A tetrazine cyanine compound, a preparation method and application thereof

Abstract

The application discloses a tetrazine cyanine compound and a preparation method and application thereof. The skeleton of the tetrazine cyanine compound is directly connected with a BFI part of cyanine, can better affect the overall fluorescence opening multiple of the tetrazine cyanine compound by adjusting an R1 group, can also realize high-resolution imaging of a lysosome, an endoplasmic reticulum and a mitochondrial membrane by selecting a suitable R1 group and cooperating with a predetermined site molecule, can realize high fluorescence opening multiple in a near-infrared region, can significantly enhance a fluorescence emission signal from an extremely low background level after reacting with BCN, and can also have excellent light stability, wherein the fluorescence intensity of a TBCy3P addition product generated in situ is basically maintained unchanged in the whole irradiation period under continuous 100-frame laser scanning excitation, and the cell morphology is normal.

Description

Technical Field

[0001] This invention relates to the field of fluorescent dyes, specifically to a tetraazine anthocyanin compound, its preparation method, and its applications. Background Technology

[0002] In high-resolution imaging, high-resolution images can be obtained by using appropriate bioorthogonal fluorescent probes, such as SiR-DBCO and HMSiRTz, for click labeling. However, although untargeted and "constantly bright" bioorthogonal fluorescent molecules exhibit versatility in bioimaging, they typically require rigorous washing steps to reduce background signal. Therefore, designing bioorthogonally activated optical probes is crucial. These probes selectively activate their fluorescence signal only after a bioorthogonal reaction occurs at the target biological site. This enables precise tracking of biological processes and conditional control of target function through activated photosensitization.

[0003] To obtain activatable tetrazine probes, pioneering research has linked tetrazines to fluorophores including BODIPY, aza-BODIPY, coumarin, fluorescein, rhodamine, and SiR-rhodamine. Recent efforts have focused on developing fluorescent tetrazine-cyanine probes, as cyanine dyes have shown great potential in optical imaging and phototherapy applications (Chem. Soc. Rev., 2025, 54, 2984). However, cyanine carries a charge, and bioorthogonal systems for targeting organelle membranes remain limited.

[0004] In the prior art, the inventors proposed a fluorescent red to near-infrared tetrazine-cyanine probe in patent CN116444451B. Based on a bioorthogonal platform constructed from tetrazine and boron difluoride cyanine, it achieves efficient fluorescence quenching and activation after the bioorthogonal reaction. However, in the backbone of this type of tetrazine-cyanine probe, the tetrazine and cyanine are linked by an amide group. The relative independence of the two groups results in a weak impact of the modification groups on the tetrazine group on the overall fluorescence folding factor of the probe. Therefore, the fluorescence folding factor and other properties of this type of tetrazine-cyanine probe need further improvement. Summary of the Invention

[0005] One object of the present invention is to provide a tetrazine cyanine compound that, by changing the skeletal structure of tetrazine cyanine compounds in the prior art, allows the tetrazine to be directly connected to the cyanine, thereby effectively enhancing geometric and electronic interactions, and thus significantly improving the fluorescence activation effect of the probe, enabling it to be better used for cell membrane imaging.

[0006] This invention is achieved through the following technical solution:

[0007] A tetraazine cyanine compound having the structural formula shown in Formula I:

[0008] Formula I: ;

[0009] In Formula I, group R1 is selected from... , , or Group R2 is selected from C1-C6 alkyl groups, or The group R3 is selected from H or cyano; the group X is selected from H or halogen; where n=1~5, m=1~8, p=1~8.

[0010] In this technical solution, the backbone of the tetraazine cyanine compound consists of two parts: tetraazine and cyanine. By directly linking the tetraazine to the BFI portion of the cyanine, it is possible to better influence the overall fluorescence fold of the tetraazine cyanine compound by adjusting the R1 group. Furthermore, by selecting an appropriate R1 group, it can interact with pre-site molecules to achieve high-resolution imaging of lysosomes, endoplasmic reticulum, and mitochondrial membranes. Moreover, these tetraazine cyanine compounds exhibit excellent photostability. Under continuous laser scanning excitation for 100 frames, the fluorescence intensity of the in-situ generated TBCy3P addition product remains essentially unchanged throughout the irradiation cycle, and the cell morphology remains normal.

[0011] In this technical solution, the R1 group can be , , or Where m = 1~8. In some preferred embodiments, the R1 group may be... , ,or , where m = 1~7.

[0012] In this technical solution, R2 is selected from C1~C6 alkyl groups, or Where p = 1 to 8. In some preferred embodiments, R2 is selected from C1 to C4 alkyl groups, or For example, R2 can be methyl, ethyl, tert-butyl, etc., where p=1~7.

[0013] In this technical solution, R3 is selected from H or cyano, and group X is selected from H or halogen, such as bromine or chlorine. In this technical solution, by adjusting the length of the conjugated chain, full spectral coverage of the tetraazine cyanide compound can be achieved to meet different imaging requirements. Therefore, in some embodiments, n=1~5, preferably n=1~4.

[0014] Furthermore, the tetraazine cyanide compound is selected from the following compounds:

[0015] , , , , , , , , , , , , , , , .

[0016] Another object of the present invention is to provide a method for preparing any of the aforementioned tetraazine anthocyanin compounds, specifically comprising the following steps:

[0017] The tetraazine cyanide precursor shown in Formula II is dissolved with the compound shown in Formula III, and then acetic acid and piperidine are added. The mixture is then heated to react and yield the tetraazine cyanide compound shown in Formula I; or

[0018] The tetraazine cyanine precursor shown in Formula II was dissolved with the compound shown in Formula IV, and N,N-diisopropylethylamine was added. The mixture was then heated to prepare the tetraazine cyanine compound shown in Formula I.

[0019] Formula I: Formula II: Formula III: Formula IV: ;

[0020] In Formulas I to IV, the group R1 is selected from... , , or Group R2 is selected from C1-C6 alkyl groups, or The group R3 is selected from H or cyano; the group X is selected from H or halogen; where n=1~5, m=1~8, p=1~8.

[0021] In some embodiments, the tetraazine cyanine precursor can be directly reacted with a compound of formula III or formula IV in one step to obtain a tetraazine cyanine compound having corresponding R1, R2 and other groups.

[0022] In one or more embodiments, the tetraazine cyanide precursor is dissolved with the compound shown in Formula III, and acetic acid and piperidine are added to obtain a mixture. The mixture is then reacted at 40-100°C to obtain the tetraazine cyanide compound.

[0023] In one or more embodiments, the tetraazine cyanide precursor is dissolved with the compound shown in Formula IV, and N,N-diisopropylethylamine is added. The reaction system is refluxed or reacted at 40-100°C to obtain the tetraazine cyanide compound.

[0024] Furthermore, group R1 is The tetraazine cyanide precursor is reacted with the compound shown in Formula IV to prepare a first intermediate. The first tetraazine intermediate is dissolved with N-hydroxysuccinimide, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is added to react and prepare a second intermediate. The second intermediate is then reacted with... After mixing, N,N-diisopropylethylamine was added, and the reaction yielded group R1. Tetraazine anthocyanin compounds.

[0025] In this technical solution, for group R1 as The tetraazine cyanide precursor can be reacted with the compound shown in Formula IV to prepare the first intermediate, wherein the first intermediate can also be a tetraazine cyanide compound. Next, the first intermediate undergoes an amide condensation reaction with NHS and EDCI, where the carboxyl group on the alkyl chain reacts with the nitrogen atom in the NHS molecule to form a stable amide bond, yielding the second intermediate. Subsequently, the second intermediate is mixed with polyethylene glycolamine and DIPEA, and then undergoes an amino-active ester coupling reaction to finally obtain R1. Tetraazine anthocyanin compounds.

[0026] Furthermore, the preparation method of the tetraazine cyanide precursor shown in Formula II includes the following steps:

[0027] The tetrazine compound shown in Formula V was dissolved with the tetrazine compound shown in Formula VI, and Pd(PPh3)4 and CuTc were added. The mixture was heated to prepare the tetrazine anthocyanin precursor shown in Formula II.

[0028] Formula V: Formula VI: .

[0029] Furthermore, the tetraazine cyanine precursor is prepared by halogenation to obtain a tetraazine cyanine precursor with a halogenated group X.

[0030] Furthermore, group R1 is After dissolving the tetraazine cyanide precursor, trifluoroacetic acid was added, and the reaction was carried out at room temperature to obtain the group R1. Tetraazine anthocyanin precursor; or group R1 is After dissolving the tetraazine cyanide precursor, trifluoroacetic acid was added, and the reaction was carried out at room temperature to obtain the group R1. Tetraazine anthocyanin precursor, wherein the group R1 is The tetraazine cyanide precursor was reacted with a mixture of 1-hydroxybenzotriazole and N,N'-carbonyldiimidazole, followed by the addition of The reaction continued to produce group R1. Tetraazine anthocyanin precursor.

[0031] Another object of the present invention is to provide a reagent for cell membrane fluorescence imaging comprising any of the aforementioned tetraazine cyanine compounds.

[0032] Furthermore, the reagent also includes a pre-positioning molecule for targeting the cell membranes of mitochondria, lysosomes, and endoplasmic reticulum. In one or more embodiments, the pre-positioning molecule may be MemLyso for labeling lysosomal membranes, MemER for labeling endoplasmic reticulum membranes, or MemMito for labeling mitochondrial membranes.

[0033] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0034] 1. The tetrazine cyanine compound skeleton of the present invention directly links the tetrazine to the BFI part of the cyanine, which not only allows for better control of the overall fluorescence fold of the tetrazine cyanine compound by adjusting the R1 group, but also allows for interaction with the prepositioning molecule by selecting a suitable R1 group to achieve high-resolution imaging of lysosomes, endoplasmic reticulum and mitochondrial membranes.

[0035] 2. The tetraazine cyanine compound of the present invention can achieve a high fluorescence on-hook rate in the near-infrared region, and after reacting with BCN, the fluorescence emission signal is significantly enhanced from an extremely low background level.

[0036] 3. The tetraazine cyanine compound of the present invention has excellent photostability. Under continuous laser scanning excitation of 100 frames, the fluorescence intensity of the in-situ generated TBCy3P addition product remains basically unchanged throughout the entire irradiation cycle, and the cell morphology remains normal.

[0037] 4. The method for preparing tetraazine cyanide compounds of the present invention has a short synthesis route and mild conditions, which is conducive to large-scale production. Attached Figure Description

[0038] The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and form part of this application, do not constitute a limitation thereof. In the drawings:

[0039] Figure 1 This is a flowchart illustrating the preparation method of tetraazine anthocyanin compounds in a specific embodiment of the present invention;

[0040] Figure 2 The absorption (Abs) and emission (Em) spectra of six tetraazine anthocyanin compounds TBCy5-1, TBCy5-3, TBCy5-4, TBCy3-1, TBCy7-1, and TBCy7-3 before and after bioorthogonal reaction with BCN in specific embodiments of the present invention.

[0041] Figure 3The absorption (A) and emission (B) spectra of tetraazine cyanine compound TBCy5-2 in DMSO are shown in a specific embodiment of the present invention, showing its condition before (black) and after (red) reaction with BCN, respectively;

[0042] Figure 4 The absorption (A) and emission (B) spectra of tetraazine cyanine compound TBCy3-2 in DMSO are shown in a specific embodiment of the present invention, showing its condition before (black) and after (red) reaction with BCN, respectively;

[0043] Figure 5 The absorption (A) and emission (B) spectra of the tetraazine cyanine compound TBCy3-3 in DMSO are shown in a specific embodiment of the present invention, showing its condition before (black) and after (red) reaction with BCN, respectively;

[0044] Figure 6 The absorption (A) and emission (B) spectra of tetraazine cyanine compound TBCy7-2 in DMSO are shown in a specific embodiment of the present invention, showing its condition before (black) and after (red) reaction with BCN, respectively;

[0045] Figure 7 The results of specific targeting colocalization imaging and quantitative analysis of the tetraazine cyanide compound TBCy5P in A549 cells are shown in a specific embodiment of the present invention.

[0046] Figure 8 The results of specific targeting colocalization imaging and quantitative analysis of the tetraazine cyanide compound TBCy3P in A549 cells are shown in a specific embodiment of the present invention.

[0047] Figure 9 The results of specific targeting colocalization imaging and quantitative analysis of the tetraazine cyanide compound TBCy7P in A549 cells are shown in a specific embodiment of the present invention.

[0048] Figure 10 The following are live-cell no-wash imaging results of tetraazine cyanide compounds TBCy3P and TBCy5P in specific embodiments of the present invention. (AD) Confocal microscopy images of mitochondria and lysosomes using a specified TBCy probe for in situ bioorthogonal imaging under conditions of added (+) or unadded (-) targeting pretreatment molecules, and quantitative difference analysis of imaging contrast (signal-to-noise ratio) between the experimental and control groups; (E) Co-localization imaging of the endoplasmic reticulum labeled with TBCy5P in the presence of MemER and ER-tracker Green.

[0049] Figure 11The cytotoxicity of three tetraazine cyanide compounds, TBCy5P (A), TBCy3P (B), and TBCy7P (C), at different concentrations is shown in specific embodiments of the present invention.

[0050] Figure 12 The accompanying illustration shows a comparison of the anti-photobleaching performance monitoring curves of the tetraazine cyanine compound TBCy3P with commercial dyes MTR and LTR under continuous laser irradiation excitation in a specific embodiment of the present invention.

[0051] Figure 13 The illustration shows STED imaging of lysosomal membranes labeled with TBCy3-CN and MemLyso in a specific embodiment of the present invention, along with the corresponding intensity distribution curves plotted along the dashed lines; magnified views are shown within the selected regions of interest (ROIs).

[0052] Figure 14 The STED imaging and intensity distribution curves of endoplasmic reticulum membranes labeled with TBCy3-CN and MemER are shown in a specific embodiment of the present invention.

[0053] Figure 15 The illustration shows STED imaging of mitochondrial membranes labeled with TBCy3-CN and MemMito, along with intensity distribution curves plotted along the dashed line, in a specific embodiment of the present invention. Detailed Implementation

[0054] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0055] The sources of all raw materials used in this invention are not particularly limited; they can be purchased commercially or prepared using conventional methods well-known to those skilled in the art. The purity of all raw materials used in this invention is not particularly limited; however, analytical grade or the purity requirements conventional in the field of fluorescent dyes are preferred. The designations and abbreviations of all raw materials used in this invention are conventional designations and abbreviations in the art, and each designation and abbreviation is clearly defined within its relevant application. Those skilled in the art can obtain these materials from commercially available sources or prepare them using conventional methods based on the designation, abbreviation, and corresponding application.

[0056] The present invention does not impose any particular restrictions on the expression of the substituents, and all expressions are well known to those skilled in the art. Based on common sense, those skilled in the art can correctly understand their meaning according to their expression.

[0057] The terms "first," "second," etc., used in this document (e.g., first intermediate, second intermediate, etc.) are merely for clarity of description and are not intended to restrict any order or emphasize importance. Furthermore, the term "link" used herein, unless otherwise specified, can refer to a direct link or an indirect link via other groups.

[0058] I. Preparation of Tetraazine Cyanide Precursor (TBFI)

[0059] In this embodiment, the tetrazine anthocyanin precursor TBFI of Formula II was prepared by Stille coupling reaction based on the compound BFI-Sn of Formula III and the tetrazine compound of Formula IV.

[0060] Formula II: ;

[0061] Formula III: ;

[0062] Formula IV: .

[0063] In some embodiments, group R 1 Selected from or In one or more embodiments, it may further be based on group R 1 for The tetraazine anthocyanin precursor was used to prepare the group R. 1 for or Tetraazine anthocyanin precursors, where m = 1~7.

[0064] In some embodiments, group X is a halogen or hydrogen; for example, group X can be chlorine, bromine, or hydrogen. In one or more embodiments, the tetraazine cyanine precursor with hydrogen group X can be further prepared into a tetraazine cyanine precursor with halogen X via a halogenation reaction.

[0065] Examples 1 to 5 illustrate the preparation of six tetrazine anthocyanin precursors TBFI.

[0066]

Example 1

[0067]

[0068]

[0069]

[0070] (a) Under argon protection, boron trifluoride diethyl ether salt (4 mL, 27.2 mmol) was added to 30 mL of acetic anhydride (Ac₂O) solution of 5-iodo-2,3,3-trimethyl-3H-indole (5.7 g, 20 mmol). After stirring at 120 °C for 4 hours, the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give the yellow solid product BFI-I in 61% yield.

[0071] (b) Under argon protection, anhydrous 1,4-dioxane (60 mL) and hexamethyldistinane (4.9 g, 15 mmol) were added to a mixture of compound BFI-I (3.74 g, 10 mmol) and Pd(PPh3)4 (577 mg, 0.5 mmol). The mixture was heated at 90 °C for 6 hours. After evaporation of the solvent under reduced pressure, the residue was purified by silica gel column chromatography to give the intermediate BFI-Sn as a yellow solid in 95% yield.

[0072] (c) Under argon protection, anhydrous 1,4-dioxane (25 mL) and intermediate BFI-Sn (619 mg, 1.5 mmol) were added to a reaction flask containing compound 1 (498 mg, 1.95 mmol), Pd(PPh3)4 (259 mg, 0.225 mmol), and CuTc (569 mg, 3.0 mmol). The reaction system was heated and stirred at 70 °C for 90 min. After the reaction was completed, the reaction solution was filtered through a diatomaceous earth filter to remove insoluble matter, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by silica gel column chromatography to finally obtain the target product, a red solid, namely the tetraazine cyanide precursor TBFI-1, in 37% yield.

[0073] The tetraazine anthocyanin precursor TBFI-1 was characterized as follows:

[0074] 1 H NMR (400 MHz, CDCl3) δ 8.68 (dd, J = 8.4, 1.7 Hz, 1H), 8.60 (d, J =1.7 Hz, 1H), 7.83 (d, J = 8.5 Hz, 1H), 5.81 (s, 1H), 3.65 (t, J = 7.1 Hz,2H), 3.02 (t, J = 7.1 Hz, 2H), 2.35 (s, 3H), 1.55 (s, 6H), 1.43 (s, 9H).

[0075] 13C NMR (101 MHz, CDCl3) δ 182.63, 181.79, 171.17, 168.77, 163.84,146.82, 141.74, 129.31, 129.29, 121.54, 116.69, 91.82, 81.13, 49.76, 32.25,29.99, 28.06, 24.63, 23.92.

[0076] HRMS [M+H] + m / z calcd. for [C 22 H 27 BF2N5O3] + 458.2170 found: 458.2148

[0077]

Example 2

[0078] In this embodiment, based on the tetraazine cyanine precursor TBFI-1 prepared in Example 1, the tetraazine cyanine precursor TBFI-2 with Cl-substituted group X is prepared by halogenation reaction. The synthetic route is as follows:

[0079]

[0080] Specifically, dimethyl sulfoxide (DMSO, 11 μL, 0.15 mmol) was added to a chloroform (2.5 mL) solution of tetraazine cyanide precursor TBFI-1 (228 mg, 0.5 mmol) and N-chlorosuccinimide (NCS, 120 mg, 0.9 mmol). The reaction system was heated and stirred at 40 °C for 4 h. After the reaction was completed, the solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography to finally obtain the target product, a red solid, namely tetraazine cyanide precursor TBFI-2, in 87% yield. Its characterization is as follows:

[0081] 1 H NMR (400 MHz, CDCl3) δ 8.68 (dd, J = 8.4, 1.7 Hz, 1H), 8.59 (d, J =1.7 Hz, 1H), 7.84 (d, J = 6.8 Hz, 1H), 3.66 (t, J = 7.1 Hz, 2H), 3.02 (t, J =7.1 Hz, 2H), 2.49 (s, 3H), 1.81 (s, 6H), 1.43 (s, 9H).

[0082] 13C NMR (101 MHz, CDCl3) δ 178.65, 177.71, 171.15, 168.89, 163.71,145.77, 142.67, 130.10, 129.29, 121.24, 117.10, 81.16, 52.72, 32.22, 30.01,28.06, 22.13.

[0083] HRMS [M+H] + m / z calcd. for [C 22 H 26 [BClF2N5O3] + 492.1780 found: 492.1789

[0084]

Example 3

[0085] In this embodiment, based on the compound BFI-Sn obtained in Example 1, the group R1 is used as... Compound 2 replaces compound 1 to prepare the tetraazine cyanine precursor TBFI-3 based on a palladium-catalyzed metal coupling reaction. The synthetic route is as follows:

[0086]

[0087] Specifically, in step (c), under argon protection, anhydrous 1,4-dioxane (20 mL) and intermediate BFI-Sn (287.7 mg, 0.7 mmol) were added to a reaction flask containing compound 2 (280 mg, 0.84 mmol), Pd(PPh3)4 (121.1 mg, 0.105 mmol), and CuTc (266 mg, 1.4 mmol). The reaction system was heated and stirred at 80 °C for 90 min. After the reaction, the reaction solution was filtered through a diatomaceous earth filter to remove insoluble matter, and the solvent was removed from the filtrate by vacuum distillation. The resulting residue was purified by silica gel column chromatography, finally yielding the red solid target product, namely tetraazine cyanide precursor TBFI-3, in 42% yield, characterized as follows:

[0088] 1H NMR (400 MHz, CDCl3) δ 8.71 (dd, J = 8.4, 1.6 Hz, 1H), 8.64 (s,1H), 8.59 (d, J = 8.1 Hz, 2H), 7.83 (d, J = 8.3 Hz, 1H), 7.53 (d, J = 8.1 Hz,2H), 5.83 (s, 1H), 5.08 (s, 1H), 4.45 (d, J = 6.2 Hz, 2H), 2.36 (s, 3H), 1.56(s, 6H), 1.49 (s, 9H).

[0089] 13 C NMR (101 MHz, CDCl3) δ 182.62, 181.86, 163.63, 163.43, 155.97,141.81, 130.63, 129.30, 129.23, 128.20, 128.13, 121.48, 116.72, 91.88, 79.83,49.76, 44.39, 28.42, 24.64, 23.93.

[0090] HRMS [M+H] + m / z calcd. for [C 27 H 30 BF2N6O3] + 535.2435 found: 535.2458

[0091]

Example 4

[0092] In this embodiment, based on the tetraazine anthocyanin precursor TBFI-3 prepared in Example 3, the tetraazine anthocyanin precursor TBFI-4 with Cl-substituted group X is prepared by halogenation reaction. The synthetic route is as follows:

[0093]

[0094] Specifically, dimethyl sulfoxide (4.2 μL, 0.06 mmol) was added to a chloroform (1 mL) solution of compound TBFI-3 (106.8 mg, 0.2 mmol) and N-chlorosuccinimide (40 mg, 0.3 mmol). The reaction system was stirred overnight at room temperature. After the reaction was completed, the solvent was removed by vacuum distillation. The resulting residue was purified by silica gel column chromatography to finally obtain the target product, a red solid, namely the tetraazine cyanide precursor TBFI-4, in 80% yield. Its characterization is as follows:

[0095] 1 H NMR (400 MHz, CDCl3) δ 8.70 (dd, J = 8.4, 1.7 Hz, 1H), 8.64 – 8.58(m, 3H), 7.83 (d, J = 8.4 Hz, 1H), 7.53 (d, J = 8.1 Hz, 2H), 5.07 (s, 1H), 4.45 (d, J = 6.2 Hz, 2H), 2.49 (s, 3H), 1.83 (s, 6H), 1.49 (s, 9H).

[0096] 13 C NMR (101 MHz, CDCl3) δ 178.70, 177.68, 163.70, 163.31, 155.96,145.81, 144.38, 142.73, 130.58, 130.09, 129.29, 129.22, 128.33, 128.26,128.15, 121.17, 117.12, 102.26, 79.85, 52.71, 44.39, 28.42, 22.15.

[0097] HRMS [M+H] + m / z calcd. for [C 27 H 29 [BClF2N6O3] + 569.2045 found: 569.2059

[0098]

Example 5

[0099] In this embodiment, based on the tetraazine anthocyanin precursor TBFI-2 prepared in Example 2, an R1 group was prepared by a two-step method. The synthetic route for the tetraazine cyanide precursor TBFI-6 (compound 21) is as follows:

[0100]

[0101]

[0102] Specifically, compound TBFI-2 (491 mg, 1 mmol) was dissolved in 5 mL of DCM, followed by the addition of trifluoroacetic acid (TFA, 5 mL). The reaction system was stirred at room temperature for 2 h. After the reaction was completed, the solvent and excess acid were removed by vacuum distillation to finally obtain the tetraazine cyanide precursor TBFI-5.

[0103] In a solution of TBFI-5 (87 mg, 0.2 mmol) and 1-hydroxybenzotriazole (HOBt, 33.8 mg, 0.25 mmol) dissolved in 2 mL of anhydrous dichloromethane, N,N'-carbonyldiimidazole (CDI, 40.5 mg, 0.25 mmol) was added. The reaction was stirred at room temperature for 90 minutes. Then, Me-PEG4-NH2 (45 mg, 0.22 mmol) was added. The reaction was continued to be stirred at room temperature for 3 hours. After purification by silica gel column chromatography, the tetraazine cyanide precursor TBFI-6 (compound 21) was obtained as a red solid in a yield of 32%, characterized as follows:

[0104] 1 H NMR (400 MHz, CDCl3) δ 8.58 (ddt, J = 6.9, 3.5, 1.8 Hz, 1H), 8.51(t, J = 2.0 Hz, 1H), 7.78 – 7.70 (m, 1H), 7.24 (t, J = 5.3 Hz, 1H), 3.64 (td,J = 7.1, 2.6 Hz, 2H), 3.60 – 3.53 (m, 10H), 3.51 – 3.45 (m, 4H), 3.38 (dd, J= 6.6, 3.7 Hz, 2H), 3.30 (q, J = 1.8 Hz, 3H), 2.92 (td, J = 7.2, 2.0 Hz, 2H), 2.41 (d, J = 2.4 Hz, 3H), 1.74 (d, J = 2.1 Hz, 6H).

[0105] 13 C NMR (101 MHz, CDCl3) δ 178.54, 177.68, 171.01, 169.26, 163.55,145.65, 142.62, 130.22, 129.18, 121.24, 117.02, 102.21, 71.86, 70.52, 70.49,70.47, 70.36, 70.13, 69.88, 58.95, 52.70, 39.36, 32.58, 30.25, 22.11, 22.06.

[0106] II. Tetraazine cyanide compounds

[0107]

Example 6

[0108] In this embodiment, tetraazine cyanide compound TBCy5-1 was directly prepared based on tetraazine cyanide precursor TBFI-2 and compound 3.

[0109]

[0110] Specifically, the tetraazine cyanide precursor TBFI-2 (19.6 mg, 0.04 mmol) and compound 3 (11.8 mg, 0.052 mmol) were dissolved in 1 mL of toluene, followed by the addition of a mixture of acetic acid (8 μL) and piperidine (16 μL). The reaction mixture was heated and stirred at 50 °C for 2 h. After the reaction was complete, the solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography to finally obtain the tetraazine cyanide compound TBCy5-1 in 85% yield, which was characterized as follows:

[0111] 1 H NMR (400 MHz, CDCl3) δ 8.62 (dd, J = 8.4, 1.7 Hz, 1H), 8.51 (d, J =1.7 Hz, 1H), 7.93 (dd, J = 13.9, 12.1 Hz, 1H), 7.74 (d, J = 8.3 Hz, 1H), 7.51(t, J = 13.1 Hz, 1H), 7.24 (d, J = 7.3 Hz, 2H), 7.06 – 6.97 (m, 1H), 6.79 (d,J = 7.8 Hz, 1H), 6.56 (d, J = 14.0 Hz, 1H), 6.40 – 6.26 (m, 1H), 5.60 (d, J =12.7 Hz, 1H), 3.63 (t, J = 7.1 Hz, 2H), 3.28 (s, 3H), 3.01 (t, J = 7.1 Hz, 2H), 1.81 (s, 6H), 1.64 (s, 6H), 1.43 (s, 9H).

[0112] 13C NMR (101 MHz, CDCl3) δ 173.85, 171.19, 169.40, 168.41, 165.07,164.00, 150.41, 147.15, 145.30, 143.88, 142.39, 139.65, 129.25, 128.05,127.97, 123.35, 121.95, 121.88, 121.03, 115.72, 112.50, 107.56, 100.85,98.22, 81.10, 51.77, 47.11, 32.34, 29.99, 29.60, 28.26, 28.07, 22.84.

[0113] HRMS [M+H] + m / z calcd. for [C 37 H 41 [BClF2N6O3] + 701.2984 found: 701.2943

[0114]

Example 7

[0115] In this embodiment, tetraazine cyanide compound TBCy5-2 was directly prepared based on tetraazine cyanide precursor TBFI-1 and compound 3.

[0116]

[0117] Tetraazine cyanide precursor TBFI-1 (18.3 mg, 0.04 mmol) and compound 3 (11.8 mg, 0.052 mmol) were dissolved in 1 mL of toluene, followed by the addition of a mixture of acetic acid (8 μL) and piperidine (16 μL). The reaction mixture was heated and stirred at 80 °C for 2 h. After the reaction was complete, the solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography to finally obtain tetraazine cyanide compound TBCy5-1 in 77% yield, which was characterized as follows:

[0118] 1H NMR (400 MHz, CDCl3) δ 8.64 (dd, J = 8.4, 1.7 Hz, 1H), 8.54 (d, J =1.7 Hz, 1H), 7.84 – 7.74 (m, 2H), 7.39 (t, J = 13.1 Hz, 1H), 7.24 – 7.17 (m,2H), 6.96 (t, J = 7.4 Hz, 1H), 6.73 (d, J = 7.8 Hz, 1H), 6.26 – 6.16 (m, 1H), 5.99 (d, J = 14.3 Hz, 1H), 5.70 (s, 1H), 5.52 (d, J = 12.5 Hz, 1H), 3.63 (t,J = 7.1 Hz, 2H), 3.22 (s, 3H), 3.00 (t, J = 7.1 Hz, 2H), 1.62 (s, 6H), 1.53(s, 6H), 1.43 (s, 9H).

[0119] 13 C NMR (101 MHz, CDCl3) δ 179.73, 173.57, 171.20, 168.40, 164.06,163.22, 147.88, 147.12, 144.18, 142.62, 141.68, 139.39, 129.28, 127.94,127.69, 122.58, 121.79, 121.39, 121.27, 117.23, 115.71, 107.06, 97.40, 91.89,48.93, 46.66, 32.34, 29.98, 29.39, 28.27, 28.07, 25.50.

[0120] HRMS [M+H] + m / z calcd. for [C 37 H 42 BF2N6O3] + 667.3374 found: 667.3341

[0121]

Example 8

[0122] In this embodiment, tetraazine cyanide compound TBCy5-3 was directly prepared based on tetraazine cyanide precursor TBFI-4 and compound 3.

[0123]

[0124] Tetraazine cyanide precursor TBFI-4 (22.7 mg, 0.04 mmol) and compound 3 (11.8 mg, 0.052 mmol) were dissolved in 1 mL of toluene, followed by the addition of a mixture of acetic acid (8 μL) and piperidine (16 μL). The reaction mixture was heated and stirred at 40 °C for 2 h. After the reaction was complete, the solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography to finally obtain tetraazine cyanide compound TBCy5-3 in 76% yield, which was characterized as follows:

[0125] 1 H NMR (400 MHz, CDCl3) δ 8.67 (dd, J = 8.4, 1.7 Hz, 1H), 8.60 (d, J =8.3 Hz, 2H), 8.56 (d, J = 1.6 Hz, 1H), 7.93 (dd, J = 14.0, 12.1 Hz, 1H), 7.76(d, J = 8.3 Hz, 1H), 7.55 – 7.48 (m, 3H), 7.24 (d, J = 7.6 Hz, 2H), 7.01 (t,J = 7.4 Hz, 1H), 6.79 (d, J = 7.8 Hz, 1H), 6.56 (d, J = 14.0 Hz, 1H), 6.39 –6.28 (m, 1H), 5.61 (d, J = 12.7 Hz, 1H), 5.01 (d, J = 7.9 Hz, 1H), 4.45 (d, J= 6.1 Hz, 2H), 3.28 (s, 3H), 1.82 (s, 6H), 1.64 (s, 6H), 1.49 (s, 9H).

[0126] 13 C NMR (101 MHz, CDCl3) δ 173.73, 169.41, 165.16, 163.60, 163.39,155.94, 150.47, 147.26, 145.41, 144.00, 143.86, 142.47, 139.66, 130.85,129.25, 128.13, 128.06, 127.93, 123.38, 121.99, 121.88, 120.98, 115.77,112.47, 107.59, 100.91, 98.27, 79.84, 51.75, 47.14, 44.44, 29.62, 28.43, 28.26, 22.87.

[0127] HRMS [M] + m / z calcd. for [C 42 H 43 [BClF2N7O3] + 777.3172 found: 777.3157

[0128]

Example 9

[0129] In this embodiment, tetraazine cyanide compound TBCy5-4 was directly prepared based on tetraazine cyanide precursor TBFI-3 and compound 3.

[0130]

[0131] Tetraazine cyanide precursor TBFI-3 (21.4 mg, 0.04 mmol) and compound 3 (11.8 mg, 0.052 mmol) were dissolved in 1 mL of toluene, followed by the addition of a mixture of acetic acid (8 μL) and piperidine (16 μL). The reaction mixture was heated and stirred at 70 °C for 2 h. After the reaction was complete, the solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography to finally obtain tetraazine cyanide compound TBCy5-4 in 77% yield, which was characterized as follows:

[0132] 1 H NMR (400 MHz, CDCl3) δ 8.68 (dd, J = 8.4, 1.7 Hz, 1H), 8.62 – 8.55(m, 3H), 7.85 – 7.75 (m, 2H), 7.52 (d, J = 8.1 Hz, 2H), 7.39 (t, J = 13.1 Hz,1H), 7.20 (d, J = 7.7 Hz, 2H), 6.95 (t, J = 7.4 Hz, 1H), 6.72 (d, J = 7.8 Hz,1H), 6.26 – 6.15 (m, 1H), 5.99 (d, J = 14.3 Hz, 1H), 5.71 (s, 1H), 5.51 (d, J= 12.5 Hz, 1H), 5.04 (d, J = 6.5 Hz, 1H), 4.44 (d, J = 6.1 Hz, 2H), 3.21 (s, 3H), 1.62 (s, 6H), 1.54 (s, 6H), 1.49 (s, 9H).

[0133] 13C NMR (101 MHz, CDCl3) δ 179.65, 173.58, 163.65, 163.38, 163.28,155.97, 147.97, 147.17, 144.16, 144.03, 142.70, 141.77, 139.39, 130.82,129.27, 128.12, 128.09, 127.95, 127.67, 122.61, 121.78, 121.35, 121.29,117.21, 115.75, 107.09, 97.44, 91.97, 79.81, 48.91, 46.68, 44.42, 29.40, 28.44, 28.27, 25.53.

[0134] HRMS [M+H] + m / z calcd. for [C 42 H 45 BF2N7O3] + 744.3640 found: 744.3640

[0135]

Example 10

[0136] Based on Example 6, this example uses compound 4 instead of compound 3 to react with the tetraazine cyanide precursor TBFI-2 to prepare tetraazine cyanide compounds TBCy3-1 with different conjugated carbon chain lengths.

[0137]

[0138] Tetraazine cyanide precursor TBFI-2 (19.3 mg, 0.03 mmol) and compound 4 (10.5 mg, 0.052 mmol) were dissolved in 1 mL of toluene, followed by the addition of a mixture of acetic acid (8 μL) and piperidine (16 μL). The reaction mixture was heated and stirred at 40 °C for 2 h. After the reaction was complete, the solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography to finally obtain tetraazine cyanide compound TBCy3-1 in 87% yield, which was characterized as follows:

[0139] 1H NMR (400 MHz, CDCl3) δ 8.61 (d, J = 9.3 Hz, 1H), 8.49 (s, 1H), 8.38 (t, J = 13.3 Hz, 1H), 7.70 (d, J = 8.3 Hz, 1H), 7.29 (t, J = 7.5 Hz, 2H),7.09 (t, J = 7.4 Hz, 1H), 6.88 (d, J = 8.0 Hz, 1H), 6.50 (d, J = 13.4 Hz,1H), 5.81 (d, J = 13.3 Hz, 1H), 3.63 (t, J = 7.1 Hz, 2H), 3.37 (s, 3H), 3.00(t, J = 7.1 Hz, 2H), 1.80 (s, 6H), 1.69 (s, 6H), 1.43 (s, 9H).

[0140] 13 C NMR (101 MHz, CDCl3) δ 172.93, 171.21, 169.69, 169.30, 168.31,164.08, 147.50, 145.82, 143.41, 142.16, 140.12, 129.25, 128.13, 127.40,122.92, 122.04, 120.99, 115.29, 109.96, 108.25, 99.89, 98.24, 81.09, 51.44,47.94, 32.37, 29.98, 29.90, 28.73, 28.07, 22.97.

[0141] HRMS [M+H] + m / z calcd. for [C 35 H 39 [BClF2N6O3] + 675.2828 found: 675.2818

[0142]

Example 11

[0143] Based on Example 7, this example uses compound 4 instead of compound 3 to react with the tetraazine cyanide precursor TBFI-1 to prepare tetraazine cyanide compound TBCy3-2 with different conjugated carbon chain lengths.

[0144]

[0145] Tetraazine cyanide precursor TBFI-1 (18.3 mg, 0.04 mmol) and compound 4 (10.5 mg, 0.052 mmol) were dissolved in 1 mL of toluene, followed by the addition of a mixture of acetic acid (8 μL) and piperidine (16 μL). The reaction mixture was heated and stirred at 80 °C for 2 h. After the reaction was complete, the solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography to finally obtain tetraazine cyanide compound TBCy3-2 in 66% yield, which was characterized as follows:

[0146] 1 H NMR (400 MHz, CDCl3) δ 8.63 (dd, J = 8.4, 1.8 Hz, 1H), 8.52 (d, J =1.6 Hz, 1H), 8.24 (t, J = 13.4 Hz, 1H), 7.74 (d, J = 8.3 Hz, 1H), 7.27 – 7.23(m, 2H), 7.03 (t, J = 7.4 Hz, 1H), 6.81 (d, J = 8.0 Hz, 1H), 5.90 (d, J =13.7 Hz, 1H), 5.64 – 5.58 (m, 2H), 3.63 (t, J = 7.3 Hz, 2H), 3.29 (s, 3H), 3.00 (t, J = 7.1 Hz, 2H), 1.68 (s, 6H), 1.52 (s, 6H), 1.43 (s, 9H).

[0147] 13 C NMR (101 MHz, CDCl3) δ 178.97, 173.91, 171.22, 168.29, 167.10,164.14, 148.22, 143.78, 142.44, 141.51, 139.81, 129.29, 127.98, 127.14,122.08, 121.93, 121.54, 121.35, 115.34, 114.50, 107.61, 96.54, 91.07, 81.09,48.60, 47.40, 32.37, 29.97, 29.62, 28.72, 28.07, 25.76.

[0148] HRMS [M+H] + m / z calcd. for [C 35 H 40 BF2N6O3] +641.3218 found: 641.3226

[0149]

Example 12

[0150] Based on Example 8, this example uses compound 4 instead of compound 3 to react with the tetraazine cyanide precursor TBFI-4 to prepare tetraazine cyanide compounds TBCy3-3 with different conjugated carbon chain lengths.

[0151]

[0152] Tetraazine cyanide precursor TBFI-4 (22.7 mg, 0.04 mmol) and compound 4 (10.0 mg, 0.05 mmol) were dissolved in 1 mL of toluene, followed by the addition of a mixture of acetic acid (8 μL) and piperidine (16 μL). The reaction mixture was heated and stirred at 40 °C for 2 h. After the reaction was complete, the solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography to finally obtain tetraazine cyanide compound TBCy3-3 in 71% yield, which was characterized as follows:

[0153] 1 H NMR (400 MHz, CDCl3) δ 8.68 – 8.53 (m, 4H), 8.40 (d, J = 13.5 Hz,1H), 7.71 (d, J = 8.3 Hz, 1H), 7.52 (d, J = 7.9 Hz, 2H), 7.29 (d, J = 7.4 Hz,2H), 7.08 (t, J = 7.4 Hz, 1H), 6.88 (d, J = 7.9 Hz, 1H), 6.50 (d, J = 13.3Hz, 1H), 5.82 (d, J = 13.2 Hz, 1H), 5.05 (s, 1H), 4.46 (s, 2H), 3.37 (s, 3H),1.81 (s, 6H), 1.69 (s, 6H), 1.49 (s, 9H).

[0154] 13C NMR (101 MHz, CDCl3) δ 172.78, 169.68, 169.41, 163.65, 163.32,155.97, 147.62, 145.92, 143.97, 143.38, 142.23, 140.12, 130.88, 129.25,128.08, 127.36, 122.98, 122.03, 120.95, 115.32, 109.91, 108.33, 99.95, 98.33,79.81, 51.41, 47.97, 44.43, 29.93, 28.72, 28.43, 23.01.

[0155] HRMS [M+H] + m / z calcd. for [C 40 H 42 [BClF2N7O3] + 752.3093 found: 752.3083

[0156]

Example 13

[0157] In this embodiment, based on Example 6, compound 5 was used to replace compound 3 to prepare tetraazine cyanine compound TBCy7-1 with different conjugated carbon chain lengths.

[0158]

[0159] Under argon protection, DIPEA (21 μL, 0.12 mmol) was added to a solution of tetraazin cyanide precursor TBFI-2 (19.3 mg, 0.04 mmol) and compound 5 (37.4 mg, 0.075 mmol) in anhydrous dichloromethane (0.5 mL). The reaction system was stirred under reflux for 1 h. After the reaction, the crude product was purified by silica gel column chromatography to finally obtain tetraazin cyanide compound TBCy7-1 in a yield of 41%, which was characterized as follows:

[0160] 1H NMR (400 MHz, CDCl3) δ 8.63 (dd, J = 8.5, 1.7 Hz, 1H), 8.53 (d, J =1.7 Hz, 1H), 7.86 – 7.73 (m, 2H), 7.24 – 7.15 (m, 3H), 7.03 – 6.91 (m, 2H),6.72 (d, J = 7.9 Hz, 1H), 6.66 (d, J = 14.2 Hz, 1H), 6.46 – 6.36 (m, 1H),6.27 – 6.16 (m, 1H), 5.51 (d, J = 12.4 Hz, 1H), 3.64 (t, J = 7.1 Hz, 2H),3.21 (s, 3H), 3.01 (t, J = 7.1 Hz, 2H), 1.81 (s, 6H), 1.62 (s, 6H), 1.43 (s,9H).

[0161] 13 C NMR (101 MHz, CDCl3) δ 174.79, 171.19, 169.10, 168.51, 163.94,161.93, 148.84, 148.36, 146.87, 144.34, 142.61, 139.30, 139.18, 129.27,128.47, 127.94, 126.22, 124.63, 121.74, 121.06, 121.00, 116.12, 115.57,106.91, 101.53, 97.81, 81.12, 52.05, 46.48, 32.32, 29.99, 29.37, 28.33, 28.07, 22.69.

[0162] HRMS [M] + m / z calcd. for [C 39 H 42 [BClF2N6O3] + 726.3063 found: 726.3040

[0163]

Example 14

[0164] In this embodiment, based on Example 8, compound 5 was used to replace compound 3 to prepare tetraazine cyanine compound TBCy7-2 with different conjugated carbon chain lengths.

[0165]

[0166] Under argon protection, DIPEA (26 μL) was added to a solution of tetraazin cyanide precursor TBFI-4 (22.7 mg, 0.04 mmol) and compound 5 (37.4 mg, 0.075 mmol) in anhydrous dichloromethane (0.5 mL). The reaction system was stirred under reflux for 1 h. After the reaction was completed, the solvent was removed by vacuum distillation, and the residue was purified by silica gel column chromatography to finally obtain tetraazin cyanide compound TBCy7-2 in 61% yield, which was characterized as follows:

[0167] 1 H NMR (400 MHz, CDCl3) δ 8.75 – 8.55 (m, 4H), 7.89 – 7.73 (m, 2H), 7.53 (d, J = 8.0 Hz, 2H), 7.26 – 7.14 (m, 3H), 6.96 (dt, J = 13.8, 9.4 Hz,2H), 6.79 – 6.62 (m, 2H), 6.48 – 6.34 (m, 1H), 6.28 – 6.14 (m, 1H), 5.50 (d,J = 12.4 Hz, 1H), 5.00 (s, 1H), 4.46 (s, 2H), 3.21 (s, 3H), 1.82 (s, 6H), 1.62 (s, 6H), 1.49 (s, 9H).

[0168] 13 C NMR (101 MHz, CDCl3) δ 174.70, 169.12, 163.55, 163.46, 161.97,155.93, 148.89, 148.42, 146.98, 144.33, 144.05, 142.68, 139.25, 130.82,129.26, 128.45, 128.17, 127.94, 126.24, 124.65, 121.74, 121.01, 116.19,115.56, 106.92, 101.59, 97.85, 79.85, 52.03, 46.50, 44.45, 29.37, 28.42, 28.34, 22.72.

[0169] HRMS [M] + m / z calcd. for [C 44 H 45 [BClF2N7O3] +803.3328 found: 803.3301

[0170]

Example 15

[0171] In this embodiment, based on Example 9, compound 5 was used to replace compound 3 to prepare tetraazine cyanine compound TBCy7-3 with different conjugated carbon chain lengths.

[0172]

[0173] Under argon protection, acetic acid (5 μL) and piperidine (10 μL) were added to a toluene (0.8 mL) solution of tetraazine cyanide precursor TBFI-3 (16.0 mg, 0.03 mmol) and compound 5 (30 mg, 0.06 mmol). The reaction system was heated and stirred at 80 °C for 2 h. After the reaction was completed, the solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography to finally obtain tetraazine cyanide compound TBCy7-3 in 46% yield. Its characterization was as follows:

[0174] 1 H NMR (400 MHz, CDCl3) δ 8.71 (dd, J = 8.4, 1.7 Hz, 1H), 8.63 – 8.59 (m, 3H), 7.82 (d, J = 8.2 Hz, 1H), 7.68 (dd, J = 14.4, 11.8 Hz, 1H), 7.53 (d,J = 8.1 Hz, 2H), 7.24 – 7.16 (m, 2H), 7.15 – 7.07 (m, 1H), 6.91 (t, J = 7.4Hz, 2H), 6.68 (d, J = 7.8 Hz, 1H), 6.29 (dd, J = 14.2, 11.8 Hz, 1H), 6.17(dd, J = 13.9, 11.5 Hz, 1H), 6.08 (d, J = 14.4 Hz, 1H), 5.74 (s, 1H), 5.44(d, J = 12.3 Hz, 1H), 4.99 (s, 1H), 4.46 (d, J = 6.1 Hz, 2H), 3.18 (s, 3H), 1.61 (s, 6H), 1.59 (s, 6H), 1.49 (s, 9H).

[0175] 13C NMR (101 MHz, CDCl3) δ 180.30, 173.27, 163.64, 163.45, 160.72,155.94, 147.77, 146.14, 145.73, 144.55, 141.88, 139.16, 137.33, 130.83,129.32, 128.16, 128.07, 127.86, 125.68, 124.48, 121.69, 121.38, 120.55,120.26, 116.11, 106.58, 97.31, 92.42, 79.88, 49.14, 46.21, 46.01, 44.44, 29.25, 28.42, 28.34, 28.22, 25.33.

[0176] HRMS [M+H] + m / z calcd. for [C 44 H 47 BF2N7O3] + 770.3796 found: 770.3768

[0177]

Example 16

[0178] In this embodiment, the tetraazine cyanide compound TBCy5P was prepared by a three-step method based on the tetraazine cyanide precursor TBFI-5, through addition reaction, amide condensation reaction and amino-active ester coupling reaction.

[0179]

[0180]

[0181]

[0182] (a) Under argon protection, tetraazine cyanide precursor TBFI-5 (43.5 mg, 0.1 mmol) and compound 9 (79.5 mg, 0.15 mmol) were added to anhydrous dichloromethane (1 mL), followed by the addition of N,N-diisopropylethylamine (DIPEA, 0.4 mmol). The reaction mixture was heated and stirred at 50 °C for 1 h. After the reaction was complete, the mixture was cooled to room temperature and slowly added to 30 mL of tert-butyl methyl ether (TBME), and allowed to stand at 4 °C overnight to precipitate. The precipitate was collected by centrifugation to obtain intermediate compound 10, which was used directly in the next reaction without further purification.

[0183] (b) Intermediate compound 10 (42 mg, 0.06 mmol) and N-hydroxysuccinimide (NHS, 28.8 mg, 0.25 mmol) were dissolved in anhydrous dichloromethane (1 mL), followed by the addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 57 mg, 0.3 mmol). The reaction mixture was stirred at room temperature for 3 h. After the reaction was complete, glacial acetic acid (20 μL) was added to quench the reaction. The crude product was purified by silica gel column chromatography to finally obtain compound 11 in 53% yield, which was characterized as follows:

[0184] 1 H NMR (400 MHz, C2D6SO) δ 8.63 (d, J = 1.7 Hz, 1H), 8.54 (dd, J =8.4, 1.5 Hz, 1H), 8.07 (t, J = 13.0 Hz, 1H), 7.95 (t, J = 12.9 Hz, 1H), 7.56(d, J = 7.9 Hz, 1H), 7.43 (d, J = 7.3 Hz, 1H), 7.27 (t, J = 7.6 Hz, 1H), 7.16(d, J = 8.0 Hz, 1H), 7.05 (t, J = 7.4 Hz, 1H), 6.50 (t, J = 13.6 Hz, 2H),5.98 (d, J = 13.0 Hz, 1H), 4.25 (s, 2H), 3.70 (t, J = 6.8 Hz, 2H), 3.44 (t, J= 7.0 Hz, 2H), 3.17 (t, J = 6.7 Hz, 2H), 2.80 (s, 4H), 2.78 (s, 4H), 1.79 (s,6H), 1.61 (s, 6H).

[0185] (c) N,N-diisopropylethylamine (DIPEA, 13 μL, 0.075 mmol) was added to an anhydrous dichloromethane (0.5 mL) solution of compound 11 (22.4 mg, 0.025 mmol) and methyl-PEG8-Amine (Me-PEG8-Amine, 28.7 mg, 0.075 mmol). The reaction system was stirred at room temperature for 2 h. After the reaction was completed, the solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography to finally obtain the tetraazine cyanide compound TBCy5P in 74% yield, which was characterized as follows:

[0186] 1H NMR (400 MHz, CDCl3) δ 8.61 (dd, J = 8.4, 1.7 Hz, 1H), 8.50 (d, J =1.8 Hz, 1H), 7.91 (dd, J = 14.0, 12.1 Hz, 1H), 7.73 (d, J = 8.2 Hz, 1H), 7.50(t, J = 13.1 Hz, 1H), 7.23 (d, J = 3.1 Hz, 1H), 7.21 (d, J = 2.6 Hz, 1H),7.00 (t, J = 7.4 Hz, 1H), 6.92 (d, J = 7.8 Hz, 1H), 6.78 (t, J = 5.1 Hz, 1H),6.69 (t, J = 5.7 Hz, 1H), 6.56 (d, J = 14.0 Hz, 1H), 6.36 (t, J = 12.8 Hz,1H), 5.71 (d, J = 12.6 Hz, 1H), 4.09 (t, J = 7.2 Hz, 2H), 3.71 (t, J = 7.2Hz, 2H), 3.69 – 3.57 (m, 50H), 3.54 (dd, J = 5.9, 3.4 Hz, 8H), 3.44 (dt, J =14.4, 4.9 Hz, 6H), 3.37 (d, J = 3.1 Hz, 6H), 2.97 (t, J = 7.2 Hz, 2H), 2.57(t, J = 7.1 Hz, 2H), 1.81 (s, 6H), 1.63 (s, 6H).

[0187] 13C NMR (101 MHz, CDCl3) δ 173.96, 171.05, 170.01, 169.33, 168.75, 163.90, 163.72, 150.26, 147.04, 145.12, 143.07, 142.40, 139.52, 129.18,128.14, 123.68, 122.01, 121.82, 121.06, 115.72, 112.72, 108.14, 100.90,98.34, 71.93, 70.52, 70.47, 70.43, 70.22, 70.13, 69.87, 69.65, 59.04, 51.81, 47.12, 39.39, 33.20, 32.73, 30.20, 28.33, 22.82.

[0188] HRMS [M+2H] 2+ m / z calcd. for [C 69 H 106 BClF2N8O 19 ] 2+ 717.3657 found: 717.3627

[0189]

Example 17

[0190] In this embodiment, based on Example 16, compound 26 was used instead of compound 9 to prepare tetraazine cyanine compound TBCy3P with different conjugated carbon chain lengths.

[0191]

[0192]

[0193] (a) Under argon protection, DIPEA (260 μL, 1.5 mmol) was added to a solution of tetraazine cyanide precursor TBFI-5 (130.7 mg, 0.3 mmol) and compound 26 (302 mg, 0.6 mmol) in anhydrous dichloromethane (3 mL). The reaction system was heated and stirred at 40 °C for 60 min. After the system cooled to room temperature, it was added to 10 mL of TBME and allowed to stand at 4 °C overnight to precipitate. The precipitate was collected by centrifugation to obtain intermediate compound 12, which was used directly in the next reaction.

[0194] (b) Intermediate compound 12 was dissolved in anhydrous dichloromethane (3 mL) with NHS (172.5 mg, 1.5 mmol), followed by the addition of EDCI (286 mg, 1.5 mmol). The reaction mixture was stirred at room temperature for 3 h. After the reaction was complete, glacial acetic acid (20 μL) was added to quench the reaction. The crude product was purified by silica gel column chromatography to finally obtain compound 13. The yield was 29%, and its characterization was as follows:

[0195] 1 H NMR (400 MHz, CDCl3) δ 8.63 (dd, J = 8.4, 1.7 Hz, 1H), 8.52 (d, J =1.7 Hz, 1H), 8.35 (t, J = 13.3 Hz, 1H), 7.72 (d, J = 8.3 Hz, 1H), 7.29 (ddd,J = 7.5, 6.4, 1.5 Hz, 2H), 7.13 – 7.06 (m, 1H), 6.91 (d, J = 7.7 Hz, 1H), 6.59 (d, J = 13.6 Hz, 1H), 5.82 (d, J = 13.0 Hz, 1H), 4.21 (t, J = 7.5 Hz,2H), 3.81 (t, J = 7.3 Hz, 2H), 3.43 (t, J = 7.3 Hz, 2H), 3.12 – 3.01 (m, 2H), 2.88 (s, 4H), 2.83 (s, 4H), 1.81 (s, 6H), 1.70 (s, 6H).

[0196] (c) DIPEA (17.4 μL, 0.1 mmol) was added to a solution of compound 13 (26.1 mg, 0.03 mmol) and Me-PEG8-Amine (28.6 mg, 0.075 mmol) in anhydrous dichloromethane (0.3 mL). The reaction system was stirred at room temperature for 1 h. After the reaction was completed, the crude product was purified by silica gel column chromatography to finally obtain the tetraazine cyanide compound TBCy3P in 61% yield, which was characterized as follows:

[0197] 1H NMR (400 MHz, CDCl3) δ 8.60 (dd, J = 8.4, 1.7 Hz, 1H), 8.48 (d, J= 1.7 Hz, 1H), 8.37 (t, J = 13.4 Hz, 1H), 7.69 (d, J = 8.3 Hz, 1H), 7.30 –7.28 (m, 1H), 7.08 (t, J = 7.5 Hz, 1H), 7.03 (d, J = 8.1 Hz, 1H), 6.82 (s,1H), 6.61 (t, J = 5.7 Hz, 1H), 6.52 (d, J = 13.4 Hz, 1H), 5.90 (d, J = 13.2Hz, 1H), 4.18 (t, J = 7.2 Hz, 2H), 3.71 (t, J = 7.2 Hz, 2H), 3.69 – 3.46 (m,64H), 3.37 (d, J = 4.5 Hz, 6H), 2.97 (t, J = 7.2 Hz, 2H), 2.61 (t, J = 7.2Hz, 2H), 1.80 (s, 6H), 1.68 (s, 6H).

[0198] 13 C NMR (101 MHz, CDCl3) δ 173.01, 171.03, 169.61, 168.62, 168.21,163.99, 147.39, 145.75, 142.62, 142.15, 140.01, 129.19, 128.24, 127.56,122.95, 121.97, 121.01, 115.29, 110.20, 108.91, 99.92, 98.26, 71.93, 70.56,70.52, 70.44, 70.25, 70.15, 69.85, 69.61, 59.04, 51.46, 47.96, 39.40, 33.22,32.75, 30.18, 28.81, 22.96.

[0199] HRMS [M+Na] + m / z calcd. for [C 67 H 102 BClF2N8NaO 19 ] + 1429.6903 found:1429.6907

[0200]

Example 18

[0201] In this embodiment, based on Example 16, compound 6 was used instead of compound 9 to prepare tetraazine cyanine compound TBCy7P with different conjugated carbon chain lengths.

[0202]

[0203]

[0204] (a) Tetraazine cyanide precursor TBFI-5 (26 mg, 0.06 mmol) and compound 6 (55.6 mg, 0.1 mmol) were dissolved in dichloromethane (0.6 mL), followed by the addition of DIPEA (52 μL, 0.3 mmol). The reaction mixture was heated and stirred at 50 °C for 1 h. After the reaction was complete, the system was cooled to room temperature, and the solvent was removed by vacuum distillation. Methyl tert-butyl ether (MTBE) was added to the residue to induce precipitation. After centrifugation and drying, solid intermediate compound 14 was obtained and used directly in the next reaction.

[0205] (b) Intermediate compound 14 (approximately 0.06 mmol) and NHS (28.8 mg, 0.25 mmol) were dissolved in anhydrous dichloromethane (1 mL), followed by the addition of EDCI (57 mg, 0.3 mmol). The reaction mixture was stirred at room temperature for 3 h. The crude product was purified by silica gel column chromatography to obtain compound 15.

[0206] (c) DIPEA (8.7 μL, 0.05 mmol) was added to a solution of compound 15 (13.8 mg, 0.015 mmol) and Me-PEG8-Amine (15.3 mg, 0.04 mmol) in anhydrous dichloromethane (0.3 mL). The reaction system was stirred at room temperature for 1 h. After the reaction was completed, the crude product was purified by silica gel column chromatography to finally obtain the tetraazine cyanide compound TBCy7P in 56% yield, which was characterized as follows:

[0207] 1H NMR (400 MHz, CDCl3)δ 8.62 (dd, J = 8.4, 1.7 Hz, 1H), 8.52 (d, J =1.6 Hz, 1H), 7.85 – 7.73 (m, 2H), 7.22 – 7.16 (m, 2H), 6.98 – 6.90 (m, 2H), 6.85 – 6.78 (m, 2H), 6.68 (d, J = 14.2 Hz, 1H), 6.46 – 6.38 (m, 1H), 6.29 –6.18 (m, 1H), 5.60 (d, J = 12.4 Hz, 1H), 4.03 (t, J = 7.3 Hz, 2H), 3.66 –3.59 (m, 52H), 3.55 (dt, J = 5.8, 2.7 Hz, 8H), 3.48 – 3.41 (m, 6H), 3.37 (d,J = 1.8 Hz, 6H), 2.98 (t, J = 7.2 Hz, 2H), 2.59 – 2.52 (m, 2H), 1.81 (s, 6H),1.61 (s, 6H).

[0208] HRMS [M+2H] 2+ m / z calcd. for [C 71 H 108 BClF2N8O 19 ] 2+ 730.3735 found: 730.3745

[0209]

Example 19

[0210] In this embodiment, tetraazine cyanide compound TBCy3-CN with different R2 and R3 was directly prepared based on tetraazine cyanide precursor TBFI-6 and compound 20.

[0211]

[0212] Specifically, the tetraazine cyanide precursors TBFI-6 (compound 21, 18.0 mg, 0.03 mmol) and 20 (14.1 mg, 0.03 mmol) were dissolved in 0.6 mL of toluene, and 8 μL of glacial acetic acid and 15 μL of piperidine (precisely weighed) were added. The mixture was stirred at 40°C for 2 hours. After complete conversion of the starting material, 5 μL of glacial acetic acid was added to terminate the reaction. After purification by silica gel thin-layer chromatography, the tetraazine cyanide compound TBCy3-CN was finally obtained in 53% yield, and its characterization was as follows:

[0213] 1 H NMR (400 MHz, CDCl3) δ 8.63 (dd, J = 8.4, 1.7 Hz, 1H), 8.52 (d, J =1.6 Hz, 1H), 8.25 (t, J = 13.3 Hz, 1H), 7.75 (d, J = 8.3 Hz, 1H), 7.56 (dd, J= 8.3, 1.5 Hz, 1H), 7.46 (d, J = 1.5 Hz, 1H), 7.03 (dd, J = 15.8, 6.8 Hz,2H), 6.68 – 6.58 (m, 2H), 5.88 (d, J = 12.9 Hz, 1H), 4.15 (t, J = 7.0 Hz,2H), 3.72 (t, J = 7.2 Hz, 2H), 3.69 – 3.63 (m, 10H), 3.62 (s, 4H), 3.57 (ddt,J = 9.6, 6.3, 3.7 Hz, 11H), 3.51 (dd, J = 5.8, 3.1 Hz, 2H), 3.47 (t, J = 6.2Hz, 4H), 3.42 (t, J = 5.1 Hz, 2H), 3.37 (s, 3H), 3.30 (s, 3H), 2.98 (t, J =7.2 Hz, 2H), 2.60 (t, J = 6.9 Hz, 2H), 2.27 (s, 2H), 1.81 (s, 6H), 1.68 (s,6H).

[0214] 13C NMR (101 MHz, CDCl3) δ 174.77, 171.00, 169.48, 169.36, 168.80,165.00, 163.84, 146.82, 146.60, 143.47, 142.43, 140.45, 133.48, 129.21,128.49, 125.27, 121.08, 119.40, 115.96, 113.41, 108.94, 104.63, 100.68,99.48, 71.89, 71.82, 70.52, 70.45, 70.42, 70.36, 70.26, 70.22, 70.09, 69.84, 69.60, 59.00, 58.84, 51.97, 47.07, 39.46, 39.40, 32.94, 32.69, 30.19, 28.73, 22.71.

[0215] HRMS [M+H] + m / z calcd. for [C 52 H 70 BClF2N9O 11 ] + 1080.4939 found: 1080.4951

[0216]

Example 20

[0217] In this embodiment, the tetraazine cyanide compound TBCy5-CN was prepared by a three-step method based on the tetraazine cyanide precursor TBFI-5.

[0218]

[0219]

[0220] (a) Compound 23 (51 mg, 0.18 mmol) and the tetraazine cyanide precursor TBFI-5 (63 mg, 0.15 mmol) were dissolved in 2 mL of a mixed solvent consisting of toluene, acetic acid (20 µL), and piperidine (55 µL). The resulting mixture was stirred at 60 °C for 2 hours. Subsequently, the solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography to give direct product 24 for subsequent steps.

[0221] (b) Compound 24 (weighed 0.1 mmol) was mixed with NHS (29.5 mg, 0.26 mmol) in anhydrous dichloromethane (0.6 mL), followed by the addition of EDCI (66.5 mg, 0.35 mmol). The reaction mixture was stirred at room temperature for 2 hours. Subsequently, the residue was purified by silica gel column chromatography to give compound 25 in a yield of 26%, characterized as follows:

[0222] 1 H NMR (400 MHz, CDCl3) δ 8.67 (dd, J = 8.5, 1.7 Hz, 1H), 8.57 (d, J =1.6 Hz, 1H), 7.90 – 7.79 (m, 2H), 7.54 (dd, J = 8.3, 1.6 Hz, 1H), 7.42 (d, J= 1.5 Hz, 1H), 7.39 – 7.31 (m, 1H), 6.84 (d, J = 8.3 Hz, 1H), 6.71 (d, J =14.3 Hz, 1H), 6.43 (dd, J = 13.9, 11.8 Hz, 1H), 5.63 (d, J = 12.2 Hz, 1H),4.11 (t, J = 7.2 Hz, 2H), 3.83 (t, J = 7.2 Hz, 2H), 3.44 (t, J = 7.2 Hz, 2H), 3.03 (t, J = 7.1 Hz, 2H), 2.86 (s, 4H), 2.83 (s, 4H), 1.83 (s, 6H), 1.65 (s,6H).

[0223] (c) In a solution of compound 25 (18.4 mg, 0.02 mmol) and methyl-PEG4-Amine (Me-PEG4-Amine, 10 mg, 0.05 mmol) dissolved in dichloromethane (0.2 mL), N,N-diisopropylethylamine (10.2 μL, 0.06 mmol) was added. The mixture was stirred at room temperature for 60 min and purified by silica gel column chromatography to give the tetraazine cyanide compound TBCy5-CN in 70% yield, characterized as follows:

[0224] 1H NMR (400 MHz, CDCl3) δ 8.64 – 8.53 (m, 2H), 7.92 (dd, J = 14.2,12.0 Hz, 1H), 7.66 – 7.55 (m, 4H), 7.01 (d, J = 8.8 Hz, 1H), 6.69 (d, J =14.2 Hz, 1H), 6.47 (dd, J = 13.7, 12.0 Hz, 1H), 5.85 (d, J = 12.7 Hz, 1H),4.04 (t, J = 6.5 Hz, 2H), 3.59 – 3.51 (m, 18H), 3.49 – 3.42 (m, 10H), 3.28(dd, J = 7.1, 4.7 Hz, 10H), 3.19 (t, J = 5.4 Hz, 2H), 2.87 (t, J = 7.2 Hz,2H), 2.49 (t, J = 6.6 Hz, 2H), 1.81 (s, 6H), 1.61 (s, 6H).

[0225] 13 C NMR (101 MHz, CD3CN) δ 176.23, 172.01, 171.92, 171.00, 170.91,170.20, 169.87, 164.77, 163.20, 150.06, 148.28, 147.34, 145.32, 143.98,141.56, 134.21, 130.24, 129.78, 126.48, 126.39, 122.33, 120.65, 116.28,115.01, 109.85, 104.01, 101.97, 101.47, 72.58, 71.15, 71.11, 71.07, 71.00,70.96, 70.91, 70.88, 70.26, 70.04, 58.88, 53.20, 47.35, 39.87, 39.86, 33.91,33.87, 33.37, 33.32, 31.07, 28.18, 22.77.

[0226] HRMS [M+H] + m / z calcd. for [C 54 H 71 BClF2N9O 11 ] +1106.5095 found: 1106.5103

[0227] The synthetic route for preparing compound 23 is as follows:

[0228]

[0229]

[0230] Anhydrous acetic acid (3 mL) was added dropwise to dimethylaminopropenal (35 μL, 3.5 mmol), and the mixture was stirred at room temperature for 15 minutes. Then, a solution of compound 18 (284 mg, 1 mmol) and CH₂Cl₂ (1 mL) was added. The reaction mixture was stirred at room temperature for 48 hours, and then the solvent was removed under reduced pressure. The residue was dissolved in THF (8 mL), and a saturated solution of K₂CO₃ (12 mL) was carefully added. After stirring the mixture at room temperature for 30 minutes, water (30 mL) was added, and the aqueous layer was extracted with ethyl acetate (2 × 20 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give compound 22 as a yellow oil (220 mg, 65%).

[0231] To a THF solution (156 mg, 0.5 mmol) of compound 22 (4 mL), an aqueous solution (4 mL) of LiOH·H₂O (25.2 mg, 0.6 mmol) was added. The mixture was stirred at 25 °C for 2 hours. After the reaction was complete, the pH was adjusted to 3 with 1 M hydrochloric acid, 20 mL of water was added, and the mixture was then extracted with acetic acid-diethyl ether (EA). The mixture was dried over sodium sulfate. The solvent was removed under reduced pressure to give compound 23, which can be used without purification for the reaction with the tetraazine cyanide precursor TBFI-5.

[0232] III. Performance Testing of Tetraazine Cyanide Compounds

[0233]

Example 21

[0234] In this embodiment, the optical properties and bioorthogonal reactions of each tetrazine cyanide compound (TBCy series probes) were detected.

[0235] Specifically, take appropriate amounts of the stock solutions (TBCy3-1, TBCy3-2, TBCy3-3, TBCy5-1, TBCy5-2, TBCy5-3, TBCy5-4, TBCy7-1, TBCy7-2, TBCy7-3) and dilute them with different solvents (dichloromethane, tetrahydrofuran, methanol, and dimethyl sulfoxide) to a final concentration of 0.5 μM. Take 1 ml of the test solution and add an appropriate amount of bicyclic [6.1.0]nonyne (BCN) stock solution (10.0 mM) to make the final concentration of BCN in the system 5 μM (i.e., the molar equivalent ratio of probe to BCN is 1:10, to ensure that the reaction proceeds completely). After adding, mix quickly and place the cuvette in a constant temperature and light-protected environment, set the reaction temperature to 37°C, and incubate for 2 hours. After incubation, the absorption and fluorescence emission spectra of the product (pyridazine adduct) were recorded again using the same UV and fluorescence spectrophotometers under the same instrument parameters.

[0236] The test results are as follows Figure 2 As shown, each tetrazine cyanine compound exhibits sharp and typical BODIPY-type characteristic absorption peaks in the visible to near-infrared region. Furthermore, by adjusting the length of the conjugated carbon chain, full spectral coverage was achieved, meeting different imaging requirements. Specifically, the maximum absorption wavelengths of TBCy3-1, TBCy5-1, and TBCy7-1 are located at 610 nm, 700 nm, and 732 nm, respectively. After introducing excess BCN into the system, although the tetrazine unit underwent a chemical transformation from tetrazine to dalazine, the maximum absorption wavelength and peak shape of each probe remained essentially constant.

[0237] Compared to the relatively stable absorption spectrum, the fluorescence emission spectrum of the probe before and after the reaction exhibits a significant intensity contrast, such as... Figures 3 to 6 As shown, before the addition of BCN, the emission signal of the probe is at a very low background fluorescence signal; however, once the IEDDA cycloaddition reaction occurs with BCN, a strong fluorescence emission peak is generated at the corresponding wavelength.

[0238]

Example 22

[0239] In this embodiment, the wash-free specific imaging of various tetrazine cyanine compounds on organelles such as mitochondria, lysosomes, and endoplasmic reticulum was verified.

[0240] Specifically, A549 cells (human lung cancer cells) were used as a biological model and cultured in intact DMEM medium containing 10% FBS (37 °C, 5% CO2). Cells were co-incubated for 1 h with 1 μM of targeted pretreatment probes (MemER, MemMito, or MemER) and their corresponding commercial tracking agents (Mito-tracker Green, Lyso-tracker Green, or ER-trackerGreen). After washing twice with PBS to remove free molecules, 1 μM of TBCy series probes were added and incubated for 60 min. No further washing was required after the reaction; images were directly acquired using a confocal laser scanning microscope (CLSM). The excitation / emission wavelengths for TBCy5P were set to 660 / 675–799 nm; for TBCy3P, 610 / 627–711 nm; and for TBCy7P, 701 / 720–841 nm.

[0241] The test results are as follows Figures 7 to 10 As shown, the fluorescence signals of tetraazine cyanide compounds highly overlap spatially with those of commercially available tracers, and their Pearson correlation coefficient (R0) is similar. 2 The fluorescence intensity profiles (IMPs) were between 0.84 and 0.95, and the peak phases of the IMPs were highly consistent, demonstrating that the probe system has extremely high positioning accuracy.

[0242] In the bioorthogonal performance validation, the pretreated group (+) rapidly generated a strong fluorescence signal after the addition of the probe, while the control group (-) without pre-targeting molecules showed extremely low background fluorescence. Quantitative analysis showed that the imaging contrast was improved by approximately 3 to 6 times compared to the control group. Imaging validation targeting the endoplasmic reticulum further confirmed that, guided by TER, the probe could clearly depict the typical reticulum structure and maintain an excellent signal-to-noise ratio even at pretreatment concentrations as high as 5 μM.

[0243]

Example 23

[0244] In this embodiment, the biocompatibility of each tetrazine cyanide compound with cells at the in vitro level was tested.

[0245] Specifically, A549 cells in the logarithmic growth phase were digested with trypsin and collected, then divided into 5 × 10⁶ cells per well. 3Cells were seeded at a density of 100 μL in 96-well plates, with 100 μL of DMEM complete medium containing 10% fetal bovine serum (FBS) added to each well. The plates were pre-cultured at 37 °C with 5% CO2 for 24 h. After cell attachment, the original medium was aspirated from the wells, and different concentration gradient probes (0, 0.5, 1.0, 2.0, and 4.0 μM) were added to each well. The plates were then incubated in the dark for another 24 h. After incubation, the drug-containing medium was aspirated, and 100 μL of DMEM complete medium containing 10% CCK-8 working solution was added to each well. The plates were then incubated in the dark for another 2 h. Finally, the absorbance (OD) of each well was measured at 450 nm using a microplate reader.

[0246] The results are as follows Figure 11 As shown, the cell viability of the TBCy3P, TBCy5P, and TBCy7P treatment groups remained above 95%. Even at the highest concentration gradient (4.0 μM) set in the experiment, the cell viability of the TBCy3P and TBCy5P treatment groups was still close to 100%, while the cell viability of TBCy7P remained at around 95%, and none of them showed any significant inhibition of cell proliferation or cytotoxicity, indicating that the TBCy series probes have excellent biocompatibility.

[0247]

Example 24

[0248] In this embodiment, TBCy3P after the targeting reaction was used as a representative molecule, and it was compared in parallel with commercial mitochondrial dye Mito-Tracker Red (MTR) and lysosomal dye Lyso-Tracker Red (LTR) in the A549 live cell model.

[0249] Specifically, CLSM was used to apply 100 consecutive frames of laser scanning irradiation to the cell regions loaded with the corresponding fluorescent molecules. Fluorescence emission signals at each time point were extracted, and the fluorescence intensity of the initial first frame was normalized to 100% to plot the signal decay kinetics curve.

[0250] Experimental results are as follows Figure 12As shown, commercial dyes and TBCy series probes exhibit drastically different decay kinetics under the same light stress. Under continuous excitation, the fluorescence intensity of MTR and LTR rapidly decreased precipitously; after 100 consecutive scans, the fluorescence signals of both commercial dyes were almost completely quenched, with a relative retention rate of only about 15% of the initial intensity. Conversely, the in-situ generated TBCy3P addition product exhibited excellent optical toughness throughout the entire continuous irradiation cycle, maintaining its fluorescence emission signal at approximately the initial value after 100 frames of irradiation. Microscopic morphological observation revealed that MTR induced severe photodamage to cells during irradiation, resulting in significant cell shrinkage and morphological deformation in A549 cells within the observation area. Cells treated with TBCy series probes, however, maintained normal adherent and extended morphology after being subjected to the same dose of continuous light irradiation, and no acute morphological changes induced by the probes were observed.

[0251]

Example 25

[0252] In this embodiment, the lysosomal membrane imaging performance was tested.

[0253] Specifically, A549 cells were seeded in 35 mm glass-bottomed culture dishes and cultured for 24 hours, followed by probe treatment according to the specified protocol. Cell cultures were imaged using a STELLARIS 5 STED microscope (Leica DMI8) equipped with an HC PL APO CS2 100x / 1.40 oil immersion objective at the specified excitation wavelength. The excitation source was a pulsed white laser. Fluorescence intensity of regions of interest (ROIs) over time was measured using Leica's built-in analysis software, LAXS X. The intensity of ROIs in each frame is expressed as a percentage of the intensity of the first frame, defined as 100%. The fluorescence intensity curves and distances of lysosomes were analyzed using ImageJ software, and the full width at half maximum (FWHM) of lysosomes was processed using Gaussian fitting with Origin 2015 software.

[0254] TBCy3-CN: Cells were labeled with the 350 nM lysosomal membrane pre-targeting probe MemLyso for 1 hour (37 °C, 5% CO2) in DMEM medium containing 10% FBS, washed twice with PBS, and then 250 nM TBCy3-CN or TBCy5-CN was added to DMEM medium containing 10% FBS. The cells were incubated at 37 °C, 5% CO2 for 40 minutes and then directly imaged without washing.

[0255] The test results are as follows Figure 13As shown, after adding MemLyso-pretreated A549 cells to TBCy3-CN, STED microscopy imaging clearly revealed numerous ring-shaped, tortuous, and irregular membrane structures associated with lysosomes. In region of interest 3, two adjacent lysosomes spaced 120 nm apart were clearly observed, while these fine membrane structures could not be resolved by confocal microscopy. The transverse intensity distribution curves of the lysosomes showed a full width at half maximum (FWHM) value of 125–181 nm.

[0256]

Example 26

[0257] In this embodiment, the endoplasmic reticulum imaging performance of TBCy3-CN was tested.

[0258] Specifically, A549 cells were seeded in 35 mm glass-bottomed culture dishes and cultured for 24 hours, followed by probe treatment according to the specified protocol. Cell cultures were imaged using a STELLARIS 5 STED microscope (Leica DMI8) equipped with an HC PL APO CS2 100x / 1.40 oil immersion objective at the specified excitation wavelength. The excitation source was a pulsed white laser. Fluorescence intensity of regions of interest (ROIs) over time was measured using Leica's built-in analysis software, LAXS X. The intensity of ROIs in each frame is expressed as a percentage of the intensity of the first frame, defined as 100%. The fluorescence intensity curves and distances of the endoplasmic reticulum (ER) were analyzed using ImageJ software, and the full width at half maximum (FWHM) of the ER was processed using Gaussian fitting with Origin 2015 software.

[0259] TBCy3-CN: Cells were labeled with the 3 μM endoplasmic reticulum membrane pre-targeting probe MemER for 1 hour (37 °C, 5% CO2) in DMEM medium containing 10% FBS, washed twice with PBS, and then 250 nM TBCy3-CN or TBCy5-CN was added to DMEM medium containing 10% FBS. The cells were incubated at 37 °C, 5% CO2 for 40 minutes and then directly imaged without washing.

[0260] The results are as follows Figure 14 As shown, STED imaging of the endoplasmic reticulum using TBCy3-CN achieved a spatial resolution of 172 nm.

[0261]

Example 27

[0262] In this embodiment, the STED imaging performance of the TBCy3-CN mitochondrial membrane was tested.

[0263] Specifically, HEUVC cells were seeded in 35 mm glass-bottomed culture dishes and cultured for 24 hours, followed by probe treatment according to the specified protocol. The cell cultures were imaged using a STELLARIS 5 STED microscope (Leica DMI8) equipped with an HC PL APO CS2 100x / 1.40 oil immersion objective at the specified excitation wavelength. The excitation source was a pulsed white laser. The fluorescence intensity of regions of interest (ROIs) over time was measured using Leica's built-in analysis software, LAXS X. The intensity of ROIs in each frame is expressed as a percentage of the intensity of the first frame, defined as 100%. The fluorescence intensity curves and distances of mitochondria were analyzed using ImageJ software, and the full width at half maximum (FWHM) of mitochondria was processed using Gaussian fitting with Origin 2015 software.

[0264] TBCy3-CN: Cells were labeled with the 350 nM mitochondrial membrane pre-targeting probe MemMito for 1 hour (37 °C, 5% CO2) in DMEM medium containing 10% FBS, washed twice with PBS, and then 250 nM TBCy3-CN or TBCy5-CN was added to DMEM medium containing 10% FBS. The cells were incubated at 37 °C, 5% CO2 for 40 minutes and then directly imaged without washing.

[0265] The results are as follows Figure 15 As shown, a 167 nm resolution image of the mitochondrial membrane was obtained in HEUVC cells, capturing the ongoing morphological changes. In contrast, confocal images failed to resolve these fine structures.

[0266] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A tetraazine anthocyanin compound, characterized in that, It has the structural formula shown in Equation I: Formula I: ; In Formula I, group R1 is selected from... , , or Group R2 is selected from C1-C6 alkyl groups, or The group R3 is selected from H or cyano; the group X is selected from H or halogen; where n=1~5, m=1~8, p=1~8.

2. The tetraazine anthocyanin compound according to claim 1, characterized in that, The selection is selected from , ,or The group R2 is selected from C1-C4 alkyl groups, or The group R3 is selected from H or cyano; the group X is selected from H or halogen; where n=1~4, m=1~7, p=1~7.

3. A tetraazine anthocyanin compound according to claim 1 or 2, characterized in that, The tetraazine anthocyanin compound is selected from the following compounds: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 4. A method for preparing a tetraazine anthocyanin compound, characterized in that, Includes the following steps: The tetraazine cyanine precursor shown in Formula II was dissolved with the compound shown in Formula III, and acetic acid and piperidine were added. The mixture was then heated to prepare the tetraazine cyanine compound shown in Formula I. or The tetraazine cyanine precursor shown in Formula II was dissolved with the compound shown in Formula IV, and N,N-diisopropylethylamine was added. The mixture was then heated to prepare the tetraazine cyanine compound shown in Formula I. Formula I: Formula II: Formula III: Formula IV: ; In Formulas I to IV, group R1 is selected from... , , or Group R2 is selected from C1-C6 alkyl groups, or The group R3 is selected from H or cyano; the group X is selected from H or halogen; where n=1~5, m=1~8, p=1~8.

5. The method for preparing a tetraazine anthocyanin compound according to claim 4, characterized in that, Group R1 is The tetraazine cyanide precursor is reacted with the compound shown in Formula IV to prepare a first intermediate. The first tetraazine intermediate is dissolved with N-hydroxysuccinimide, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is added to react and prepare a second intermediate. The second intermediate is then reacted with... After mixing, N,N-diisopropylethylamine was added, and the reaction yielded group R1. Tetraazine anthocyanin compounds.

6. A method for preparing a tetraazine anthocyanin compound according to claim 4 or 5, characterized in that, The preparation method of the tetraazine cyanide precursor shown in Formula II includes the following steps: The tetrazine compound shown in Formula V was dissolved with the tetrazine compound shown in Formula VI, and Pd(PPh3)4 and CuTc were added. The mixture was heated to prepare the tetrazine anthocyanin precursor shown in Formula II. Formula V: Formula VI: .

7. The method for preparing a tetraazine anthocyanin compound according to claim 6, characterized in that, The tetraazine anthocyanin precursor was prepared by halogenation to obtain a tetraazine anthocyanin precursor with a halogen group X.

8. The method for preparing a tetraazine anthocyanin compound according to claim 6, characterized in that, Group R1 is After dissolving the tetraazine cyanide precursor, trifluoroacetic acid was added, and the reaction was carried out at room temperature to obtain the group R1. Tetraazine cyanine precursor; or Group R1 is After dissolving the tetraazine cyanide precursor, trifluoroacetic acid was added, and the reaction was carried out at room temperature to obtain the group R1. Tetraazine anthocyanin precursor, wherein the group R1 is The tetraazine cyanide precursor was reacted with a mixture of 1-hydroxybenzotriazole and N,N'-carbonyldiimidazole, followed by the addition of The reaction continued to produce group R1. Tetraazine anthocyanin precursor.

9. A reagent for cell membrane fluorescence imaging, characterized in that, Includes the tetraazine cyanine compound according to any one of claims 1 to 3.

10. A reagent for cell membrane fluorescence imaging according to claim 9, characterized in that, It also includes prepositioning molecules for targeting the cell membranes of mitochondria, lysosomes, and endoplasmic reticulum.

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