion site at the living level, and the luminescence on-off ratio is as high as 4210 times, demonstrating its potential for high-contrast molecular imaging applications in complex biological systems. Attached Figure Description

[0095] Figures 1 to 16 The images show the nuclear magnetic resonance (NMR) spectrum, high-resolution mass spectrometry (HRMS), mass-assisted laser desorption / ionization-time-of-flight (MALDI-TOF-MS), and high-performance liquid chromatography (HPLC) spectra of the bioorthogonal chemiluminescent probe AD-Alloc, the ruthenium metal complex catalyst [Ru], and its key intermediates synthesized in this invention; among them, Figure 6 , Figure 7 , Figure 15 a and Figure 16 In the figure, 'a' represents the proton NMR spectrum of the probe mentioned above. 1 H NMR, carbon spectrum ( 13 chromatograms of C1NMR, HRMS and HPLC; Figure 14 , Figure 15 b in Figure 16 b in the above catalysts are respectively 1 ¹H NMR, MALDI-TOF-MS, and HPLC chromatograms were used together to confirm the exact chemical structure and purity of the target probe, catalyst, and intermediates.

[0096] Figure 17 a in Figure 17 In the figure, b represents the HPLC comparison of the probe (100 μM) prepared in this invention in phosphate buffer at pH 7.4 at the initial time of 0 hours and after 24 hours of incubation, as well as the probe decomposition rate within 24 hours, which is used to illustrate the stability of the probe.

[0097] Figure 17 c in Figure 17 In the figure, d represents the HPLC comparison of the probe (100 μM) prepared in this invention after incubation in phosphate buffer at pH 7.4 for 24 hours with and without the addition of a transition metal ruthenium complex catalyst (10 μM, 0.1 eq.), and the probe decomposition rate over 24 hours, which is used to confirm the occurrence of the catalytic reaction.

[0098] Figure 18 a in Figure 18 In the figure, b represents the chemiluminescence spectra and signal enhancement ratio of the probe (50 μM) prepared in this invention in phosphate buffer at pH 7.4, with and without the addition of a transition metal ruthenium complex catalyst (5 μM, 0.1 eq.), which are used to demonstrate the characteristics of the probe transitioning from a "silent" state to an "on" state and its emission wavelength.

[0099] Figure 19 The graph shows the cell viability assay results after A549 cells were co-incubated with different concentrations of probes prepared in this invention for 24 hours, which was used to evaluate the cytotoxicity of the probes.

[0100] Figure 20 This is a chemiluminescence imaging image of the probe prepared in this invention after co-incubation in A549 cells for 2 hours, used to demonstrate that the probe can achieve specific activation and imaging at the cellular level in the presence of a transition metal complex catalyst.

[0101] Figure 21 and Figure 22 The images shown are in vivo imaging and quantitative images obtained after applying the probe (100 μM) prepared in this invention and the transition metal ruthenium complex catalyst (10 μM, 0.1 eq.), respectively, to demonstrate the feasibility of achieving bioimaging at the in vivo level using the probe. Detailed Implementation

[0102] The probe AD-Alloc synthesized in this invention will be described in detail below.

[0103]

[0104] 1. Synthesis of Compound 1

[0105] Compound 1 is a previously reported compound, prepared according to the method described in the reference, and obtained as a white solid compound in 8 steps with an overall yield of 15%.

[0106] 1H NMR (400 MHz, CDCl3) δ (ppm): 8.03 (d, J = 16.1 Hz, 1H), 7.41 (d, J= 8.0 Hz, 1H), 6.88 (d, J = 8.0 Hz, 1H), 6.63 (d, J = 16.1 Hz, 1H), 3.32 (s,3H), 3.27 (s, 1H), 2.12 (s, 1H), 1.97 – 1.73 (m, 12H).

[0107] MS (ESI): m / z [M+H] + Calculated value C 21 H 23 ClO4, 375.1; Measured value 375.1.

[0108] 2. Synthesis of compound 1a

[0109] Compound 1 (50 mg, 0.13 mmol) was dissolved in N,N-dimethylformamide (5 mL), and allyl bromide (45 μL, 0.52 mmol) and potassium carbonate (108 mg, 0.78 mmol) were added. The reaction was carried out at 50 °C, and the reaction progress was monitored by thin-layer chromatography. After 2 hours, the insoluble salt was filtered off and the filtrate was concentrated and purified by column chromatography (petroleum ether / ethyl acetate = 10:1, v / v) to give a yellow oily liquid (47 mg, 79% yield).

[0110] 1 H NMR (400 MHz, CDCl3) δ (ppm): 7.99 (d, J = 16.2 Hz, 1H), 7.45 (d, J= 8.0 Hz, 1H), 7.06 (d, J = 7.9 Hz, 1H), 6.52 (d, J = 16.2 Hz, 1H), 6.13(ddt, J = 17.1, 10.3, 5.9 Hz, 1H), 5.99 (ddt, J = 17.2, 10.5, 5.6 Hz, 1H), 5.46 – 5.35 (m, 2H), 5.29 (ddq, J = 10.1, 8.8, 1.2 Hz, 2H), 4.72 (dt, J =5.6, 1.4 Hz, 2H), 4.55 – 4.49 (m, 2H), 3.31 (s, 3H), 3.27 (s, 1H), 2.07 (s,1H), 1.96 – 1.63 (m, 12H).

[0111] 13 C NMR (101 MHz, CDCl3) δ (ppm): 166.5.1, 154.25, 139.27, 132.49,132.33, 129.91, 129.55, 127.75, 120.10, 119.04, 118.29, 39.30, 39.15, 38.71,37.17, 33.04, 29.81, 28.47, 28.31.

[0112] MS (ESI): m / z [M+H] + Calculated value C 27 H 31 ClO4, 455.2; measured value, 455.0.

[0113] 3. Synthesis of compound 1b

[0114] Compound 1a (45 mg, 0.10 mmol) was dissolved in 1M lithium hydroxide solution / tetrahydrofuran / methanol (1:1:1, v / v / v, 1 mL). The reaction was carried out at room temperature for 10 minutes, and the reaction was monitored by LC-MS to indicate completion. The reaction solution was concentrated, and the mixture was extracted with 1M hydrochloric acid and ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product. The crude product was then purified by column chromatography (petroleum ether / ethyl acetate = 1:4, v / v) to give a yellow solid (37 mg, 90% yield).

[0115] 1 H NMR (400 MHz, CDCl3) δ (ppm): 8.04 (d, J = 16.1 Hz, 1H), 7.46 (d, J= 8.0 Hz, 1H), 7.05 (d, J = 8.0 Hz, 1H), 6.54 (d, J = 16.1 Hz, 1H), 6.17 –6.06 (m, 1H), 5.38 (s, 1H), 5.27 (d, J = 11.6 Hz, 1H), 4.50 (s, 2H), 3.30 (s,3H), 3.27 (s, 1H), 2.06 (s, 1H), 1.97 – 1.66 (m, 12H).

[0116] 13C NMR (101 MHz, CDCl3) δ (ppm): 172.56, 154.35, 140.76, 139.51,138.53, 132.83, 132.62, 129.93, 129.39, 127.80, 125.46, 120.24, 119.20,75.36, 57.36, 39.31, 39.15, 38.72, 37.17, 33.06, 28.47.

[0117] MS (ESI): m / z [M+H] + Calculated value C 24 H 27 ClO4, 414.2; measured value, 414.9.

[0118] 4. Synthesis of compound AD-Alloc

[0119] Compound 1b (30 mg, 0.07 mmol) was dissolved in dichloromethane (5 mL), and a catalyst amount of methylene blue was added. The reaction mixture was placed in an oxygen atmosphere and irradiated with a yellow LED lamp at room temperature for 3 hours. The reaction was monitored by LC-MS. After the reaction was completed, the reaction solution was concentrated and purified by column chromatography under light-protected conditions (dichloromethane / methanol = 50:1, v / v) to give a white solid (28 mg, yield 87%).

[0120] 1 H NMR (400 MHz, CDCl3) δ (ppm): 8.06 (d, J = 16.2 Hz, 1H), 7.92 (d, J= 8.4 Hz, 1H), 7.62 (d, J = 8.4 Hz, 1H), 6.58 (d, J = 16.2 Hz, 1H), 3.02 (s, 1H), 2.32 (d, J =12.0 Hz, 1H), 2.03 – 1.32 (m, 12H).

[0121] 13C NMR (101 MHz, CDCl3) δ (ppm): 171.41, 154.88, 140.71, 135.76,132.51, 131.14, 129.00, 127.98, 125.66, 120.76, 119.64, 111.87, 96.53, 75.53,49.86, 36.72, 34.02, 33.73, 32.77, 32.36, 31.71, 31.65, 26.30, 25.96.

[0122] HRMS (ESI): m / z [MH] - Calculated value C 24 H 27 ClO6, 445.1418; Measured value, 445.1454.

[0123] The catalyst [Ru] synthesized in this invention will be described in detail below.

[0124]

[0125] 5. Synthesis of compound 2a

[0126] Compound 2 was synthesized according to the reference. Compound 2 (110 mg, 0.44 mmol) was dissolved in N,N-dimethylformamide (3 mL), and allyl bromide (146 μL, 1.3 mmol) and sodium bicarbonate (138 mg, 1.3 mmol) were added. The reaction was carried out at 50 °C, and the reaction progress was monitored by thin-layer chromatography. After 2 hours, the insoluble salt was filtered off and the filtrate was concentrated. Purification was performed by column chromatography (petroleum ether / ethyl acetate = 4:1, v / v) to give a white solid (108 mg, 85% yield).

[0127] MS (ESI): m / z [M+H] + Calculated value C 13 H 10 BrNO2, 292.0; measured value, 292.0.

[0128] 6. Synthesis of compound 2b

[0129] Compound 2a (72 mg, 0.25 mmol) was dissolved in dimethyl sulfoxide (2 mL), and tert-butyl 2-(methylamino)propylcarbamate (148 μL, 0.75 mmol) was added. The reaction was carried out at 100 °C for 18 hours, and the reaction was monitored by thin-layer chromatography. After concentrating the reaction solution, the solid residue was diluted with saturated ammonium chloride aqueous solution (5 mL), and then extracted with dichloromethane (10 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. Purification was carried out by column chromatography (petroleum ether / ethyl acetate = 7:3, v / v) to give a yellow oily liquid (79 mg, yield 80%).

[0130] 1 H NMR (400 MHz, CDCl3) δ (ppm): 8.19 (d, J = 8.4 Hz, 1H), 7.99 (d, J= 8.5 Hz, 1H), 7.65 (t, J = 7.0 Hz, 1H), 7.57 (s, 1H), 7.49 (t, J = 7.7 Hz,1H), 6.14 – 6.01 (m, 1H), 5.42 (d, J = 17.1 Hz, 1H), 5.28 (d, J = 10.4 Hz,1H), 4.93 (d, J = 5.9 Hz, 2H), 4.74 (s, 1H), 3.34 (t, J = 7.3 Hz, 2H), 3.15(d, J = 6.8 Hz, 2H), 3.00 (s, 3H), 1.89 (p, J = 7.0 Hz, 2H), 1.36 (s, 9H).

[0131] 13 C NMR (101 MHz, CDCl3) δ (ppm): 165.80, 158.38, 155.99, 149.26,148.12, 132.01, 131.34, 129.62, 126.78, 124.31, 124.01, 119.18, 108.53,79.27, 66.78, 53.31, 41.46, 38.50, 28.40, 27.89.

[0132] MS (ESI): m / z [M+H] + Calculated value C 22 H 29 N3O4, 399.2; measured value, 400.2.

[0133] 7. Synthesis of compound 2c

[0134] Compound 2b (66 mg, 0.17 mmol) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (3 mL) and water (1 mL) were added. The reaction was carried out at room temperature. The reaction progress was monitored by thin-layer chromatography, and the reaction was completed after 1 hour. The reaction solution was concentrated under reduced pressure, and the crude product was purified by column chromatography (dichloromethane / methanol = 5:1, v / v) to give a yellow oil (47 mg, 95% yield).

[0135] 1 H NMR (400 MHz, CD3OD) δ (ppm): 8.35 (d, J = 8.7 Hz, 1H), 8.18 (d, J= 8.6 Hz, 1H), 7.97 (t, J = 7.7 Hz, 1H), 7.76 – 7.69 (m, 1H), 7.56 (s, 1H), 6.14 (ddt, J = 16.7, 10.3, 6.0 Hz, 1H), 5.51 (d, J = 17.3 Hz, 1H), 5.39 (d, J= 10.4 Hz, 1H), 5.03 (s, 2H), 3.99 (t, J = 7.7 Hz, 2H), 3.61 (s, 3H), 3.12(t, J = 7.6 Hz, 2H), 2.30 (p, J = 7.6 Hz, 2H).

[0136] 13 C NMR (101 MHz, CD3OD) δ (ppm): 162.36, 161.47, 142.17, 140.41,135.35, 132.39, 127.95, 127.83, 122.47, 120.59, 120.31, 105.46, 69.22, 53.87,53.83, 43.43, 38.02, 26.02.

[0137] MS (ESI): m / z [M+H] + Calculated value C 17 H 21 N3O2, 299.2; measured value, 300.2.

[0138] 8. Synthesis of compound 2d

[0139] Compound 2c (99 mg, 0.33 mmol) was dissolved in 1M sodium bicarbonate solution / acetonitrile (1:1, v / v, 4 mL), and N-(methoxycarbonyl)maleimide (56 mg, 0.36 mmol) was added. The reaction was carried out in an ice-water bath, and the reaction progress was monitored by LC-MS. The reaction was completed after 30 minutes. The reaction solution was concentrated under reduced pressure to remove most of the acetonitrile. The pH of the solution was adjusted to 2-3 with 1M hydrochloric acid solution, and then water was removed by freeze-drying. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 1:1, v / v) to give a white solid (36 mg, yield 29%).

[0140] 1 H NMR (400 MHz, CDCl3) δ (ppm): 8.23 ​​(d, J = 8.5 Hz, 1H), 8.01 (d, J= 9.9 Hz, 1H), 7.69 (ddd, J = 8.4, 6.7, 1.4 Hz, 1H), 7.61 (s, 1H), 7.54 (ddd,J = 8.2, 6.7, 1.3 Hz, 1H), 6.67 (s, 2H), 6.12 (ddt, J = 16.3, 10.4, 5.9 Hz,1H), 5.46 (dq, J = 17.2, 1.5 Hz, 1H), 5.33 (dq, J = 10.4, 1.3 Hz, 1H), 4.97(dt, J = 5.9, 1.3 Hz, 2H), 3.59 (t, J = 7.1 Hz, 2H), 3.37 – 3.32 (m, 2H), 3.04 (s, 3H), 2.09 – 2.01 (m, 2H).

[0141] 13 C NMR (101 MHz, CDCl3) δ (ppm): 170.68, 165.76, 158.29, 149.25,148.14, 134.15, 132.02, 131.37, 129.69, 126.85, 124.36, 123.98, 119.20,108.67, 66.83, 52.87, 41.44, 35.57, 26.37.

[0142] MS (ESI): m / z [M+H] + Calculated value C 21 H 21 N3O4, 379.2; measured value, 380.0.

[0143] 9. Synthesis of compound [Ru]

[0144] Compound 2d (1 mg, 0.003 mmol) was dissolved in anhydrous dichloromethane (1 mL), and tris(acetonitrile)cyclopentadiene ruthenium hexafluorophosphate (1.7 mg, 0.004 mmol) was added. The mixture was reacted under a nitrogen atmosphere for 30 minutes. After the reaction was complete, the crude product was washed with diethyl ether (1 mL × 3) to give a brownish-yellow solid (1.7 mg, yield 83%).

[0145] 1 H NMR (400 MHz, DMSO-d6) δ (ppm): 8.09 (d, J = 8.6 Hz, 1H), 7.92 –7.88 (m, 1H), 7.72 (d, J = 8.9 Hz, 1H), 7.68 (d, J = 7.9 Hz, 1H), 7.65 – 7.59(m, 1H), 7.00 – 6.97 (m, 2H), 6.37 (d, J = 9.7 Hz, 5H), 6.04 – 5.86 (m, 1H), 5.19 (s, 2H), 4.96 (s, 2H), 3.45 (s, 2H), 3.43 (s, 2H), 3.23 (s, 3H), 2.06(s, 2H).

[0146] MALDI-TOF-MS: m / z [M+H] + Calculated value C 26 H 26 N3O4Ru + :, 546.097; Measured value, 546.056.

[0147] Example 1: Probe stability test

[0148] 1. Testing Methods

[0149] The AD-Alloc probe prepared according to this invention was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution. 10 μL of the stock solution and 40 μL of DMSO were added to 950 μL of pH 7.4 phosphate buffer and mixed thoroughly to obtain a 100 μM probe test solution (5% DMSO). The test solution was incubated at room temperature under ambient light conditions. Samples were taken at 0, 1, 2, 3, 4, 5, and 24 hours and analyzed by liquid chromatography-mass spectrometry (LC-MS).

[0150] 2. Test Results

[0151] Test results are as follows Figure 17 As shown in a and b in the figure. The results show that after incubation at room temperature and under normal ambient light for 24 hours, the decomposition rate of the probe AD-Alloc is less than 5%. This result indicates that the probe of the present invention has high stability in PBS, and under the protection of the allyl group, its chemiluminescent active center, namely the dioxane structure, does not undergo significant non-specific decomposition. This characteristic ensures that the chemiluminescent probe protected by the bioorthogonal group can remain in a "silent" state in the absence of a matching catalyst, which is an important basis for achieving low background and high on-ratio chemiluminescent imaging.

[0152] Example 2: Verification Test of Probe-Based Bioorthogonal Catalytic Reaction

[0153] 1. Testing Methods

[0154] The [Ru] catalyst prepared in this invention was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 1 mM. 10 μL of the AD-Alloc probe stock solution (10 mM), 10 μL of the [Ru] catalyst stock solution (1 mM), and 30 μL of DMSO were added to 950 μL of pH 7.4 PBS and mixed thoroughly to obtain a test solution (5% DMSO) with a final substrate concentration of 100 μM and a final catalyst concentration of 10 μM. This test solution was incubated in a 37°C incubator under controlled light conditions. Samples were taken at 0, 1, 2, 3, 4, 6, and 24 hours and analyzed by liquid chromatography-mass spectrometry (LC-MS).

[0155] 2. Test Results

[0156] Test results are as follows Figure 17 Figures c and d are shown in the diagram. LC-MS results show that, with increasing time, the probe gradually decomposes under the action of the catalyst (t = 7.0 min), generating the product shown in the figure (t = 4.5 min). After 24 hours, the substrate decomposition rate is approximately 80%.

[0157] Example 3: Test of the catalytic activation chemiluminescence performance of the probe

[0158] 1. Testing Methods

[0159] Set up the following two parallel test systems:

[0160] Experimental group: Take 5 μL of probe stock solution (10 mM) and 5 μL of catalyst stock solution (1 mM), add them to 990 μL of pH7.4 PBS, mix well, and obtain a test solution (1% DMSO) with a final substrate concentration of 50 μM and a final catalyst concentration of 5 μM.

[0161] Control group: Take 5 μL of probe stock solution (10 mM) and 5 μL of DMSO, add them to 990 μL of PBS at pH 7.4, mix well, and obtain a test solution (1% DMSO) with a final substrate concentration of 50 μM.

[0162] Immediately transfer the mixed test solution to a cuvette in a fluorescence spectrophotometer and start a spectral scan at room temperature. Set the instrument parameters to Luminescence scanning mode with an emission wavelength range of 400 nm to 650 nm. Starting from the moment the mixing is completed (0 hours), continuously collect data for 10 minutes at 1-minute intervals.

[0163] 2. Test Results

[0164] Test results are as follows Figure 18 As shown in a and b in the figure, the chemiluminescence spectra show that the control group only detected an extremely weak background luminescence signal, while the experimental group rapidly generated a strong chemiluminescence signal after the substrate and catalyst were mixed, with an emission peak at 510 nm, and the signal intensity was 50 times stronger than that of the control group. This test confirms that the AD-Alloc probe of the present invention can be efficiently activated in the presence of a specific catalyst to generate a strong chemiluminescence signal, thereby achieving a transition from "silent" to "on".

[0165] Example 4: In vitro cytotoxicity test of the probe

[0166] 1. Testing Methods

[0167] A549 cells were seeded in 96-well plates at a density of 1 × 10⁶ cells per well. 4 Cells were cultured for 24 hours to allow them to adhere. After 24 hours, the original culture medium was discarded and replaced with fresh complete culture medium (DMEM) containing different concentrations of AD-Alloc probe or [Ru] catalyst (0, 6.25, 12.5, 25, 50, 100 μM). Cells were cultured for another 24 hours, with five replicates for each concentration. After culturing, 100 μL of 10% CCK8 solution was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance at 450 nm was then measured using a microplate reader to calculate cell viability.

[0168] 2. Test Results

[0169] The results are as follows Figure 19As shown, at concentrations up to 100 μM, A549 cells treated with the AD-Alloc probe for 24 hours maintained a survival rate above 80%, showing no significant difference compared to the control group, indicating that the probe has no significant cytotoxicity within the tested concentration range and possesses good biocompatibility. For the [Ru] catalyst, although a statistically significant decrease was observed compared to the control group when its concentration exceeded 50 μM, its cell viability remained at a high level (>95%), indicating good biocompatibility.

[0170] Example 5: Specific activation imaging assay of probes at the cellular level

[0171] 1. Testing Methods

[0172] A549 cells were seeded in 96-well plates at a density of 1 × 10⁶ cells per well. 4 Cells were cultured for 24 hours to allow them to adhere. After 24 hours, the original culture medium was discarded, and the cells were divided into two groups for further treatment:

[0173] Experimental group: Fresh culture medium containing [Ru] catalyst (10 μM, 0.1 eq.) was added.

[0174] Control group: Add an equal amount of culture medium without catalyst.

[0175] Both groups were incubated at 37℃ and 5% CO2 for 2 hours.

[0176] After incubation, the culture medium was aspirated, and the cells were washed three times with PBS solution. Immediately afterwards, PBS solution containing AD-Alloc probe (100 μM) was added to both groups of cells, and the 96-well plates were quickly placed in an in vivo imaging system (IVIS) to acquire chemiluminescence signals in real time.

[0177] 2. Test Results

[0178] Test results are as follows Figure 20 As shown, no obvious chemiluminescence signal was detected in the control group; however, the experimental group immediately exhibited a strong chemiluminescence signal after the probe was added. This result indicates that the pre-entered bioorthogonal catalyst can effectively activate the subsequently added AD-Alloc probe, specifically generating a chemiluminescence signal, thus verifying the feasibility of the probe of this invention for specific chemiluminescence imaging at the cellular level.

[0179] Example 6: In vivo chemiluminescence imaging test of the probe

[0180] 1. Testing Methods

[0181] Four-week-old male BALB / c nude mice were selected, and A549 (2×10⁻⁶) was subcutaneously injected into the lower right back. 6 A subcutaneous xenograft mouse model was established (one xenograft per mouse). Tumor growth was monitored regularly, and when the tumor volume reached approximately 150 mm... 3 At that time, tumor-bearing mice that met the experimental conditions were randomly divided into the following two groups (n = 3):

[0182] Experimental group: The AD-Alloc probe and [Ru] catalyst were premixed in PBS buffer at pH 7.4 to prepare a mixed reaction solution (2% DMSO) with a final probe concentration of 100 μM and a final catalyst concentration of 10 μM. Subsequently, the mixture was immediately injected into the tumor site of mice via intratumoral injection.

[0183] Control group: An equal volume of PBS solution containing the same concentration of AD-Alloc probe (100 μM) but without the catalyst was injected into the tumor at the same location.

[0184] 2. Test Results

[0185] Live imaging situation as follows Figure 21 and Figure 22 As shown. During the 60-minute observation period, no significant chemiluminescent signal was detected at the tumor site in the control group mice. Figure 21 , Figure 22 (a) in the middle; while in the experimental group, a strong luminescent signal was immediately generated at the tumor site after injection (a). Figure 22 (b) has a signal enhancement ratio of up to 4210 times ( Figure 22 (d in the original text). Subsequently, although the signal gradually weakened, it remained significantly higher than the background level ( ). Figure 22 (c) The above results show that the AD-Alloc probe exhibits extremely low background signal and good stability in vivo; under the action of a specific catalyst [Ru], it can transition from a "silent" state to a luminescent "on" state, generating a high-contrast chemiluminescence signal that lasts for up to 60 minutes. This signifies that the bioorthogonal catalytic chemiluminescence probe constructed in this invention has successfully achieved a functional leap from in vitro to in vivo levels, demonstrating its broad prospects for application in the field of molecular imaging.

Claims

1. A dioxane chemiluminescent probe, characterized in that, The structure is as follows: 。 2. The method for synthesizing a dioxane chemiluminescent probe according to claim 1, characterized in that, Includes the following steps: S1 and compound 1 undergo a substitution reaction with an allylating agent under alkaline and heated conditions to produce compound 1a, as shown in the following reaction formula: S2. Compound 1a undergoes hydrolysis under alkaline conditions to produce compound 1b, as shown in the following reaction equation: Compound S3 and Formula 1b undergo a [2+2] cycloaddition reaction in the presence of a photosensitizer, under light and aerobic conditions, to generate compound AD-Alloc, as shown in the following reaction formula: 。 3. The method for preparing a dioxane chemiluminescent probe according to claim 2, characterized in that, In step S1: The alkaline reagent used to provide an alkaline reaction environment is selected from inorganic bases, preferably lithium carbonate, sodium carbonate, potassium carbonate or cesium carbonate; The allylating agent is selected from allyl halides, preferably allyl chloride, allyl bromide or allyl iodide; The reaction is carried out in an organic solvent, which is selected from aprotic polar solvents, preferably N,N-dimethylformamide, dimethyl sulfoxide or acetonitrile; The reaction temperature range is from room temperature to the reflux temperature of the solvent used, preferably 25°C to 80°C; The reaction time is from 1 hour to 24 hours; The molar ratio of the compound of Formula 1, the base reagent, and the allylating reagent is 1.0:(0.5-8.0):(0.5-8.0).

4. The method for preparing a dioxane chemiluminescent probe according to claim 2, characterized in that, In step S2: The alkali is selected from alkali metal hydroxides or alkaline earth metal hydroxides, preferably lithium hydroxide, sodium hydroxide or potassium hydroxide; The solvent for the reaction is water and at least one water-miscible organic solvent, preferably methanol or ethanol; The reaction is carried out at a temperature of 0°C to 50°C, preferably at 25°C. The reaction time ranges from 5 minutes to 3 hours.

5. The method for preparing a dioxane chemiluminescent probe according to claim 2, characterized in that, In step S3: The reaction is carried out under illumination, with the light source being visible light, preferably light in the wavelength range of 400-700 nm; The photosensitizer is selected from organic dyes capable of generating singlet oxygen, preferably methylene blue, Bengal rose red, tetraphenylporphyrin or its derivatives; The reaction is carried out in an organic solvent, which is selected from halogenated hydrocarbons, alcohols or ethers, preferably dichloromethane, methanol or tetrahydrofuran; The reaction temperature range is from 0°C to the reflux temperature of the solvent used, preferably at 25°C; The reaction time is 2 to 24 hours.

6. A transition metal complex catalyst, characterized in that, The structure is as follows: 。 7. The method for synthesizing a transition metal complex catalyst according to claim 6, characterized in that, Includes the following steps: S4 and compound 2 undergo a substitution reaction with an allylating agent under basic conditions to produce compound 2a, as shown in the following reaction equation: S5. Compound 2a undergoes a substitution reaction with an amine compound to produce compound 2b, as shown in the following reaction equation: S6. Compound 2b undergoes hydrolysis under acidic conditions to produce compound 2c, as shown in the following reaction equation: S7, compound 2c, undergoes a substitution reaction with an acylation reagent under basic conditions to produce compound 2d, as shown in the following reaction equation: S8, the 2d compound, undergoes a coordination reaction with the ruthenium precursor under an inert atmosphere to form the compound [Ru], as shown in the following reaction formula: 。 8. The method for preparing a transition metal complex catalyst according to claim 7, characterized in that, In step S4: The alkaline reagent used to provide an alkaline reaction environment is selected from inorganic bases, preferably sodium bicarbonate or potassium bicarbonate; The allylating agent is selected from allyl halides, preferably allyl chloride, allyl bromide or allyl iodide; The reaction is carried out in an organic solvent, which is an aprotic polar solvent, preferably N,N-dimethylformamide, dimethyl sulfoxide or acetonitrile; The reaction temperature range is from room temperature to the reflux temperature of the solvent used, preferably 25°C to 80°C; The reaction time is from 0.5 hours to 24 hours; The molar ratio of the compound of formula 2, the base reagent, and the allylating reagent is 1.0 : (0.5-8.0 : (0.5-8.0). In step S5: The amine compound is tert-butyl 2-(methylamino)propylcarbamate or an analogue thereof; The reaction is carried out in an organic solvent, which is an aprotic polar solvent, preferably dimethyl sulfoxide or N,N-dimethylformamide; The reaction temperature range is from room temperature (50°C) to 150°C, preferably from 80°C to 120°C. The reaction time is from 1 hour to 48 hours; The molar ratio of the compound of formula 2a to the amine compound is 1.0:(0.5-6.0). In step S6: The acid used in the acidic conditions is selected from organic or inorganic acids, preferably strong organic acids such as trifluoroacetic acid or p-toluenesulfonic acid; The hydrolysis reaction is carried out in an organic solvent, which is selected from halogenated hydrocarbons or ether solvents, preferably dichloromethane, chloroform or tetrahydrofuran; The hydrolysis reaction is carried out at a temperature range of 0°C to 40°C, preferably at 25°C. The hydrolysis reaction takes from 5 minutes to 24 hours; In step S7: The alkaline reagent used to provide an alkaline reaction environment is selected from inorganic bases, preferably sodium bicarbonate or potassium bicarbonate; The acylation reagent is an active carboxylic acid derivative that can react with amines to form amide bonds in an aqueous phase or a water-organic phase mixture, preferably in the form of an active ester of N-(methoxycarbonyl)maleimide or N-hydroxysuccinimide; The reaction is carried out in a mixed solution of water and at least one organic solvent mixed with water, wherein the organic solvent is a nitrile, ether, ketone or sulfoxide solvent, preferably acetonitrile, tetrahydrofuran, acetone or dimethyl sulfoxide; The temperature range of the reaction is 0°C to 35°C, preferably 0°C to 10°C; The reaction time is from 5 minutes to 3 hours; The molar ratio of the compound of formula 2c to the acylation reagent is 1.0:(0.5-3.0). In step S8: The ruthenium precursor is tri(acetonitrile)cyclopentadiene ruthenium hexafluorophosphate or an analogue thereof; The reaction is carried out in an inert atmosphere, preferably nitrogen or argon. The reaction is carried out in an organic solvent, which is selected from halogenated hydrocarbons, ethers or nitrile solvents, preferably dichloromethane, tetrahydrofuran or acetonitrile; The reaction is carried out in a temperature range of 0°C to 35°C, preferably at 25°C. The reaction time is from 10 minutes to 12 hours; The molar ratio of the 2d compound to the ruthenium precursor is 1.0:(0.5-3.0).

9. The application of the dioxane chemiluminescent probe according to claim 1 in bioimaging.

10. The application of a dioxane chemiluminescent probe according to claim 9 in bioimaging, characterized in that, The dioxane chemiluminescent probe undergoes a specific bioorthogonal cleavage reaction and emits a chemiluminescent signal in the presence of the transition metal complex catalyst described in claim 6.

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