Dye with up-conversion performance as well as synthesis method and application thereof

Abstract

The invention discloses a dye with up-conversion performance, and a synthesis method and application thereof. The dye generally has up-conversion performance, can emit fluorescent light (short wavelength) under irradiation of light (long wavelength) of 750-900 nm, and can avoid the problem of photobleaching caused by short wavelength excitation. Meanwhile, the dye has a high extinction coefficient and a high fluorescence quantum yield in an aqueous solvent and a polar protic solvent, so that the dye is very beneficial to fluorescence imaging, gene sequencing, biomacromolecule recognition and the like. Meanwhile, the upconversion mechanism also has deeper tissue penetrating ability, is beneficial to in-situ imaging and photodynamic therapy of deep pathological tissues of a living body, and has a very good application prospect.

Description

[0001] This application is a divisional application of the following original application: Application date of the original application: December 26, 2023; Application number of the original application: 2023118072978; Title of the original application: A dye with upconversion properties, its synthesis method and application. Technical Field

[0002] This invention belongs to the field of fine chemical technology and relates to the synthesis of a dye with upconversion properties and its application in bioimaging, gene sequencing and photodynamic therapy. Background Technology

[0003] Fluorescent dyes can absorb light of specific wavelengths and emit bright fluorescence, offering advantages such as high efficiency, convenience, tunable wavelength, and visualization. Therefore, fluorescence imaging technology based on fluorescent dyes has developed rapidly in recent decades. However, with the continuous upgrading of lasers and recognition devices used in imaging, higher-performance fluorescent dyes are needed to suit complex application scenarios in fields such as biomolecular recognition, gene sequencing, and super-resolution imaging.

[0004] Currently, commonly used fluorescent dyes face challenges including short wavelengths and poor photostability. While fluorescent dyes with short-wavelength excitation and emission typically offer relatively high imaging resolution, short-wavelength light has poor tissue penetration and is highly susceptible to interference from the autofluorescence of biomolecules, resulting in poor imaging performance. Simultaneously, short-wavelength lasers are usually high-energy, leading to photobleaching of the dye after multiple exposures, thus losing imaging and recognition capabilities. Therefore, the development of novel fluorescent dyes is necessary to overcome these problems. Upconversion is an unconventional luminescence phenomenon, referring to the emission of short-wavelength fluorescence from compounds excited by long-wavelength light. If applied to fluorescence imaging, low-energy but high-tissue-penetration long-wavelength lasers can excite dyes to obtain short-wavelength fluorescence, achieving high-resolution imaging and effectively avoiding photobleaching. However, currently, materials with upconversion properties are mainly inorganic noble metals, such as lanthanides, which are difficult to modify specifically, hindering practical applications. Therefore, the development of fluorescent dyes with upconversion properties is of great significance. Summary of the Invention

[0005] To address the problem of short-wavelength excitation in current fluorescent dyes, this invention provides a dye with upconversion properties, its synthesis method, and its applications. The dye of this invention is synthesized from indole with specific substituents, a quaternary ammonium salt, and a condensing agent. This dye possesses excellent optical properties and can be applied in fields such as fluorescent labeling, bioimaging, gene sequencing, biomolecule recognition, and photodynamic therapy. It exhibits significant advantages, particularly in bioimaging, long-wavelength excited gene sequencing, and photodynamic therapy.

[0006] The technical solution of this application is as follows: The first aspect of this application is to protect a dye having upconversion properties, said dye having the structure of general formula I.

[0007]

[0008] I In general formula I, A1 is selected from any one or more of the groups described in i-iv;

[0009] A2 is selected from any one or more of the groups described in vx;

[0010] R1 is selected from any one or more of hydrogen, aryl, alkyl with 1-18 carbons, carboxyl alkyl with 1-18 carbons, sulfonic acid group or sulfonate with 1-18 carbons, hydroxyalkyl with 1-18 carbons, aminoalkyl with 1-18 carbons, and aryl carboxylic acids; more preferably from any one or more of hydrogen, alkyl with 1-6 carbons, carboxyl alkyl with 1-6 carbons, sulfonic acid group or sulfonate with 1-6 carbons, aryl, and aryl carboxylic acids. R2 is a substituent at any position on the benzene ring, selected from any one or more of alkyl, carboxyl, amino, nitro, methoxy, halogen, hydroxy, ester, amide, and sulfonate groups with 1 to 6 carbons; more preferably, selected from any one or more of alkyl, carboxyl, halogen, methoxy, amide, ester, and sulfonate groups with 1 to 3 carbons. R3 is selected from one or more of hydrogen, alkyl groups with 1-18 carbons, carboxyl groups with 1-18 carbons, sulfonic acid groups with 1-18 carbons, and aryl carboxylic acids; more preferably from one or more of hydrogen, aryl carboxylic acids, and carboxyl groups with 1-8 carbons. Y is selected from halide ions, ClO4 - CF3COO - or OTs - Any one or more of the following.

[0011] R4 is selected from any one or more of alkyl, carboxyl, amino, methoxy, halogen, aryl, ester, and amide groups with 1 to 6 carbons; more preferably from any one or more of alkyl, carboxyl, halogen, and aryl groups with 1 to 3 carbons. Wherein, R5 is a substituent at any position on the benzene ring, selected from any one or more of alkyl, carboxyl, amino, nitro, methoxy, halogen, hydroxy, ester, amide, and sulfonate groups with 1 to 6 carbons; more preferably, selected from any one or more of alkyl, carboxyl, halogen, methoxy, amide, ester, and sulfonate groups with 1 to 3 carbons. R6 is selected from any one or more of hydrogen, aryl, alkyl with 1-18 carbons, carboxyl alkyl with 1-18 carbons, sulfonic acid group or sulfonate with 1-18 carbons, hydroxyalkyl with 1-18 carbons, aminoalkyl with 1-18 carbons, and aryl carboxylic acid; more preferably from any one or more of hydrogen, alkyl with 1-6 carbons, carboxyl alkyl with 1-6 carbons, sulfonic acid group or sulfonate with 1-6 carbons, aryl, and aryl carboxylic acid. R7 is selected from hydrogen, alkyl groups of 1-18 carbons, carboxyl groups of 1-18 carbons, sulfonic acid groups of 1-18 carbons, and aryl carboxylic acids; more preferably from any one or more of hydrogen, aryl carboxylic acids, and carboxyl groups of 1-8 carbons. The second aspect of this application is to protect a method for synthesizing a dye with upconversion properties, comprising the following steps: Step 1: Synthesis of Indole

[0012] 2-hydrazinopyridine with R2 substituent and ethyl ketone with R3 substituent are added to an organic solvent in a molar ratio of 1:1-5. After reacting at 50-120℃, the solvent is removed, and then added to an organic acid solvent. After reacting at 80-120℃, the temperature is lowered, the pH is adjusted to 1-5, and the mixture is extracted and purified to obtain indole with substituents. More preferably, in step one, the molar ratio of the 2-hydrazinopyridine with the R2 substituent to the ethyl ketone with the R3 substituent is more preferably 1:1-3. More preferably, in step one, the organic solvent is selected from one or more mixed solvents selected from toluene, o-dichlorobenzene, and benzene; More preferably, in step one, the organic acid is selected from one or a mixture of several of acetic acid, polyphosphoric acid, trifluoroacetic acid, and benzoic acid. More preferably, in step one, the pH is adjusted to 1-3; Step 2: Synthesis of Quaternary Ammonium Salts

[0013] Indole with R2 and R3 is mixed with N-alkylating agent with R1 substituent in a molar ratio of 1:1-10, added to a polar organic solvent, and reacted at 40-100℃ to obtain solid powder quaternary ammonium salt. More preferably, in step two, the molar ratio of the indole with R2 and R3 to the N-alkylating agent with R1 substituent is 1:1-5. More preferably, in step two, the polar organic solvent is selected from one or more mixed solvents selected from methanol, ethanol, acetonitrile, and acetone; Step 3: Dye Synthesis

[0014] The quaternary ammonium salt obtained in step 2 is mixed with a condensing agent containing A1 and A2 at a molar ratio of 1:0.5-8. Solvent A is added to the mixture, followed by a catalytic reagent B. The mixture is reacted at 50-130°C until the reaction product no longer increases. The solvent is removed, and the product is obtained by chromatography with a metallic luster.

[0015] In step three, reagent A is selected from at least one of ethanol, acetic anhydride, n-butanol, isopropanol, and acetic acid; In step three, reagent B is selected from at least one of sodium acetate, pyridine, potassium carbonate, and triethylamine.

[0016] For the technical solution described above, in step three, the molar ratio of the quaternary ammonium salt and the condensing agent containing A1 and A2 is preferably 1:0.5-4; more preferably 1:1-3.

[0017] The third aspect of this application is to protect the application of a dye having upconversion absorption properties in the upper heat zone.

[0018] Furthermore, the dye with upconversion properties can emit fluorescence when irradiated with light of wavelengths of 750-900 nm.

[0019] Furthermore, the dyes with upconversion properties can be applied to fields such as fluorescent labeling, cell imaging, gene sequencing, biomolecule recognition, and photosensitizers as photodynamic therapy; more preferably, they can be used for cell imaging with upconversion properties, gene sequencing with long-wavelength excitation, and as photosensitizers in the field of photodynamic therapy.

[0020] Compared with the prior art, the beneficial effects of this application are as follows: The dyes described in this application generally possess upconversion properties due to heat absorption, enabling them to emit fluorescence (short wavelength) under irradiation with 750-900 nm light (long wavelength) that the compound does not absorb. This process relies on the driving energy provided by ambient temperature, avoiding the photobleaching problem caused by short-wavelength excitation. Furthermore, these dyes exhibit high extinction coefficients and fluorescence quantum yields in aqueous and polar protic solvents, making them highly advantageous for fluorescence imaging, gene sequencing, and the recognition of biomolecules. This upconversion mechanism also allows for deeper tissue penetration, which is beneficial for in-situ imaging of deep lesions in living organisms and for photodynamic therapy. Attached Figure Description

[0021] Figure 1 These are the UV-Vis absorption spectra of compounds 5-6, 8-9, and comparative examples 1-2; Figure 2These are the normalized fluorescence emission spectra of the UV-Vis absorption spectra of compounds 5-6, 8-9, and comparative examples 1-2; Figure 3 ao is the upconversion fluorescence emission spectrum of compounds 5-6 and compounds 8-9; Figure 4 abcd are upconversion photodynamic effects of compounds 1, 5, 6 and control compound 1; Figure 5 This is a liquid phase diagram of activated compound 2 labeled with L-lysine; Figure 6 Photostability of Compound 1 under different laser irradiations; Detailed Implementation The present invention will now be described in further detail. Unless otherwise stated, the terms used herein have the following meanings. The term "halogen" as used in this article includes fluorine, chlorine, bromine, and iodine. The term "alkyl" as used in this article includes both straight-chain alkyl and branched-chain alkyl. Y is used in this article - This refers to negative ions, which can be any suitable negative ion, including inorganic and organic negative ions. Examples include, but are not limited to, halide ions and ClO4. - PF6 - BF4 - CH3COO - CF3COO - or OTs - . The instruments and equipment used in the embodiments are as follows: In the column chromatography process of this invention, 200-300 mesh and 100-200 mesh silica gel columns purchased from Qingdao Meigao Group Co., Ltd., and 20-40 mesh analytical grade quartz sand purchased from Tianjin University Chemical Reagent Factory were used. The reversed-phase purification chromatograph was a CHEETAH rapid purification preparative chromatograph manufactured by Bona Electronics Technology Co., Ltd. The absorption and emission spectra of the dyes were measured using an Agilent Cary 60 UV-Vis spectrophotometer and a CaryEclipse fluorescence spectrophotometer.

[0022] Cytotoxicity assays were performed using the Varioskan LUX Multimode Microplate Reader instrument from Thermofisher, Inc.

[0023] The following are specific examples of compounds represented by general formula I, but the present invention is not limited to these specific examples.

[0024]

[0025] The compounds represented by general formula I can be synthesized by the methods described below.

[0026] Example 1: Preparation of Compound 1 The structural formula of compound 1:

[0027] Intermediate azaindole 1.1

[0028] 2-Hydroxy-5-bromopyridine (1 g, 1 eq) was dissolved in 20 mL of toluene, and 3-methyl-2-butanone (0.55 g, 1.5 eq) was added. The mixture was stirred at 100 °C for 6 h, and then the solvent was evaporated under reduced pressure. Polyphosphoric acid (2 g) was added to the residue, followed by 10 mL of toluene as a dissolving agent. The mixture was reacted at 110 °C for 3 h. The mixed solution was then introduced into ice water, and the pH was adjusted to 1 with NaOH. The mixture was then extracted with ethyl acetate. Several layers were collected, washed with saturated brine, dried over anhydrous sodium sulfate, and then concentrated to remove the solvent. The solution was purified by silica gel chromatography to obtain a brownish-yellow intermediate, azaindole 1.1 (0.84 g, Y = 66%).

[0029] Intermediate quaternary ammonium salt 1.2

[0030] Azaindole 1.1 (0.5 g, 1 eq) was dissolved in 20 mL of ethanol, and then iodoethane (1.3 g, 4 eq) was added. The reaction mixture was stirred at 70 °C under nitrogen protection for 12 h, after which the reaction was stopped. Most of the ethanol was removed under reduced pressure, and the mixture was recrystallized from ethyl acetate and n-hexane to give a yellow powder intermediate, quaternary ammonium salt 1.2 (0.72 g, Y=87%). Quaternary ammonium salt 1.2 can be used in subsequent reactions without further purification.

[0031] Manufacturing compound 1

[0032] Intermediate quaternary ammonium salt 1.2 (0.2 g, 1 eq) and condensing agent 1.3 (0.19 g, 1.1 eq) were dissolved in 10 mL of ethanol. 0.1 g of sodium acetate was added to catalyze the reaction. After stirring for 12 h, the reaction was stopped. After the reaction solution cooled to room temperature, the solvent was concentrated and extracted with dichloromethane. The crude product obtained after concentration was purified by silica gel chromatography to give blue solid compound 1 (0.17 g, Y=50%). 1H NMR (500 MHz, DMSO-d6) δ 9.25 (d, J = 1.6 Hz, 1H), 8.50 (d, J =1.8 Hz, 1H), 7.11 – 7.03 (m, 2H), 6.97 (dd, J = 14.8, 1.0 Hz, 1H), 6.85 –6.77 (m, 2H), 6.59 – 6.51 (m, 1H), 6.53 – 6.49 (m, 1H), 6.51 – 6.44 (m, 1H), 4.60 (q, J = 6.1 Hz, 2H), 3.81 (s, 2H), 3.51 (s, 2H), 1.56 (t, J = 6.1 Hz, 3H). 13 C NMR (125 MHz, DMSO-d6) δ 182.71, 162.89, 157.62, 155.98, 149.34,143.93, 141.86, 140.60, 136.91, 134.81, 134.16, 133.37, 116.10, 115.72,115.70, 115.39, 111.73, 105.17, 55.50, 52.96, 52.05, 35.89, 26.06, 12.91 ppm. Example 2: Preparation of Compound 2 The structural formula of compound 2:

[0033] Intermediate azaindole 2.1

[0034] 2-Hydroxy-5-carboxy-pyridine (1 g, 1 eq) was dissolved in 20 mL of toluene, and 3-methyl-2-butanone (1.12 g, 2 eq) was added. The mixture was stirred at 100 °C for 6 h, and then the solvent was evaporated under reduced pressure. Polyphosphoric acid (2 g) was added to the residue, followed by 10 mL of toluene as a dissolving agent. The mixture was reacted at 120 °C for 5 h. The mixed solution was introduced into ice water, and the pH was adjusted to 1 with NaOH. The solution was then extracted with ethyl acetate. Several layers were collected, washed with saturated brine, dried over anhydrous sodium sulfate, and then concentrated to remove the solvent. The solution was purified by silica gel chromatography to obtain the brownish-yellow intermediate azidoindole 2.1 (0.79 g, Y=59%).

[0035] Intermediate quaternary ammonium salt 2.2

[0036] Azaindole 2.1 (0.5 g, 1 eq) was dissolved in 20 mL of acetonitrile, and then iodopropane (1.25 g, 3 eq) was added. The reaction mixture was stirred at 65 °C under nitrogen protection for 10 h, after which the reaction was stopped. Most of the ethanol was removed under reduced pressure, and the mixture was recrystallized from ethyl acetate and n-hexane to give a yellow powder intermediate, quaternary ammonium salt 2.2 (0.64 g, Y=70%). Quaternary ammonium salt 2.2 can be used in subsequent reactions without further purification.

[0037] Manufacturing compound 2

[0038] Quaternary ammonium salt 2.2 (0.2 g, 1 eq) and condensing agent 2.3 (0.41 g, 1.2 eq) were dissolved in 10 mL of DMF. 0.1 g of potassium carbonate was added to catalyze the reaction. The reaction was stopped after stirring at 60 °C for 10 h. After the reaction solution cooled to room temperature, the solvent was concentrated and extracted with dichloromethane. The crude product obtained after concentration was purified by silica gel chromatography to give blue solid compound 2 (0.16 g, Y=38 %). 1 H NMR (500 MHz, DMSO-d6) δ 9.28 (d, J = 1.6 Hz, 1H), 8.84 (d, J =1.6 Hz, 1H), 8.40 (d, J = 7.1 Hz, 1H), 8.19 (s, 1H), 8.06 (d, J = 2.0 Hz, 1H), 7.98 (d, J = 2.3 Hz, 1H), 7.95 – 7.89 (m, 2H), 7.35 – 7.27 (m, 3H), 7.13 (d, J = 7.1 Hz, 1H), 6.79 (dt, J = 14.8, 1.3 Hz, 1H), 6.62 (dd, J = 8.3, 1.4Hz, 1H), 1.51 (s, 4H),1.12 (t, J = 8.2 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ 182.46, 173.28, 168.16,162.11, 159.03, 152.54, 141.54, 139.30, 139.22, 139.21, 138.48, 136.45,135.80, 135.44, 134.84, 134.41, 132.58, 131.18, 130.57, 130.48, 130.29,128.02, 127.73, 126.16, 125.33, 120.17, 111.06, 96.77, 60.25, 51.98, 49.43,49.12, 29.79, 26.06, 23.87, 13.71 ppm. Manufacturing activated compound 2

[0039] Compound 2 (0.05 g) was added to 3 mL of DMF solution, followed by 2-(5-norbornene-2,3-dicarboximide)-1,1,3,3-tetramethylurea tetrafluoroboric acid (0.025 g), and then triethylamine (0.1 mL). The reaction was carried out at 25 °C for 2 h, and the reaction was stopped. The reaction solution was poured into ethyl acetate, centrifuged at 9000 rpm, and the precipitate was collected. The precipitate was purified by HPLC to obtain activated compound 3 (0.052 g, Y=88%). 1H NMR (500 MHz, DMSO-d6) δ 9.28 (d, J = 1.6 Hz,1H), 8.84 (d, J = 1.6 Hz, 1H), 8.40 (d, J = 7.1 Hz, 1H), 8.06 (d, J = 2.0 Hz,1H), 8.01 – 7.95 (m, 3H), 7.39 – 7.27 (m, 2H), 7.13 (d, J = 7.1 Hz, 1H), 6.79 (dt, J = 14.8, 1.3 Hz, 1H), 6.62 (dd, J = 8.3, 1.4 Hz, 1H), 6.14 – 6.05 (m,3H), 5.06 (t, J = 1.0 Hz, 2H), 4.94 (s, 1H), 4.61 (td, J = 5.2, 1.6 Hz, 2H), 3.38 (dtdd, J = 7.7, 4.7, 3.2, 1.9 Hz, 2H), 3.33 – 3.26 (m, 2H), 2.25 – 2.09(m, 2H), 1.62 (d, J = 9.5 Hz, 1H), 1.57 (s, 2H), 1.53 (s, 2H), 1.48 (s, 2H), 1.12 (t, J = 8.2 Hz, 3H). 13 C NMR (125 MHz, DMSO-d6) δ 182.46, 170.14, 168.16,164.00, 162.11, 159.03, 152.54, 141.54, 139.30, 139.22, 139.21, 138.89,136.45, 135.80, 135.44, 134.84, 134.77, 134.41, 132.58, 131.60, 130.57,130.29, 128.45, 128.02, 127.49, 126.16, 125.33, 120.17, 111.06, 96.77, 60.25,51.98, 50.49, 49.43, 49.12, 44.68, 43.12, 29.79, 26.06, 23.87, 13.71 ppm. Example 3: Preparation of Compound 3 The structural formula of compound 3:

[0040] Intermediate azaindole 3.1

[0041] 2-Hydroxypyridine (1 g, 1 eq) was dissolved in 20 mL of toluene, and 4-(2-methyl-3-oxobutyl)benzoic acid (2.27 g, 1.2 eq) was added. The mixture was stirred at 100 °C for 6 h, and then the solvent was evaporated under reduced pressure. Polyphosphoric acid (2 g) was added to the residue, followed by 10 mL of toluene as a dissolving agent. The mixture was reacted at 120 °C for 5 h. The mixed solution was introduced into ice water, and the pH was adjusted to 1 with NaOH. The mixture was then extracted with ethyl acetate. Several layers were collected, washed with saturated brine, dried over anhydrous sodium sulfate, and then concentrated to remove the solvent. The solution was purified by silica gel chromatography to obtain the brownish-yellow intermediate azaindole 3.1 (1.45 g, Y = 56%).

[0042] Intermediate quaternary ammonium salt 3.2

[0043] Azaindole 3.1 (0.5 g, 1 eq) was dissolved in 20 mL of ethanol, and then iodomethane (1.01 g, 4 eq) was added. The reaction mixture was stirred at 50 °C under nitrogen protection for 10 h, after which the reaction was stopped. Most of the ethanol was removed under reduced pressure, and the mixture was recrystallized from ethyl acetate and n-hexane to give a yellow powder intermediate, quaternary ammonium salt 3.2 (0.56 g, Y=74%). Quaternary ammonium salt 3.2 can be used in subsequent reactions without further purification.

[0044] Manufacturing compound 3

[0045] Quaternary ammonium salt 3.2 (0.2 g, 1 eq) and condensing agent 3.3 (0.16 g, 1.2 eq) were dissolved in 10 mL of DMF. 0.1 g of potassium carbonate was added to catalyze the reaction. The reaction was stopped after stirring at 45 °C for 10 h. After the reaction solution cooled to room temperature, the solvent was concentrated and extracted with dichloromethane. The crude product obtained after concentration was purified by silica gel chromatography to give blue solid compound 3 (0.11 g, Y=38%). 1H NMR (500 MHz, DMSO-d6) δ 8.77 (dq, J = 5.1, 0.8 Hz, 1H), 8.19(s, 1H), 8.01 (dd, J = 7.8, 1.4 Hz, 1H), 7.96 – 7.91 (m, 2H), 7.91 (dd, J =7.9, 5.1 Hz, 1H), 7.23 – 7.16 (m, 3H), 7.12 – 7.03 (m, 1H), 6.70 (dddd, J =6.1, 4.1, 2.6, 1.4 Hz, 1H), 6.70 – 6.62 (m, 2H), 6.29 (ddt, J = 16.3, 2.8,1.3 Hz, 2H), 4.12 (s, 2H), 3.89 (q, J = 6.8 Hz, 2H), 3.22 (q, J = 0.8 Hz, 2H), 2.22 (d, J = 1.3 Hz, 6H), 1.17 (t, J = 6.9 Hz, 3H). 13 C NMR (125 MHz, DMSO-d6) δ 183.53, 173.27, 160.38, 142.49, 142.47, 139.82, 136.92, 136.85,135.85, 135.52, 134.56, 131.95, 130.60, 130.58, 129.07, 123.80, 123.79,116.62, 109.04, 107.92, 53.56, 44.24, 43.01, 42.71, 24.01, 18.76, 12.95 ppm. Manufacturing activated compound 3

[0046] Compound 3 (0.05 g) was added to 3 mL of DMF solution, followed by 2-(5-norbornene-2,3-dicarboximide)-1,1,3,3-tetramethylurea tetrafluoroboric acid (0.025 g), and then triethylamine (0.1 mL). The reaction was carried out at 25 °C for 2 h, and the reaction was stopped. The reaction solution was poured into ethyl acetate, centrifuged at 9000 rpm, and the precipitate was collected. The activated compound 3 (0.051 g, Y=81%) was purified by HPLC. 1H NMR (500 MHz, DMSO-d6) δ 8.76 (ddt, J = 5.2, 1.4,0.9 Hz, 1H), 8.01 (dd, J = 7.8, 1.4 Hz, 1H), 7.99 – 7.94 (m, 2H), 7.91 (dd, J= 7.9, 5.1 Hz, 1H), 7.27 (dt, J = 7.5, 1.0 Hz, 2H), 7.21 – 7.13 (m, 1H), 6.76– 6.59 (m, 4H), 6.29 (ddt, J = 16.3, 2.8, 1.3 Hz, 2H), 6.14 – 6.06 (m, 2H),4.12 (s, 2H), 3.89 (q, J = 6.8 Hz, 2H), 3.42 – 3.34 (m, 2H), 3.33 – 3.26 (m,2H), 3.22 (q, J = 0.8 Hz, 2H), 2.21 (dd, J = 3.5, 1.3 Hz, 6H), 1.62 (t, J =4.7 Hz, 2H), 1.17 (t, J = 6.9 Hz, 3H). 13 C NMR (125 MHz, DMSO-d6) δ 183.30,170.14, 164.01, 160.38, 142.53, 142.49, 142.47, 139.81, 136.92, 136.48,135.85, 134.94, 134.77, 134.56, 131.59, 130.60, 129.70, 127.62, 124.00,123.80, 123.79, 109.03, 107.91, 53.56, 50.49, 44.68, 44.24, 43.12, 43.01,42.71, 24.01, 18.76, 12.95 ppm. Example 4: Preparation of Compound 4 Structural formula of compound 4:

[0047] Intermediate aza-indole 4.1

[0048] 2-Hydroxypyridine (1 g, 1 eq) was dissolved in 20 mL of toluene, and 8-methyl-9-oxodecanoic acid (5.51 g, 3 eq) was added. The mixture was stirred at 100 °C for 6 h, and then the solvent was evaporated under reduced pressure. Polyphosphoric acid (2 g) was added to the residue, followed by 10 mL of toluene as a dissolving agent. The mixture was reacted at 120 °C for 5 h. The mixed solution was introduced into ice water, and the pH was adjusted to 1 with NaOH. The mixture was then extracted with ethyl acetate. Several layers were collected, washed with saturated brine, dried over anhydrous sodium sulfate, and then concentrated to remove the solvent. The solution was purified by silica gel chromatography to obtain the brownish-yellow intermediate azidoindole 4.1 (1.64 g, Y=65%).

[0049] Intermediate quaternary ammonium salt 4.2

[0050] Azaindole 4.1 (0.5 g, 1 eq) was dissolved in 20 mL of ethanol, and then iodopropane (0.93 g, 3 eq) was added. The reaction mixture was stirred at 50 °C under nitrogen protection for 10 h, after which the reaction was stopped. Most of the ethanol was removed under reduced pressure, and the mixture was recrystallized from ethyl acetate and n-hexane. Ion exchange was performed using a counterion exchange column to obtain a yellow powder intermediate, quaternary ammonium salt 4.2 (0.43 g, Y=56%). Quaternary ammonium salt 4.2 can be used in subsequent reactions without further purification.

[0051] Manufacturing compound 4

[0052] Quaternary ammonium salt 4.2 (0.25 g, 1 eq) and condensing agent 4.3 (0.15 g, 1.1 eq) were dissolved in 10 mL of DMF. 0.1 g of potassium carbonate was added to catalyze the reaction. The reaction was stopped after stirring at 60 °C for 10 h. After the reaction solution cooled to room temperature, the solvent was concentrated and extracted with dichloromethane. The crude product obtained after concentration was purified by silica gel chromatography to give blue solid compound 4 (0.16 g, Y=56 %). 1H NMR (500 MHz, DMSO-d6) δ 8.66 (dd, J = 5.2, 1.4 Hz, 1H), 8.00 (dd, J = 8.0, 1.4 Hz, 1H), 7.89 (dd, J = 8.1, 5.1 Hz, 1H), 7.35 (dd, J = 7.7,1.3 Hz, 1H), 7.22 (ddd, J = 8.6, 7.2, 1.4 Hz, 1H), 7.14 – 7.03 (m, 2H), 6.67 (ddt, J = 8.6, 1.6, 0.8 Hz, 1H), 6.65 – 6.50 (m, 2H), 6.43 (td, J = 7.5, 0.7Hz, 1H), 5.94 (dq, J = 7.6, 1.1 Hz, 1H), 4.60 (t, J = 5.2 Hz, 2H), 4.03 –3.89 (m, 2H), 2.29 (t, J = 8.9 Hz, 2H), 2.23 – 2.00 (m, 4H), 1.58 (tt, J =8.7, 7.6 Hz, 2H), 1.44 (pd, J = 7.0, 0.7 Hz, 2H), 1.39 – 1.29 (m, 7H), 1.12(t, J = 8.2 Hz, 3H). 13 C NMR (125 MHz, DMSO-d6) δ 184.57, 180.45, 158.96,146.17, 140.26, 139.82, 135.81, 135.40, 134.79, 133.95, 130.09, 126.06,124.14, 117.53, 116.21, 112.56, 111.55, 59.06, 51.70, 45.94, 37.63, 34.36,28.91, 28.48, 25.36, 24.57, 24.44, 23.93, 14.15, 13.71 ppm. Manufacturing activated compound 4

[0053] Compound 4 (0.05 g) was added to 3 mL of DMF solution, followed by 2-(5-norbornene-2,3-dicarboximide)-1,1,3,3-tetramethylurea tetrafluoroboric acid (0.025 g), and then triethylamine (0.1 mL). The reaction was carried out at 25 °C for 2 h, and the reaction was stopped. The reaction solution was poured into ethyl acetate, centrifuged at 9000 rpm, and the precipitate was collected. The activated compound 4 (0.049 g, Y=75%) was purified by HPLC. 1 H NMR (500 MHz, DMSO-d6) δ 8.66 (dd, J = 5.2, 1.4Hz, 1H), 8.00 (dd, J = 8.0, 1.4 Hz, 1H), 7.89 (dd, J = 8.1, 5.1 Hz, 1H), 7.35(dd, J = 7.7, 1.3 Hz, 1H), 7.22 (ddd, J = 8.6, 7.2, 1.4 Hz, 1H), 7.11 (dd, J= 9.2, 1.0 Hz, 1H), 6.72 (ddt, J = 9.0, 7.7, 1.0 Hz, 1H), 6.69 – 6.64 (m,1H), 6.64 (tt, J = 7.7, 0.8 Hz, 1H), 6.54 (ddt, J = 16.1, 8.1, 1.0 Hz, 1H), 6.43 (td, J = 7.5, 0.7 Hz, 1H), 6.14 – 6.06 (m, 2H), 5.94 (dq, J = 8.1, 1.1Hz, 1H), 4.60 (t, J = 5.2 Hz, 2H), 4.03 – 3.89 (m, 2H), 3.43 – 3.34 (m, 2H), 3.33 – 3.26 (m, 2H), 2.54 (t, J = 8.8 Hz, 2H), 2.23 – 2.00 (m, 4H), 1.65 –1.54 (m, 4H), 1.44 (pd, J = 7.0, 0.7 Hz, 2H), 1.39 – 1.28 (m, 7H), 1.12 (t, J= 8.2 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ 184.35, 171.41, 170.39, 158.96,146.17, 140.26, 139.82, 135.53, 134.91, 134.79, 134.77, 133.95, 130.09,126.06, 124.14, 123.41, 117.53, 112.56, 111.55, 59.06, 51.70, 50.49, 45.94,44.69, 43.12, 37.63, 33.05, 28.90, 28.30, 24.57, 24.44, 24.24, 23.93, 14.15,13.71 ppm. Example 5: Preparation of Compound 5 The structural formula of compound 5:

[0054] Intermediate quaternary ammonium salt 5.1

[0055] The azidoindole 1.1 (0.5 g, 1 eq) obtained in Example 1 was dissolved in 20 mL of ethanol, and then iodopropane (1.07 g, 3 eq) was added. The reaction system was heated to 50 °C and stirred under nitrogen protection, and the reaction was stopped after 10 h. Most of the ethanol was removed under reduced pressure, and the mixture was recrystallized from ethyl acetate and n-hexane to give a yellow powder intermediate, quaternary ammonium salt 5.1 (0.39 g, Y=46%). Quaternary ammonium salt 5.1 can be used in subsequent reactions without further purification.

[0056] Manufacturing compound 5

[0057] Quaternary ammonium salt 5.1 (0.2 g, 1 eq) and condensing agent 5.2 (0.59 g, 1.2 eq) were dissolved in 10 mL of DMF. 0.1 g of potassium carbonate was added to catalyze the reaction. The reaction was stopped after stirring at 50 °C for 10 h. After the reaction solution cooled to room temperature, the solvent was concentrated and extracted with dichloromethane. The crude product obtained after concentration was purified by silica gel chromatography to give blue solid compound 5 (0.17 g, Y=41 %). 1H NMR (500 MHz, DMSO-d6) δ 9.15 (d, J = 1.6 Hz, 1H), 8.58 (d, J =1.6 Hz, 1H), 7.67 (dd, J = 7.9, 2.2 Hz, 1H), 7.56 (d, J = 2.2 Hz, 1H), 7.41 –7.28 (m, 2H), 6.92 (dd, J = 8.4, 1.3 Hz, 1H), 6.54 (d, J = 7.9 Hz, 1H), 6.01(dd, J = 8.5, 0.9 Hz, 1H), 4.59 (t, J = 5.2 Hz, 2H), 3.90 (t, J = 6.1 Hz,2H), 2.17 (qt, J = 8.2, 5.1 Hz, 2H), 1.70 (qt, J = 7.6, 6.0 Hz, 2H), 1.51 (s,5H), 1.12 (t, J = 8.2 Hz, 3H), 1.02 (t, J = 7.7 Hz, 3H). 13 C NMR (125 MHz, DMSO-d6) δ 155.87, 153.70, 150.41, 145.76, 140.94, 139.07, 136.61, 136.60,129.72, 129.69, 129.47, 125.98, 123.30, 121.45, 121.23, 115.11, 112.36,89.45, 59.37, 53.57, 48.67, 48.63, 27.72, 27.70, 25.62, 23.82, 20.63, 13.70,11.50 ppm. Example 6: Preparation of Compound 6 The structural formula of compound 6:

[0058] Manufacturing compound 6

[0059] The quaternary ammonium salt 1.2 (0.2 g, 1 eq) and condensing agent 6.2 (0.59 g, 1.2 eq) obtained in Example 1 were dissolved in 10 mL of DMF. 0.1 g of potassium carbonate was added to catalyze the reaction. The reaction was stopped after stirring at 65 °C for 10 h. After the reaction solution cooled to room temperature, the solvent was concentrated and extracted with dichloromethane. The crude product obtained after concentration was purified by silica gel chromatography to obtain blue solid compound 6 (0.17 g, Y=41%).1 H NMR (500 MHz, DMSO-d6)δ = 9.25 (d, 1H), 8.51(d, 1H), 8.19 (s, 1H), 7.94 (d, 1H), 7.88 (m, 2H), 7.68 (d, 1H), 7.33 (m,2H), 7.13 (dd, 1H), 6.91 (dd, 1H), 6.31 (dddd, 2H), 6.06 (s, 1H), 4.59 (m,2H), 4.29 (tp, 1H), 3.78 (m, 2H), 1.55 (m, 5H), 1.46 (d, 4H), 1.39 (t, 3H).. 13 C NMR (125 MHz, DMSO-d6) δ 181.54, 173.33, 157.73, 149.34, 146.43, 143.95,138.21, 134.81, 132.28, 129.63, 127.71, 119.56, 115.72, 52.96, 52.28, 48.00,26.06, 12.91 ppm. Manufacturing activated compound 6

[0060] Compound 6 (0.05 g) was added to 3 mL of DMF solution, followed by 2-(5-norbornene-2,3-dicarboximide)-1,1,3,3-tetramethylurea tetrafluoroboric acid (0.025 g), and then triethylamine (0.1 mL). The reaction was carried out at 25 °C for 2 h, and the reaction was stopped. The reaction solution was poured into ethyl acetate, centrifuged at 9000 rpm, and the precipitate was collected. The activated compound 6 (0.049 g, Y=82%) was purified by HPLC. 1H NMR (500 MHz, DMSO-d6) δ = 9.25 (d, 1H), 8.51 (d,1H), 7.99 (m, 2H), 7.94 (d, 1H), 7.68 (d, 1H), 7.38 (m, 2H), 7.13 (dd, 1H), 6.91 (dd, 1H), 6.31 (dddd, 2H), 6.10 (m, 2H), 6.06 (s, 1H), 4.59 (m, 2H), 4.29 (tp, 1H), 3.78 (m, 2H), 3.38 (m, 2H), 3.29 (m, 2H), 1.62 (t, 2H), 1.55(m, 5H), 1.46 (d, 5H), 1.39 (t, 3H).. 13C NMR (125 MHz, DMSO-d6) δ 181.54,170.14, 164.05, 157.73, 149.34, 146.49, 143.95, 138.21, 134.81, 134.77,130.80, 128.41, 128.03, 119.56, 115.72, 52.96, 52.28, 50.49, 48.00, 44.68,43.12, 26.06, 12.91 ppm. Example 7: Preparation of Compound 7 Structural formula of compound 7:

[0061] Intermediate azidoindole 7.1

[0062] 2-Hydroxypyridine (1 g, 1 eq) was dissolved in 20 mL of toluene, and 3-methyl-2-butanone (3.19 g, 4 eq) was added. The mixture was stirred at 100 °C for 6 h, and then the solvent was evaporated under reduced pressure. Polyphosphoric acid (2 g) was added to the residue, followed by 10 mL of toluene as a dissolving agent. The mixture was reacted at 120 °C for 5 h. The mixed solution was introduced into ice water, and the pH was adjusted to 1 with NaOH. The mixture was then extracted with ethyl acetate. Several layers were collected, washed with saturated brine, dried over anhydrous sodium sulfate, and then concentrated to remove the solvent. The solution was purified by silica gel chromatography to obtain a brownish-yellow intermediate, azaindole 7.1 (1.21 g, Y=82%).

[0063] Intermediate quaternary ammonium salt 7.2

[0064] Azaindole 7.1 (0.5 g, 1 eq) was dissolved in 20 mL of ethanol, and then iodopropane (1.86 g, 3.5 eq) was added. The reaction mixture was stirred at 50 °C under nitrogen protection for 10 h, after which the reaction was stopped. Most of the ethanol was removed under reduced pressure, and the mixture was recrystallized from ethyl acetate and n-hexane to give a yellow powder intermediate, quaternary ammonium salt 7.2 (0.82 g, Y = 80%). Quaternary ammonium salt 7.2 can be used in subsequent reactions without further purification.

[0065] Manufacturing compound 7

[0066] Quaternary ammonium salt 7.2 (0.2 g, 1 eq) and condensing agent 7.3 (0.29 g, 0.61 mmol) were dissolved in 10 mL of ethanol. 0.1 g of sodium acetate was added to catalyze the reaction. The reaction was stopped after stirring at 60 °C for 12 h. After the reaction solution cooled to room temperature, the solvent was concentrated and extracted with dichloromethane. The crude product obtained after concentration was purified by silica gel chromatography to give blue solid compound 7 (0.26 g, Y=60%). 1 H NMR (500 MHz, DMSO-d6) δ 8.66 (dd, J = 5.2, 1.4 Hz, 1H), 8.10 (dd, J = 8.1, 1.4 Hz, 1H), 7.98 (dd, J = 8.2, 1.5 Hz, 1H), 7.91 – 7.81(m, 2H), 7.80 – 7.71 (m, 2H), 7.59 – 7.47 (m, 2H), 7.19 – 7.13 (m, 2H), 6.91(tt, J = 8.1, 2.2 Hz, 1H), 6.84 (dd, J = 7.1, 1.5 Hz, 1H), 6.70 (dt, J =15.0, 1.2 Hz, 1H), 6.19 (dd, J = 7.1, 0.9 Hz, 1H), 4.60 (t, J = 5.2 Hz, 2H), 4.06 (t, J = 6.3 Hz, 2H), 2.16 (qt, J = 8.2, 5.3 Hz, 2H), 1.76 (qt, J = 7.6,6.2 Hz, 2H), 1.12 (t, J = 8.2 Hz, 3H), 1.01 (t, J = 7.7 Hz, 3H). 13C NMR (125MHz, DMSO-d6) δ 183.16, 163.21, 163.15, 161.20, 161.13, 159.80, 150.57,147.92, 146.17, 141.54, 139.12, 139.10, 139.07, 136.16, 134.67, 133.89,132.26, 132.20, 132.13, 130.36, 128.22, 127.90, 126.45, 126.24, 124.44,124.42, 124.28, 124.01, 123.85, 121.68, 111.80, 111.77, 111.64, 111.61,105.93, 105.77, 105.61, 100.96, 59.06, 52.15, 50.37, 26.01, 23.93, 20.92,13.71, 11.51 ppm. Example 8: Preparation of Compound 8 Structural formula of compound 8:

[0067] Intermediate azidoindole 8.1

[0068] The azaindole 2.1 (0.4 g, 1 eq) obtained in Example 2 was dissolved in 10 mL of DMF, and TBTU (0.828 g, 1.3 eq) and DIPEA (0.33 g, 1.3 eq) were added. The mixture was stirred at 40 °C for 0.5 h, and then cooled to room temperature. Propanolamine (0.24 g, 2 eq) was added, and the reaction was allowed to proceed for 1.5 h. The mixture was filtered to remove impurity salts, and purified on a silica gel column to obtain the intermediate azaindole 8.1 (0.36 g, Y=73%).

[0069] Intermediate quaternary ammonium salt 8.2

[0070] Azaindole 8.1 (0.5 g, 1 eq) was dissolved in 20 mL of acetonitrile, and then 1,4-butyrylolactone (0.74 g, 3 eq) was added. The reaction mixture was stirred at 50 °C under nitrogen protection for 10 h, after which the reaction was stopped. Most of the acetonitrile was removed under reduced pressure, and the mixture was recrystallized from ethyl acetate to obtain a yellow powder intermediate, quaternary ammonium salt 8.2 (0.43 g, Y=52%). Quaternary ammonium salt 8.2 can be used in subsequent reactions without further purification.

[0071] Manufacturing compound 8

[0072] Quaternary ammonium salt 8.2 (0.2 g, 1 eq) and condensing agent 8.3 (0.070 g, 1.3 eq) were dissolved in 10 mL of ethanol. 0.1 g of sodium acetate was added to catalyze the reaction. The reaction was stopped after stirring at 60 °C for 12 h. After the reaction solution cooled to room temperature, the solvent was concentrated and extracted with dichloromethane. The crude product obtained after concentration was purified by silica gel chromatography to give blue solid compound 8 (0.18 g, Y=40%). 1 H NMR (500 MHz, DMSO-d6) δ = 9.40 (d, 1H), 8.85 (t, 1H), 8.77 (d,1H), 8.19 (s, 1H), 7.94 (m, 2H), 7.87 (d, 1H), 7.69 (dd, 1H), 7.18 (dt, 2H),7.07 (m, 2H), 6.97 (dd, 1H), 6.55 (m, 2H), 5.98 (dt, 1H), 4.66 (t, 2H), 3.85(t, 2H), 3.53 (q, 2H), 3.37 (qd, 2H), 3.28 (t, 1H), 3.05 (dt, 1H), 2.97 (dt,1H), 2.70 (t, 2H), 2.15 (m, 2H), 1.87 (m, 2H), 1.73 (m, 1H), 1.71 (d, 1H),1.69 (m, 2H), 1.53 (s, 2H), 1.48 (s, 2H), 1.02 (t, 3H). 13 C NMR (125 MHz, DMSO-d6) δ = 183.02, 173.27, 166.55, 160.61, 160.45, 151.22, 145.49, 144.39,139.21, 138.86, 138.59, 136.68, 135.45, 135.37, 133.32, 131.95, 130.58,129.21, 129.00, 128.95, 124.21, 115.97, 112.30, 100.88, 59.18, 56.56, 53.41,51.83, 49.73, 49.17, 45.77, 37.36, 31.66, 29.03, 26.05, 25.77, 21.00, 20.72,11.51 ppm. Manufacturing activated compound 8

[0073] Compound 8 (0.05 g) was added to 3 mL of DMF solution, followed by 2-(5-norbornene-2,3-dicarboximide)-1,1,3,3-tetramethylurea tetrafluoroboric acid (0.025 g), and then triethylamine (0.1 mL). The reaction was carried out at 25 °C for 2 h, and the reaction was stopped. The reaction solution was poured into ethyl acetate, centrifuged at 9000 rpm, and the precipitate was collected. The activated compound 8 (0.047 g, Y=78%) was purified by HPLC. 1 H NMR (500 MHz, DMSO-d6) δ = 9.40 (d, 1H), 8.85 (t,1H), 8.77 (d, 1H), 7.98 (m, 2H), 7.87 (d, 1H), 7.69 (dd, 1H), 7.27 (dt, 2H), 7.11 (d, 1H), 7.07 (ddt, 1H), 6.97 (dd, 1H), 6.55 (m, 2H), 6.10 (m, 2H), 5.98(dt, 1H), 4.66 (t, 2H), 3.85 (t, 2H), 3.53 (q, 2H), 3.38 (m, 4H), 3.29 (m,3H), 3.05 (dt, 1H), 2.97 (dt, 1H), 2.70 (t, 2H), 2.15 (m, 2H), 1.87 (m, 2H), 1.72 (m, 2H), 1.70 (m, 2H), 1.62 (t, 2H), 1.53 (s, 2H), 1.48 (s, 2H), 1.02(t, 3H). 13C NMR (125 MHz, DMSO-d6) δ = 183.02, 170.14, 166.55, 164.01, 160.61,160.45, 151.22, 145.49, 144.39, 139.48, 138.86, 138.59, 136.68, 135.45,135.37, 134.77, 133.32, 131.59, 129.66, 129.21, 128.95, 127.62, 124.21,115.97, 112.30, 100.88, 59.18, 56.56, 53.41, 51.83, 50.49, 49.73, 49.17,45.77, 44.68, 43.12, 37.36, 31.66, 29.03, 26.05, 25.77, 21.00, 20.72, 11.51ppm. Example 9: Preparation of Compound 9 Structural formula of compound 9:

[0074] Intermediate quaternary ammonium salt 9.1

[0075] The azaindole 7.1 (0.5 g, 1 eq) obtained in Example 7 was dissolved in 20 mL of ethanol, and then p-carboxybenzyl bromide (3.76 g, 2.8 eq) was added. The reaction mixture was stirred at 70 °C under nitrogen protection for 10 h, after which the reaction was stopped. Most of the ethanol was removed under reduced pressure, and the mixture was recrystallized from ethyl acetate and n-hexane to obtain a yellow powder, quaternary ammonium salt 9.1 (1.52 g, Y = 65%). Quaternary ammonium salt 9.1 can be used in subsequent reactions without further purification.

[0076] Manufacturing compound 9

[0077] Compound 9.1 (0.2 g, 1 eq) and condensing agent 9.2 (0.31 g, 0.75 mmol) were dissolved in 10 mL of ethanol. 0.1 g of sodium acetate was added to catalyze the reaction. The reaction was stopped after stirring at 30 °C for 12 h. After the reaction solution cooled to room temperature, the solvent was concentrated and extracted with dichloromethane. The crude product obtained after concentration was purified by liquid chromatography to give blue solid compound 9 (0.09 g, Y=24%). 1H NMR (500 MHz, DMSO-d6) δ 9.66 (s, 1H), 8.67 (dd, J = 5.1,1.3 Hz, 1H), 8.10 (dd, J = 8.1, 1.5 Hz, 1H), 8.03 – 7.97 (m, 2H), 7.90 (dd, J= 8.1, 5.1 Hz, 1H), 7.51 (dd, J = 8.0, 5.0 Hz, 1H), 7.46 (dt, J = 8.5, 1.1Hz, 2H), 7.43 – 7.29 (m, 3H), 6.97 (dd, J = 8.7, 1.2 Hz, 1H), 6.12 (t, J =1.0 Hz, 2H), 5.62 (dd, J = 8.6, 0.9 Hz, 1H), 4.14 (q, J = 7.3 Hz, 2H), 1.27 (t, J = 7.2 Hz, 3H). 13 C NMR (125 MHz, DMSO-d6) δ 182.54, 173.26, 158.67,150.88, 150.72, 149.25, 148.86, 148.73, 148.70, 148.57, 146.72, 146.56,145.31, 144.05, 140.33, 139.41, 139.39, 139.35, 139.32, 138.24, 133.89,131.35, 130.22, 129.98, 127.79, 124.23, 123.02, 123.00, 122.96, 122.93,122.10, 117.90, 112.71, 112.65, 112.55, 112.49, 104.26, 104.20, 104.10,104.04, 102.82, 55.03, 52.15, 41.73, 26.01, 13.24 ppm. Manufacturing activated compound 9

[0078] Compound 9 (0.05 g) was added to 3 mL of DMF solution, followed by 2-(5-norbornene-2,3-dicarboximide)-1,1,3,3-tetramethylurea tetrafluoroboric acid (0.025 g), and then triethylamine (0.1 mL). The reaction was carried out at 25 °C for 2 h, and the reaction was stopped. The reaction solution was poured into ethyl acetate, centrifuged at 9000 rpm, and the precipitate was collected. The activated compound 9 (0.041 g, Y=67%) was purified by HPLC. 1 H NMR (500 MHz, DMSO-d6) δ 8.67 (dd, J = 5.1, 1.3Hz, 1H), 8.13 – 8.05 (m, 3H), 7.90 (dd, J = 8.1, 5.1 Hz, 1H), 7.55 (dt, J =8.7, 1.0 Hz, 2H), 7.51 (dd, J = 8.0, 5.0 Hz, 1H), 7.43 – 7.33 (m, 2H), 6.97 (dd, J = 8.7, 1.2 Hz, 1H), 6.55 – 6.49 (m, 1H), 6.16 – 6.06 (m, 4H), 4.31(qd, J = 7.2, 2.2 Hz, 2H), 3.38 (ddtd, J = 8.7, 4.8, 2.9, 2.4, 1.4 Hz, 2H), 3.33 – 3.26 (m, 2H), 1.62 (t, J = 4.7 Hz, 2H), 1.46 (s, 2H), 1.39 (t, J = 7.2Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ 182.54, 170.14, 163.97, 158.67, 150.88,150.72, 149.27, 148.86, 148.73, 148.70, 148.57, 146.72, 146.56, 145.31,144.05, 140.33, 139.41, 139.39, 139.35, 139.32, 139.17, 134.77, 133.89,131.18, 128.73, 128.56, 127.66, 124.23, 123.02, 123.00, 122.96, 122.93,122.10, 117.90, 112.71, 112.65, 112.55, 112.49, 104.26, 104.20, 104.10,104.04, 102.82, 55.03, 52.15, 50.49, 44.68, 43.12, 41.73, 26.01, 13.24 ppm. Example 10: Preparation of Compound 10 Structural formula of compound 10:

[0079] Intermediate azidoindole 10.1

[0080] The azaindole 2.1 (0.4 g, 1 eq) obtained in Example 2 was dissolved in 10 mL of DMF, and TBTU (0.828 g, 1.3 eq) and DIPEA (0.33 g, 1.3 eq) were added. The mixture was stirred at 0 °C for 0.5 h, and then cooled to room temperature. Ethylamine (0.18 g, 2 eq) was added, and the reaction was allowed to proceed for 1.5 h. The mixture was filtered to remove impurity salts, and purified on a silica gel column to obtain the intermediate azaindole 10.1 (0.31 g, Y=68%).

[0081] Intermediate quaternary ammonium salt 10.2

[0082] Azaindole 10.1 (0.5 g, 1 eq) was dissolved in 20 mL of acetonitrile, and then 1,3-propanesulfonic acid lactone (0.92 g, 3.5 eq) was added. The reaction mixture was stirred at 75 °C under nitrogen protection for 10 h, after which the reaction was stopped. Most of the acetonitrile was removed under reduced pressure, and the mixture was recrystallized from ethyl acetate to give a yellow powder intermediate, quaternary ammonium salt 10.2 (0.47 g, Y=62%). Quaternary ammonium salt 10.2 can be used in subsequent reactions without further purification.

[0083] Manufacturing compound 10

[0084] Quaternary ammonium salt 10.1 (0.2 g, 1 eq) and condensing agent 10.2 (0.31 g, 0.47 mmol) were dissolved in 10 mL of ethanol. 0.1 g of sodium acetate was added to catalyze the reaction. The reaction was stopped after stirring at 60 °C for 12 h. After the reaction solution cooled to room temperature, the solvent was concentrated and extracted with dichloromethane. The crude product obtained after concentration was purified by liquid chromatography to give green solid compound 10 (0.319 g, Y=62%). 1H NMR (500 MHz, DMSO-d6) δ 9.40 (d, J = 1.8 Hz, 1H), 8.79 –8.74 (m, 2H), 8.19 (s, 1H), 7.97 – 7.91 (m, 3H), 7.69 (dd, J = 8.1, 2.2 Hz,1H), 7.24 – 7.15 (m, 3H), 7.19 – 7.11 (m, 2H), 6.83 (dd, J = 15.2, 7.3 Hz,1H), 6.61 (dt, J = 7.3, 1.6 Hz, 0H), 6.56 – 6.47 (m, 2H), 6.26 (dd, J = 7.2,1.4 Hz, 1H), 4.63 (tt, J = 6.9, 3.4 Hz, 2H), 4.00 (td, J = 4.6, 2.0 Hz, 2H),3.40 (qdd, J = 6.2, 4.1, 0.9 Hz, 2H), 3.05 (dt, J = 12.6, 1.1 Hz, 1H), 2.97(dt, J = 12.6, 1.0 Hz, 1H), 2.87 – 2.77 (m, 2H), 2.76 (td, J = 4.6, 3.0 Hz,2H), 2.37 – 2.26 (m, 2H), 2.04 (t, J = 3.0 Hz, 1H), 1.53 (s, 2H), 1.48 (s,2H), 1.25 (t, J = 6.2 Hz, 3H). 13 C NMR (125 MHz, DMSO-d6) δ 182.83, 173.27,166.53, 160.85, 159.88, 151.39, 145.19, 139.21, 138.59, 137.16, 137.10,135.61, 133.32, 132.29, 132.22, 131.95, 130.72, 130.58, 129.15, 129.00,124.44, 119.48, 119.32, 117.98, 117.82, 117.39, 117.37, 111.14, 100.88,100.86, 80.18, 70.67, 56.38, 52.53, 51.75, 49.15, 48.16, 45.77, 36.34, 26.05,25.77, 24.04, 17.92, 14.69 ppm. Manufacturing activated compound 10

[0085] Compound 10 (0.05 g) was added to 3 mL of DMF solution, followed by 2-(5-norbornene-2,3-dicarboximide)-1,1,3,3-tetramethylurea tetrafluoroboric acid (0.025 g), and then triethylamine (0.1 mL). The reaction was carried out at 25 °C for 2 h, and the reaction was stopped. The reaction solution was poured into ethyl acetate, centrifuged at 9000 rpm, and the precipitate was collected. The precipitate was purified by HPLC to obtain activated compound 9 (0.048 g, Y=82%). 1 H NMR (500 MHz, DMSO-d6) δ 9.40 (d, J = 1.8 Hz,1H), 8.79 – 8.74 (m, 2H), 8.00 – 7.93 (m, 3H), 7.69 (dd, J = 8.1, 2.2 Hz,1H), 7.27 (dt, J = 7.5, 1.0 Hz, 2H), 7.24 – 7.11 (m, 3H), 6.57 (dd, J = 9.8,7.2 Hz, 1H), 6.49 – 6.44 (m, 1H), 6.47 – 6.39 (m, 1H), 6.26 (dd, J = 7.3, 1.4Hz, 1H), 6.14 – 6.06 (m, 2H), 4.63 (tt, J = 6.9, 3.4 Hz, 2H), 4.00 (td, J =4.6, 2.0 Hz, 2H), 3.46 – 3.34 (m, 4H), 3.33 – 3.26 (m, 2H), 3.05 (dt, J =12.6, 1.1 Hz, 1H), 2.97 (dt, J = 12.6, 1.0 Hz, 1H), 2.87 – 2.77 (m, 2H), 2.76 (td, J = 4.6, 3.0 Hz, 2H), 2.37 – 2.26 (m, 2H), 2.04 (t, J = 3.0 Hz, 1H),1.62 (t, J = 4.8 Hz, 2H), 1.53 (s, 2H), 1.48 (s, 2H), 1.25 (t, J = 6.2 Hz,3H). 13C NMR (125 MHz, DMSO-d6) δ 182.83, 170.14, 166.53, 164.01, 160.85,159.88, 158.03, 156.01, 151.39, 145.19, 139.48, 138.59, 136.81, 136.75,135.61, 134.77, 133.32, 132.01, 131.94, 131.59, 130.72, 129.66, 129.15,127.62, 124.44, 118.60, 118.47, 118.44, 118.31, 117.39, 117.37, 111.14,100.18, 100.16, 80.18, 70.67, 56.38, 52.53, 51.75, 50.49, 49.15, 48.16,45.77, 44.68, 43.12, 36.34, 26.05, 25.77, 24.04, 17.92, 14.69 ppm. Example 11: Preparation of Compound 11 The structural formula of compound 11:

[0086] Intermediate quaternary ammonium salt 11.1

[0087] The azaindole 2.1 (0.4 g, 1 eq) obtained in Example 2 was dissolved in 20 mL of acetonitrile, and then benzyl bromide (1.47 g, 3.5 eq) was added. The reaction mixture was stirred at 65 °C under nitrogen protection for 10 h, after which the reaction was stopped. Most of the acetonitrile was removed under reduced pressure, and the mixture was recrystallized from ethyl acetate to obtain a yellow powder intermediate, quaternary ammonium salt 11.1 (0.74 g, Y=81%). Quaternary ammonium salt 11.1 can be used in subsequent reactions without further purification.

[0088] Manufacturing compound 11

[0089] Quaternary ammonium salt 11.1 (0.2 g, 1 eq) and condensing agent 11.2 (0.34 g, 1.3 eq) were dissolved in 10 mL of ethanol. 0.1 g of sodium acetate was added to catalyze the reaction. The reaction was stopped after stirring at 60 °C for 12 h. After the reaction solution cooled to room temperature, the solvent was concentrated and extracted with dichloromethane. The crude product obtained after concentration was purified by liquid chromatography to obtain green solid compound 11 (0.17 g, Y=38%). 1H NMR (500 MHz, DMSO-d6) δ 9.36 (d, J = 1.6 Hz, 1H), 8.84 (d, J =1.6 Hz, 1H), 8.02 – 7.96 (m, 1H), 7.83 (d, J = 7.8 Hz, 1H), 7.79 – 7.70 (m,2H), 7.57 (ddd, J = 8.1, 7.1, 1.3 Hz, 1H), 7.56 – 7.51 (m, 1H), 7.53 – 7.45(m, 2H), 7.48 – 7.38 (m, 7H), 7.38 – 7.30 (m, 2H), 7.34 – 7.26 (m, 4H), 7.29– 7.23 (m, 1H), 6.44 (d, J = 14.5 Hz, 1H), 6.39 (dt, J = 8.4, 1.1 Hz, 1H), 6.25 (dt, J = 8.4, 1.1 Hz, 1H), 6.15 (d, J = 0.9 Hz, 2H), 5.14 (d, J = 0.9Hz, 2H), 2.85 – 2.71 (m, 4H), 1.56 (qt, J = 7.6, 5.7 Hz, 2H). 13 C NMR (125 MHz, DMSO-d6) δ 182.60, 168.18, 158.96, 151.54, 148.69, 142.99, 139.75, 139.35,138.34, 137.91, 137.02, 135.74, 135.50, 135.24, 135.17, 134.64, 131.38,129.19, 129.16, 129.04, 128.47, 128.40, 128.20, 128.18, 128.08, 127.90,127.78, 127.08, 126.93, 126.36, 126.27, 124.49, 124.28, 123.98, 115.83,97.89, 56.18, 52.12, 50.83, 29.25, 28.23, 26.06, 23.62 ppm. Example 12: Preparation of Compound 12 The structural formula of compound 12:

[0090] Intermediate azaindole 12.1

[0091] The azaindole 2.1 (0.4 g, 1 eq) obtained in Example 2 was dissolved in 10 mL of DMF, and TBTU (0.8 g) and DIPEA (0.3 g) were added. The mixture was stirred at 0 °C for 0.5 h. After cooling to room temperature, (methoxy-methoxy)methylamine (0.36 g) was added, and the reaction was carried out for 1.5 h. The mixture was filtered to remove impurity salts and purified on a silica gel column to obtain the intermediate azaindole 12.1 (0.37 g, Y=68%).

[0092] Intermediate quaternary ammonium salt 12.2

[0093] Azaindole 12.1 (0.3 g, 1 eq) was dissolved in 20 mL of acetonitrile, and then 1,3-propanesulfonic acid lactone (0.33 g, 2.5 eq) was added. The reaction mixture was stirred at 65 °C under nitrogen protection for 10 h, after which the reaction was stopped. Most of the acetonitrile was removed under reduced pressure, and the mixture was recrystallized from ethyl acetate to obtain a yellow powder intermediate, quaternary ammonium salt 12.2 (0.27 g, Y=62%). Quaternary ammonium salt 12.2 can be used in subsequent reactions without further purification.

[0094] Intermediate azaindole 12.3

[0095] The azaindole 2.1 (0.4 g, 1 eq) obtained in Example 2 was dissolved in 10 mL of DMF, and TBTU (0.8 g) and DIPEA (0.3 g) were added. The mixture was stirred at 0 °C for 0.5 h. After cooling to room temperature, propanol (0.18 g) was added, and the reaction was carried out for 1.5 h. The mixture was filtered to remove impurity salts and purified on a silica gel column to obtain the intermediate azaindole 12.3 (0.52 g, Y=86%).

[0096] Intermediate quaternary ammonium salt 12.4

[0097] Azaindole 12.3 (0.5 g, 1 eq) was dissolved in 20 mL of acetonitrile, and then 1,3-propanesulfonic acid lactone (0.74 g, 3 eq) was added. The reaction mixture was stirred at 65 °C under nitrogen protection for 10 h, after which the reaction was stopped. Most of the acetonitrile was removed under reduced pressure, and the mixture was recrystallized from ethyl acetate to obtain a yellow powder intermediate, quaternary ammonium salt 12.4 (0.48 g, Y=64%). Quaternary ammonium salt 12.4 can be used in subsequent reactions without further purification.

[0098] Manufacturing compound 12

[0099] Quaternary ammonium salt 12.2 (0.2 g, 1 eq), quaternary ammonium salt 12.4 (0.18 g, 1 eq), and condensing agent 12.5 (0.14 g, 1 eq) were dissolved in 10 mL of ethanol. 0.1 g of sodium acetate was added to catalyze the reaction. The reaction was stopped after stirring at 30 °C for 12 h. After the reaction solution cooled to room temperature, the solvent was concentrated and extracted with dichloromethane. The crude product obtained after concentration was purified by liquid chromatography to obtain green solid compound 12 (0.07 g, Y=15%). 1 H NMR (500 MHz, DMSO-d6) δ 9.40 (d, J =1.8 Hz, 1H), 8.82 (d, J = 1.8 Hz, 1H), 8.75 (t, J = 3.8 Hz, 1H), 8.26 (d, J =1.3 Hz, 1H), 7.98 – 7.92 (m, 2H), 7.39 – 7.33 (m, 2H), 7.23 (dt, J = 8.4, 1.0Hz, 1H), 7.13 – 7.07 (m, 2H), 6.68 (d, J = 14.6 Hz, 1H), 6.44 (dt, J = 8.4,1.0 Hz, 1H), 4.68 (d, J = 3.8 Hz, 2H), 4.63 (t, J = 6.8 Hz, 2H), 4.53 (s,1H), 4.27 (s, 1H), 4.12 (td, J = 5.5, 4.1 Hz, 4H), 3.31 (s, 2H), 2.82 (dd, J= 11.8, 11.2 Hz, 2H), 2.79 – 2.72 (m, 3H), 2.75 – 2.68 (m, 3H), 2.31 (tt, J =11.4, 6.7 Hz, 2H), 1.91 (tt, J = 6.8, 5.4 Hz, 2H), 1.71 – 1.51 (m, 4H), 1.33(s, 4H), 0.90 (t, J = 8.0 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ 182.43, 172.99,169.39, 166.61, 166.22, 161.07, 159.91, 152.01, 151.47, 147.92, 138.95,137.68, 137.19, 137.06, 134.39, 133.49, 131.75, 128.69, 128.60, 127.32,126.53, 123.57, 120.01, 119.54, 110.94, 104.46, 94.70, 68.36, 67.58, 56.38,55.66, 55.07, 52.53, 51.75, 48.54, 35.74, 27.69, 26.59, 26.55, 26.05, 25.43,24.04, 23.54, 21.91, 10.69 ppm. Manufacturing activated compound 12

[0100] Compound 12 (0.05 g) was added to 3 mL of DMF solution, followed by 2-(5-norbornene-2,3-dicarboximide)-1,1,3,3-tetramethylurea tetrafluoroboric acid (0.025 g), and then triethylamine (0.1 mL). The reaction was carried out at 25 °C for 2 h, and the reaction was stopped. The reaction solution was poured into ethyl acetate, centrifuged at 9000 rpm, and the precipitate was collected. The activated compound 12 (0.043 g, Y=74%) was purified by HPLC. 1H NMR (500 MHz, DMSO-d6) δ 8.91 (s, 1H), 8.26(d, J = 1.3 Hz, 1H), 8.03 – 7.97 (m, 2H), 7.81 (t, J = 3.8 Hz, 1H), 7.36 (d,J = 1.3 Hz, 1H), 7.23 (d, J = 9.2 Hz, 1H), 7.20 – 7.15 (m, 2H), 7.09 (dt, J =8.4, 0.9 Hz, 1H), 6.89 (d, J = 9.3 Hz, 1H), 6.57 (dt, J = 8.4, 1.0 Hz, 1H),6.14 – 6.06 (m, 2H), 4.66 (t, J = 3.8 Hz, 2H), 4.55 (d, J = 3.8 Hz, 1H), 4.51(d, J = 3.8 Hz, 1H), 4.29 – 4.20 (m, 2H), 4.19 – 4.13 (m, 1H), 4.15 – 4.10(m, 1H), 4.12 (s, 1H), 4.11 (d, J = 5.3 Hz, 2H), 3.42 – 3.34 (m, 2H), 3.33 –3.26 (m, 4H), 3.04 (td, J = 11.5, 4.8 Hz, 2H), 2.80 – 2.67 (m, 6H), 2.02 –1.86 (m, 4H), 1.71 – 1.65 (m, 1H), 1.68 – 1.62 (m, 1H), 1.62 (t, J = 4.8 Hz,2H), 1.62 – 1.55 (m, 1H), 1.58 – 1.50 (m, 1H), 1.39 (s, 2H), 1.35 (s, 2H),1.30 (s, 2H), 0.90 (t, J = 8.0 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ 183.34,170.14, 169.22, 167.57, 166.22, 163.82, 160.15, 154.34, 151.61, 149.09,147.92, 147.13, 138.82, 137.68, 137.19, 134.77, 133.18, 132.89, 132.76,128.74, 126.15, 123.57, 123.19, 120.66, 119.11, 118.91, 112.54, 104.46,94.70, 68.20, 67.58, 56.02, 55.66, 54.28, 53.81, 51.70, 50.49, 48.54, 44.68,43.12, 35.74, 30.44, 27.69, 26.55, 25.58, 25.43, 23.54, 21.91, 20.66, 10.69ppm. Example 13: Preparation of Compound 13 The structural formula of compound 13:

[0101] Manufacturing compound 13

[0102] The quaternary ammonium salt 5.1 (0.2 g, 1 eq) and condensing agent 13.1 (0.21 g, 1.5 eq) obtained in Example 5 were dissolved in 10 mL of ethanol. 0.1 g of sodium acetate was added to catalyze the reaction. The reaction was stopped after stirring at 90 °C for 12 h. After the reaction solution cooled to room temperature, the concentrated solvent was extracted with dichloromethane. The crude product obtained after concentration was purified by liquid chromatography to obtain blue solid compound 13 (0.19 g, Y=57%). 1H NMR (500 MHz, DMSO-d6) δ 9.25 (d, J = 1.6 Hz, 1H), 8.89 (dd, J = 4.1, 2.3 Hz, 1H), 8.58 (d, J = 1.6 Hz, 1H), 8.46 (dd, J =7.1, 2.2 Hz, 1H), 7.96 (d, J = 8.1 Hz, 1H), 7.70 (d, J = 8.1 Hz, 1H), 7.57 –7.48 (m, 2H), 7.50 – 7.46 (m, 1H), 6.66 (d, J = 14.1 Hz, 1H), 4.60 (q, J =6.1 Hz, 2H), 2.71 (t, J = 6.3 Hz, 2H), 2.71 – 2.61 (m, 2H), 1.65 – 1.53 (m,5H). 13 C NMR (125 MHz, DMSO-d6) δ 182.65, 157.81, 149.59, 149.34, 146.17,145.17, 144.12, 141.06, 140.08, 136.80, 135.71, 134.81, 131.99, 128.39,128.20, 127.81, 126.76, 122.18, 121.83, 120.62, 115.72, 52.96, 51.99, 31.08,27.90, 26.06, 24.27, 12.91 ppm. Example 14: Preparation of Compound 14 The structural formula of compound 14:

[0103] Intermediate quaternary ammonium salt 14.1

[0104] The azaindole 1.1 (0.5 g, 1 eq) obtained in Example 1 was dissolved in 20 mL of acetonitrile, and then iodomethane (1.19 g, 4 eq) was added. The reaction mixture was stirred at 50 °C under nitrogen protection for 8 h, after which the reaction was stopped. Most of the acetonitrile was removed under reduced pressure, and the mixture was recrystallized from ethyl acetate to obtain a yellow powder intermediate, quaternary ammonium salt 14.1 (0.35 g, Y=66%). Quaternary ammonium salt 14.1 can be used in subsequent reactions without further purification.

[0105] Manufacturing compound 14

[0106] Quaternary ammonium salt 14.1 (0.2 g, 1 eq) and condensing agent 14.2 (0.15 g, 1.2 eq) were dissolved in 10 mL of ethanol. 0.1 g of sodium acetate was added to catalyze the reaction. The reaction was stopped after stirring at 40 °C for 12 h. After the reaction solution cooled to room temperature, the solvent was concentrated. The residue was dissolved in anhydrous dichloromethane, and 0.3 mL of BBr3 was added. The reaction was carried out at 0 °C for 2 h. The solvent was concentrated under reduced pressure. The crude product obtained after concentration was purified by liquid chromatography to give blue solid compound 14 (0.08 g, Y=26%). 1 H NMR(500 MHz, DMSO-d6) δ 8.87 (dd, J = 1.5, 0.9 Hz, 1H), 8.09 (d, J = 1.6 Hz,1H), 7.45 (d, J = 9.3 Hz, 1H), 7.40 (d, J = 14.5 Hz, 1H), 6.92 – 6.88 (m,1H), 6.71 – 6.63 (m, 2H), 6.54 (d, J = 2.1 Hz, 1H), 4.15 (s, 2H), 2.73 – 2.63(m, 3H), 2.59 (dddd, J = 12.5, 7.7, 5.1, 0.9 Hz, 1H), 1.68 – 1.56 (m, 2H). 13 CNMR (125 MHz, DMSO-d6) δ 181.68, 158.88, 158.58, 155.72, 147.13, 145.65,140.41, 136.44, 135.74, 131.30, 130.43, 128.34, 126.79, 119.49, 114.49,113.93, 111.95, 100.76, 51.96, 42.05, 30.57, 27.91, 26.05, 24.18 ppm. Example 15: Preparation of Compound 15 The structural formula of compound 15:

[0107] Intermediate indole 15.1

[0108] 5-Carboxymethyl ester-1-hydrazinopyridine (1 g, 1 eq) was dissolved in 20 mL of toluene, and 8-methyl-9-oxodecanoic acid (3.92 g, 3 eq) was added. The mixture was stirred at 100 °C for 6 h, and then the solvent was evaporated under reduced pressure. Polyphosphoric acid (2 g) was added to the residue, followed by 10 mL of toluene as a solubilizer. The mixture was reacted at 120 °C for 5 h. The mixed solution was introduced into ice water, and the pH was adjusted to 1 with NaOH. The mixture was then extracted with ethyl acetate. Several layers were collected, washed with saturated brine, dried over anhydrous sodium sulfate, and then concentrated to remove the solvent. The solution was purified by silica gel chromatography to obtain the brownish-yellow azaindole intermediate indole 15.1 (1.49 g, Y=72%).

[0109] Intermediate quaternary ammonium salt 15.2

[0110] Azaindole 15.1 (0.5 g, 1 eq) was dissolved in 20 mL of acetonitrile, and then iodoethane (0.94 g, 4 eq) was added. The reaction mixture was stirred at 50 °C under nitrogen protection for 8 h, after which the reaction was stopped. Most of the acetonitrile was removed under reduced pressure, and the mixture was recrystallized from ethyl acetate to obtain a yellow powder intermediate, quaternary ammonium salt 15.2 (0.56 g, Y=76%). Quaternary ammonium salt 15.2 can be used in subsequent reactions without further purification.

[0111] Manufacturing compound 15

[0112] Quaternary ammonium salt 15.2 (0.2 g, 1 eq) and condensing agent 15.3 (0.15 g, 1.2 eq) were dissolved in 10 mL of ethanol. 0.1 g of sodium acetate was added to catalyze the reaction. The reaction was stopped after stirring at 40 °C for 12 h. After the reaction solution cooled to room temperature, the solvent was concentrated. The crude product obtained after concentration was purified by liquid chromatography to obtain blue solid compound 15 (0.11 g, Y=35%). 1HNMR (500 MHz, DMSO-d6) δ 9.47 (d, J = 1.6 Hz, 1H), 8.84 (d, J = 1.8 Hz, 1H), 7.53 (t, J = 1.2 Hz, 1H), 7.35 (d, J = 14.5 Hz, 1H), 6.89 (s, 1H), 6.72 (t, J= 1.1 Hz, 1H), 6.63 (d, J = 14.5 Hz, 1H), 4.73 – 4.58 (m, 2H), 3.88 (s, 2H), 3.39 (dd, J = 6.5, 3.8 Hz, 1H), 3.33 (dd, J = 6.5, 3.8 Hz, 1H), 2.96 (s, 2H),2.79 (td, J = 7.0, 1.1 Hz, 2H), 2.71 (t, J = 6.3 Hz, 2H), 2.71 – 2.61 (m,2H), 2.29 (t, J = 8.9 Hz, 2H), 2.12 (dt, J = 12.7, 7.1 Hz, 1H), 2.04 (dt, J =12.7, 7.1 Hz, 1H), 1.92 (dqd, J = 17.4, 6.8, 3.7 Hz, 2H), 1.65 – 1.53 (m,7H), 1.44 (pd, J = 7.0, 0.7 Hz, 2H), 1.39 – 1.29 (m, 4H). 13 C NMR (125 MHz, DMSO-d6) δ 184.45, 180.45, 163.75, 158.91, 150.78, 149.00, 145.86, 141.36, 140.00, 139.31, 135.06, 133.20, 132.76, 126.82, 126.25, 125.87, 125.15,123.02, 120.85, 106.92, 52.97, 52.17, 51.78, 50.63, 39.44, 37.51, 34.36,31.12, 29.27, 28.91, 28.48, 27.72, 25.36, 24.57, 24.50, 24.27, 22.57, 12.89ppm. Manufacturing activated compound 15

[0113] Compound 15 (0.05 g) was added to 3 mL of DMF solution, followed by 2-(5-norbornene-2,3-dicarboximide)-1,1,3,3-tetramethylurea tetrafluoroboric acid (0.025 g), and then triethylamine (0.1 mL). The reaction was carried out at 25 °C for 2 h, and the reaction was stopped. The reaction solution was poured into ethyl acetate, centrifuged at 9000 rpm, and the precipitate was collected. The activated compound 15 (0.053 g, Y=88%) was purified by HPLC. 1 H NMR (500 MHz, DMSO-d6) δ 9.47 (d, J = 1.6 Hz,1H), 8.84 (d, J = 1.8 Hz, 1H), 7.53 (t, J = 1.2 Hz, 1H), 7.20 (d, J = 9.0 Hz,1H), 6.89 (s, 1H), 3.88 (s,2H), 3.42 – 3.34 (m, 3H), 3.33 (dd, J = 6.5, 3.7 Hz, 1H), 3.24 – 3.17 (m,2H), 2.96 (s, 2H), 2.79 (td, J = 7.0, 1.1 Hz, 2H), 2.71 (t, J = 6.3 Hz, 2H), 2.71 – 2.61 (m, 2H), 2.37 (t, J = 8.8 Hz, 2H), 2.12 (dt, J = 12.7, 7.1 Hz,1H), 2.04 (dt, J = 12.7, 7.1 Hz, 1H), 1.92 (dqd, J = 17.4, 6.8, 3.7 Hz, 2H),1.65 – 1.49 (m, 11H), 1.49 – 1.39 (m, 4H), 1.39 – 1.29 (m, 4H). 13C NMR (125MHz, DMSO-d6) δ 203.09, 184.25, 172.99, 163.75, 158.91, 150.78, 149.00,145.69, 141.36, 140.00, 139.31, 135.93, 135.06, 133.20, 132.76, 126.82,126.25, 125.87, 125.15, 123.02, 121.49, 106.92, 85.52, 52.97, 52.17, 51.78,50.63, 48.52, 47.91, 42.34, 39.44, 37.51, 34.08, 31.12, 29.27, 28.91, 28.32,27.72, 25.60, 24.57, 24.50, 24.27, 22.57, 12.89 ppm. Example 16 Preparation of Comparative Compound 1 Compare the structural formula of compound 1:

[0114] Intermediate quaternary ammonium salt 16.1

[0115] 5-Iodo-2,3,3-trimethyl-3H-indole (1 g, 1 eq) was dissolved in 20 mL of acetonitrile, and then iodoethane (2.19 g, 4 eq) was added. The reaction mixture was stirred at 50 °C under nitrogen protection for 8 h, after which the reaction was stopped. Most of the acetonitrile was removed under reduced pressure, and the mixture was recrystallized from ethyl acetate to give a yellow powder intermediate, quaternary ammonium salt 16.1 (1.24 g, Y=80%). Quaternary ammonium salt 16.1 can be used in subsequent reactions without further purification.

[0116] Manufacturing Comparative Compound 1

[0117] Quaternary ammonium salt 16.1 (0.5 g, 2 eq) and condensing agent 16.2 (0.15 g, 1.2 eq) were dissolved in 10 mL of ethanol. 0.1 g of sodium acetate was added to catalyze the reaction. The reaction was stopped after stirring at 40 °C for 12 h. After the reaction solution cooled to room temperature, the solvent was concentrated. The crude product obtained after concentration was purified by liquid chromatography to obtain blue solid comparative compound 1 (0.31 g, Y=69%). 1HNMR (500 MHz, DMSO-d6) δ 7.95 (dd, J = 8.2, 2.0 Hz, 1H), 7.83 (d, J = 1.9 Hz, 1H), 7.67 (dd, J = 7.9, 2.2 Hz, 1H), 7.56 (d, J = 2.2 Hz, 1H), 7.39 (dd, J =14.2, 1.0 Hz, 1H), 7.17 (s, 0H), 7.00 (ddt, J = 14.3, 7.9, 1.0 Hz, 1H), 6.62– 6.53 (m, 2H), 6.49 (ddd, J = 15.2, 7.4, 1.0 Hz, 1H), 6.15 (dt, J = 7.4, 1.0Hz, 1H), 4.61 (q, J = 7.0 Hz, 2H), 3.98 (q, J = 7.0 Hz, 2H), 1.72 (s, 5H), 1.55 (t, J = 7.0 Hz, 3H), 1.29 (t, J = 6.9 Hz, 3H). 13 C NMR (125 MHz, DMSO-d6)δ 165.44, 155.67, 144.43, 143.32, 141.20, 139.07, 138.06, 136.96, 136.65,135.11, 133.82, 130.19, 129.67, 121.58, 112.43, 111.31, 101.70, 89.63, 89.45,49.06, 48.00, 41.37, 40.89, 27.68, 27.04, 13.24, 13.16 ppm. Example 17 Preparation of Comparative Compound 2 Compare the structural formula of compound 2:

[0118] Intermediate quaternary ammonium salt 17.1

[0119] Potassium 5-sulfonic acid-2,3,3-trimethyl-3H-indole (1 g, 2 eq) was dissolved in 20 mL of acetonitrile, and then iodomethane (2.56 g, 5 eq) was added. The reaction mixture was stirred at 50 °C under nitrogen protection for 8 h, after which the reaction was stopped. Most of the acetonitrile was removed under reduced pressure, and the mixture was recrystallized from ethyl acetate to give a yellow powder intermediate, quaternary ammonium salt 17.1 (1.23 g, Y=81%). Quaternary ammonium salt 17.1 can be used in subsequent reactions without further purification.

[0120] Intermediate quaternary ammonium salt 17.2

[0121] Potassium 5-sulfonic acid-2,3,3-trimethyl-3H-indole (1 g, 2 eq) was dissolved in 20 mL of acetonitrile, followed by the addition of 6-bromohexanoic acid (2.26 g, 3 eq). The reaction mixture was stirred at 50 °C under nitrogen protection for 8 h, after which the reaction was stopped. Most of the acetonitrile was removed under reduced pressure, and the mixture was recrystallized from ethyl acetate to obtain a yellow powder intermediate, quaternary ammonium salt 17.2 (1.11 g, Y=63%). Quaternary ammonium salt 17.2 can be used in subsequent reactions without further purification.

[0122] Manufacturing Comparative Compound 2

[0123] Quaternary ammonium salt 17.1 (0.2 g, 1 eq), quaternary ammonium salt 17.2 (0.23 g, 1 eq), and condensing agent 16 (0.12 g, 1 eq) were dissolved in 10 mL of ethanol. 0.1 g of sodium acetate was added to catalyze the reaction. The reaction was stopped after stirring at 40 °C for 12 h. After the reaction solution cooled to room temperature, the solvent was concentrated. The crude product obtained after concentration was purified by liquid chromatography to obtain blue solid contrast compound 2 (0.07 g, Y=21%). 1H NMR (500 MHz, DMSO-d6) δ 8.03 (d, J = 1.9 Hz, 1H), 7.97 (dd, J = 8.0, 1.8 Hz, 1H), 7.91 (d, J = 2.2 Hz, 1H), 7.74 (d, J = 8.1Hz, 1H), 7.69 (dd, J=8.1, 2.2 Hz, 1H), 7.39 (dd, J=14.3, 1.1 Hz, 1H),7.29 (ddt, J=14.3, 7.9, 1.1 Hz, 1H), 6.58 (ddt, J=15.4, 7.7, 1.0 Hz, 1H),6.49 (ddd, J = 15.3, 7.4, 1.0 Hz, 1H), 6.18 (dt, J = 7.4, 1.0 Hz, 1H), 4.25(s, 2H), 3.78 (t, J = 5.4 Hz, 2H), 2.29 (t, J = 8.9 Hz, 2H), 1.76 (tt, J =6.9, 5.5 Hz, 2H), 1.69 (d, J = 17.8 Hz, 9H), 1.56 – 1.46 (m, 2H), 1.42 – 1.26 (m, 4H). 13 C NMR (125 MHz, DMSO-d6) δ 180.45, 168.56, 160.30, 150.90, 146.05,144.27, 140.65, 140.55, 138.38, 134.82, 133.88, 131.74, 131.45, 129.16,125.17, 123.60, 120.23, 114.10, 110.87, 101.29, 45.69, 43.59, 43.00, 36.67,34.36, 28.43, 28.32, 27.57, 27.24, 27.11, 25.36 ppm. Manufacturing activated contrast compound 2

[0124] Comparative compound 2 (0.05 g) was added to 3 mL of DMF solution, followed by 2-(5-norbornene-2,3-dicarboximide)-1,1,3,3-tetramethylurea tetrafluoroboric acid (0.025 g), and then triethylamine (0.1 mL). The reaction was carried out at 25 °C for 2 h, and the reaction was stopped. The reaction solution was poured into ethyl acetate, centrifuged at 9000 rpm, and the precipitate was collected. The precipitate was purified by HPLC to obtain activated comparative compound 2 (0.053 g, Y=87%). 1 H NMR (500 MHz, DMSO-d6) δ 8.03 (d, J = 1.8Hz, 1H), 7.97 (dd, J = 8.1, 1.8 Hz, 1H), 7.91 (d, J = 2.2 Hz, 1H), 7.76 –7.66 (m, 2H), 7.39 (dd, J = 14.3, 1.1 Hz, 1H), 7.29 (ddt, J = 14.3, 7.9, 1.1Hz, 1H), 6.58 (ddt, J = 15.4, 7.7, 1.0 Hz, 1H), 6.49 (ddd, J = 15.3, 7.4, 1.0Hz, 1H), 6.18 (dt, J = 7.4, 1.0 Hz, 1H), 6.14 – 6.06 (m, 2H), 4.25 (s, 2H), 3.84 – 3.72 (m, 2H), 3.38 (dpt, J = 4.0, 2.9, 1.8 Hz, 2H), 3.33 – 3.26 (m,2H), 2.54 (t, J = 8.8 Hz, 2H), 1.80 – 1.54 (m, 16H), 1.42 – 1.26 (m, 4H). 13 CNMR (125 MHz, DMSO-d6) δ 171.41, 170.39, 168.56, 160.30, 150.90, 146.05,144.27, 140.65, 140.55, 138.38, 134.82, 134.77, 133.88, 131.74, 131.45,129.16, 125.17, 123.60, 120.23, 114.10, 110.87, 101.29, 50.49, 45.69, 44.69,43.59, 43.12, 43.00, 36.67, 33.05, 28.32, 28.25, 27.57, 27.24, 27.11, 24.24ppm. Example 18 Preparation of Comparative Compound 3 Compare the structural formula of compound 3:

[0125] Manufacturing comparative compound 3

[0126] Quaternary ammonium salt 7.2 (0.2 g, 2 eq) and condensing agent 18 (0.03 g, 1 eq) were dissolved in 10 mL of ethanol. 0.1 g of sodium acetate was added to catalyze the reaction. The reaction was stopped after stirring at 40 °C for 12 h. After the reaction solution cooled to room temperature, the solvent was concentrated. The crude product obtained after concentration was purified by liquid chromatography to obtain blue solid comparative compound 3 (0.05 g, Y=30%). 1 HNMR (500 MHz, DMSO-d6) δ = 8.66 (dd, 1H), 8.05 (dd, 1H), 7.87 (dd, 1H), 7.59(dd, 1H), 7.38 (m, 1H), 7.36 (m, 1H), 7.13 (dd, 1H), 6.67 (m, 1H), 6.63 (dd,1H), 4.60 (t, 2H), 3.95 (t, 2H), 2.16 (qt, 2H), 1.66 (qt, 2H), 1.29 (s, 4H),1.12 (t, 3H), 0.94 (t, 3H). 13 C NMR (125 MHz, DMSO-d6) δ = 182.63, 169.89,159.59, 149.71, 146.17, 141.35, 138.96, 134.13, 133.89, 131.12, 124.01,121.57, 116.07, 113.25, 109.31, 59.06, 52.86, 51.86, 51.60, 26.01, 25.47,23.93, 21.82, 13.71, 10.93 ppm. Example 19: Determination of UV-Vis absorption and fluorescence spectra of compounds 5-6, 8-9, and comparative compounds 1-2 Accurately weigh the vacuum-dried dye using a 0.01 g / L balance to prepare a 2 mmol / L DMSO dye stock solution. Store in brown sample vials at 4°C for later use. For testing UV-Vis absorption and fluorescence spectra, 3 μL of the dye stock solution was measured using a micropipette and dissolved in a quartz cuvette containing 3 mL of dichloromethane solvent. The mixture was thoroughly mixed to obtain a dye concentration of 2.0 μmol / L, which was then used for testing the absorption and fluorescence emission spectra. All tests were performed at 25°C.

[0127] Since the structures of compounds 5, 6, 8, and 9 are similar to those of comparative compounds 1-2, these compounds were primarily selected for comparative studies. Figure 1 As shown, the maximum absorption wavelengths of nitrogen-containing cyanine dyes with upconversion properties (compounds 5, 6, 8, and 9) are significantly longer than those of ordinary cyanine dyes (comparative compounds 1-2). Meanwhile, as... Figure 2 As shown, the maximum emission wavelengths of nitrogen-containing cyanine dyes with upconversion properties are all above 700 nm, longer than those of ordinary cyanine dyes. This property allows for compatibility with long-wavelength lasers and detectors during imaging and photodynamic therapy, which helps reduce the photobleaching rate of the dye and increase the depth of tissue penetration.

[0128] Example 20: Upconversion fluorescence spectroscopy of compounds 1-15 and comparative examples 1-2 First, the tail emission wavelength of the dye was determined by fluorescence spectroscopy. Then, using this wavelength as the excitation wavelength, the fluorescence spectrum of the dye was measured. All compounds involved in the examples were tested, and the results are shown in Table 1 and... Figure 3 As shown in AO, compounds 1-15 of general formula I all exhibit upconversion fluorescence, while the molecular structures of comparative compounds 1-3, although very similar to those of general formula I, did not show upconversion fluorescence in fluorescence spectroscopy. In fact, the experimental design phase of this application predicted that the molecular structures of comparative compounds 1-3, being similar to those of general formula I, might exhibit similar fluorescence properties. However, the experimental results completely overturned expectations; these comparative compounds did not exhibit upconversion fluorescence. This unexpected discovery fully demonstrates the unpredictability that frequently exists in experimental science.

[0129] Table 1:

[0130] (Note: "+" indicates that it has upconversion performance; "-" indicates that it does not have upconversion performance) Example 21: Upconversion photodynamic effect test of compound 1, compound 5, compound 6 and control compound 1 Since the substitution of heavy atoms can enhance the photodynamic effect of dyes, compound 1 with heavy atom substitution and control compound 1 were selected in the examples for the study of upconversion photodynamic effects. 1,3-Diphenylisobenzofuran (DPBF) was used as a singlet oxygen scavenger, and the singlet oxygen generation efficiency of the compounds was detected by spectrophotometry.

[0131] Procedure: Add 3 mL of dichloromethane to a 1×1 cm quartz cuvette, and then add 3 μL of the dye stock solution to bring the dye concentration to 2 μM. After mixing thoroughly, irradiate the cuvette with a 760 nm laser source, maintaining a uniform light power of 20 mW / cm². 2 The absorbance of DPBF at 415 nm was measured at different time points. The rate of decrease in DPBF absorbance was used to determine the singlet oxygen production capacity, thereby demonstrating the photodynamic effect.

[0132] like Figure 4 As shown in the diagram, compounds 1, 5, and 6 can generate singlet oxygen under 760 nm light irradiation. This leads to a decrease in DPBF absorbance. In contrast, compound 1 does not exhibit upconversion photodynamic effects. Therefore, photosensitizers designed with dyes according to general formula I can generate a large amount of singlet oxygen under long-wavelength light excitation, thereby achieving good photodynamic effects and showing potential for treating deep lesions.

[0133] Example 22: Fluorescent labeling of lysine with activated compounds A 10 mmol / L L-lysine stock solution was prepared using PBS (pH = 7.4) in a 5 mL brown sample vial. 50 μL of the 10 mmol / L L-lysine stock solution was added to a 0.5 mL centrifuge tube, followed by 100 mL of a 5 mmol / L stock solution of the activated compound. After reacting in a benchtop constant temperature shaker for 6 h, methanol and ultrapure water were used as the mobile phase, and a C18-packed reverse-phase column was used as the separation column. The activated compound 1 and the L-lysine-labeled compound 1 were characterized by liquid chromatography at a wavelength of 254 nm.

[0134] Taking compound 2 as an example, such as Figure 5As shown, the retention time of activated compound 2 was 11.4 min. However, after co-incubation with L-lysine, liquid chromatography characterization under the same conditions revealed that the 11.4 min peak almost disappeared, and a new absorption peak with a retention time of 14.3 min appeared, with the new peak area accounting for 91%. This indicates that activated compound 1 can react well with L-lysine, successfully linking L-lysine to the dye compound 2. The labeling of the remaining compounds is shown in Table 2. The conventional dye control compound 2 was also successfully labeled, as was the structurally modified aza dye. This demonstrates that structural changes do not affect the ability of activated dyes to label biomolecules.

[0135] Table 2:

[0136] Example 23: Photostability Test of Compound 5 To verify that this type of dye with upconversion ability can have better photostability under long-wavelength irradiation, compound 1 was selected for verification.

[0137] Procedure: Add 3 mL of water to a 1×1 cm quartz cuvette, and add 3 μL of dye stock solution to bring the dye concentration to 2 μM. After mixing thoroughly, irradiate the cuvette using a 760 nm laser source and a 660 nm laser source, maintaining a uniform optical power of 20 mW / cm². 2 The absorption spectrum of compound 1 was measured at different time points, and the photobleaching rate of compound 1 was determined by the rate of decrease at its maximum absorption wavelength.

[0138] like Figure 6 As shown, the photobleaching rate of the compound under 760 nm laser irradiation is significantly lower than that under 660 nm laser irradiation, indicating that longer wavelength irradiation is beneficial for improving the photostability of the dye. Since both 660 nm and 760 nm lasers can induce bright fluorescence in compound 5 at 720 nm, the compounds involved in general formula I exhibit better stability in imaging and therapeutic applications compared to ordinary dyes.

[0139] The above description, in conjunction with specific preferred embodiments of the present invention, further illustrates the invention and should not be construed as limiting the scope of the invention to these descriptions. Any modifications or alterations made by those skilled in the art without departing from the technical scope of the invention will be considered to be covered within the scope of the claims of the present invention.

Claims

1. A dye with upconversion properties, characterized in that, The dye has the structure of general formula I: I In general formula I, A1 is selected from any one or more of the groups described in ii or iii; A2 is selected from any one or more of the groups described in vii or viiii; R1 is selected from any one or more of hydrogen, aryl, alkyl with 1-18 carbons, carboxyl alkyl with 1-18 carbons, sulfonic acid group or sulfonate with 1-18 carbons, hydroxyalkyl with 1-18 carbons, aminoalkyl with 1-18 carbons, and aryl carboxylic acids; more preferably from any one or more of hydrogen, alkyl with 1-6 carbons, carboxyl alkyl with 1-6 carbons, sulfonic acid group or sulfonate with 1-6 carbons, aryl, and aryl carboxylic acids. R2 is a substituent at any position on the benzene ring, selected from any one or more of alkyl, carboxyl, amino, nitro, methoxy, halogen, hydroxy, ester, amide, and sulfonate groups with 1 to 6 carbons; more preferably, selected from any one or more of alkyl, carboxyl, halogen, methoxy, amide, ester, and sulfonate groups with 1 to 3 carbons. R3 is selected from one or more of hydrogen, alkyl groups with 1-18 carbons, carboxyl groups with 1-18 carbons, sulfonic acid groups with 1-18 carbons, and aryl carboxylic acids; more preferably from one or more of hydrogen, aryl carboxylic acids, and carboxyl groups with 1-8 carbons. Y is selected from halide ions, ClO4 - CF3COO - or OTs - Any one or more of the following; R4 is selected from any one or more of alkyl, carboxyl, amino, methoxy, halogen, aryl, ester, and amide groups with 1 to 6 carbons; more preferably from any one or more of alkyl, carboxyl, halogen, and aryl groups with 1 to 3 carbons. R5 is a substituent at any position on the benzene ring, selected from any one or more of alkyl, carboxyl, amino, nitro, methoxy, halogen, hydroxy, ester, amide, and sulfonate groups with 1 to 6 carbons; more preferably, selected from any one or more of alkyl, carboxyl, halogen, methoxy, amide, ester, and sulfonate groups with 1 to 3 carbons. R6 is selected from any one or more of hydrogen, aryl, alkyl with 1-18 carbons, carboxyl alkyl with 1-18 carbons, sulfonic acid group or sulfonate with 1-18 carbons, hydroxyalkyl with 1-18 carbons, aminoalkyl with 1-18 carbons, and aryl carboxylic acids; more preferably from any one or more of hydrogen, alkyl with 1-6 carbons, carboxyl alkyl with 1-6 carbons, sulfonic acid group or sulfonate with 1-6 carbons, aryl, and aryl carboxylic acids. R7 is selected from hydrogen, alkyl groups of 1-18 carbons, carboxyl groups of 1-18 carbons, sulfonic acid groups of 1-18 carbons, and aryl carboxylic acids; more preferably from any one or more of hydrogen, aryl carboxylic acids, and carboxyl groups of 1-8 carbons.

2. The method for synthesizing the dye as described in claim 1, characterized in that, Includes the following steps: Step 1: Synthesis of Indole 2-hydrazinopyridine with R2 substituent and ethyl ketone with R3 substituent are added to an organic solvent in a molar ratio of 1:1-5. After reacting at 50-120℃, the solvent is removed, and then added to an organic acid solvent. After reacting at 80-120℃, the temperature is lowered, the pH is adjusted to 1-5, and the mixture is extracted and purified to obtain indole with substituents. The organic solvent is selected from one or more mixed solvents selected from toluene, o-dichlorobenzene, and benzene; Step 2: Synthesis of Quaternary Ammonium Salts Indole with R2 and R3 is mixed with N-alkylating agent with R1 substituent in a molar ratio of 1:1-10, added to a polar organic solvent, and reacted at 40-100℃ to obtain solid powder quaternary ammonium salt. Step 3: Dye Synthesis The quaternary ammonium salt obtained in step 2 is mixed with a condensing agent containing A1 and A2 at a molar ratio of 1:0.5-8. Solvent A is added to the mixture, followed by a catalytic reagent B. The mixture is reacted at 50-130°C until the reaction product no longer increases. The solvent is removed, and the product is obtained by chromatography. The reagent A is selected from at least one of ethanol, acetic anhydride, n-butanol, isopropanol, and acetic acid; The reagent B is selected from at least one of sodium acetate, pyridine, potassium carbonate, and triethylamine.

3. The method according to claim 2, characterized in that, In step one, the molar ratio of 2-hydrazinopyridine with R2 substituent to ethyl ketone with R3 substituent is 1:1-3.

4. The method according to claim 2, characterized in that, In step two, the molar ratio of the indole with R2 and R3 to the N-alkylating agent with R1 substituent is 1:1-5.

5. The method according to claim 2, characterized in that, In step three, the molar ratio of the quaternary ammonium salt to the condensing agent containing A1 and A2 is 1:0.5-4.

6. The method according to claim 2, characterized in that, In step two, the organic acid is selected from one or a mixture of several of acetic acid, polyphosphoric acid, trifluoroacetic acid, and benzoic acid.

7. The method according to claim 2, characterized in that, In step three, the polar organic solvent is selected from one or more mixed solvents selected from methanol, ethanol, acetonitrile, and acetone.

8. The application of the dye with upconversion absorption properties in the upper heat zone as described in claim 1.

9. The application according to claim 8, characterized in that, The dye fluoresces at room temperature when irradiated with light of wavelength 750-900 nm.

10. The application according to claim 8, characterized in that, The dyes described herein are used in fluorescent labeling, cell imaging, gene sequencing, biomolecule recognition, and photosensitizers; more preferably, they are used in cell imaging with self-upconversion properties, gene sequencing with long-wavelength excitation, and as photosensitizers.

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