A class of dihydroxanthones chemiluminescent groups, their preparation methods and applications

Abstract

This invention relates to a class of dihydroxanthine chemiluminescent groups, their preparation methods, and applications. The dihydroxanthine chemiluminescent group developed in this invention, compared to the traditional phenolic dioxane chemiluminescent group which uses two rigid six-membered rings to fix the double bond conformation, exhibits a higher luminescence quantum yield and a longer emission wavelength. More importantly, it possesses a reactive aldehyde group, which, while maintaining high rigidity, extends the π-electron conjugated system, causing its emission wavelength to redshift to the near-infrared region. This allows it to be extended to chemiluminescence detection and in vivo imaging in small animal models.

Description

Technical Field

[0001] This invention relates to the field of biological sample detection technology, specifically to a class of dihydroxanthin chemiluminescent groups, their preparation methods, and applications. Background Technology

[0002] In recent years, a series of phenol-based dioxane luminescent groups have been widely used in the design of various chemiluminescent groups to extend their luminescence intensity and wavelength in aqueous conditions. These phenol groups are linked to electron-withdrawing chromophores (EWGs) via double bonds. However, these phenol-based dioxane luminescent groups currently suffer from low quantum yields and short wavelengths.

[0003] Therefore, this invention selects dihydroxanthene as the chemiluminescent parent nucleus to design a class of chemiluminescent groups with high luminescence quantum yield and high emission wavelength to solve the problem. Summary of the Invention

[0004] The purpose of this invention is to provide a class of dihydroxanthine chemiluminescent groups, their preparation methods, and applications, thereby addressing the problems mentioned in the background art. This invention conducts an in-depth study of the structural details and excitation mechanism of the phenol-based dioxane chemiluminescent group. Two configurations (cis / trans) exist between the double bond between the phenolic core (donor) and the electron-withdrawing chromophore (acceptor). This structural design is similar to the donor-acceptor design strategy of the cyanine fluorophore (Cy3), as follows:

[0005]

[0006] Among the optical properties of cyanine fluorophores, the cis / trans photoisomerization phenomenon is highly characteristic. In the absence of excitation light, the ground-state cyanine fluorophore exists in the trans form. Upon light absorption, the singlet excited state relaxes to the ground state, releasing energy through fluorescence, conformational changes around the C / C bond, or internal conversion. Bond rotation produces the cis isomer. For Cy3, photoisomerization results in a 2:1 cis / trans ratio. Due to the lower fluorescence efficiency of the cis isomer, a "dark state" is formed, and the fluorescence intensity is weakened. Cy3B has a structure similar to Cy3; its rigid structure prevents photoisomerization, increasing the fluorescence quantum yield from 0.09 to 0.85.

[0007] In the photoisomerization of cyanine fluorophores, when the fluorophore attaches to DNA or a protein (e.g., BSA), the interaction between the biomolecule and the probe imposes spatial confinement, providing a rigid environment that reduces the efficiency of photoisomerization and thus increases the fluorescence quantum yield of the cyanine fluorophore. Recent studies have shown an increase in the luminescence signal of chemiluminescent fluorophores designed based on donor-acceptor structures within BSA. Researchers believe that the hydrophobic microenvironment and conformational confinement provided by BSA increase the luminescence quantum yield of the chemiluminescent fluorophore, a phenomenon similar to that observed in cyanine fluorophores. Therefore, this invention proposes a chemiluminescent chemiluminescence mechanism based on a donor-acceptor design strategy, similar to the rigid structure of Cy3B, utilizing ring-fusing to increase structural rigidity, reduce chemiluminescent chemiluminescence, and thereby improve the luminescence quantum yield. This invention selects dihydroxanthene as the chemiluminescent core, which is a modified rhodamine skeleton with a rigid molecular structure. Its 4-position has a reactive aldehyde group that can flexibly extend the π-conjugated system, and its 6-position has a phenolic group that can act as a signal switch, as shown in the figure below:

[0008]

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] This invention provides a class of dihydroxanthones chemiluminescent groups, the general structural formula of which is shown below:

[0011]

[0012] Specifically, R1 is a bioactive small molecule and enzyme reactive group, Ln is a linker group, R2 is an electron-withdrawing group, R3 is an electron-withdrawing chromophore, R4 is oxacyclohexane, azacyclohexane, cyclohexane or cyclopentane, and R5 is an adamantane modifying group.

[0013] Where the structural formula of R1 is any of the following:

[0014] The structural formula of Ln is any of the following:

[0015] The structural formula of R2 is any of the following: The structural formula of R3 is any of the following:

[0016] The structural formula of R4 is any of the following:

[0017] The structural formula for R5 is: n = 1, 2, 3, 4, 5.

[0018] The present invention also provides a class of dihydroxanthones chemiluminescent groups, characterized in that the structural formula of the dihydroxanthones chemiluminescent group is as follows:

[0019]

[0020] The present invention also provides a class of dihydroxanthones chemiluminescent groups, characterized in that the structural formula of the dihydroxanthones chemiluminescent group is as follows:

[0021]

[0022] The present invention also provides a class of dihydroxanthones chemiluminescent groups, characterized in that the structural formula of the dihydroxanthones chemiluminescent group is as follows:

[0023]

[0024] R1 is selected independently from TIPS and H, and R2 is selected independently from CN, CO2Et and CONHPEG3.

[0025] The present invention also provides a class of dihydroxanthones chemiluminescent groups, characterized in that the structural formula of the dihydroxanthones chemiluminescent group is as follows:

[0026]

[0027] Where R1 = TIPS, and R2 is derived independently from CN, CO2Et, and Selected from the options.

[0028] This invention provides a chemical synthesis method for a class of dihydroxanthones chemiluminescent groups, comprising the following steps:

[0029] Step 1) Using known compound 2.1 as the starting material, the methoxy meta-C-H bond borate is catalyzed by the transition metal Ir. Under alkaline conditions, the borate is oxidized by hydrogen peroxide to generate phenolic hydroxy compound 2.2.

[0030] Step 2) Compound 2.2 undergoes a formylation reaction with paraformaldehyde under anhydrous magnesium chloride catalysis to generate a pair of isomers 2.3a and 2.3b;

[0031] Step 3) The mixture of 2.3a and 2.3b is reacted with the bromounsaturated aldehyde 2.3c in a tandem reaction of Oxa-Michael, Retro-Michael and Aldol to generate compound 2.4;

[0032] Step 4) Compound 2.4 is reacted with sodium ethanethiol under alkaline conditions to remove the methyl protecting group, producing compound 2.5;

[0033] Step 5) React compound 2.5 with TIPSCl to protect the phenolic hydroxyl group and generate compound 2.6;

[0034] In step 6), the obtained compound 2.6 is sensitized with the photosensitizer methylene blue (MB) under light to generate singlet oxygen. The singlet oxygen undergoes a [2+2] cycloaddition to the electron-rich olefin of 2.6 to generate dioxane, thus obtaining the chemiluminescent group.

[0035] The present invention relates to the use of any of the dihydroxanthine chemiluminescent groups, which enables chemiluminescence detection and in vivo imaging in biological samples.

[0036] Compared with the prior art, the dihydroxanthones chemiluminescent groups, their preparation methods, and applications provided by this invention have at least the following beneficial effects:

[0037] 1. Compared with traditional phenolic dioxane chemiluminescent groups, the rigid dihydroxanthin dioxane chemiluminescent group designed in this invention fixes the double bond conformation through two rigid six-membered rings, resulting in higher luminescence quantum yield and longer emission wavelength. More importantly, it has a reactive aldehyde group, which, while maintaining high rigidity, extends the π-electron conjugation system, causing its emission wavelength to redshift to the near-infrared region, thus enabling its application in small animal models for chemiluminescence detection and in vivo imaging.

[0038] 2. This invention investigates the influence of the chemical structure of the rigid dihydroxanthine chemiluminescent group on its luminescence performance, and screens out chemiluminescent groups 4a, 5c, and 6c with excellent chemiluminescence performance. It solves the problems of low quantum yield and short emission wavelength of the phenol-dioxane chemiluminescent group, laying a theoretical and experimental foundation for the subsequent development of chemiluminescent probes for in vivo imaging. Attached Figure Description

[0039] Figure 1 : Chemical structural formula and in vitro characterization diagram of the lead chemiluminescent group 1a;

[0040] Figure 2 Synthetic route diagram of lead chemiluminescent group 1a;

[0041] Figure 3 Schematic diagram of a pair of rotational isomers, syn-1a and anti-1a, of the lead chemiluminescent group 1a;

[0042] Figure 4In vitro characterization of the lead chemiluminescent group 1a. (A) Chemiluminescence kinetics curves of 1a and the reference luminescent group (Ref.). (B) Chemiluminescence spectrum of 1a and fluorescence spectrum of 1a-de. (C) Chemiluminescence quantum yields of 1a, 1b, and Ref. (D) Chemiluminescence wavelengths of 1a, 1b, and Ref. Reaction conditions: PBS buffer solution (pH = 7.4, 10 mM, 20% ACN); luminescent group (10 M); 2 eq TBAF; T = 37 °C; ex = 460 nm.

[0043] Figure 5 Design strategy diagram of dihydroxanthon chemiluminescent groups 2a, 3a, and 4a based on 1a;

[0044] Figure 6 Synthetic routes and structural diagrams of the corresponding chemically excited products of dihydroxanthones chemiluminescent groups 2a, 3a, and 4a;

[0045] Figure 7 In vitro characterization of luminescent groups 2a, 3a, and 4a. (A) Chemiluminescence kinetic curves of 1a-4a. (B) Chemiluminescence spectra of 1a-4a. (C) Fluorescence spectra of 1a-4a. (D) Chemical quantum yield of 1a-4a. (E) Luminescent half-life of 1a-4a. (F) Fluorescence quantum yield of 1a-4a. Reaction conditions: PBS buffer solution (pH = 7.4, 10 mM, 20% ACN); luminescent group (10 μM); 2 eq TBAF; T = 37℃; λex = 460 nm;

[0046] Figure 8 : Chemical structural diagrams of near-infrared dioxane chemiluminescent groups 5a and 6a;

[0047] Figure 9 Synthetic routes for near-infrared dioxane chemiluminescent groups 5a and 6a;

[0048] Figure 10 In vitro characterization of luminescent groups 5a and 6a. (A) Chemiluminescence dynamics curve of 5a. (B) Chemiluminescence spectrum of 5a and fluorescence spectrum of 5a-de. (C) Chemiluminescence dynamics curve of 6a. (D) Chemiluminescence spectrum of 6a and fluorescence spectrum of 6a-de. Reaction conditions: PBS buffer solution (pH = 7.4, 10 mM, 20% ACN); luminescent group (10 M); 2 eq TBAF; T = 37℃; ex = 460 / 520 / 690 nm;

[0049] Figure 11 Chemical structure diagram of chemiluminescent groups 5b, 5c, 6b, and 6c in water-soluble near-infrared dioxane;

[0050] Figure 12Synthetic route diagram of water-soluble near-infrared dioxane chemiluminescent groups 5b, 5c, 6b, and 6c;

[0051] Figure 13 In vitro characterization of luminescent groups 5a, 5b, and 5c. (A) Chemiluminescence dynamics curve of 5a-5c (20% ACN). (B) Luminescent quantum yield of 5a-5c. (C) Luminescent half-life of 5a-5c. (D) Chemiluminescence dynamics curve of 5b and 5c (1% DMSO). (E) Chemiluminescence spectrum of 5a-5c. (F) Fluorescence spectra of 5a-de, 5b-de, and 5c-de. Reaction conditions: PBS buffer solution (pH = 7.4, 10mM, 20% ACN); luminescent group (10M); 2 eq TBAF; T = 37℃; ex = 520nm;

[0052] Figure 14 In vitro characterization of luminescent groups 6a, 6b, and 6c. (A) Chemiluminescence dynamics curve of 6a-6c (0-160 min). (B) Chemiluminescence dynamics curve of 6a-6c (0-840 min). (C) Quantum yield of 6a-6c. (D) Half-life of 6a-6c. (E) Chemiluminescence spectrum of 6a-6c. (F) Fluorescence spectra of 6a-de, 6b-de, and 6c-de. Reaction conditions: PBS buffer solution (pH = 7.4, 10 mM, 20% ACN); luminescent group (10 μM); 2 eq TBAF; T = 37℃; λ ex =690nm. Detailed Implementation

[0053] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased on the market.

[0054] Definitions of some terms

[0055] Unless otherwise defined below, all technical and scientific terms used in the specific embodiments of this invention are intended to have the same meaning as commonly understood by those skilled in the art. While it is believed that the following terms will be well understood by those skilled in the art, the following definitions are set forth to better explain the invention.

[0056] As used in this invention, the terms “comprising,” “including,” “having,” “containing,” or “involving” are inclusive or open-ended and do not exclude other unlisted elements or method steps. The term “consisting of” is considered a preferred embodiment of the term “comprising.” If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists only of those embodiments.

[0057] When referring to a singular noun, the indefinite or definite article used, such as "a" or "a kind of," "the," includes the plural form of the noun.

[0058] The term "approximately" in this invention refers to an accuracy range that, as would be understood by those skilled in the art, still guarantees the technical effects of the features in question. This term typically indicates a deviation from the indicated value of ±10%, preferably ±5%.

[0059] A lead compound, or lead compound for short, is a compound with a certain biological activity and chemical structure obtained through various pathways and means. It is used for further structural modification and is the starting point for modern new drug research.

[0060] Furthermore, the terms first, second, third, (a), (b), (c), and similar terms used in the specification and claims are for distinguishing similar elements and are not necessary for the order of description or chronological sequence. It should be understood that such terms are interchangeable in appropriate contexts, and the embodiments described in this invention can be implemented in a different order than that described or illustrated in this invention.

[0061] The following terms or definitions are provided merely to aid in understanding the invention. These definitions should not be construed as having a scope less than that understood by those skilled in the art.

[0062] The invention is further described with reference to the accompanying drawings and the following embodiments, which are merely illustrative of specific implementations of the invention and should not be construed as limiting the scope of the invention in any way.

[0063] Example 1: Design, Synthesis, and In Vitro Characterization of Lead Chemiluminescent Clusters

[0064] 1.1 Design and Synthesis of Lead Chemiluminescent Clusters

[0065] To verify the design strategy of the chemiluminescent group mentioned above, this invention uses dihydroxanthine as the parent structure and uses the TIPS protecting group to protect the phenolic hydroxyl group to obtain the lead chemiluminescent group 1a. Figure 1The TIPS group can be rapidly removed via the TBAF reaction, triggering a chemical excitation reaction to generate the excited-state intermediate 1a-de, followed by a chemiluminescent signal. The protection and deprotection of the TIPS group is simple and convenient, does not affect the chemical synthesis and in vitro characterization of dioxane, and is a good protecting group for in vitro verification of chemiluminescent group performance.

[0066] The synthetic route of the lead chemiluminescent group 1a is as follows: Figure 2 As shown, using known compound 2.1 as the starting material, 2-(methoxy(3-methoxyphenyl)methylene)adamantane, the methoxy-meta-carbon-hydrogen bond is borated under transition metal Ir catalysis. Under alkaline conditions, the borate ester is oxidized by hydrogen peroxide to generate phenolic hydroxyl compound 2.2. 2.2 undergoes formylation with paraformaldehyde under anhydrous magnesium chloride catalysis to generate a pair of isomers 2.3a and 2.3b. The two isomers cannot be separated by column chromatography. A mixture of 2.3a and 2.3b is used to react with the bromounsaturated aldehyde 2.3c in a tandem Oxa-Michael, Retro-Michael, and Aldol reaction to generate 2.4. 2.4 reacts with sodium ethanethiol under alkaline conditions to remove the methyl protecting group, generating 2.5. Subsequently, 2.5 reacts with TIPSCl to protect the phenolic hydroxyl group and generate 2.6. Under light irradiation, 2.6 sensitizes oxygen with the photosensitizer methylene blue (MB) to generate singlet oxygen. The singlet oxygen undergoes a [2+2] cycloaddition to the electron-rich olefin of 2.6 to generate dioxane, i.e., the lead chemiluminescent group 1a.

[0067] 1 1H NMR revealed a pair of rotomers, syn-1a and anti-1a, in the lead chemiluminescent group 1a. These rotomers originate from the conjugated hydrogen atom in the aromatic ring of dihydroxanthrene. Figure 3 The interaction between the hydrogen atom (labeled in the middle) and the adamantyl group. According to the naming rules for rotational isomers, the rotational isomer on the same side as the phenolic hydroxyl group in dioxane is named syn-1a, and the one on the opposite side is named anti-1a. Because the dioxane moiety is spatially closer to the phenolic group, the chemical shift of the green-labeled hydrogen atom in syn-1a is greater than that in anti-1a, thus... 1 The chemical shift pattern caused by the syn and anti isomers is consistent with the steric hindrance of the adjacent adamantyl group and the conjugated hydrogen of the aromatic ring, which makes it difficult for the aromatic ring to rotate, thus producing a pair of rotational isomers. The two isomers are distinguished by their different interactions with the conjugated hydrogen of the aromatic ring in their spatial structure.

[0068] Synthesis of compound 2.2:

[0069]

[0070] Compound 2.1 was synthesized via an HWE reaction of dimethyl(methoxy(3-methoxyphenyl)methyl)phosphonate with adamantane. Compound 2.1 (2.66 g, 9.35 mmol), pinacol diboronate (4.27 g, 16.83 mmol), (1,5-cyclooctadiene)(methoxy)iridium(I) dimer (122 mg, 0.18 mmol), and 4,4-di-tert-butyl-2,2-bipyridine (100 mg, 0.37 mmol) were dissolved in anhydrous tetrahydrofuran (20 mL). The reaction was carried out in a sealed tube under nitrogen protection, heated to 80 °C, and stirred for 2 h. After the reaction was complete, the reaction solution was allowed to cool to room temperature and transferred to a 500 mL flask. The flask was then cooled to 0°C in an ice bath. Sodium hydroxide (1 M, 22.4 mL, 22.4 mmol) was carefully added dropwise, followed by the careful addition of 30% hydrogen peroxide (7.5 mL, 74.8 mmol). The mixture was stirred for 15 min. The reaction solution was diluted with saturated ammonium chloride solution and extracted three times with ethyl acetate. The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate solid, and filtered. The filtrate was concentrated under reduced pressure using a vacuum pump and separated by rapid column chromatography (ethyl acetate / petroleum ether = 1 / 10) to give 2.2 g (2.05 g, 73%) of a white solid. 1 H NMR (400MHz, CDCl3) δ6.47(s,1H),6.45(s,1H),6.37(s,1H),5.94(s,1H),3.77(s,3H),3.33(s,3H),3.22(s,1H),2.68(s,1H),1.98–1.75(m,12H). 13 C NMR (101MHz, CDCl3) δ160.58,156.85,142.85,137.47,132.42,108.76,107 .74,100.69,57.84,55.36,39.21,39.07,37.18,32.33,30.33,28.31.HRMS calcd for C 19 H 25 O3 + [M+H] + :301.17982; found:301.17984.

[0071] Synthesis of compounds 2.3a and 2.3b

[0072]

[0073] Compound 2.2 (300 mg, 1 mmol), anhydrous magnesium chloride (285 mg, 3 mmol), and paraformaldehyde (405 mg, 13.5 mmol) were dissolved in anhydrous acetonitrile (20 mL), followed by the addition of anhydrous triethylamine (1 mL, 7.5 mmol). The reaction mixture was heated to 90 °C under nitrogen protection. Heating was stopped and the mixture was cooled to room temperature when the color of the reaction mixture changed from white to bright yellow. The reaction mixture was diluted with saturated ammonium chloride solution and extracted three times with ethyl acetate. The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate solid, and filtered. The filtrate was concentrated under reduced pressure using a vacuum pump and separated by rapid column chromatography (ethyl acetate / petroleum ether = 1 / 100) to give a mixture of 2.3a and 2.3b (275 mg, 84%). 2.3a / 2.3b = 2 / 1. 2.3a 1 HNMR(400MHz, CDCl3)δ11.99(s,1H),10.26(s,1H),6.45(s,1H),6.39(s,1H), 3.87(s,3H),3.32(s,3H),3.22(s,1H),2.74(s,1H),1.99–1.68(m,12H).2.3b 1 H NMR (400MHz, CDCl3) δ12.23(s,1H),9.94(s,1H),6.37(d,J=2.4Hz,1H),6.33(d,J=2 .4Hz,1H),3.83(s,3H),3.28(s,3H),3.26(s,1H),2.27(s,1H),2.02–1.67(m,12H). 13 C NMR (101MHz, CDCl3) δ195.40,193.62,165.94,165.87,163.23,162.25,146.27,142.74,141.74,138.61,135.37,134.38,113.28,111.10, 110.76,110.01,101.76,100.18,58.21,57.30,55.85,55.69,39.23,39.09,38.80,37.05,36.97,32.69,32.49,30.53,29.88,28.20.HRMS calcd forC 20 H 25 O4 + [M+H] + :329.17474; found:329.17471.

[0074] Synthesis of compound 2.4

[0075]

[0076] Mixtures 2.3a and 2.3b (281 mg, 0.86 mmol), and anhydrous cesium carbonate (835 mg, 2.57 mmol) were dissolved in anhydrous N,N-dimethylformamide (20 mL), followed by the addition of 2.3c.

[242] (194 mg, 1.03 mmol) The reaction was carried out under nitrogen protection with stirring at room temperature for 16 h. The reaction was monitored by TLC (ethyl acetate / petroleum ether = 1 / 4). After the reaction was complete, the reaction solution was diluted with saturated ammonium chloride solution and extracted three times with ethyl acetate. The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate solid, and filtered. The filtrate was concentrated under reduced pressure using a vacuum pump and separated by rapid column chromatography (ethyl acetate / petroleum ether = 1 / 20) to give 2.4 g of yellow foamy solid (153 mg, 65%). 1 H NMR (400MHz, CDCl3) δ10.31(s,1H),6.87(s,1H),6.63(s,1H),6.55(s,1H),3.85 (s,3H),3.27(s,4H),2.56(s,2H),2.44(s,2H),2.22(s,1H),1.98–1.67(m,14H). 13 C NMR (101MHz, CDCl3) δ187.63,160.86,160.39,153.80,139.43,133.92,133.31,126.69,125.50,114.17,1 13.16,112.48,99.97,57.28,55.71,39.24,38.97,37.06,32.68,30.12,29.89,28.31,21.58,20.48.HRMS calcd for C 27 H 31 O4 + [M+H] + :419.22169; found:419.22171.

[0077] Synthesis of compound 2.5

[0078]

[0079] Compound 2.4 (175 mg, 0.42 mmol) and anhydrous cesium carbonate (547 mg, 1.68 mmol) were dissolved in anhydrous N,N-dimethylformamide (20 mL), followed by the addition of sodium ethanethiol (108 mg, 1.26 mmol). The reaction was heated to 90 °C and stirred for 6 h under nitrogen protection. The reaction was monitored by TLC (ethyl acetate / petroleum ether = 1 / 2). After the reaction was complete, the reaction solution was diluted with saturated ammonium chloride solution and extracted three times with ethyl acetate. The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate solid, and filtered. The filtrate was concentrated under reduced pressure using a vacuum pump and separated by rapid column chromatography (ethyl acetate / petroleum ether = 1 / 4) to give a yellow solid 2.5 (113 mg, 67%). 1 H NMR (400MHz, CDCl3) δ10.32(s,1H),9.70(br,1H),7.03(s,1H),6.65(s,1H),6.64(s,1H),3 .28(s,4H),2.59(t,J=6.0Hz,2H),2.48(t,J=6.1Hz,2H),2.26(s,1H),2.04–1.61(m,14H). 13 C NMR (101MHz, CDCl3) δ187.03,163.50,158.90,153.74,139.38,134.56,133.64,128.08,125.38,115.3 3,113.31,111.56,102.18,57.38,39.25,39.04,37.05,32.65,29.92,29.86,28.30,21.49,20.43.HRMS calcdfor C 26 H 27 O4 - [MH] - :403.19148; found:403.19135.

[0080] Synthesis of compound 2.6

[0081]

[0082] Compound 2.5 (60 mg, 0.14 mmol) and imidazole (20 mg, 0.27 mmol) were dissolved in anhydrous dichloromethane (20 mL), followed by the addition of triisopropylchlorosilane (40 μL, 0.19 mmol). The reaction was stirred at room temperature for 1 h. The reaction was monitored by TLC (ethyl acetate / petroleum ether = 1 / 5). After the reaction was complete, the reaction solution was concentrated under reduced pressure using a vacuum pump and separated by rapid column chromatography (ethyl acetate / petroleum ether = 1 / 20) to give a yellow solid 2.6 (65 mg, 85%).1 H NMR (400MHz, CDCl3) δ10.31(s,1H),6.86(s,1H),6.61(d,J=2.4Hz,1H),6.51(d,J=2.4Hz,1H),3.28(s,1H),3.25(s,3H) ,2.61–2.51(m,2H),2.43(t,J=6.0Hz,2H),2.20(s,1H),1.99–1.67(m,14H),1.32–1.21(m,3H),1.10(d,J=7.4Hz,18H). 13 C NMR (101MHz, CDCl3) δ187.71,160.76,157.07,153.58,139.51,133.67,132.94,126.88,125.42,118.24,114.68, 112.51,106.79,57.18,39.21,38.93,37.04,32.71,30.15,29.84,28.38,28.21,21.56,20.50,17.89,12.68.HRMS calcdfor C 35 H 49 O4Si + [M+H] + :561.33946; found:561.33911.

[0083] Synthesis of lead chemiluminescent group 1a

[0084]

[0085] Compound 2.6 (10 mg, 0.018 mmol) was dissolved in deuterated chloroform (5 mL), followed by the addition of a catalytic amount of methylene blue. The reaction was placed under a yellow light source with a color temperature of 3000 K, where oxygen participated in the reaction in a bubbling manner. The reaction was measured by TLC and... 1 After the reaction was complete (approximately 3 minutes) as monitored by ¹H NMR, the reaction solution was concentrated under reduced pressure using a vacuum pump and separated by preparative thin-layer chromatography on silica gel plates (ethyl acetate / petroleum ether = 1 / 5) to obtain a pair of inseparable rotational isomers, syn-1a and anti-1a (4.2 mg, 40%). The ratio of rotational isomers syn-1a to anti-1a was 3:1. 11H NMR (400 MHz, CDCl3) δ 10.31 (s, 1H), 8.05 (s, 1H), 6.74 (s, 1H), 6.70 (s, 1H), 3.29 (s, 3H), 3.05 (s, 1H), 2.70–2.39 (m, 4H), 2.24–1.38 (m, 15H), 1.36–1.23 (m, 3H), 1.11 (d, J = 7.5 Hz, 18H). syn-isomer 1 1H NMR (400 MHz, CDCl3) δ 10.31 (s, 1H), 7.42 (s, 1H), 7.29 (d, J = 2.5 Hz, 1H), 6.70 (s, 1H), 3.22 (s, 3H), 3.05 (s, 1H), 2.69–2.42 (m, 4H), 2.23–1.40 (m, 15H), 1.36–1.24 (m, 3H), 1.11 (d, J = 7.5 Hz, 18H). 13 13C NMR (101 MHz, CDCl3) δ 187.66, 159.72, 157.44, 153.84, 131.50, 127.87, 126.05, 124.54, 119.83, 116.52, 112.92, 112.60, 112.24, 108.94, 108.53, 96.01, 49.90, 49.75, 47.02, 39.31, 36.47, 36.35, 36.31, 34.95, 34.79, 33.54, 33.36, 33.20, 32.31, 32.24, 32.16, 31.72, 31.56, 30.47, 30.35, 29.76, 27.49, 26.06, 26.01, 25.90, 21.52, 21.47, 20.53, 17.90, 17.76, 12.70, 12.65, 12.33. HRMS calcd for C 35 H 49 O6Si + [M + H] + : 593.32929; found: 593.32947.

[0086] Synthesis of Compounds 1a - de

[0087]

[0088] The synthesis of compound 1a-de was performed as follows: A chemiluminescent group (0.025 mmol) was dissolved in tetrahydrofuran (5 mL), followed by the addition of tetrabutylammonium fluoride (30 L, 0.03 mmol, 1 M in THF). The reaction was stirred at room temperature for 1 h. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was diluted with saturated ammonium chloride solution and extracted three times with ethyl acetate. The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate solid, and filtered. The filtrate was concentrated under reduced pressure using a vacuum pump and separated by rapid column chromatography (methanol / dichloromethane = 1 / 10) to obtain a yellow solid 1a-de (6 mg, 85%). 1 H NMR (400MHz, DMSO-d6) δ10.55(s,1H),10.20(s,1H),7.58(s,1H),7.10(d,J=2.5Hz,1H),6.84(d ,J=2.5Hz,1H),3.85(s,3H),2.56(t,J=6.0Hz,2H),2.28(t,J=6.0Hz,2H),1.62(p,J=6.0Hz,2H). 13 C NMR (101MHz, DMSO-d6) δ186.51,165.83,159.08,158.49,153.30,127.19,127. 13,124.41,113.69,112.69,111.84,106.16,52.48,29.34,21.14,19.94.HRMS calcdfor C 16 H 13 O5 – [M–H] – :285.07685; found:285.07693.

[0089] 1.2 In vitro characterization of the lead chemiluminescent group

[0090] The synthesized chemiluminescent group 1a underwent a chemical excitation reaction in PBS solution, producing a very strong chemiluminescent signal. The luminescence intensity of 1a reached its maximum at 10 min, and then slowly decayed over 80 min, exhibiting a typical chemiluminescence kinetic curve. This luminescence curve was significantly higher than that of the reference luminescent group (Ref.). Figure 4 A). The chemiluminescence spectrum of 1a is 555 nm ( Figure 4 B), the fluorescence spectrum of the chemically excited product 1a-de of 1a is 555 nm ( Figure 4 B) The emission spectrum is very similar to the fluorescence spectrum.

[0091] This invention integrates the chemiluminescence kinetic curve and calculates the luminescence quantum yield of the leader chemiluminescent group 1a using the luminescence quantum yield of the reference luminescent group (Ref.). For example... Figure 4 As shown in C, 1a exhibits a chemiluminescence quantum yield of 3.63%, which is 1100 times higher than the reference compound. Compared to the phenol-based dioxane 1b developed by Shabat, 1a demonstrates a higher luminescence quantum yield and a longer chemiluminescence wavelength. Figure 4 D) and the extensibility of conjugate structures.

[0092] In summary, this embodiment provides the synthesis and in vitro characterization of the lead chemiluminescent group 1a, demonstrating the superiority of the rigid dihydroxanthine chemiluminescent group over phenolic chemiluminescent groups derived from non-rigid π-conjugated structures in terms of emission wavelength and emission quantum yield. This provides feasibility for the design of chemiluminescent groups based on the rigid dihydroxanthine core structure.

[0093] Example 2: Design, synthesis, and in vitro characterization of a chemiluminescent molecular library

[0094] 2.1 Design and Synthesis of Chemiluminescent Group Molecular Library

[0095] To further optimize the various parameters of chemiluminescence, this invention designs three strategies based on the leader chemiluminescent group structure (see...). Figure 5 ).

[0096] (A) Chlorine atom at the ortho position of the phenolic hydroxyl group: The chlorine atom acts as an electron-withdrawing group, lowering the pKa of phenol and generating more phenolic anions at physiological pH. This is beneficial for in vitro detection and in vivo imaging, and also increases the photostability of the chemiluminescent group. Therefore, this invention designs and synthesizes chemiluminescent group 2a based on 1a.

[0097] (B) Replacement of the five-membered ring in the dihydroxanthonium core: Compared to six-membered rings, five-membered rings are more compact and have higher rigidity, which can further restrict the molecular conformational change from singlet excited state relaxation to ground state, thereby improving the luminescence quantum yield. Therefore, this invention designs and synthesizes chemiluminescent group 3a based on 2a.

[0098] (C) Substitution of the oxygen atom in the dihydroxanthine parent nucleus: In the design strategy of phenolic dioxane, the introduction of electron-withdrawing substituents (EWGs) can improve the luminescence quantum yield. In the dihydroxanthine structure, the carbon atom at position 2 is far from the π-conjugated system. By substituting it with a non-conjugated oxygen atom, this invention aims to more finely tune the electron density of the conjugated system. Therefore, this invention designs and synthesizes chemiluminescent group 4a based on 2a.

[0099] like Figure 6As shown, similar to the synthetic route of the lead chemiluminescent group 1a, compound 2.7 is used as the starting material. The methoxy-meta-C-H bond is borated under transition metal Ir catalysis. Under alkaline conditions, the borate ester is oxidized by hydrogen peroxide to generate phenolic hydroxyl compound 2.8. Due to the occupancy effect of the chlorine atom, 2.8 undergoes formylation in high yield to generate a single compound 2.9. 2.9 reacts with bromounsaturated aldehydes 2.3c, 2.3d, and 2.3e to generate 2.10, 2.13, and 2.16, respectively. Subsequently, sodium ethanethiol is used to remove the methyl protecting group, generating 2.11, 2.14, and 2.17. Then, TIPSCl is used to protect the hydroxyl group to generate 2.12, 2.15, and 2.18. Finally, in the presence of a photosensitizer, a [2+2] cycloaddition reaction occurs with singlet oxygen to generate dioxane luminescent groups 2a, 3a, and 4a. Synthesis of chemiluminescent group 2a.

[0100] Synthesis of compound 2.8

[0101]

[0102] Compound 2.7 was synthesized via an HWE reaction of dimethyl((4-chloro-3-methoxyphenyl)(methoxy)methyl)phosphonate with adamantane. Compound 2.7 (5.55 g, 17.1 mmol), pinacol diboronate (10.6 g, 41.78 mmol), (1,5-cyclooctadiene)(methoxy)iridium(I) dimer (400 mg, 0.69 mmol), and 4,4-di-tert-butyl-2,2-bipyridine (374 mg, 1.39 mmol) were dissolved in anhydrous tetrahydrofuran (20 mL). The reaction was carried out in a sealed tube under nitrogen protection, heated to 80 °C, and stirred for 12 h. After the reaction was complete, the reaction solution was allowed to cool to room temperature and transferred to a 500 mL flask. The flask was then cooled to 0 °C in an ice bath. Sodium hydroxide (1 M, 42 mL, 42 mmol) was carefully added dropwise, followed by the careful addition of 30% hydrogen peroxide (14.3 mL, 140 mmol). The mixture was stirred for 15 min. The reaction solution was diluted with saturated ammonium chloride solution and extracted three times with ethyl acetate. The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate solid, and filtered. The filtrate was concentrated under reduced pressure using a vacuum pump and separated by rapid column chromatography (ethyl acetate / petroleum ether = 1 / 10) to give 2.8 g (4.08 g, 70%) of a white, foamy solid. 1 H NMR (400MHz, CDCl3) δ6.62(d,J=1.7Hz,1H),6.52(d,J=1.7Hz,1H),6.10(br, 1H),3.86(s,3H),3.31(s,3H),3.22(s,1H),2.66(s,1H),2.01–1.68(m,12H). 13C NMR (101MHz, CDCl3) δ155.36,152.14,142.59,135.17,132.81,109.89,107 .68,104.65,57.89,56.32,39.17,39.03,37.14,32.37,30.34,28.28.HRMS calcd forC 19 H 24 ClO3 + [M+H] + :335.14085; found:335.14084.

[0103] Synthesis of compound 2.9

[0104]

[0105] Compound 2.8 (3.41 g, 10.19 mmol), anhydrous magnesium chloride (2.91 g, 30.57 mmol), and paraformaldehyde (4.12 g, 137.6 mmol) were dissolved in anhydrous acetonitrile (60 mL), followed by the addition of anhydrous triethylamine (10.62 mL, 76.43 mmol). The reaction mixture was heated to 90 °C under nitrogen protection. Heating was stopped and the mixture was cooled to room temperature when the color of the reaction mixture changed from white to bright yellow. The reaction mixture was diluted with saturated ammonium chloride solution and extracted three times with ethyl acetate. The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate solid, and filtered. The filtrate was concentrated under reduced pressure using a vacuum pump and separated by rapid column chromatography (ethyl acetate / petroleum ether = 1 / 100) to give a yellow solid 2.9 (3.01 g, 84%). 1 H NMR (500MHz, CDCl3) δ12.50(s,1H),9.97(s,1H),6.43(s,1H),3.96(s,3H),3.29(s,3H),3.26(s,1H),2.23(s,1H),1.97–1.55(m,12H). 13 C NMR (126MHz, CDCl3) δ195.57,160.96,160.01,140.28,138.08,135.50,114.12,109.03,1 05.99,57.40,56.72,39.22,39.05,38.84,38.73,36.85,32.78,29.98,28.21,28.00.HRMS calcd for C 20 H 23 ClNaO4 + [M+Na] + :385.11771; found:385.11771.

[0106] Synthesis of compound 2.10

[0107]

[0108] Compound 2.9 (1.2 g, 3.30 mmol) and anhydrous cesium carbonate (3.23 g, 9.9 mmol) were dissolved in anhydrous N,N-dimethylformamide (20 mL), followed by the addition of 2.3c (1.75 g, 9.24 mmol). The reaction was stirred at room temperature for 16 h under nitrogen protection. The reaction was monitored by TLC (ethyl acetate / petroleum ether = 1 / 4). After the reaction was complete, the reaction solution was diluted with saturated ammonium chloride solution and extracted three times with ethyl acetate. The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate solid, and filtered. The filtrate was concentrated under reduced pressure using a vacuum pump and separated by rapid column chromatography (ethyl acetate / petroleum ether = 1 / 20) to give a yellow foamy solid 2.10 (970 mg, 65%). 1 H NMR (400MHz, CDCl3) δ10.31(s,1H),6.87(s,1H),6.63(s,1H),6.55(s,1H),3.85 (s,3H),3.27(s,4H),2.56(s,2H),2.44(s,2H),2.22(s,1H),1.98–1.67(m,14H). 13 C NMR (101MHz, CDCl3) δ187.63,160.86,160.39,153.80,139.43,133.92,133.31,126.69,125.50,114.17,1 13.16,112.48,99.97,57.28,55.71,39.24,38.97,37.06,32.68,30.12,29.89,28.31,21.58,20.48.HRMS calcd for C 27 H 31 O4 + [M+H] + :419.22169; found:419.22171.

[0109] Synthesis of Compound 2.11

[0110]

[0111] Compound 2.10 (650 mg, 1.14 mmol) and anhydrous cesium carbonate (1.03 g, 3.168 mmol) were dissolved in anhydrous N,N-dimethylformamide (20 mL), followed by the addition of sodium ethanethiol (250 mg, 2.88 mmol). The reaction was heated to 90 °C and stirred for 6 h under nitrogen protection. The reaction was monitored by TLC (ethyl acetate / petroleum ether = 1 / 2). After the reaction was complete, the reaction solution was diluted with saturated ammonium chloride solution and extracted three times with ethyl acetate. The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate solid, and filtered. The filtrate was concentrated under reduced pressure using a vacuum pump and separated by rapid column chromatography (ethyl acetate / petroleum ether = 1 / 4) to give a yellow solid 2.11 (428 mg, 68%). 1 H NMR (500MHz, CDCl3) δ10.42(s,1H),6.86(s,1H),6.75(br,1H),6.70(s,1H),3.27(s ,4H),2.58(d,J=5.9Hz,2H),2.46(t,J=6.1Hz,2H),2.23(s,1H),1.99–1.71(m,14H). 13 C NMR (126MHz, CDCl3) δ188.30,160.16,152.59,148.69,138.87,134.09,131.92,127.06,125.04,11 4.84,113.53,113.47,107.07,57.35,39.20,36.98,32.68,29.91,29.87,28.22,21.47,20.35.HRMS calcd forC 26 H 26 ClO4 - [MH] - :437.15251; found:437.15256.

[0112] Synthesis of compound 2.12

[0113]

[0114] Compound 2.11 (60 mg, 0.14 mmol) and imidazole (14 mg, 0.21 mmol) were dissolved in anhydrous dichloromethane (20 mL), followed by the addition of triisopropylchlorosilane (46 μL, 0.22 mmol). The reaction was stirred at room temperature for 1 h. The reaction was monitored by TLC (ethyl acetate / petroleum ether = 1 / 5). After the reaction was complete, the reaction solution was concentrated under reduced pressure using a vacuum pump and separated by rapid column chromatography (ethyl acetate / petroleum ether = 1 / 20) to give a yellow solid 2.12 (61 mg, 75%). 1H NMR (400MHz, CDCl3) δ10.46(s,1H),6.82(s,1H),6.54(s,1H),3.27(s,1H),3.25(s,3H),2.58(d,J=3.7Hz ,2H),2.45(t,J=6.0Hz,2H),2.20(s,1H),2.04–1.66(m,14H),1.35–1.27(m,3H),1.12(d,J=7.4Hz,18H). 13 C NMR (101MHz, CDCl3) δ188.48,159.92,152.96,149.35,139.22,133.48,130.74,127.41,124.65,117.06,115.30, 113.58,112.28,57.21,39.21,38.95,36.97,32.76,29.94,29.87,28.33,28.16,21.47,20.45,17.91,12.97.HRMS calcd for C 35 H 48 ClO4Si + [M+H] + :595.30049; found:595.30066.

[0115] Synthesis of chemiluminescent group 2a

[0116]

[0117] Compound 2.12 (10 mg, 0.017 mmol) was dissolved in deuterated chloroform (5 mL), followed by the addition of a catalytic amount of methylene blue. The reaction was placed under a yellow light source with a color temperature of 3000 K, where oxygen participated in the reaction in a bubbling manner. The reaction was measured by TLC and... 1 After the reaction was complete (approximately 3 minutes) as monitored by ¹H NMR, the reaction solution was concentrated under reduced pressure using a vacuum pump and separated by preparative thin-layer chromatography on silica gel plates (ethyl acetate / petroleum ether = 1 / 5) to obtain a pair of inseparable rotational isomers, syn-2a and anti-2a (4.8 mg, 45%). The ratio of rotational isomers syn-2a to anti-2a was 2:1. (anti-isomer:) 1H NMR (400MHz, CDCl3) δ10.47(s,1H),8.02(s,1H),6.81(s,1H),3.28(s,3H),3.05(s,1H),2.6 9–2.43(m,4H),2.11–1.75(m,15H),1.41–1.32(m,3H),1.15(d,J=7.4Hz,18H).syn-isomer: 1 H NMR (400MHz, CDCl3) δ10.47(s,1H),7.37(s,2H),3.20(s,3H),3.05(s,1H),2.69–2. 43(m,4H),2.11–1.75(m,15H),1.41–1.32(m,3H),1.14(d,J=7.4Hz,18H).Endo-and exo-isomer: 13 CNMR(101MHz, CDCl3)δ188.33,188.23,158.70,153.25,149.37,128.52,128.28,127.78, 125.07,123.54,118.13,115.03,114.19,113.64,113.49,111.79,96.11,95.87,49.62,46 .92,39.20,36.32,36.24,36.14,34.88,33.41,33.25,32.14,31.84,31.69,31.54,30.13, 30.02,27.39,25.93,25.79,21.28,20.37,17.79,17.75,17.71,17.63,12.82,12.78.HRMS calcd for C 35 H 48 ClO6Si + [M+H] + :627.29032; found:627.29053.

[0118] Synthesis of compound 2.13

[0119]

[0120] Compound 2.9 (200 mg, 0.55 mmol) and tetrabutylammonium fluoride (1.65 mL, 1.65 mmol, 1 M in THF) were dissolved in anhydrous dimethyl sulfoxide (10 mL), followed by the addition of 2.3 d. The reaction was stirred at room temperature for 16 h under nitrogen protection. The reaction was monitored by TLC (ethyl acetate / petroleum ether = 1 / 4). After the reaction was complete, the reaction solution was diluted with saturated ammonium chloride solution and extracted three times with ethyl acetate. The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate solid, and filtered. The filtrate was concentrated under reduced pressure using a vacuum pump and separated by rapid column chromatography (ethyl acetate / petroleum ether = 1 / 20) to give a yellow solid 2.13 (144 mg, 60%). 1 H NMR(400MHz, CDCl3)δ10.10(s,1H),6.79(s,1H),6.58(s,1H),3.92(s,3H),3 .28(s,1H),3.26(s,3H),2.82–2.66(m,4H),2.16(s,1H),2.05–1.67(m,12H). 13 C NMR (101MHz, CDCl3) δ184.84,162.75,155.29,148.60,139.20,138.23,133.63,131.05,119.90,117.49,116.33, 109.91,109.42,57.21,56.65,39.32,39.17,39.00,38.85,36.99,32.76,29.90,28.32,28.12,24.49,23.67.HRMS calcd for C 26 H 28 ClO4 + [M+H] + :439.16706; found:439.16714.

[0121] Synthesis of compounds 2.14 and 2.15

[0122]

[0123] Compound 2.13 (92 mg, 0.21 mmol) and anhydrous cesium carbonate (136.8 mg, 0.42 mmol) were dissolved in anhydrous N,N-dimethylformamide (20 mL), followed by the addition of sodium ethanethiol (30.7 mg, 0.36 mmol). The reaction was heated to 90 °C and stirred for 6 h under nitrogen protection. The reaction was monitored by TLC (ethyl acetate / petroleum ether = 1 / 2). After the reaction was complete, the reaction solution was diluted with saturated ammonium chloride solution and extracted three times with ethyl acetate. The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate solid, and filtered. The filtrate was concentrated under reduced pressure using a vacuum pump and separated by rapid column chromatography (ethyl acetate / petroleum ether = 1 / 4) to obtain the crude product 2.14. Compound 2.14 (10 mg, 0.024 mmol) and imidazole (4.9 mg, 0.072 mmol) were dissolved in anhydrous dichloromethane (5 mL), followed by the addition of triisopropylchlorosilane (10 μL, 0.048 mmol). The reaction was stirred at room temperature for 1 h. The reaction was monitored by TLC (ethyl acetate / petroleum ether = 1 / 5). After the reaction was complete, the reaction solution was concentrated under reduced pressure using a vacuum pump and separated by rapid column chromatography (ethyl acetate / petroleum ether = 1 / 20) to give a yellow solid 2.15 (10.4 mg, two-step yield 7.5%). 1 H NMR (400MHz, CDCl3) δ10.12(s,1H),6.77(s,1H),6.56(s,1H),3.27(s,1H),3.24(s,3H),2.74(dd ,J=8.8,3.6Hz,4H),2.19(s,1H),2.05–1.63(m,12H),1.35–1.29(m,3H),1.13(d,J=6.3Hz,18H). 13 C NMR (101MHz, CDCl3) δ184.82,163.09,152.49,148.90,139.32,138.13,133.38,130.50,120.11,117.67,117.19, 116.28,113.06,57.15,39.18,38.90,36.96,32.75,29.81,29.38,28.34,28.12,24.51,23.64,17.91,12.94.HRMS calcdfor C 34 H 46 ClO4Si + [M+H] + :581.28484; found:581.28510.

[0124] Synthesis of chemiluminescent group 3a

[0125]

[0126] Compound 2.15 (10 mg, 0.017 mmol) was dissolved in deuterated chloroform (5 mL), followed by the addition of a catalytic amount of methylene blue. The reaction was placed under a yellow light source with a color temperature of 3000 K, where oxygen participated in the reaction in a bubbling manner. The reaction was measured by TLC and... 1 After the reaction was complete (approximately 3 minutes) as monitored by ¹H NMR, the reaction solution was concentrated under reduced pressure using a vacuum pump and separated by preparative thin-layer chromatography on silica gel plates (ethyl acetate / petroleum ether = 1 / 8) to obtain a pair of inseparable rotational isomers, syn-3a and anti-3a (3.2 mg, 30%). The ratio of rotational isomers syn-3a to anti-3a was 3:1. 1 H NMR (400MHz, CDCl3) δ10.14(s,1H),8.02(s,1H),6.86(s,1H),3.29(s,3H),3.05(s,1H),2.89–2. 72(m,4H),2.25–1.61(m,13H),1.37(dt,J=12.1,6.1Hz,3H),1.15(d,J=7.4Hz,18H).syn-isomer 1 H NMR (400MHz, CDCl3) δ10.14(s,1H),7.43(s,1H),7.35(s,1H),3.21(s,3H),3.05(s,1H),2. 89–2.72(m,4H),2.25–1.61(m,13H),1.37(dt,J=12.1,6.1Hz,3H),1.15(d,J=7.4Hz,18H). 13 C NMR (101MHz, CDCl3) δ184.76,161.91,152.91,139.17,128.27,120.59,118.9 7,118.76,117.41,115.71,114.60,111.99,96.04,49.81,49.69,39.29,36.39 ,36.21,34.96,34.80,33.49,33.29,32.24,31.92,31.76,31.57,29.74,29.3 6,25.99,25.84,24.88,24.72,23.67,17.90,17.85,17.81,12.89,12.85.HRMS calcd forC 34 H 46 ClO6Si + [M+H] +:613.27467; found:613.27484.

[0127] Synthesis of compound 2.16

[0128]

[0129] Compound 2.9 (700 mg, 1.93 mmol) was dissolved in anhydrous cesium carbonate (3.14 g, 9.65 mmol) in anhydrous N,N-dimethylformamide (20 mL), followed by the addition of 2.3e (1.47 g, 7.73 mmol). The reaction was stirred at room temperature for 16 h under nitrogen protection. The reaction was monitored by TLC (ethyl acetate / petroleum ether = 1 / 5). After the reaction was complete, the reaction solution was diluted with saturated ammonium chloride solution and extracted three times with ethyl acetate. The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate solid, and filtered. The filtrate was concentrated under reduced pressure using a vacuum pump and separated by rapid column chromatography (ethyl acetate / petroleum ether = 1 / 10) to give a yellow foamy solid 2.16 (560 mg, 65%). 1 H NMR (400MHz, CDCl3) δ10.30(s,1H),6.89(s,1H),6.61(s,1H),4.55(s,2H),4.49 (s,2H),3.94(s,3H),3.28(s,1H),3.26(s,3H),2.19(s,1H),2.03–1.71(m,12H). 13 C NMR (101MHz, CDCl3) δ185.79,156.61,156.33,149.00,138.90,134.50,132.34,123.86,122.58,114.63,111.04,109 .48,109.31,67.64,64.16,57.36,56.68,39.33,39.20,39.09,38.86,36.93,32.78,29.98,29.74,28.27,28.06.HRMS calcdfor C 26 H 28 ClO5 + [M+H] + :455.16198; found:455.16208.

[0130] Synthesis of compound 2.17

[0131]

[0132] Compound 2.16 (196 mg, 0.43 mmol) and anhydrous cesium carbonate (308 mg, 0.95 mmol) were dissolved in anhydrous N,N-dimethylformamide (20 mL), followed by the addition of sodium ethanethiol (75 mg, 0.86 mmol). The reaction was heated to 90 °C and stirred for 6 h under nitrogen protection. The reaction was monitored by TLC (ethyl acetate / petroleum ether = 1 / 2). After the reaction was complete, the reaction solution was diluted with saturated ammonium chloride solution and extracted three times with ethyl acetate. The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate solid, and filtered. The filtrate was concentrated under reduced pressure using a vacuum pump and separated by rapid column chromatography (ethyl acetate / petroleum ether = 1 / 4) to give a yellow solid 2.17 (121 mg, 64%). 1 H NMR (400MHz, CDCl3) δ10.28(s,1H),6.91(s,1H),6.74(s,1H),4.58(s,2H),4.50(s,2H),3.26(s,4H),2.24(s,1H),2.06–1.71(m,12H). 13 C NMR (101MHz, CDCl3) δ185.66,157.05,153.16,148.75,138.63,134.93,132.94,123.47,123.14,114.22,114. 05,110.77,107.34,67.65,64.17,57.48,39.25,38.95,36.93,32.69,29.96,29.75,29.37,28.22,27.26.HRMS calcd for C 25 H 24 ClO5 – [MH] – :439.13178; found:439.13199.

[0133] Synthesis of compound 2.18

[0134]

[0135] Compound 2.17 (30 mg, 0.067 mmol) and imidazole (7.8 mg, 0.12 mmol) were dissolved in anhydrous dichloromethane (5 mL), followed by the addition of triisopropylchlorosilane (22 μL, 0.10 mmol). The reaction was stirred at room temperature for 1 h. The reaction was monitored by TLC (ethyl acetate / petroleum ether = 1 / 5). After the reaction was complete, the reaction solution was concentrated under reduced pressure using a vacuum pump and separated by rapid column chromatography (ethyl acetate / petroleum ether = 1 / 20) to give a yellow solid 2.18 (34 mg, 85%). 1H NMR (400MHz, CDCl3) δ10.32(s,1H),6.87(s,1H),6.58(s,1H),4.57(s,2H),4.49(s,2H),3.27(s ,1H),3.24(s,3H),2.21(s,1H),2.02–1.62(m,12H),1.36–1.29(m,3H),1.13(d,J=7.4Hz,18H). 13 C NMR (101MHz, CDCl3) δ185.83,156.92,153.67,149.41,138.99,134.26,131.72,123.74,122.81,117.58,114.60, 112.63,110.76,67.67,64.21,57.30,39.24,38.92,36.90,32.77,29.89,29.76,28.29,28.07,17.89,12.96.HRMS calcd for C 34 H 46 ClO5Si + [M+H] + :597.27976; found:597.28003.

[0136] Synthesis of chemiluminescent group 4a

[0137]

[0138] Compound 2.18 (10 mg, 0.017 mmol) was dissolved in deuterated chloroform (5 mL), followed by the addition of a catalytic amount of methylene blue. The reaction was placed under a yellow light source with a color temperature of 3000 K, where oxygen participated in the reaction in a bubbling manner. The reaction was measured by TLC and... 1 After the reaction was complete (approximately 3 minutes) as monitored by ¹H NMR, the reaction solution was concentrated under reduced pressure using a vacuum pump and separated by preparative thin-layer chromatography on silica gel plates (ethyl acetate / petroleum ether = 1 / 5) to obtain a pair of inseparable rotational isomers, syn-4a and anti-4a (3.2 mg, 30%). The ratio of rotational isomers syn-4a to anti-4a was 2:1. 1 H NMR (400MHz, CDCl3) δ10.36(s,1H),8.12(s,1H),6.90(s,1H),4.70–4.48(m,4H),3.32(s,3H),3. 08(s,1H),2.14–1.58(m,13H),1.40(dt,J=15.1,7.4Hz,3H),1.18(d,J=7.5Hz,18H).syn-isomer1 H NMR(400MHz, CDCl3)δ10.36(s,1H),7.46(s,2H),4.70–4.48(m,4H),3.23(s,3H),3. 08(s,1H),2.14–1.58(m,13H),1.40(dt,J=15.1,7.4Hz,3H),1.18(d,J=7.5Hz,18H). 13 CNMR(101MHz, CDCl3)δ185.82,185.77,155.85,154.10,149.87,149.50,130.00,129.57,124.75,124.18,123 .45,121.63,118.81,116.83,115.69,114.67,112.82,112.57,111.77,110.86,110.75,96.22,95.97,68.01, 67.77,64.17,49.92,49.76,47.02,39.31,36.33,36.17,35.10,34.84,33.62,33.50,33.45,33.41,32.24,31 .89,31.74,31.62,31.54,29.76,27.49,25.96,25.90,25.85,25.78,17.90,17.86,17.82,12.91,12.89.HRMS calcdfor C 34 H 46 ClO7Si + [M+H] + :629.26958; found:629.26978.

[0139] Synthesis of compound 2a-de

[0140]

[0141] The synthesis of compound 2a-de was the same as that of compound 1a-de, and the yellow solid 2a-de (6.8 mg, 85%) was obtained by rapid column chromatography (methanol / dichloromethane = 1 / 10). 1 H NMR(500MHz,DMSO-d6)δ11.25(br,1H),10.21(s,1H),7.56(s,1H),7.26(s,1H) ,3.84(s,3H),2.58–2.52(m,2H),2.28(t,J=6.0Hz,2H),1.62(p,J=6.1Hz,2H). 13C NMR (126MHz, DMSO-d6) δ186.68,165.80,158.77,154.81,149.09,128.26,124. 73,124.19,114.10,113.70,113.40,111.82,52.98,29.60,21.53,20.26.HRMS calcd for C 16 H 12 ClO5 – [M–H] – :319.03787; found:319.03784.

[0142] Synthesis of compound 3a-de

[0143]

[0144] The synthesis of compound 3a-de was the same as that of compound 1a-de, and the yellow solid 3a-de (5.7 mg, 75%) was obtained by rapid column chromatography (methanol / dichloromethane = 1 / 10). 1 H NMR (400MHz, DMSO-d6) δ11.29(s,1H),9.95(s,1H),7.54(s,1H),7.32(s,1H),3.86(s,3H),2.77(t,J=4.5Hz,2H),2.59–2.55(m,2H). 13 C NMR(101MHz,DMSO-d6)δ183.55,174.86,166.15,161.81,154.46,148.88,139.44,1 30.24,125.00,119.63,117.37,115.33,114.08,112.67,53.23,29.60,29.42.HRMS calcdfor C 15 H 10 ClO5 – [M–H] – :305.02222; found:305.02203.

[0145] Synthesis of compound 4a-de

[0146]

[0147] The synthesis of compound 4a-de was the same as that of compound 1a-de, and the yellow solid 4a-de (6.4 mg, 80%) was obtained by rapid column chromatography (methanol / dichloromethane = 1 / 10). 1H NMR (400MHz, DMSO-d6) δ10.10(s,1H),7.70(s,1H),7.34(s,1H),4.51(s,2H),4.38(s,2H),3.86(s,3H). 13 C NMR (101MHz, DMSO-d6) δ184.46, 165.75, 156.18, 155.56, 149.17, 125.69, 1 24.75, 122.63, 114.25, 113.43, 112.05, 110.63, 67.10, 63.46, 53.20.HRMS calcd for C 15 H 10 ClO6 - [MH] - :321.01714; found:321.01733.

[0148] 2.2 In vitro characterization analysis of chemiluminescent molecular libraries

[0149] This experiment tested the luminescence dynamics of chemiluminescent groups 2a, 3a, and 4a. Figure 7 A). Compared to the leader luminescent group, 2a, 3a, and 4a all exhibited higher chemiluminescence kinetics. Regarding chemiluminescence kinetics, luminescent group 3a reached its maximum emission value in just 4 minutes, followed by 2a (6 minutes), 1a (10 minutes), and 4a (20 minutes). Regarding the maximum chemical excitation intensity, the order from highest to lowest was 3a, 4a, 2a, and 1a. Regarding the chemiluminescence spectrum (… Figure 7 B), the chemiluminescence spectra of 2a, 3a, and 4a are similar, indicating that the effect of structural modification on the chemiluminescence wavelength is negligible. For the fluorescence spectra of the chemically excited products ( Figure 7 C). The fluorescence spectra of 2a-de, 3a-de, and 4a-de are extremely similar to their corresponding chemiluminescence spectra, thus verifying that... Figure 1 The chemiluminescence mechanism.

[0150] like Figure 7 As shown in Figure D, the chemiluminescence quantum yield was calculated using chemiluminescence kinetic curves. For 2a, 3a, and 4a, the chemiluminescence quantum yield is significantly increased compared to the leader chemiluminescent group 1b. Figure 7 As shown in E, the luminescence half-life is calculated from the chemiluminescence kinetic curve. The half-life is the time required to reach half of the integral area of ​​the chemiluminescence curve, and it is inversely proportional to the chemical excitation rate of the luminescent group. The excitation rate of 3a is the fastest, followed by 2a and 1a, while 4a exhibits the slowest chemical excitation rate.

[0151] Compared to 1a, 2a showed improvements in both chemiluminescence quantum yield and chemiluminescence excitation rate. Figure 7 D、 Figure 7 E). Due to the electron-withdrawing effect of the chlorine atom, the pKa of the phenolic hydroxyl group decreases after deprotection of the TIPS protecting group, resulting in the generation of more phenolic salt anions at physiological pH. This leads to a faster chemiluminescence mechanism, causing the 2-year luminescence half-life (12 min) to be less than that of the 1-year (20 min). The increase in chemiluminescence quantum yield is due to two reasons: firstly, the introduction of the chlorine atom increases the fluorescence quantum yield (…). Figure 7 D); Secondly, the fluorescence quantum yield increased by about 1.5 times, while the chemiluminescence quantum yield increased by about 3.3 times, according to the formula for calculating the chemiluminescence quantum yield as follows. This indicates that there may be other gain pathways, such as the chlorine atom increasing the efficiency of single-electron transfer (SET) attacking the peroxy bond, thereby increasing the singlet state excitation ratio and thus improving the chemiluminescence quantum yield.

[0152] Φ CL =Φ C ×Φ S ×Φ FL

[0153] Φ CL For chemiluminescence quantum yield; Φ C The ratio of excitable molecules; Φ S The singlet excitation ratio; Φ FL The fluorescence quantum yield is given.

[0154] Compared to 2a, 3a exhibits an increased chemical excitation rate (8 min for 3a, 12 min for 2a). The only structural difference between the two is that one is a five-membered ring and the other a six-membered ring, with the five-membered ring structure exhibiting greater rigidity.

[0155] Compared to 2a, the chemiluminescence quantum yield of 3a is slightly improved, with 3a (13.6%) and 2a (12.2%). The corresponding fluorescence quantum yields of the chemically excited products are 3a-de (75.1%) and 2a-de (47.5%). While the fluorescence quantum yield increases by approximately 1.6 times, the luminescence quantum yield only increases by approximately 1.1 times, according to the aforementioned formula for calculating chemiluminescence quantum yield. This indicates the presence of factors that reduce the singlet excitation ratio. Combined with the conclusions of chemical excitation kinetics, this may be due to the steric effect of the five-membered ring, which increases the electron transfer rate while reducing the electron transfer efficiency.

[0156] Compared to 2a, the chemical excitation rate of 4a decreases to some extent (30 min for 4a, 12 min for 2a). Both have a six-membered ring structure; the only difference is that in 4a, the carbon atom is replaced by an oxygen atom.

[0157] Compared to 2a, 4a exhibits a significant increase in chemiluminescence quantum yield, approximately three times higher than 2a (35.4%). Correspondingly, the fluorescence quantum yields of the chemically excited products, 4a-de (74.5%) and 2a-de (47.5%), are approximately 1.5 times higher, as indicated by the aforementioned chemiluminescence quantum yield calculation formula. This suggests the presence of factors increasing the singlet excitation ratio. Combined with the conclusions from chemical excitation kinetics, this could be due to the inductive / field effect of oxygen atoms, which reduces the electron transfer rate while increasing the efficiency of single-electron transfer or reverse electron transfer. Notably, 4a (35.4%, 560 nm) is currently the second highest chemiluminescent group after 1.25a (55%, 465 nm), and boasts the highest reported luminescence quantum yield above 500 nm, approaching that of most luciferin / luciferase systems (41.0 ± 7.4%).

[0158] Through structure-property relationship analysis of 2a, 3a, and 4a, this invention observes that the rigid five-membered ring space effect promotes the singlet state excitation rate while reducing the singlet state excitation efficiency; the inductive effect / field effect of oxygen atoms reduces the singlet state excitation rate while increasing the singlet state excitation efficiency.

[0159] Example 3: Design, Synthesis, and In Vitro Characterization of Near-Infrared Chemiluminescent Clusters

[0160] 3.1 Design and Synthesis of Near-Infrared Chemiluminescent Clusters

[0161] Through Example 2, dihydrogutane luminescent group 4a was screened out, which has a high chemiluminescence quantum yield (35.4%) and a long luminescence half-life (30 min). However, its emission wavelength is 560 nm. Compared with the visible light band (380-650 nm), the near-infrared band has stronger tissue penetration and a higher signal-to-noise ratio. Therefore, developing near-infrared luminescent groups is more suitable for in vivo imaging.

[0162] Currently, there are two main strategies known to extend the emission wavelength of the luminescent group within its parent structure, as shown in the figure:

[0163] The first strategy involves using donor-acceptor design to extend the π-conjugated system through electron-withdrawing chromophores, thereby reducing the energy gap between the singlet excited state and the ground state, and thus achieving...

[0164]

[0165] Near-infrared emission is one approach. The second approach involves replacing the oxygen atom in the luminescent group with a heavy atom (sulfur or selenium). The increased atomic radius and change in electronegativity lead to a smaller band gap between the singlet excited state and the ground state, resulting in near-infrared emission. However, the introduction of heavy atoms increases the rate of intersystem crossing, reducing the emission quantum yield. In contrast, the first strategy is simpler and more versatile, but the introduction of conjugated groups reduces molecular water solubility, increasing aggregation-induced emission quenching (ACQ) effects. Furthermore, the reduced rigidity of the structure also leads to a decrease in the emission quantum yield.

[0166] like Figure 8 As shown, this invention selects strategy one as the starting point, and designs and successfully synthesizes near-infrared luminescent groups 5a and 6a by introducing two electron-withdrawing chromophores into the reactive aldehyde group of luminescent group 4a.

[0167] The specific synthetic route is as follows: Figure 9 As shown, the near-infrared luminescent group uses 2.18 as the starting material and undergoes a Knoevenagel condensation reaction to condense with malononitrile (2.18a) or tricyanofuran chromophore (2.18b) to generate compounds 2.19 and 2.21. Then, TIPS protects the hydroxyl groups to generate 2.20 and 2.22, respectively. The photosensitizer generates singlet oxygen and undergoes a [2+2] cycloaddition reaction to generate near-infrared luminescent groups 5a and 6a.

[0168] Synthetic steps of chemiluminescent groups 5a and 6a:

[0169] Synthesis of compound 2.19

[0170]

[0171] Compound 2.18 (50 mg, 0.11 mmol) and a catalytic amount of piperidine were dissolved in anhydrous ethanol (5 mL), followed by the addition of compound 2.18a (22.4 mg, 0.34 mmol). The reaction was heated to 90 °C and stirred for 3 h. The reaction was monitored by TLC (methanol / dichloromethane = 1 / 50). After the reaction was complete, the reaction solution was concentrated under reduced pressure using a vacuum pump and separated by rapid column chromatography (methanol / dichloromethane = 1 / 200) to give a red solid 2.19 (43 mg, 78%). 1 H NMR (400MHz, CDCl3) δ7.96(s,1H),7.09(s,1H),6.80(s,1H),4.95(s,2H),4.53(s,2H),3.26(s,4H),2.23(s,1H),1.90(d,J=68.3Hz,12H). 13C NMR (101MHz, CDCl3) δ154.65,153.61,149.20,147.42,138.33,135.93,133.56,125.83,123.53,116.31,115.0 0,114.57,108.18,107.19,72.58,67.09,65.39,57.65,39.28,36.86,32.71,30.03,29.76,29.38,28.12.HRMS calcd for C 28 H 24 ClN2O4 – [M–H] – :487.14301; found:487.14301.

[0172] Synthesis of compound 2.20

[0173]

[0174] Compound 2.19 (32 mg, 0.065 mmol) and imidazole (6.6 mg, 0.097 mmol) were dissolved in anhydrous dichloromethane (5 mL), followed by the addition of triisopropylchlorosilane (14 μL, 0.065 mmol). The reaction was stirred at room temperature for 1 h. The reaction was monitored by TLC (ethyl acetate / petroleum ether = 1 / 10). After the reaction was complete, the reaction solution was concentrated under reduced pressure using a vacuum pump and separated by rapid column chromatography (ethyl acetate / petroleum ether = 1 / 20) to give a red solid 2.20 (31 mg, 75%). 1 H NMR (400MHz, CDCl3) δ7.99(s,1H),7.08(s,1H),6.65(s,1H),4.94(s,2H),4.52(s,2H),3.27(s ,1H),3.23(s,3H),2.21(s,1H),2.04–1.66(m,12H),1.35–1.28(m,3H),1.13(d,J=7.5Hz,18H). 13 C NMR (101MHz, CDCl3) δ154.95,154.49,149.93,147.62,138.71,135.29,132.32,125.90,123.56,118.61,116.37,115.25, 114.80,112.65,108.07,72.07,67.09,65.39,57.50,39.26,38.90,36.82,32.78,29.97,29.76,28.06,17.86,12.96.HRMS calcd for C 37 H46 ClN2O4Si + [M+H] + :645.29099; found:645.29120.

[0175] Synthesis of chemiluminescent group 5b

[0176]

[0177] Compound 2.20 (10 mg, 0.016 mmol) was dissolved in deuterated chloroform (5 mL), followed by the addition of a catalytic amount of methylene blue. The reaction was placed under a red light source at a wavelength of 650 nm, with oxygen participating in the reaction in a bubbling manner. The reaction was measured by TLC and... 1 After the reaction was completed (approximately 6 minutes) as monitored by ¹H NMR, the reaction solution was concentrated under reduced pressure using a vacuum pump and separated by preparative thin-layer chromatography on silica gel plates (ethyl acetate / petroleum ether = 1 / 5) to obtain a pair of inseparable rotational isomers, syn-5a and anti-5a (4.7 mg, 45%). The ratio of rotational isomers syn-5a to anti-5a was 2:1. 1 H NMR(400MHz, CDCl3)δ8.26(s,1H),8.00(s,1H),6.94(s,1H),5.02–4.90(m,2H),4.66–4.48(m,2H),3. 27(s,3H),3.05(s,1H),2.09–1.55(m,13H),1.38(q,J=7.4Hz,3H),1.14(d,J=7.3Hz,18H).syn-isomer 1 H NMR (400MHz, CDCl3) δ7.99(s,1H),7.60(s,1H),7.50(s,1H),5.02–4.90(m,2H),4.66–4.48(m,2 H),3.19(s,3H),3.05(s,1H),2.09–1.55(m,13H),1.38(q,J=7.4Hz,3H),1.14(d,J=7.3Hz,18H). 13C NMR (101MHz, CDCl3) δ154.87,154.00,153.78,150.24,149.96,147.64,147.58,130.57,130.12,126.35,124. 46,124.31,124.01,119.88,116.91,116.77,116.05,115.00,114.68,112.99,112.40,111.59,108.23,108.13 ,96.19,95.95,73.22,72.78,67.43,67.20,65.36,49.97,49.82,39.31,36.26,36.10,35.17,34.86,33.73,3 3.53,32.27,32.17,31.83,31.72,31.65,31.54,29.38,25.90,25.77,17.87,17.83,17.78,12.91,12.88.HRMS calcd for C 37 H 46 ClN2O6Si + [M+H] + :677.28082; found:677.28101.

[0178] Synthesis of Compound 2.21

[0179]

[0180] Compound 2.18 (20 mg, 0.046 mmol) and compound 2.18b were used. [ (10 mg, 0.050 mmol) was dissolved in anhydrous ethanol (5 mL), and the reaction was heated to 90 °C and stirred for 3 h. The reaction was monitored by TLC (methanol / dichloromethane = 1 / 50). After the reaction was complete, the reaction solution was concentrated under reduced pressure by a vacuum pump and separated by rapid column chromatography (methanol / dichloromethane = 1 / 200) to give 2.21 (19 mg, 68%) of a purple-black solid. 1 H NMR(400MHz, CDCl3)δ8.12(d,J=16.0Hz,1H),7.01(s,1H),6.78(s,1H),6.10(d,J=1 6.0Hz,1H),4.64(s,2H),4.56(s,2H),3.26(s,4H),2.24(s,1H),2.03–1.77(m,18H). 13C NMR (101MHz, CDCl3) δ176.23,173.61,153.65,153.60,149.40,138.45,137.53,135.62,133.30,124.33,114.97,114.85,112.68,111.89,111 .22,110.35,109.60,107.16,96.90,95.03,68.24,67.33,65.18,57.6 1,55.03,39.27,36.89,32.73,30.01,29.75,28.97,28.14,26.99.HRMS calcd for C 36 H 31 ClN3O5 – [M–H] – :620.19577; found:620.19574.

[0181] Synthesis of compound 2.22

[0182]

[0183] Compound 2.21 (70 mg, 0.11 mmol) and imidazole (13 mg, 0.19 mmol) were dissolved in anhydrous dichloromethane (5 mL), followed by the addition of triisopropylchlorosilane (36 μL, 0.17 mmol). The reaction was stirred at room temperature for 1 h. The reaction was monitored by TLC (ethyl acetate / petroleum ether = 1 / 2). After the reaction was complete, the reaction solution was concentrated under reduced pressure using a vacuum pump and separated by rapid column chromatography (ethyl acetate / petroleum ether = 1 / 2) to give a purple-black solid 2.22 (66 mg, 75%). 1 H NMR (400MHz, CDCl3) δ8.03(d,J=16.1Hz,1H),6.98(s,1H),6.63(s,1H),6.17(d,J=16.1Hz,1H),4.64(s,2H),4.5 6(s,2H),3.27(s,1H),3.24(s,3H),2.21(s,1H),2.06–1.73(m,18H),1.36–1.30(m,3H),1.14(d,J=7.5Hz,18H). 13C NMR (101MHz, CDCl3) δ176.05,173.87,154.24,153.75,150.02,138.78,1 37.51,135.05,132.23,124.45,124.18,118.20,115.26,112.73,112.23 ,111.94,111.06,110.35,109.74,96.92,95.57,67.32,65.14,57.44,54 .97,39.26,36.83,32.81,29.95,29.76,28.23,27.17,17.90,12.97.HRMS calcd for C 45 H 53 ClN3O5Si + [M+H] + :778.34375; found:778.34381.

[0184] Synthesis of chemiluminescent group 6a

[0185]

[0186] Compound 2.22 (10 mg, 0.013 mmol) was dissolved in deuterated chloroform (5 mL), followed by the addition of a catalytic amount of methylene blue. The reaction was placed under a red light source at a wavelength of 650 nm, with oxygen participating in the reaction in a bubbling manner. The reaction was measured by TLC and... 1 After the reaction was completed (approximately 10 min) as monitored by ¹H NMR, the reaction solution was concentrated under reduced pressure using a vacuum pump and separated by preparative thin-layer chromatography on silica gel plates (ethyl acetate / petroleum ether = 1 / 1.8) to yield a pair of inseparable rotational isomers, syn-6a and anti-6a (3.7 mg, 35%). The ratio of rotational isomers syn-6a to anti-6a was 2:1. 1 H NMR(400MHz, CDCl3)δ8.21(s,1H),8.17–8.00(m,1H),6.95(s,1H),6.22(dd,J=16.1,7.9Hz,1H),4.76–4.54( m,4H),3.32(s,3H),3.09(s,1H),2.17–1.66(m,19H),1.49–1.39(m,3H),1.19(d,J=7.5Hz,18H).syn-isomer 1H NMR (400MHz, CDCl3) δ8.17–8.00(m,1H),7.53(s,1H),7.51(s,1H),6.22(dd,J=16.1,7.9Hz,1H),4.76– 4.54(m,4H),3.23(s,3H),3.09(s,1H),2.17–1.66(m,19H),1.49–1.39(m,3H),1.19(d,J=7.5Hz,18H). 13 C NMR (101MHz, CDCl3) δ175.86,173.74,154.56,153.75,152.65,152.39,150.38,150.04,137.36,137.11,130.46,130.02,125.33,124.84,124. 56,122.44,119.38,117.40,116.31,114.54,114.19,113.45,112.61,1 12.51,112.45,111.83,111.75,111.64,110.99,110.89,110.37,110.3 2,110.05,96.99,96.95,96.44,96.23,95.97,67.67,67.44,65.09,49. 96,49.80,36.29,36.12,35.15,34.85,33.73,33.55,33.52,32.27,32. 21,31.85,31.73,31.64,31.52,29.76,29.38,27.13,26.94,25.91,25. 86,25.81,22.75,17.91,17.87,17.83,17.76,14.19,12.91,12.32.HRMS calcd for C 45 H 53 ClN3O7Si + [M+H] + :810.33358; found:810.33380.

[0187] Synthesis of compound 5a-de

[0188]

[0189] The synthesis of compound 5a-de was the same as that of compound 1a-de, and the red solid 5a-de (4.6 mg, 50%) was obtained by rapid column chromatography (methanol / dichloromethane = 1 / 10). 1H NMR (400MHz, DMSO-d6 with CD2Cl2) δ8.17(s,1H),8.05(s,1H),7.66(s,1H),5.03(s,2H),4.73(s,2H),4.05(s,3H). 13 C NMR(101MHz,DMSO-d6 with CD2Cl2)δ175.21,165.53,156.37,154.78,149.88,147.23,129.97,126.17,125 .82,124.62,116.70,115.61,113.87,112.24,108.14,71.06,66.77,64.82.HRMS calcdfor C 18 H 10 ClN2O5 – [M–H] – :369.02837; found:369.02863.

[0190] Synthesis of compound 6a-de

[0191]

[0192] The synthesis of compound 6a-de was the same as that of compound 1a-de, and the purplish-black solid 6a-de (8.8 mg, 70%) was obtained by rapid column chromatography (methanol / dichloromethane = 1 / 10). 1 H NMR(500MHz,DMSO-d6)δ8.20(d,J=15.8Hz,1H),7.79(s,1H),7.38(s,1H),6 .21(d,J=15.8Hz,1H),4.64(s,2H),4.57(s,2H),3.86(s,3H),1.69(s,6H). 13 C NMR(126MHz,DMSO-d6)δ177.96,174.44,165.64,155.90,152.99,150.09,137.63,130.12,126.04,125.58,123.82,115.0 8,114.42,113.83,112.92,112.39,112.13,111.57,110.89,98.71,92.93,66.97,65.11,53.16,52.42,25.98.HRMScalcd for C 26 H 17 ClN3O6 – [M–H] –:502.08114; found:502.08115.

[0193] 3.12 In vitro characterization analysis of near-infrared chemiluminescent groups

[0194] After the synthesis of near-infrared chemiluminescent groups 5a and 6a was completed, the luminescence kinetics of the chemiluminescent groups were analyzed. Figure 10 A and Figure 10 C), both exhibited typical chemiluminescence kinetic curves, with 5a reaching its maximum emission value at 18 min and 6a at 15 min. From the chemiluminescence spectra, the emission wavelength of luminescent group 5a, due to the introduction of the malononitrile structure, red-shifted by 80 nm to 640 nm. Figure 10 B) Approaching the near-infrared band (650nm-900nm), the emission wavelength of luminescent group 6a is red-shifted by 220nm due to the introduction of the tricyanofuran chromophore, reaching 780nm. Figure 10 D). Regarding the fluorescence spectra of the chemically excited products, this invention observed differences compared to the 1a-4a luminescent groups. Compared to the emission spectra, the fluorescence spectra of 5a-de (630nm) differed by 10nm and 6a-de (760nm) by 20nm. This may be due to the different forms of chemiluminescent and fluorescent emitters under aqueous conditions.

[0195] The chemiluminescence quantum yield was calculated using chemiluminescence kinetic curves. The 5a (0.72%) and 6a (1.2%) yields showed a significant decrease compared to 4a, consistent with the near-exponential increase of the nonradiative rate constant as the bandgap decreases (emission wavelength increases) (bandgap law). Simultaneously, this invention observed precipitation of 5a and 6a in PBS buffer solution after prolonged storage during testing. This phenomenon led this invention to consider that the absence of any water-soluble groups in the 5a and 6a molecular structures, and their poor water solubility, could lead to intermolecular π-π interactions, resulting in aggregation-induced emission quenching (ACQ) and consequently a decrease in luminescence quantum yield.

[0196] In summary, this invention successfully synthesized near-infrared luminescent groups 5a and 6a by introducing two electron-withdrawing chromophores into the reactive aldehyde group of chemical group 4a. This demonstrates the good scalability of the chemical structure of the dihydroxanthonium core, allowing for the derivation of chemiluminescent groups with different emission wavelengths through simple π-conjugated structures. Analysis of theoretical and experimental phenomena suggests that reducing the aggregation-induced emission quenching (ACQ) effect of the chemiluminescent groups may further improve the luminescence quantum yield.

[0197] Example 4: Design, Synthesis, and In Vitro Characterization of Water-Soluble Near-Infrared Chemiluminescent Clusters

[0198] 4.1 Design and Synthesis of Water-Soluble Near-Infrared Chemiluminescent Clusters

[0199] This embodiment improves solubility and reduces intermolecular π-π interactions by modifying water-soluble groups, thereby avoiding the ACQ effect. To avoid the structural modification affecting the chemiluminescence wavelength, this invention uses ethyl esters or amides with conjugated electron-withdrawing effects to replace the electron-withdrawing cyano groups in the chromophore, such as... Figure 11 As shown, this invention synthesizes ethyl ester-derived 5b and 6b luminescent groups, and polyethylene glycol chain-derived amide 5c and ethyl ester 6c luminescent groups.

[0200] The specific synthetic route is as follows Figure 12 As shown, water-soluble near-infrared luminescent groups 5b and 5c are synthesized from 2.18 via a Knoevenagel condensation reaction, reacting with ethyl cyanoacetate (2.18c) and cyanotris(polyethylene glycol)amide (2.18d) to generate 2.23 and 2.25. The hydroxyl group of 2.17 is protected by a TIPS substituent before reacting with singlet oxygen to generate 5b. 2.18 reacts directly with singlet oxygen without TIPS protection to generate 5c.

[0201] The water-soluble near-infrared luminescent groups 6b and 6c are also synthesized using compound 2.18 as a starting material. Through a Knoevenagel condensation reaction, they react with tricyanofuran chromophore derivatives 2.18e and 2.18f to generate 2.26 and 2.28. The hydroxyl group of 2.26 is protected by a TIPS substituent, and then reacts with singlet oxygen to generate 6b. 2.28 reacts with azide octadecyl (polyethylene glycol) via a copper ion-catalyzed Click reaction to generate 2.29. Since 2.29 does not yield the expected product with TIPSCl, this invention uses 2.29 to directly react with singlet oxygen to generate 6c. Because the protecting groups of 5c and 6c are different from the other luminescent groups, this invention uses 2.23 to directly react with singlet oxygen to generate 5b-OH, ensuring that the variables for 5b-OH, 5c, and 6c are the same in chemical kinetic experiments.

[0202] The steps for synthesizing chemiluminescent groups 5b, 5c, 6b, and 6c are as follows:

[0203] Synthesis of compound 2.23

[0204]

[0205] Compound 2.18 (30 mg, 0.068 mmol) and a catalytic amount of piperidine were dissolved in anhydrous ethanol (5 mL), followed by the addition of compound 2.18c (7.2 μL, 0.082 mmol). The reaction was heated to 90 °C and stirred for 3 h. The reaction was monitored by TLC (methanol / dichloromethane = 1 / 50). After the reaction was complete, the reaction solution was concentrated under reduced pressure using a vacuum pump and separated by rapid column chromatography (ethyl acetate / dichloromethane = 1 / 10) to give a red solid 2.23 (33 mg, 92%). 1 H NMR (400MHz, CDCl3) δ8.63(s,1H),6.94(s,1H),6.75(s,1H),5.01(s,2H),4.51(s,2H),4. 33(q,J=7.1Hz,3H),3.26(s,4H),2.23(s,1H),2.02–1.76(m,12H),1.37(t,J=7.1Hz,3H). 13 C NMR (101MHz, CDCl3) δ164.15,154.00,153.22,149.28,144.25,138.54,135.22,133.02,124.05,123.68,117.57,114.56,114.31 ,107.67,107.30,94.88,67.23,65.97,62.10,57.54,39.26,36.91,32.69,29.98,29.76,29.38,28.16,22.75,14.30,14.19.HRMS calcd for C 30 H 29 ClNO6 – [M–H] – :534.16889; found:534.16907.

[0206] Synthesis of compound 2.24

[0207]

[0208] Compound 2.23 (33 mg, 0.062 mmol) and imidazole (10 mg, 0.16 mmol) were dissolved in anhydrous dichloromethane (5 mL), followed by the addition of triisopropylchlorosilane (26 μL, 0.12 mmol). The reaction was stirred at room temperature for 1 h. The reaction was monitored by TLC (ethyl acetate / petroleum ether = 1 / 10). After the reaction was complete, the reaction solution was concentrated under reduced pressure using a vacuum pump and separated by rapid column chromatography (ethyl acetate / petroleum ether = 1 / 20) to give red solid 2.24 (36 mg, 85%). 1H NMR(400MHz, CDCl3)δ8.67(s,1H),6.92(s,1H),6.60(s,1H),5.01(s,2H),4.51(s,2H),4.36–4.23(m,2H ),3.26(s,1H),3.23(s,3H),2.21(s,1H),2.03–1.62(m,12H),1.39–1.30(m,6H),1.12(d,J=7.4Hz,18H). 13 C NMR (101MHz, CDCl3) δ164.09,154.18,153.99,149.97,144.42,138.93,134.57,131.78,124.18,123.61,118.05,117.71,114 .86,112.76,107.58,94.67,67.23,65.96,61.99,57.37,39.24,38.94,36.88,32.76,29.92,28.26,17.87,14.32,12.94.HRMS calcdfor C 39 H 51 ClNO6Si + [M+H] + :692.31687; found:692.31702.

[0209] Synthesis of chemiluminescent group 5b

[0210]

[0211] Compound 2.24 (10 mg, 0.014 mmol) was dissolved in deuterated chloroform (5 mL), followed by the addition of a catalytic amount of methylene blue. The reaction was placed under a red light source at a wavelength of 650 nm, with oxygen participating in the reaction in a bubbling manner. The reaction was measured by TLC and... 1 After the reaction was complete (approximately 6 minutes) as monitored by ¹H NMR, the reaction solution was concentrated under reduced pressure using a vacuum pump and separated by preparative thin-layer chromatography on silica gel plates (ethyl acetate / petroleum ether = 1 / 5) to obtain a pair of inseparable rotational isomers, syn-5b and anti-5b (5.5 mg, 53%). The ratio of rotational isomers syn-5b to anti-5b was 2:1. 1H NMR (400MHz, CDCl3) δ8.11(d,J=5.5Hz,1H),7.96(d,J=8.4Hz,1H),6.87(s,1H),5.09–4.96(m,2H),4.65–4.47(m,2H),4 .35–4.24(m,2H),3.27(s,3H),3.04(s,1H),2.13–1.58(m,13H),1.40–1.33(m,6H),1.13(d,J=7.5Hz,18H).syn-isomer 1 H NMR(400MHz, CDCl3)δ8.67(d,J=7.7Hz,1H),7.45(s,1H),7.43(s,1H),5.09–4.96(m,2H),4.65–4.47(m,2H), 4.35–4.24(m,2H),3.19(s,3H),3.04(s,1H),2.13–1.58(m,13H),1.40–1.33(m,6H),1.13(d,J=7.5Hz,18H). 13 C NMR (101MHz, CDCl3) δ163.85,154.39,153.56,153.05,150.38,150.05,144.88,144.34,144.21,129.62,125.11,124.59,1 24.13,122.20,121.99,119.27,117.50,116.16,114.80,113.08,112.53,111.72,107.77,107.64,96.23,95.97,95.54,95 .15,68.40,67.72,67.57,67.34,65.92,62.11,62.06,61.95,49.94,49.79,36.30,36.15,35.12,34.84,33.64,33.50,33. 42,32.24,31.88,31.73,31.62,31.54,29.76,25.94,25.85,25.77,17.89,17.84,17.80,14.31,14.19,12.91,12.87.HRMS calcd for C 39 H 51 ClNO8Si + [M+H] + :724.30670; found:724.30682.

[0212] Synthesis of the chemiluminescent group 5b-OH

[0213]

[0214] Compound 2.23 (10 mg, 0.019 mmol) was dissolved in deuterated chloroform (5 mL), followed by the addition of a catalytic amount of methylene blue. The reaction was placed under a red light source at a wavelength of 650 nm, with oxygen participating in the reaction in a bubbling manner. The reaction was measured by TLC and... 1 After the reaction was completed (approximately 6 minutes) as monitored by ¹H NMR, the reaction solution was concentrated under reduced pressure using a vacuum pump and separated by preparative thin-layer chromatography on silica gel plates (ethyl acetate / dichloromethane = 1 / 10) to yield a pair of inseparable rotational isomers, syn-5b-OH and anti-5b-OH (5.6 mg, 52%). The ratio of syn-5b-OH to anti-5b-OH was 3:1. 1 H NMR(500MHz, CDCl3)δ8.62(s,1H),8.12(s,1H),7.00(s,1H),6.46(br s,1H),5.05–4.97(m,2H),4.61–4.48(m,2H),4.33(q,J=7.1Hz,2H),3.27(s,3H),3.03(s,1H),2.10–1.46(m,13H),1.36(t,J=7.1Hz,3H). anti-isomer 1 H NMR(500MHz, CDCl3)δ8.62(s,1H),7.54(s,1H),7.47(s,1H),6.69(br s,1H),5.05–4.97(m,2H),4.61–4.48(m,2H),4.33(q,J=7.1Hz,2H),3.19(s,3H),3.03(s,1H),2.10–1.46(m,13H),1.36(t,J=7.1Hz,3H). 13 C NMR (126MHz, CDCl3) δ163.84,153.79,152.82,149.49,143.95,130.85,125.00,122.14,117.21,115.98,112.81,111.62,109.64,107.92,9 5.95,95.85,67.27,65.88,62.13,49.74,47.01,39.33,39.24,36.32,35.12,33.40,33.33,32.06,31.67,27.50,25.97,25.80,14.19.HRMS calcdfor C 30 H 29 ClNO8 – [M–H]– :566.15872; found:566.15887.

[0215] Synthesis of compound 2.25

[0216]

[0217] Compound 2.18 (80 mg, 0.18 mmol) and a catalytic amount of piperidine were dissolved in anhydrous ethanol (5 mL), followed by the addition of compound 2.18d (125 mg, 0.55 mmol). The reaction was heated to 90 °C and stirred for 3 h. The reaction was monitored by TLC (ethyl acetate / dichloromethane = 1 / 1). After the reaction was complete, the reaction solution was concentrated under reduced pressure using a vacuum pump and separated by rapid column chromatography (ethyl acetate / dichloromethane = 1 / 1) to give a red solid 2.25 (100 mg, 85%). 1 H NMR (500MHz, CDCl3) δ8.67(s,1H),6.87(s,1H),6.77(t,J=5.2Hz,1H),6.70(s,1H),4.94(s,2H ),4.48(s,2H),3.67–3.56(m,12H),3.37(s,3H),3.24(s,4H),2.22(s,1H),2.00–1.69(m,12H). 13 C NMR (126MHz, CDCl3) δ162.28,153.73,153.35,149.45,142.19,138.76,134.70,132.59,123.78,123.01,118.72,114.38,114.27 ,107.76,107.23,96.21,71.98,70.60,70.54,69.56,67.25,66.02,58.97,57.36,40.30,39.23,36.93,32.68,29.95,28.19.HRMS calcd for C 35 H 40 ClN2O8 – [M–H] – :651.24787; found:651.24890.

[0218] Synthesis of chemiluminescent group 5c

[0219]

[0220] Compound 2.25 (10 mg, 0.015 mmol) was dissolved in deuterated chloroform (5 mL), followed by the addition of a catalytic amount of methylene blue. The reaction was placed under a red light source at a wavelength of 650 nm, with oxygen participating in the reaction in a bubbling manner. The reaction was measured by TLC and... 1 After the reaction was complete (approximately 6 minutes) as monitored by ¹H NMR, the reaction solution was concentrated under reduced pressure using a vacuum pump and separated by preparative thin-layer chromatography on silica gel plates (ethyl acetate / dichloromethane = 1 / 1) to yield a pair of inseparable rotational isomers, syn-5c and anti-5c (4.5 mg, 43%). The ratio of syn-5c to anti-5c was 3:1. 1 H NMR (400MHz, CDCl3) δ8.68(s,1H),8.07(s,1H),6.96(s,1H),6.78(s,1H),5.03–4.92(m,2H),4.59–4. 46(m,2H),3.69–3.55(m,12H),3.38(s,3H),3.27(s,3H),3.02(s,1H),2.12–1.49(m,13H).syn-isomer 1 H NMR(400MHz, CDCl3)δ8.68(s,1H),7.51(s,1H),7.41(s,1H),6.78(s,1H),5.03–4.92(m,2H),4. 59–4.46(m,2H),3.69–3.55(m,12H),3.38(s,3H),3.18(s,3H),3.02(s,1H),2.12–1.49(m,13H). 13 C NMR (101MHz, CDCl3) δ162.22,154.35,153.48,152.44,149.75,142.16,131.02,130.37,124.8 3,123.83,121.82,118.67,116.16,113.26,112.55,111.83,110.13,107.56,97.00,96.61,96 .30,95.91,72.08,70.72,70.63,69.59,67.72,67.47,66.12,59.14,50.11,49.85,40.44,36. 42,35.21,34.91,33.70,33.42,33.35,32.46,32.19,31.76,31.70,29.81,26.05,25.87.HRMS calcd for C 35 H 40 ClN2O 10– [M–H] – :683.23770; found:683.23773.

[0221] Synthesis of compound 2.18e

[0222]

[0223] Compound 2.18-1 (440 mg, 2.93 mmol) was dissolved in compound 2.18c (2.2 mL). This pale yellow reaction solution was treated under vacuum in a rotary evaporator for 1 h, followed by nitrogen re-purging. The mixture was then heated to 100 °C and stirred for 48 h. After cooling to room temperature, excess compound 2.18c was removed using a rotary evaporator under mild water bath conditions. Rapid column chromatography (ethyl acetate / petroleum ether / dichloromethane = 1 / 5 / 0.1) yielded a pair of inseparable cis-trans isomers, 2.18eZ and 2.18eE (360 mg, 50%). The ratio of cis-trans isomers 2.18eZ to 2.18eE was 1.5:1. 2.18eE 1 H NMR(500MHz, CDCl3)δ4.20(q,J=7.1Hz,2H),2.29(s,3H),1.51(s,6H),1.27–1.19(m,3H).2.18eZ 1 H NMR (500MHz, CDCl3) δ4.15 (q, J = 7.1Hz, 2H), 2.27 (s, 3H), 1.52 (s, 6H), 1.27–1.19 (m, 3H). 13 CNMR (126MHz, CDCl3) δ184.04,181.62,173.41,171.30,162.17,161.53,114.36,113.94,110.84,110. 15,106.09,106.03,98.75,98.72,96.00,78.96,61.51,61.29,24.38,24.36,14.17,14.14,13.94.HRMS calcd for C 13 H 15 N2O3 + [M+H] + :247.10772; found:247.10719.

[0224] Synthesis of compound 2.18f

[0225]

[0226] Compound 2.18-1 (440 mg, 2.93 mmol) was dissolved in compound 2.18-2 (3 mL). The pale yellow reaction solution was treated under vacuum in a rotary evaporator for 1 h, followed by nitrogen re-purging. The mixture was then heated to 100 °C and stirred for 48 h. After cooling to room temperature, excess compound 2.18-2 was removed using a rotary evaporator under mild water bath conditions. Rapid column chromatography (ethyl acetate / petroleum ether = 1 / 2) yielded a pair of inseparable cis-trans isomers, 2.18fZ and 2.18fE (382 mg, 46%). The ratio of cis-trans isomers 2.18fZ to 2.18fE was 2:1. 2.18fE... 1 H NMR(400MHz, CDCl3)δ4.35(t,J=6.3Hz,2H),2.37–2.29(m,5H),1.98–1.87(m,3H),1.58(s,6H).2.18fZ 1 H NMR (400MHz, CDCl3) δ4.29 (t, J = 6.2Hz, 2H), 2.37–2.29 (m, 5H), 1.98–1.87 (m, 3H), 1.59 (s, 6H). 13 C NMR (101MHz, CDCl3) δ183.63,180.99,173.52,171.24,162.25,161.52,114.27,113.74,110.87,110. 08,106.37,98.60,95.89,82.96,69.25,69.20,64.22,63.83,27.44,24.57,24.52,15.16,13.98.HRMS calcd for C 16 H 17 N2O3 + [M+H] + :285.12337; found:285.12366.

[0227] Synthesis of compound 2.26

[0228]

[0229] Compound 2.18 (30 mg, 0.068 mmol) and compound 2.18e (20 mg, 0.082 mmol) were dissolved in anhydrous ethanol (5 mL). The reaction was heated to 90 °C and stirred for 3 h. The reaction was monitored by TLC (methanol / dichloromethane = 1 / 200). After the reaction was complete, the reaction solution was concentrated under reduced pressure by a vacuum pump and separated by rapid column chromatography (methanol / dichloromethane = 1 / 50) to give a purple-black solid 2.26 (29.5 mg, 65%). 1H NMR (400MHz, CDCl3) δ8.02(dd,J=59.2,16.3Hz,1H),6.93(d,J=24.9Hz,1H),6.79(d,J=24.2Hz,1H),6.32(dd,J=20.3,16.3Hz,1 H),4.70–4.50(m,4H),4.32(qd,J=7.1,2.7Hz,2H),3.26(s,4H),2.26(d,J=17.8Hz,1H),2.08–1.75(m,18H),1.39–1.33(m,3H). 13 C NMR (101MHz, CDCl3) δ175.53,175.34,174.18,173.61,164.60,163.12,154.98,153.61,153.42,152.39,149.85,149.38 ,138.96,138.63,137.43,136.36,135.11,134.98,133.01,132.88,124.35,123.94,123.84,122.95,116.28,114.82,114 .60,114.46,114.31,112.48,111.71,111.12,110.22,109.89,107.44,107.08,98.90,97.40,97.13,93.36,67.37,65.1 6,64.89,61.63,61.41,57.56,57.53,39.49,39.28,36.90,32.73,29.98,29.76,28.06,27.30,27.18,14.42,14.38.HRMS calcd for C 38 H 36 ClN2O7 – [M–H] – :667.22165; found:667.22284.

[0230] Synthesis of compound 2.27

[0231]

[0232] Compound 2.26 (15 mg, 0.023 mmol) and imidazole (4 mg, 0.051 mmol) were dissolved in anhydrous dichloromethane (5 mL), followed by the addition of triisopropylchlorosilane (10 μL, 0.046 mmol). The reaction was stirred at room temperature for 1 h. The reaction was monitored by TLC (ethyl acetate / petroleum ether = 1 / 2.5). After the reaction was complete, the reaction solution was concentrated under reduced pressure using a vacuum pump and separated by rapid column chromatography (ethyl acetate / petroleum ether = 1 / 3) to give a purple-black solid 2.27 (14 mg, 78%). 1 H NMR (500MHz, CDCl3) δ7.92(d,J=16.3Hz,1H),6.87(d,J=3.8Hz,1H),6.60(d,J=2.4Hz,1H),6.31(dd,J=52.2,16.3Hz,1H),4.71–4.47(m,4H),4.2 9(dq,J=36.7,7.1Hz,2H),3.27(s,1H),3.24(s,3H),2.21(s,1H),2.05– 1.69(m,18H),1.33(td,J=7.4,4.9Hz,6H),1.14(dd,J=7.5,1.7Hz,18H). 13 C NMR (126MHz, CDCl3) δ175.25,173.79,173.03,172.14,163.05,162.37,153.86,153.83,152.14,149.93,138.9 4,136.15,135.76,134.55,131.97,131.92,124.62,124.58,122.49,122.41,117.75,117.73,115.58,115.23,1 12.30,111.64,111.25,110.74,110.21,109.92,96.02,93.24,67.35,65.19,65.15,61.27,60.98,57.25,39.25 ,36.87,32.82,29.92,29.68,29.34,28.18,27.27,27.18,22.66,17.84,14.32,14.28,14.06,12.97.HRMScalcd for C 47 H 58 ClN2O7Si + [M+H] + :825.36963; found:825.36969.

[0233] Synthesis of chemiluminescent group 6b

[0234]

[0235] Compound 2.27 (10 mg, 0.012 mmol) was dissolved in deuterated chloroform (5 mL), followed by the addition of a catalytic amount of methylene blue. The reaction was placed under a red light source at a wavelength of 650 nm, with oxygen participating in the reaction in a bubbling manner. The reaction was measured by TLC and... 1 After the reaction was completed (approximately 10 min) as monitored by ¹H NMR, the reaction solution was concentrated under reduced pressure using a vacuum pump and separated by preparative thin-layer chromatography on silica gel plates (ethyl acetate / petroleum ether = 1 / 2.5) to yield a pair of inseparable rotational isomers, syn-6b and anti-6b (3.7 mg, 35%). The ratio of rotational isomers syn-6b to anti-6b was 2:1. 1 H NMR(500MHz, CDCl3)δ8.06(s,1H),7.96–7.86(m,1H),6.87(s,1H),6.34(dd,J=53.4,16.4Hz,1H),4.69–4.50(m,4H),4.35– 4.23(m,2H),3.28(s,3H),3.04(s,1H),2.14–1.58(m,19H),1.34(q,J=7.5Hz,6H),1.15(dd,J=5.7,3.4Hz,18H).syn-isomer 1 H NMR(500MHz, CDCl3)7.96–7.86(m,1H),7.44(s,1H),7.38(s,1H),6.34(dd,J=53.4,16.4Hz,1H),4.69–4.50(m,4H),4 .35–4.23(m,2H),3.19(s,3H),3.04(s,1H),2.14–1.58(m,19H),1.34(q,J=7.5Hz,6H),1.15(dd,J=5.7,3.4Hz,18H). 13C NMR (126MHz, CDCl3) δ175.19,173.74,172.94,172.05,163.05,162.35,154. 18,153.38,150.31,149.94,135.83,135.69,135.43,129.75,129.70,125.4 3,124.94,123.07,121.03,118.90,117.55,117.40,115.82,115.60,114.91,113.42,112.45,112.29,111.75,111.66,111.23,110.29,110.00,109.90, 99.55, 96.17, 95.94, 93.33, 67.68, 67.44, 65.11, 61.41, 61.12, 52.83, 49.89, 49.73, 46.98, 39.27, 36.27, 36.11, 35.08, 34.80, 33.63, 33.50, 33.43, 32 .20,31.83,31.69,31.59,31.48,29.71,27.44,27.27,27.18,25.89,25.76, 22.71,17.88,17.84,17.80,17.72,14.36,14.31,14.15,12.87,12.82.HRMS calcd for C 47 H 58 ClN2O9Si + [M+H] + :857.35946; found:857.35956.

[0236] Synthesis of compound 2.28

[0237]

[0238] Compound 2.18 (200 mg, 0.45 mmol) and compound 2.18f (193 mg, 0.68 mmol) were dissolved in anhydrous ethanol (5 mL). The reaction was heated to 90 °C and stirred for 3 h. The reaction was monitored by TLC (ethyl acetate / petroleum ether = 1 / 1). After the reaction was complete, the reaction solution was concentrated under reduced pressure by a vacuum pump and separated by rapid column chromatography (ethyl acetate / petroleum ether = 1 / 1.5) to give a purple-black solid 2.28 (212 mg, 67%). 1H NMR (400MHz, CDCl3) δ8.69(br,1H),8.02(dd,J=53.8,16.3Hz,1H),6.93(d,J=23.4Hz,1H),6.79(d,J=22.6Hz,1H),6.32(dd,J=22.4,16.3Hz,1 H),4.73–4.51(m,4H),4.35(td,J=6.3,4.9Hz,2H),3.27(s,4H),2.35(d td,J=9.5,7.0,2.6Hz,2H),2.26(d,J=17.9Hz,1H),2.03–1.68(m,21H). 13 C NMR (101MHz, CDCl3) δ175.63,175.20,174.36,173.61,164.33,163.04,154.81,153.54,153.38,152.46,149.79,149.35,138.85 ,138.58,137.35,136.43,135.16,135.00,133.05,132.91,124.37,123.90,123.01,116.01,114.85,114.57,114.46,114.39,112 .44,111.65,111.10,110.27,109.94,107.40,107.05,98.78,97.34,97.18,93.43,83.14,82.91,69.30,69.12,67.31,65.15,64. 91,63.90,63.85,57.53,39.45,39.27,38.86,36.89,32.73,29.98,29.75,28.07,27.62,27.51,27.28,27.16,15.24,15.20.HRMS calcd for C 41 H 38 ClN2O7 – [M–H] – :705.23730; found:705.23773.

[0239] Synthesis of compound 2.29

[0240]

[0241] Compound 2.28 (30 mg, 0.036 mmol) and azide-octaethylene glycol monomethyl ether (22 mg, 0.054 mmol) were dissolved in a mixed solvent (dimethyl sulfoxide / deionized water / tetrahydrofuran = 1 / 1 / 0.1, v / v / v). Tris(3-hydroxypropyltriazolylmethyl)amine (2.5 mg, 0.0058 mmol), ascorbic acid (27 mg, 0.14 mmol), and copper sulfate pentahydrate (5 mg, 0.023 mmol) were then added. The reaction was stirred at room temperature for 1 h under nitrogen protection. The reaction was monitored by TLC (methanol / dichloromethane = 1 / 15). After the reaction was complete, the reaction solution was diluted with saturated ammonium chloride solution and extracted three times with ethyl acetate. The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate solid, and filtered. After filtration, the filtrate was concentrated under reduced pressure using a vacuum pump and then separated by rapid column chromatography (methanol / dichloromethane = 1 / 15) to obtain 2.29 g (38 mg, 95%) of a purplish-black solid. 1 H NMR (400MHz, CDCl3) δ7.99 (dd, J=25.4, 16.2Hz, 1H), 7.59 (s, 1H), 6.93 (d, J=8.2 Hz,1H),6.80(s,1H),6.26(dd,J=19.0,16.2Hz,1H),4.65–4.49(m,6H),4.26(q,J =6.8Hz,2H),3.85(t,J=5.1Hz,2H),3.63–3.52(m,28H),3.35(s,3H),3.24(s,4H ),2.85(q,J=6.8Hz,2H),2.23(s,1H),2.10(t,J=7.0Hz,2H),1.98–1.65(m,18H). 13C NMR (101MHz, CDCl3) δ175.65,174.44,174.35,173.17,163.48,163.19,155.17,155.03,153.27,152.96,149.80,149.68,138.8 7,138.77,136.93,136.66,134.75,134.65,132.70,123.87,123.61,122.72,115.89,114.86,114.20,112.56,111.79,110.58,1 10.05,109.80,107.50(d,J=3.8Hz),98.18,97.30,96.86,93.42,71.92,70.57,70.54,70.48,69.53,67.34,65.18,65.04,64.2 9,64.09,59.05,57.49,57.46,50.28,39.21,38.88,36.90,32.67,29.93,29.73,28.30,28.19,27.31,27.21,22.06,21.95.HRMS calcd for C 58 H 74 ClN5NaO 15 + [M+Na] + :1138.47622; found:1138.47644.

[0242] Synthesis of chemiluminescent group 6c

[0243]

[0244] Compound 2.29 (10 mg, 0.008 mmol) was dissolved in deuterated chloroform (5 mL), followed by the addition of a catalytic amount of methylene blue. The reaction was placed under a red light source at a wavelength of 650 nm, with oxygen participating in the reaction in a bubbling manner. The reaction was measured by TLC and... 1 After the reaction was completed (approximately 10 min) as monitored by ¹H NMR, the reaction solution was concentrated under reduced pressure using a vacuum pump and separated by preparative thin-layer chromatography on silica gel plates (chloroform / acetone = 1 / 1) to obtain a pair of inseparable rotational isomers, syn-6c and anti-6c (3.7 mg, 35%). The ratio of rotational isomers syn-6c to anti-6c was 2:1. 1H NMR(500MHz, CDCl3)δ8.10(d,J=8.6Hz,1H),8.01(t,J=17.2Hz,1H),7.59(s,1H ),7.06(d,J=16.3Hz,1H),6.29(dd,J=16.2,13.1Hz,1H),4.59–4.52(m,6H),4.3 4–4.27(m,2H),3.87(t,J=5.2Hz,2H),3.65–3.53(m,28H),3.37(s,3H),3.27(d ,J=4.1Hz,3H),3.04(s,1H),2.92–2.84(m,2H),2.29–1.70(m,21H).syn-isomer 1 H NMR (500MHz, CDCl3) δ8.01(t,J=17.2Hz,1H),7.59(s,1H),7.56(s,1H),7.43(d,J=7.4Hz,1H),6.29(dd,J=16.2,13.1Hz,1H),4.59–4.52(m,6H),4.34 –4.27(m,2H),3.87(t,J=5.2Hz,2H),3.65–3.53(m,28H),3.37(s,3H),3.20 (d,J=2.7Hz,3H),3.04(s,1H),2.92–2.84(m,2H),2.29–1.70(m,21H).HRMS calcd forC 58 H 73 ClN5O 17 – [M–H] – :1146.46955; found:1146.46729.

[0245] Synthesis of compound 5b-de

[0246]

[0247] The synthesis of compound 5b-de was the same as that of compound 1a-de, and the red solid 5b-de (4.7 mg, 45%) was obtained by rapid column chromatography (methanol / dichloromethane = 1 / 10). 1 H NMR(400MHz,DMSO-d6 with CD2Cl2)δ9.02(s,1H),8.31(s,1H),7.91(s,1H),5.39(s,2H),4.99(s,2H),4.73(q,J=6.5Hz,2H),4.34(s,3H),1.32(t,J=7.1Hz,3H). 13C NMR(101MHz,DMSO-d6 with CD2Cl2)δ175.40,165.74,163.85,155.83,153.71,150.00,143.63,129.91,125.24,124.97,12 3.52,117.53,114.98,114.11,112.59,107.48,94.75,67.19,65.59,61.95,14.13,14.02.HRMS calcd forC 20 H 15 ClNO7 – [M–H] – 416.05425; found: 416.05457.

[0248] Synthesis of compound 5c-de

[0249]

[0250] The synthesis of compound 5c-de involved dissolving a chemiluminescent group (0.025 mmol) in dimethyl sulfoxide (5 mL), followed by the addition of PBS (2 mL, pH 7.4). The reaction was stirred at room temperature for 1 h. The reaction was monitored by TLC. After completion, the reaction solution was diluted with saturated ammonium chloride solution and extracted three times with ethyl acetate. The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate solid, and filtered. The filtrate was concentrated under reduced pressure using a vacuum pump and separated by rapid column chromatography (methanol / dichloromethane = 1 / 10) to obtain a red solid 5c-de (10 mg, 76%). (4.83 (s, 2H), 4.45 (s, 2H), 3.91 (s, 3H), 3.69–3.56 (m, 12H), 3.37 (s, 3H). 13 C NMR (101MHz, CDCl3) δ165.57,162.36,154.05,152.36,149.52,141.80,125.42,124.75,122.22,118.51,11 5.03,114.94,113.13,107.52,96.50,71.95,70.59,70.51,69.51,67.23,65.69,59.05,52.71,40.39.HRMS calcd forC 25 H 26 ClN2O9 – [M–H] – :533.13323; found:533.13348.

[0251] Synthesis of compound 6b-de

[0252]

[0253] The synthesis of compound 6b-de was the same as that of compound 1a-de, and the purplish-black solid 6b-de (11 mg, 80%) was obtained by rapid column chromatography (methanol / dichloromethane = 1 / 10). 1 H NMR (400MHz, DMSO-d6) δ8.06(dd,J=16.0,9.5Hz,1H),7.70(d,J=8.9Hz,1H),7.34(s,1H),6.23(dd,J=16.1,10.2Hz,1H ),4.62(s,2H),4.55(s,2H),4.16(dq,J=27.1,7.1Hz,2H),3.84(s,3H),1.68(d,J=18.4Hz,6H),1.32(t,J=7.1Hz,3H). 13 C NMR(101MHz,DMSO-d6)δ175.92,173.81,173.71,165.74,162.96,162.19,155.65,155.55,152.00,151.63,149.9 7,136.66,136.16,130.15,126.09,125.55,125.46,122.73,122.40,115.57,114.62,114.50,114.37,112.91,111 .91,111.48,111.10,111.00,97.41,96.88,95.97,94.79,79.68,75.84,66.95,65.01,64.82,61.16,60.91,55.4 3,53.23,35.60,31.79,30.32,29.53,29.33,29.22,29.07,27.07,26.59,26.34,25.61,22.61,14.68,14.47.HRMS calcd for C 28 H 22 ClN2O8 – [M–H] – :549.10702; found:549.10742.

[0254] Synthesis of compound 6c-de

[0255]

[0256] The synthesis of compound 6c-de was the same as that of compound 5c-de, and the purplish-black solid 6c-de (18 mg, 74%) was obtained by rapid column chromatography (methanol / dichloromethane = 1 / 10). 1 H NMR (400MHz, CDCl3) δ8.06–7.75(m,2H),7.71–7.44(m,2H),6.25(dd,J=16.4,6.0Hz,1H),4.73–4.42(m,6H),4.31(s,2 H),3.95–3.84(m,5H),3.66–3.53(m,28H),3.37(s,3H),2.88(t,J=8.8Hz,2H),2.14(s,2H),1.87(s,3H),1.79(s,3H). 13 C NMR (101MHz, CDCl3) δ175.20,174.07,173.93,165.68,163.24,163.00,154.86,154. 64,151.96,151.57,149.66,136.33,136.12,122.75,122.42,115.14,115.00,111.37 ,110.73,98.96,96.70,93.45,71.95,70.62,70.58,70.53,69.55,67.45,65.06,64.9 0,64.33,64.11,59.08,52.74,50.36,40.95,29.76,28.36,27.22,22.06,21.98.HRMS calcd for C 48 H 59 ClN5O 16 – [M–H] – :996.36508; found:996.36682.

[0257] 4.2 In vitro characterization analysis of water-soluble near-infrared chemiluminescent groups

[0258] After the synthesis of water-soluble near-infrared chemiluminescent groups 5b / 5b-OH and 5c, the luminescence kinetics of these chemiluminescent groups 5a, 5b, and 5c were investigated. Figure 13 A) Conduct evaluation and analysis. Since the only difference between 5b / 5b-OH is the protecting group, this invention first measures the luminescence kinetics of 5a and 5b, and then measures the luminescence kinetics of 5b-OH and 5c, using 5b / 5b-OH to ensure the uniformity of variables in the luminescence kinetics experiment.

[0259] like Figure 13As shown in Figure A, the luminescence kinetic curves of 5b and 5c are both increased to some extent compared to 5a, and the luminescence quantum yields of 5b and 5c are also higher than those of 5a. Figure 13 B) demonstrates the success of the water-soluble modification strategy. In the luminescence half-life data, 5c exhibits the fastest excitation kinetics ( Figure 14 C). Subsequently, the invention underwent repeated kinetic experiments ( Figure 13 D, 1% DMSO), with ( Figure 13 The results under conditions A (20% ACN) are opposite; the luminescence peak of 5b is lower than that of 5c. This may be because the ethyl acetate group of 5b is less water-soluble than the polyethylene glycol group of 5c, leading to aggregation under 1% DMSO conditions, which in turn causes a decrease in the luminescence peak under aqueous conditions. The chemiluminescence spectra of 5b and 5c are similar to those of 5a. Figure 2 The .10E indicates that the water-soluble modification strategy improves the luminescence quantum yield while having negligible impact on the emission wavelength. The fluorescence spectrum of the excitation product corresponding to the chemiluminescent group ( Figure 13 F) Compared to the emission spectrum, both exhibit a blue shift, which may be due to the different forms of chemiluminescent emitters and fluorescent emitters under aqueous conditions.

[0260] Although 5c has a lower luminescence quantum yield (absolute value of luminescence signal) than 5b, 5c exhibits rapid excitation and the highest luminescence extremum (relative value of luminescence signal) under aqueous conditions (1% DMSO). In target analyte detection, the sensitivity of the analyte is directly proportional to the relative value of the luminescence signal, highlighting the superior sensitivity of 5c compared to 5b in biological detection.

[0261] like Figure 14 As shown in Figure A, the luminescence kinetic curves of 6a, 6b, and 6c showed no significant difference within 0-60 min, exhibiting similar kinetic curves. However, after 60 min, the luminescence of 6a and 6b decreased to the background, while 6c exhibited persistent chemiluminescence (60-840 min). Figure 14 B) The reason may be that the polyethylene glycol chain forms a certain micelle structure, which stabilizes the single electron intermediate in the CIEEL mechanism, reduces the rate of single electron transfer or reverse electron transfer, and thus exhibits a persistent chemiluminescence phenomenon.

[0262] Due to the persistent chemiluminescence curve of 6c, its luminescent quantum yield and luminescent half-life are significantly higher than those of 6a and 6b. Figure 14 C Figure 14 D), indicating the success of the water-soluble modification strategy. The chemiluminescence spectrum of 6c shows a slight redshift to 800 nm compared to 6a and 6b. Figure 14 E). This indicates that the effect of water-soluble modification on the emission wavelength is negligible.

[0263] Fluorescence spectra of excitation products corresponding to chemiluminescent groups ( Figure 14 F) Compared to the emission spectrum, both exhibit a blue shift. For 6b, the difference between its chemiluminescence and fluorescence spectra reaches 34 nm, which is higher than that of 6a and 6b. The observed difference between the emission and fluorescence spectra of the chemiluminescent group indicates that it reflects the "non-static characteristics" of the chemiluminescent dioxane decomposition, which is different from photoexcited fluorescence.

[0264] 6c is the chemiluminescent group known to date with the largest emission wavelength (800 nm), a relatively high chemiluminescence quantum yield (3.17%), and a relatively long emission half-life (332 min) in direct excitation mode. Its persistent chemiluminescence and longest near-infrared emission spectrum are advantageous for long-term in vivo imaging detection.

[0265] As can be seen from Examples 1-4, due to the ultra-high luminescence quantum yield of 4a, the excellent chemiluminescence performance of 5c under aqueous conditions, and the persistent chemiluminescence phenomenon and near-infrared emission spectrum of 6c, the present invention selected luminescent groups 4a, 5c, and 6c as luminescent groups for subsequent in vivo imaging applications.

[0266] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.

Claims

1. A class of dihydroxanthine chemiluminescent groups, the structural formula of which is shown below: 。 2. A class of dihydroxanthin chemiluminescent groups, characterized in that, Its structural formula 2a is shown below: 。 3. A class of dihydroxanthine chemiluminescent groups, characterized in that, Its structural formula 3a is shown below: 。 4. A class of dihydroxanthine chemiluminescent groups, characterized in that, Its structural formula 4a is shown below: 。 5. The method for preparing a type of dihydroxanthine chemiluminescent group as described in claim 1, characterized in that, Includes the following steps: Step 1) Using known compound 2.1 as the starting material, 2-(methoxy(3-methoxyphenyl)methylene)adamantane, the methoxy-meta-carbon-hydrogen bond is catalyzed by Ir transition metal to boronic acid esterification. Under alkaline conditions, the boronic acid ester is oxidized by hydrogen peroxide to generate phenolic hydroxyl compound 2.

2. The specific structural formula of compound 2.2 is as follows: ; Step 2) Compound 2.2 undergoes a formylation reaction with paraformaldehyde under anhydrous magnesium chloride catalysis to generate a pair of isomers 2.3a and 2.3b. The specific structural formula of compound 2.3a is as follows: The specific structural formula of compound 2.3b is as follows: ; Step 3) A mixture of 2.3a and 2.3b is reacted with bromounsaturated aldehyde 2.3c in a tandem Oxa-Michael, Retro-Michael, and Aldol reaction to generate compound 2.

4. The specific structural formula of compound 2.4 is as follows: ; Step 4) Compound 2.4 reacts with sodium ethanethiol under alkaline conditions to remove the methyl protecting group, yielding compound 2.

5. The specific structural formula of compound 2.5 is as follows: ; Step 5) React compound 2.5 with TIPSCl to protect the phenolic hydroxyl group to generate compound 2.

6. The specific structural formula of compound 2.6 is as follows: ; In step 6), the obtained compound 2.6 is sensitized with the photosensitizer methylene blue (MB) under light to generate singlet oxygen. The singlet oxygen undergoes a [2+2] cycloaddition to the electron-rich olefin of 2.6 to generate dioxane, thus obtaining the chemiluminescent group.

6. The use of the dihydroxanthin chemiluminescent group as described in any one of claims 1-4, characterized in that, The dihydroxanthine chemiluminescent group is used as a reagent for in vivo imaging detection in the preparation of biological samples.

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