A dye with up-conversion performance, a synthetic method and application thereof

Abstract

The application discloses a dye with up-conversion performance, a synthesis method of the dye and application of the dye. The dye has up-conversion performance, can emit fluorescence (short wavelength) under irradiation of 750-900 nm light (long wavelength), and can avoid the problem of photo-bleaching caused by short wavelength excitation. Meanwhile, the dye has high extinction coefficient and fluorescence quantum yield in water-based solvents and polar protic solvents, and thus is very beneficial to fluorescence imaging, gene sequencing and biological macromolecule recognition and the like. Meanwhile, the up-conversion mechanism has deeper tissue penetration capacity, is beneficial to in-situ imaging and photodynamic therapy of deep pathological tissue of a living body, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of fine chemical industry, and relates to synthesis of a dye with upconversion performance and application thereof in biological imaging, gene sequencing and photodynamic therapy. BACKGROUND

[0002] Fluorescent dyes can absorb light of a specific wavelength and emit bright fluorescence, and have advantages of high efficiency, convenience, wavelength controllability and visualization, etc. Therefore, fluorescent imaging technology relying on fluorescent dyes has developed rapidly in recent decades. With the update and iteration of lasers and recognition devices used for imaging, fluorescent dyes with more excellent performance are needed in the fields of biological molecule recognition, gene sequencing, super-resolution imaging, etc. to be suitable for complex application scenarios.

[0003] At present, the commonly used fluorescent dyes have problems of short wavelength and poor light stability. Fluorescent dyes with short-wavelength excitation and emission usually have relatively high imaging resolution, but the short-wavelength light has poor tissue penetration depth and is very susceptible to interference of biological macromolecular autofluorescence, thereby resulting in poor imaging effect. At the same time, the short-wavelength laser usually has high energy, and the dye is prone to photobleaching after multiple exposures, thereby losing the ability of imaging and recognition. Therefore, it is necessary to develop new fluorescent dyes to overcome the above problems. Upconversion is a very unconventional luminescence phenomenon, which refers to the use of long-wavelength light to excite a compound to emit short-wavelength fluorescence. If it is applied to the field of fluorescent imaging, the dye can be excited by a long-wavelength laser with low energy but high tissue penetration depth to obtain short-wavelength fluorescence, thereby realizing high-resolution imaging effect and effectively avoiding photobleaching of the dye. However, the materials with upconversion performance at present are mainly inorganic noble metal materials such as lanthanide materials, which are difficult to modify and are not conducive to practical application. Therefore, it is of great significance to develop fluorescent dyes with upconversion performance. SUMMARY

[0004] In order to solve the problem of short-wavelength excitation of the fluorescent dye at present, the present application provides a dye with upconversion performance, a synthesis method and application thereof. The dye is synthesized and prepared by using indole with a specific substituent, quaternary ammonium salt and condensing agent. The dye has excellent optical performance and can be applied to the fields of fluorescent labeling, biological imaging, gene sequencing, biological macromolecule recognition and photodynamic therapy, etc. In particular, it has significant advantages in biological imaging, long-wavelength excitation gene sequencing and photodynamic therapy.

[0005] The technical solution of the present application is as follows:

[0006] The first aspect of the present application is to protect a dye with upconversion performance, which has the structure of general formula I.

[0007]

[0008] In general formula I,

[0009] A1 is selected from any one or more of the groups i-iv;

[0010]

[0011] A2 is selected from any one or more of the groups v-x;

[0012]

[0013] R1 is selected from any one or more of hydrogen, aryl, alkyl of 1-18 carbons, carboxyalkyl of 1-18 carbons, sulfonic acid group or sulfonate of 1-18 carbons, hydroxyalkyl of 1-18 carbons, aminoalkyl of 1-18 carbons, aryl carboxylic acid; more preferably from any one or more of hydrogen, alkyl of 1-6 carbons, carboxyalkyl of 1-6 carbons, sulfonic acid group or sulfonate of 1-6 carbons, aryl, aryl carboxylic acid;

[0014] R2 is a substituent on the phenyl ring at any position selected from any one or more of alkyl of 1-6 carbons, carboxy, amino, nitro, methoxy, halogen, hydroxy, ester, amide, sulfonate; more preferably from any one or more of alkyl of 1-3 carbons, carboxy, halogen, methoxy, amide, ester, sulfonate;

[0015] R3 is selected from any one or more of hydrogen, alkyl of 1-18 carbons, carboxyalkyl of 1-18 carbons, sulfonic acid group of 1-18 carbons, aryl carboxylic acid; more preferably from any one or more of hydrogen, aryl carboxylic acid, carboxyalkyl of 1-8 carbons;

[0016] Y is selected from any one or more of halide, CIO4 - , CF3COO - , or OTs -

[0017] wherein R4 is selected from any one or more of alkyl of 1-6 carbons, carboxy, amino, methoxy, halogen, aryl, ester, amide; more preferably from any one or more of alkyl of 1-3 carbons, carboxy, halogen, aryl;

[0018] wherein R5 is a substituent on the phenyl ring at any position selected from any one or more of alkyl of 1-6 carbons, carboxy, amino, nitro, methoxy, halogen, hydroxy, ester, amide, sulfonate; more preferably from any one or more of alkyl of 1-3 carbons, carboxy, halogen, methoxy, amide, ester, sulfonate;

[0019] ​wherein R6 is selected from any one or more of hydrogen, aryl, alkyl of 1-18 carbons, carboxyalkyl of 1-18 carbons, sulfonic acid group or sulfonic acid salt of 1-18 carbons, hydroxyalkyl of 1-18 carbons, aminoalkyl of 1-18 carbons, aryl carboxylic acid; more preferably selected from any one or more of hydrogen, alkyl of 1-6 carbons, carboxyalkyl of 1-6 carbons, sulfonic acid group or sulfonic acid salt of 1-6 carbons, aryl, aryl carboxylic acid;

[0020] wherein R7 is selected from any one or more of hydrogen, alkyl of 1-18 carbons, carboxyalkyl of 1-18 carbons, sulfonic acid group of 1-18 carbons, aryl carboxylic acid; more preferably selected from any one or more of hydrogen, aryl carboxylic acid, carboxyalkyl of 1-8 carbons;

[0021] A second aspect of the present application is to protect a method for synthesizing a dye with up-conversion performance, comprising the following steps:

[0022] Step one: synthesis of indole

[0023]

[0024] 2-hydrazinylpyridine with R2substituent and ethanone with R3substituent are added into organic solvent in a molar ratio of 1:1-5, after reaction at 50-120℃, the solvent is removed, then added into organic acid solvent, after reaction at 80-120℃, the temperature is lowered, the pH is adjusted to 1-5, extracted, purified to obtain indole with substituent;

[0025] Further preferably, in step one, the molar ratio of 2-hydrazinylpyridine with R2substituent and ethanone with R3substituent is more preferably selected from 1:1-3;

[0026] Further preferably, in step one, the organic solvent is selected from one or more of mixed solvents of toluene, o-dichlorobenzene, benzene;

[0027] Further preferably, in step one, the organic acid is selected from one or a mixture of several of acetic acid, polyphosphoric acid, trifluoroacetic acid, benzoic acid;

[0028] Further preferably, in step one, the pH is adjusted to 1-3;

[0029] Step two: synthesis of quaternary ammonium salt

[0030]

[0031] Indole with R2and R3is mixed with N-alkylating agent with R1substituent in a molar ratio of 1:1-10, added into polar organic solvent, after reaction at 40-100℃, solid powder quaternary ammonium salt is obtained;

[0032] Further preferably, in step two, the molar ratio of the indole with R2 and R3 to the N-alkylating agent with R1 is 1:1-5.

[0033] Further preferably, in step two, the polar organic solvent is selected from one or more mixed solvents of methanol, ethanol, acetonitrile, and acetone.

[0034] Step three: synthesis of the dye

[0035]

[0036] The quaternary ammonium salt obtained in step two is mixed with the condensing agent containing A1 and A2 at a molar ratio of 1:0.5-8, solvent A is added thereto, and a catalytic amount of reagent B is added, and the mixture is reacted at 50-130°C until the reaction product no longer increases; the solvent is removed, and chromatography is performed to obtain a metallic luster dye.

[0037] In step three, the reagent A is selected from at least one of ethanol, acetic anhydride, n-butanol, isopropanol, and acetic acid.

[0038] In step three, the reagent B is selected from at least one of sodium acetate, pyridine, potassium carbonate, and triethylamine.

[0039] For the technical solutions described above, in step three, the molar ratio of the quaternary ammonium salt to the condensing agent containing A1 and A2 is preferably 1:0.5-4; more preferably 1:1-3.

[0040] The third aspect of the present application is to protect the application of a dye with upconversion performance in the upper troposphere.

[0041] Further, the dye with upconversion performance can emit fluorescence under the irradiation of light with a wavelength of 750-900 nm.

[0042] Further, the dye with upconversion performance can be applied to the technical fields of fluorescent labeling, cell imaging, gene sequencing, biological macromolecule recognition, and photosensitizer as photodynamic therapy; more preferably, cell imaging under upconversion performance, gene sequencing under long-wavelength excitation, and photosensitizer in the field of photodynamic therapy.

[0043] Compared with the prior art, the present application has the following beneficial effects:

[0044] The dyes described in the present application generally have the up- conversion performance of the upper troposphere absorption, and can emit fluorescence (short wavelength) under the irradiation of 750-900 nm light (long wavelength) which the compound does not absorb, which relies on the driving energy provided by the ambient temperature, and can avoid the problem of photobleaching caused by short wavelength excitation. At the same time, the dyes have high extinction coefficient and fluorescence quantum yield in water-based solvents and polar protic solvents, so they are very advantageous for fluorescence imaging, gene sequencing and biological macromolecule recognition, etc. At the same time, the up-conversion mechanism also has a deeper tissue penetration ability, which is beneficial to in situ imaging and photodynamic therapy of deep pathological tissues in vivo. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 is the UV-Vis absorption spectrum of compound 5-6, compound 8-9 and comparative example 1-2;

[0046] Figure 2 is the UV-Vis absorption spectrum normalized fluorescence emission spectrum of compound 5-6, compound 8-9 and comparative example 1-2;

[0047] Figure 3 a-o is the up-conversion fluorescence emission spectrum of compound 5-6, compound 8-9;

[0048] Figure 4 abcd is the up-conversion photodynamic effect diagram of compound 1, compound 5, compound 6 and comparative compound 1;

[0049] Figure 5 is the liquid chromatogram of activated compound 2 labeling L-lysine;

[0050] Figure 6 is the light stability diagram of compound 1 under irradiation of different lasers; DETAILED DESCRIPTION

[0051] The present application is further described in detail below. Unless otherwise defined, the terms used herein have the following meanings.

[0052] The term "halogen" used herein includes fluorine, chlorine, bromine and iodine.

[0053] The term "alkyl" used herein includes straight-chain alkyl and branched-chain alkyl.

[0054] Y - represents a negative ion, which can be any suitable negative ion, including inorganic negative ions and organic negative ions, which can be exemplified but not limited to halogen ions, ClO4 - , PF6 - , BF4 - , CH3COO - , CF3COO -or OTs - .

[0055] Instruments and equipment employed in the examples:

[0056] In the column chromatography process of the present application, 200-300 mesh, 100-200 mesh column chromatography silica gel purchased from Qingdao Meigao Group Co., Ltd. and 20-40 mesh analytical pure quartz sand purchased from Tianda Chemical Reagent Factory were used. The reverse phase purification chromatograph was CHEETAH fast purification preparative chromatograph produced by Bonaijier Science and Technology Co., Ltd.

[0057] The dye absorption and emission spectra were measured by using a Cary 60 ultraviolet-visible spectrophotometer and a Cary Eclipse fluorescence spectrophotometer of Agilent Company.

[0058] The cytotoxicity test was measured by using a Varioskan LUX Multimode Microplate Reader instrument of Thermofisher Company of the United States.

[0059] Hereinafter, specific examples of the compound represented by the general formula I are given, but the present application is not limited to these specific examples.

[0060]

[0061] The compound represented by the general formula I of the present application can be synthesized by the method described below.

[0062] Example 1: Preparation of compound 1

[0063] Structural formula of compound 1:

[0064]

[0065] Intermediate azaindole 1.1

[0066]

[0067] Dissolve 2-hydrazinyl-5-bromo-pyridine (1 g, 1 eq) in 20 mL of toluene, add 3-methyl-2-butanone (0.55 g, 1.5 eq), stir the mixture at 100°C for 6 h, then evaporate the solvent under reduced pressure, add polyphosphoric acid (2 g) to the residue, then add toluene 10 mL as a cosolvent, react at 110°C for 3 h, introduce the mixed solution into ice water, adjust the pH to 1 by adding NaOH, then extract with ethyl acetate, wash the collected several layers with saturated brine, dry over anhydrous sodium sulfate, then concentrate to remove the solvent, and purify by silica gel chromatography to obtain brown yellow intermediate azaindole 1.1 (0.84 g, Y = 66%).

[0068] Intermediate quaternary ammonium salt 1.2

[0069]

[0070] Aza-indole 1.1 (0.5 g, 1 eq) was dissolved in 20 mL of ethanol, then ethyl iodide (1.3 g, 4 eq) was added. The reaction system was raised to 70 °C under nitrogen protection and stirred, and the reaction was stopped after 12 h. Most of the ethanol was removed under reduced pressure, and recrystallized with ethyl acetate and n-hexane to obtain yellow powder intermediate quaternary ammonium salt 1.2 (0.72 g, Y = 87%), which was used in the subsequent reaction without further purification.

[0071] Compound 1 was manufactured

[0072]

[0073] 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, the reaction was stopped after 12 h of stirring, and the solvent was concentrated after the reaction solution was lowered to room temperature and extracted with dichloromethane, and the crude product obtained after concentration was purified by silica gel chromatography to obtain blue solid compound 1 (0.17 g, Y = 50%). 1 H 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.

[0074] Compound 2 was manufactured in Example 2

[0075] The structural formula of compound 2 is:

[0076]

[0077] Intermediate azaindole 2.1

[0078]

[0079] Dissolve 2-hydrazinyl-5-carboxy-pyridine (1 g, 1 eq) in 20 mL of toluene, add 3-methyl-2-butanone (1.12 g, 2 eq), stir the mixture at 100 °C for 6 h, then evaporate the solvent under reduced pressure, add polyphosphoric acid (2 g) to the residue, then add toluene 10 mL as a cosolvent, react at 120 °C for 5 h, introduce the mixed solution into ice water, adjust the pH to 1 by adding NaOH, then extract with ethyl acetate, wash the collected several layers with saturated brine, dry over anhydrous sodium sulfate, then concentrate to remove the solvent, and purify using silica gel chromatography to obtain brownish yellow intermediate azaindole 2.1 (0.79 g, Y = 59%).

[0080] Intermediate quaternary ammonium salt 2.2

[0081]

[0082] Dissolve azaindole 2.1 (0.5 g, 1 eq) in 20 mL of acetonitrile, then add iodopropane (1.25 g, 3 eq). Raise the reaction system to 65 °C under nitrogen protection and stir, stop the reaction after 10 h of reaction. Remove most of the ethanol under reduced pressure, recrystallize with ethyl acetate and n-hexane to obtain yellow powder intermediate quaternary ammonium salt 2.2 (0.64 g, Y = 70%), which is used in subsequent reactions without further purification.

[0083] Manufacture compound 2

[0084]

[0085] Dissolve quaternary ammonium salt 2.2 (0.2 g, 1 eq) and condensing agent 2.3 (0.41 g, 1.2 eq) in 10 mL of DMF, catalyze the reaction by adding 0.1 g of potassium carbonate, stop the reaction after stirring at 60 °C for 10 h, concentrate the solvent after the reaction solution cools to room temperature, and extract with dichloromethane, concentrate the obtained crude product, and purify using silica gel chromatography to obtain blue solid compound 2 (0.16 g, Y = 38%). 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), 8.19 (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, 1.4 Hz, 1H), 6.08 (dd, J = 8.3, 1.0 Hz, 1H), 5.06 (t, J = 1.0 Hz, 2H), 4.94 (s, 1H), 4.61 (t, J = 5.2 Hz, 2H), 2.17 (qt, J = 8.2, 5.1 Hz, 2H), 1.51 (s, 4H), 1.12 (t, J = 8.2 Hz, 3H). 13 C 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.

[0086] Manufacture of activated compound 2

[0087]

[0088] Compound 2 (0.05 g) was added to 3 mL of DMF solution, 2-(5-norbornene-2,3-dicarboximidyl)-1,1,3,3-tetramethylurea tetrafluoroborate (0.025 g) was added, followed by triethylamine (0.1 mL), and reacted at 25°C for 2 h, the reaction was stopped, the reaction solution was poured into ethyl acetate, centrifuged at 9000 rpm, the precipitate was collected, and 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.

[0089] Example 3 manufacture of compound 3

[0090] Structural formula of compound 3:

[0091]

[0092] Intermediate azaindole 3.1

[0093]

[0094] Dissolve 2-hydrazinylpyridine (1 g, 1 eq) in 20 mL of toluene, add 4-(2-methyl-3-oxobutyl)benzoic acid (2.27 g, 1.2 eq), stir the mixture at 100 °C for 6 h, then evaporate the solvent under reduced pressure, add polyphosphoric acid (2 g) to the residue, then add 10 mL of toluene as a cosolvent, react at 120 °C for 5 h, introduce the mixed solution into ice water, add NaOH to adjust the pH to 1, then extract with ethyl acetate, collect several layers, wash with saturated brine, dry over anhydrous sodium sulfate, then concentrate to remove the solvent, and purify using silica gel chromatography to obtain the brown-yellow intermediate azaindole 3.1 (1.45 g, Y = 56%).

[0095] Intermediate quaternary ammonium salt 3.2

[0096]

[0097] Dissolve azaindole 3.1 (0.5 g, 1 eq) in 20 mL of ethanol, then add iodomethane (1.01 g, 4 eq). Raise the reaction system to 50 °C under nitrogen protection and stir, stop the reaction after 10 h of reaction. Remove most of the ethanol under reduced pressure, recrystallize with ethyl acetate and n-hexane to obtain the yellow powder intermediate quaternary ammonium salt 3.2 (0.56 g, Y = 74%), which is used in the subsequent reaction without further purification.

[0098] Manufacture compound 3

[0099]

[0100] Dissolve quaternary ammonium salt 3.2 (0.2 g, 1 eq) and condensing agent 3.3 (0.16 g, 1.2 eq) in 10 mL of DMF, catalyze the reaction by adding 0.1 g of potassium carbonate, stop the reaction after stirring at 45 °C for 10 h, concentrate the solvent after the reaction solution cools to room temperature, and extract with dichloromethane, then purify the obtained crude product by silica gel chromatography to obtain 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.

[0101] manufacture of activated compound 3

[0102]

[0103] Compound 3 (0.05 g) was added to 3 mL of DMF solution, 2-(5-norbornene-2,3-dicarboximidyl)-1,1,3,3-tetramethylurea tetrafluoroborate (0.025 g) was added, followed by triethylamine (0.1 mL), and reacted at 25°C for 2 h, the reaction was stopped, the reaction solution was poured into ethyl acetate, centrifuged at 9000 rpm, the precipitate was collected, and purified by HPLC to obtain activated compound 3 (0.051 g, Y = 81%). 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 Making Compound 4

[0104] Structure of Compound 4

[0105]

[0106] Intermediate Azaindole 4.1

[0107]

[0108] Dissolve 2-hydrazinylpyridine (1 g, 1 eq) in 20 mL of toluene, add 8-methyl-9-oxodecanoic acid (5.51 g, 3 eq), stir the mixture at 100 °C for 6 h, then evaporate the solvent under reduced pressure, add polyphosphoric acid (2 g) to the residue, then add 10 mL of toluene as a cosolvent, react at 120 °C for 5 h, introduce the mixed solution into ice water, add NaOH to adjust the pH to 1, then extract with ethyl acetate, collect several layers, wash with saturated brine, dry over anhydrous sodium sulfate, then concentrate to remove the solvent, and purify using silica gel chromatography to obtain the brown-yellow intermediate azaindole 4.1 (1.64 g, Y = 65%).

[0109] Intermediate quaternary ammonium salt 4.2

[0110]

[0111] Dissolve azaindole 4.1 (0.5 g, 1 eq) in 20 mL of ethanol, then add iodopropane (0.93 g, 3 eq). Stir the reaction system under nitrogen protection to 50 °C, stop the reaction after 10 h of reaction. Remove most of the ethanol under reduced pressure, recrystallize with ethyl acetate and n-hexane, and exchange ions by passing through an anion exchange column to obtain the yellow powder intermediate quaternary ammonium salt 4.2 (0.43 g, Y = 56%), which is used in the subsequent reaction without further purification.

[0112] Manufacture compound 4

[0113]

[0114] Dissolve quaternary ammonium salt 4.2 (0.25 g, 1 eq) and condensing agent 4.3 (0.15 g, 1.1 eq) in 10 mL of DMF, catalyze the reaction by adding 0.1 g of potassium carbonate, stop the reaction after stirring at 60 °C for 10 h, concentrate the solvent after the reaction solution cools to room temperature, and extract with dichloromethane, then purify the crude product obtained after concentration by silica gel chromatography to obtain 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, 0.7 Hz, 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.

[0115] Manufacture of activated compound 4

[0116]

[0117] Compound 4 (0.05 g) was added to 3 mL of DMF solution, 2-(5-norbornene-2,3-dicarboximidoyl)-1,1,3,3-tetramethylurea tetrafluoroborate (0.025 g) was added, followed by triethylamine (0.1 mL), and reacted at 25°C for 2 h, the reaction was stopped, the reaction solution was poured into ethyl acetate, centrifuged at 9000 rpm, the precipitate was collected, and purified by HPLC to obtain activated compound 4 (0.049 g, Y = 75%). 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), 7.11 (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, 1.1 Hz, 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). 13 C 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.

[0118] Example 5 Manufacture of compound 5

[0119] Structure of compound 5:

[0120]

[0121] Intermediate quaternary ammonium salt 5.1

[0122]

[0123] 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 was reduced to room temperature, the solvent was concentrated and extracted with dichloromethane, after concentration, the obtained crude product was purified by silica gel chromatography to obtain blue solid compound 5 (0.17 g, Y = 41%).

[0124] Compound 5 was manufactured

[0125]

[0126] 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 was reduced to room temperature, the solvent was concentrated and extracted with dichloromethane, after concentration, the obtained crude product was purified by silica gel chromatography to obtain blue solid compound 5 (0.17 g, Y = 41%). 1 H 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.

[0127] Compound 6 was manufactured

[0128] Structure of compound 6:

[0129]

[0130] Preparation of compound 6

[0131]

[0132] 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, after stirring at 65 °C for 10 h the reaction was stopped, after the reaction was allowed to reach room temperature the solvent was concentrated and extracted with dichloromethane, after concentration the crude product obtained was purified by silica gel chromatography to obtain compound 6 as a blue solid (0.17 g, Y = 41%). 1 H NMR (500 MHz, DMSO-d6) d = 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) d 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.

[0133] Preparation of activated compound 6

[0134]

[0135] Compound 6 (0.05 g) was added to a solution of 3 mL of DMF, 2-(5-norbornene-2,3-dicarboximidoyl)-1,1,3,3-tetramethylurea tetrafluoroborate (0.025 g) was added, followed by triethylamine (0.1 mL), the reaction was left to proceed at 25 °C for 2 h, the reaction was stopped, the reaction was poured into ethyl acetate and centrifuged at 9000 rpm, the precipitate was collected and purified by HPLC to obtain activated compound 6 (0.049 g, Y = 82%). 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.

[0136] Example 7 Preparation of compound 7

[0137] Structure of compound 7:

[0138]

[0139] Intermediate azaindole 7.1

[0140]

[0141] Dissolve 2-hydrazinylpyridine (1 g, 1 eq) in 20 mL of toluene, add 3-methyl-2-butanone (3.19 g, 4 eq), stir the mixture at 100 °C for 6 h, then evaporate the solvent under reduced pressure, add polyphosphoric acid (2 g) to the residue, then add toluene 10 mL as a cosolvent, react at 120 °C for 5 h, introduce the mixed solution into ice water, adjust the pH to 1 by adding NaOH, then extract with ethyl acetate, wash the collected several layers with saturated brine, dry over anhydrous sodium sulfate, then concentrate to remove the solvent, and purify using silica gel chromatography to obtain brown-yellow intermediate azaindole 7.1 (1.21 g, Y = 82%).

[0142] Intermediate quaternary ammonium salt 7.2

[0143]

[0144] Aza-indole 7.1 (0.5 g, 1 eq) was dissolved in 20 mL of ethanol, then iodo-propane (1.86 g, 3.5 eq) was added. The reaction was stirred at 50 °C under nitrogen protection, and stopped after 10 h. Most of the ethanol was removed under reduced pressure, and recrystallized with ethyl acetate and n-hexane to get yellow powder intermediate quaternary ammonium salt 7.2 (0.82 g, Y = 80%), which was used in the next reaction without further purification.

[0145] Compound 7 was manufactured

[0146]

[0147] 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, and the reaction was stopped after stirring at 60 °C for 12 h. After the reaction solution was cooled to room temperature, the solvent was concentrated and extracted with dichloromethane, and the obtained crude product was purified by silica gel chromatography to obtain 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 (125 MHz, 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.

[0148] Example 8 Manufacturing Compound 8

[0149] Compound 8 Structure:

[0150]

[0151] Intermediate Azaindole 8.1

[0152]

[0153] The azaindole 2.1 (0.4 g, 1 eq) obtained in Example 2 was dissolved in 10 mL of DMF, TBTU (0.828 g, 1.3 eq) and DIPEA (0.33 g, 1.3 eq) were added, stirred at 40 °C for 0.5 h, to room temperature, propylamine (0.24 g, 2 eq) was added, the reaction was carried out for 1.5 h, filtered to remove impurity salt, purified on a silica gel column to obtain the intermediate azaindole 8.1 (0.36 g, Y = 73%).

[0154] Intermediate Quaternary Ammonium Salt 8.2

[0155]

[0156] The azaindole 8.1 (0.5 g, 1 eq) was dissolved in 20 mL of acetonitrile, then 1,4-butanesultone (0.74 g, 3 eq) was added. The reaction system was raised to 50 °C under nitrogen protection and stirred, and the reaction was stopped after 10 h. Most of the acetonitrile was removed under reduced pressure, and recrystallized with ethyl acetate to obtain yellow powder intermediate quaternary ammonium salt 8.2 (0.43 g, Y = 52%), which was used in the subsequent reaction without further purification.

[0157] Manufacturing Compound 8

[0158]

[0159] Quaternary ammonium salt 8.2 (0.2 g, 1 eq) and condensing agent 8.3 (0.0.70 g, 1.3 eq) were dissolved in 10 mL of ethanol, 0.1 g of sodium acetate was added to catalyze the reaction, after stirring at 60 °C for 12 h, the reaction was stopped, after the reaction liquid was reduced to room temperature, the solvent was concentrated and extracted with dichloromethane, after concentration, the obtained crude product was purified by silica gel chromatography to obtain 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.

[0160] Manufacture of activated compound 8

[0161]

[0162] Compound 8 (0.05 g) was added to 3 mL of DMF, 2-(5-norbornene-2,3-dicarboximidoyl)-l,l,3,3-tetramethylurea tetrafluoroborate (0.025 g) was added, followed by triethylamine (0.1 mL), and the reaction was allowed to proceed at 25 °C for 2 h. The reaction was stopped, and the reaction mixture was poured into ethyl acetate and centrifuged at 9000 rpm. The precipitate was collected and purified by HPLC to give activated compound 8 (0.047 g, Y = 78%). 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). 13 CNMR (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, 11.51 ppm.

[0163] Example 9 Manufacture of compound 9

[0164] Structure of compound 9

[0165]

[0166] Intermediate quaternary ammonium salt 9.1

[0167]

[0168] To azaindole 7.1 (0.5 g, 1 eq) in Example 7 was dissolved in 20 mL of ethanol, then p-carboxybenzyl bromide (3.76 g, 2.8 eq) was added. The reaction system was raised to 70 °C under nitrogen protection and stirred, and the reaction was stopped after 10 h. Most of the ethanol was removed under reduced pressure, and recrystallized with ethyl acetate and n-hexane to obtain yellow powder quaternary ammonium salt 9.1 (1.52 g, Y = 65%), which was used in the subsequent reaction without further purification.

[0169] Manufacture of compound 9

[0170]

[0171] Compound 9.1 (0.2 g, 1 eq), 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, and the reaction was stopped after stirring at 30 °C for 12 h. After the reaction solution was cooled to room temperature, the solvent was concentrated and extracted with dichloromethane, and the crude product obtained after concentration was purified by liquid chromatography to obtain blue solid compound 9 (0.09 g, Y = 24%). 1 H 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, 1.1 Hz, 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). 13CNMR (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.

[0172] manufacture of activated compound 9

[0173]

[0174] Compound 9 (0.05 g) was added to 3 mL of DMF solution, 2-(5-norbornene-2,3-dicarboximidyl)-1,1,3,3-tetramethylurea tetrafluoroborate (0.025 g) was added, followed by triethylamine (0.1 mL), and reacted at 25°C for 2 h, the reaction was stopped, the reaction solution was poured into ethyl acetate, centrifuged at 9000 rpm, the precipitate was collected, and purified by HPLC to obtain activated compound 9 (0.041 g, Y = 67%). 1 H NMR (500 MHz, DMSO-d6) δ 8.67 (dd, J = 5.1, 1.3 Hz, 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 = 7.2 Hz, 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.

[0175] Example 10 Preparation of compound 10

[0176] Structure of compound 10:

[0177]

[0178] Intermediate azaindole 10.1

[0179]

[0180] The azaindole 2.1 obtained in example 2 (0.4 g, 1 eq) was dissolved in 10 mL of DMF, TBTU (0.828 g, 1.3 eq) and DIPEA (0.33 g, 1.3 eq) were added, stirred at 0 °C for 0.5 h, to room temperature, ethylamine (0.18 g, 2 eq) was added, the reaction was carried out for 1.5 h, filtered to remove impurity salts, purified on a silica gel column to obtain the intermediate azaindole 10.1 (0.31 g, Y = 68%).

[0181] Intermediate quaternary salt 10.2

[0182]

[0183] Dissolve azaindole 10.1 (0.5 g, 1 eq) in 20 mL acetonitrile, then add 1,3- propanesultone (0.92 g, 3.5 eq). Stir the reaction under nitrogen at 75 °C, stop the reaction after 10 h. Remove most of the acetonitrile under reduced pressure, recrystallize with ethyl acetate to get yellow powder intermediate quaternary ammonium salt 10.2 (0.47 g, Y = 62%), quaternary ammonium salt 10.2 is used in the next reaction without further purification.

[0184] Compound 10 was manufactured

[0185]

[0186] Dissolve quaternary ammonium salt 10.1 (0.2 g, 1 eq), condensing agent 10.2 (0.31 g, 0.47 mmol) in 10 mL ethanol, add 0.1 g sodium acetate to catalyze the reaction, stop the reaction after 12 h stirring at 60 °C, concentrate the solvent after the reaction solution is cooled to room temperature, extract with dichloromethane, concentrate the obtained crude product, purify by liquid chromatography to get green solid compound 10 (0.319 g, Y = 62%). 1 H 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, OH), 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). 13C 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.

[0187] manufacture of activated compound 10

[0188]

[0189] Compound 10 (0.05 g) was added to 3 mL of DMF solution, 2-(5-norbornene-2,3-dicarboximidoyl)-1,1,3,3-tetramethylurea tetrafluoroborate (0.025 g) was added, followed by triethylamine (0.1 mL), and the reaction was allowed to proceed at 25°C for 2 h. The reaction was stopped, and the reaction solution was poured into ethyl acetate and centrifuged at 9000 rpm. The precipitate was collected and purified by HPLC to obtain activated compound 9 (0.048 g, Y = 82%). 1H 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, 1.4 Hz, 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). 13 C 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.

[0190] Example 11 Manufacture of Compound 11

[0191] Structure of Compound 11:

[0192]

[0193] Intermediate quaternary ammonium salt 11.1

[0194]

[0195] The azaindole 2.1 (0.4 g, 1 eq) obtained in Example 2 was dissolved in 20 mL of acetonitrile, then benzyl bromide (1.47 g, 3.5 eq) was added. The reaction system was raised to 65 °C under nitrogen protection and stirred, and the reaction was stopped after 10 h. Most of the acetonitrile was removed under reduced pressure, and recrystallized with ethyl acetate to obtain yellow powder intermediate quaternary ammonium salt 11.1 (0.74 g, Y = 81%), which was used in the subsequent reaction without further purification.

[0196] Manufacture of compound 11

[0197]

[0198] The quaternary ammonium salt 11.1 (0.2 g, 1 eq), 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, and the reaction was stopped after stirring at 60 °C for 12 h. After the reaction solution was cooled to room temperature, the solvent was concentrated and extracted with dichloromethane, and the crude product obtained after concentration was purified by liquid chromatography to obtain green solid compound 11 (0.17 g, Y = 38%). 1 H 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 = 0.9 Hz, 2H), 2.85 - 2.71 (m, 4H), 1.56 (qt, J = 7.6, 5.7 Hz, 2H). 13C 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.

[0199] Example 12 Preparation of compound 12

[0200] Structure of compound 12:

[0201]

[0202] Intermediate azaindole 12.1

[0203]

[0204] The azaindole 2.1 (0.4 g, 1 eq) obtained in Example 2 was dissolved in 10 mL of DMF, TBTU (0.8 g) and DIPEA (0.3 g) were added, stirred at 0 °C for 0.5 h, to room temperature, (methoxy-methoxy) methylamine (0.36 g) was added, the reaction was carried out for 1.5 h, filtered to remove impurity salt, purified on a silica gel column to obtain the intermediate azaindole 12.1 (0.37 g, Y = 68%).

[0205] Intermediate quaternary ammonium salt 12.2

[0206]

[0207] The azaindole 12.1 (0.3 g, 1 eq) was dissolved in 20 mL of acetonitrile, then 1,3- propanesultone (0.33 g, 2.5 eq) was added. The reaction system was raised to 65 °C under nitrogen protection and stirred, and the reaction was stopped after 10 h. Most of the acetonitrile was removed under reduced pressure, and recrystallized with ethyl acetate to obtain the yellow powder intermediate quaternary ammonium salt 12.2 (0.27 g, Y = 62%), which was used in the subsequent reaction without further purification.

[0208] Intermediate azaindole 12.3

[0209]

[0210] The azaindole 2.1 obtained in Example 2 (0.4 g, 1 eq) was dissolved in 10 mL of DMF, TBTU (0.8 g) and DIPEA (0.3 g) were added, stirred at 0 °C for 0.5 h, to room temperature, propyl alcohol (0.18 g) was added, the reaction was carried out for 1.5 h, filtered to remove impurity salt, purified on a silica gel column to obtain the intermediate azaindole 12.3 (0.52 g, Y = 86%).

[0211] Intermediate quaternary ammonium salt 12.4

[0212]

[0213] The azaindole 12.3 (0.5 g, 1 eq) was dissolved in 20 mL of acetonitrile, then 1,3-propanesultone (0.74 g, 3 eq) was added. The reaction system was raised to 65 °C under nitrogen protection and stirred, and the reaction was stopped after 10 h. Most of the acetonitrile was removed under reduced pressure, and recrystallized with ethyl acetate to obtain yellow powder intermediate quaternary ammonium salt 12.4 (0.48 g, Y = 64%). The quaternary ammonium salt 12.4 was used in the subsequent reaction without further purification.

[0214] Manufacture of compound 12

[0215]

[0216] The 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, and the solvent was concentrated after the reaction solution was cooled to room temperature 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%). 1H 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, 1.0 Hz, 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). 13 C 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.

[0217] Manufacture of activated compound 12

[0218]

[0219] Compound 12 (0.05 g) was added to a 3 mL solution of DMF, 2-(5-norbornene-2,3-dicarboximidoyl)-1,1,3,3-tetramethylurea tetrafluoroborate (0.025 g) was added, followed by triethylamine (0.1 mL), and the reaction was allowed to proceed at 25 °C for 2 h. The reaction was stopped, and the reaction was poured into ethyl acetate and centrifuged at 9000 rpm. The precipitate was collected and purified by HPLC to give activated compound 12 (0.043 g, Y = 74%). 1 H 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, 10.69 ppm.

[0220] Example 13 Preparation of compound 13

[0221] Structure of compound 13:

[0222]

[0223] Preparation of compound 13

[0224]

[0225] The quaternary ammonium salt 5.1 (0.2 g, 1 eq) obtained in example 5, condensing agent 13.1 (0.21 g, 1.5 eq) were dissolved in 10 mL of ethanol, 0.1 g of sodium acetate was added to catalyze the reaction, after stirring at 90 °C for 12 h the reaction was stopped, after the reaction was allowed to reach room temperature the solvent was concentrated and extracted with dichloromethane, after concentration the crude product obtained was purified by liquid chromatography obtaining compound 13 as a blue solid (0.19 g, Y = 57%). 1 H 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 CNMR (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.

[0226] Example 14 Preparation of compound 14

[0227] Structure of compound 14:

[0228]

[0229] Intermediate quaternary ammonium salt 14.1

[0230]

[0231] Dissolve azaindole 1.1 (0.5 g, 1 eq) obtained in example 1 in 20 mL of acetonitrile, then add methyl iodide (1.19 g, 4 eq). Raise the reaction system to 50 °C under nitrogen protection and stir, stop the reaction after 8 h of reaction. Remove most of the acetonitrile under reduced pressure, recrystallize with ethyl acetate to obtain yellow powder intermediate quaternary ammonium salt 14.1 (0.35 g, Y = 66%), which is used in the subsequent reaction without further purification.

[0232] Preparation of compound 14

[0233]

[0234] Dissolve quaternary ammonium salt 14.1 (0.2 g, 1 eq), condensing agent 14.2 (0.15 g, 1.2 eq) in 10 mL of ethanol, add 0.1 g of sodium acetate to catalyze the reaction, stop the reaction after stirring at 40 °C for 12 h, and after the reaction solution is cooled to room temperature, concentrate the solvent, dissolve the residue in anhydrous dichloromethane, add 0.3 mL of BBr3, react at 0 °C for 2 h, concentrate the solvent under reduced pressure, and purify the crude product obtained after concentration by liquid chromatography to obtain blue solid compound 14 (0.08 g, Y = 26%). 1H 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.

[0235] Example 15 Preparation of compound 15

[0236] Structural formula of compound 15:

[0237]

[0238] Intermediate indole 15.1

[0239]

[0240] Dissolve 5-carboxymethyl ester-1-hydrazino-pyridine (1 g, 1 eq) in 20 mL of toluene, add 8-methyl-9-oxo-decanoic acid (3.92 g, 3 eq), stir the mixture at 100 °C for 6 h, then evaporate the solvent under reduced pressure, add polyphosphoric acid (2 g) to the residue, then add toluene 10 mL as a cosolvent, react at 120 °C for 5 h, introduce the mixed solution into ice water, adjust the pH to 1 by adding NaOH, then extract with ethyl acetate, collect several layers, wash with saturated brine, dry over anhydrous sodium sulfate, then concentrate to remove the solvent, purify by silica gel chromatography to obtain the brownish yellow azaindole intermediate indole 15.1 (1.49 g, Y = 72%).

[0241] Intermediate quaternary ammonium salt 15.2

[0242]

[0243] Dissolve azaindole 15.1 (0.5 g, 1 eq) in 20 mL acetonitrile, then add iodoethane (0.94 g, 4 eq). Stir the reaction under nitrogen at 50 °C, stop the reaction after 8 h. Remove most of the acetonitrile under reduced pressure, recrystallize with ethyl acetate to get yellow powder intermediate quaternary ammonium salt 15.2 (0.56 g, Y = 76%), quaternary ammonium salt 15.2 is used in the next reaction without further purification.

[0244] Manufacture compound 15

[0245]

[0246] Dissolve quaternary ammonium salt 15.2 (0.2 g, 1 eq), condensing agent 15.3 (0.15 g, 1.2 eq) in 10 mL ethanol, add 0.1 g sodium acetate to catalyze the reaction, stop the reaction after stirring at 40 °C for 12 h, concentrate the solvent after the reaction solution is cooled to room temperature, the crude product obtained after concentration is purified by liquid chromatography to obtain blue solid compound 15 (0.11 g, Y = 35%). 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), 7.35 (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). 13C 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, 12.89 ppm.

[0247] manufacture of activated compound 15

[0248]

[0249] Compound 15 (0.05 g) was added to 3 mL of DMF solution, 2-(5-norbornene-2,3-dicarboximidoyl)-1,1,3,3-tetramethylurea tetrafluoroborate (0.025 g) was added, followed by triethylamine (0.1 mL), and the reaction was allowed to proceed at 25°C for 2 h. The reaction was stopped, the reaction solution was poured into ethyl acetate, and centrifuged at 9000 rpm. The precipitate was collected and purified by HPLC to obtain activated compound 15 (0.053 g, Y = 88%). 1H 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), 6.77 (d, J = 9.0 Hz, 1H), 6.72 (d, J = 1.0 Hz, 1H), 6.16 (s, 1H), 6.09 (ddd, J = 4.2, 2.1, 0.9 Hz, 2H), 4.73 - 4.58 (m, 2H), 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). 13 C NMR (125 MHz, 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.

[0250] Example 16 Manufacture of Comparative Compound 1

[0251] Structure of Comparative Compound 1

[0252]

[0253] Intermediate Quaternary Ammonium Salt 16.1

[0254]

[0255] Dissolve 5-iodo-2,3,3-trimethyl-3H-indole (1 g, 1 eq) in 20 mL acetonitrile, then add iodoethane (2.19 g, 4 eq). Stir the reaction under nitrogen at 50 °C, stop the reaction after 8 h. Remove most of the acetonitrile under reduced pressure, recrystallize with ethyl acetate to get yellow powder intermediate quaternary ammonium salt 16.1 (1.24 g, Y = 80%), quaternary ammonium salt 16.1 can be used in the subsequent reaction without further purification.

[0256] Manufacture of Comparative Compound 1

[0257]

[0258] Dissolve quaternary ammonium salt 16.1 (0.5 g, 2 eq), condensing agent 16.2 (0.15 g, 1.2 eq) in 10 mL ethanol, add 0.1 g sodium acetate to catalyze the reaction, stop the reaction after stirring at 40 °C for 12 h, concentrate the solvent after the reaction solution is reduced to room temperature, the crude product obtained after concentration is purified by liquid chromatography to obtain blue solid comparative compound 1 (0.31 g, Y = 69%). 1 H NMR (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, OH), 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, 1.0 Hz, 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.

[0259] Example 17 Manufacture of Comparative Compound 2

[0260] Structure of Comparative Compound 2:

[0261]

[0262] Intermediate quaternary ammonium salt 17.1

[0263]

[0264] Dissolve 5-sulfonic acid-2,3,3-trimethyl-3H-indolium potassium salt (1 g, 2 eq) in 20 mL acetonitrile, then add iodomethane (2.56 g, 5 eq). Stir the reaction system under nitrogen protection at 50°C, stop the reaction after 8 h of reaction. Remove most of the acetonitrile under reduced pressure, recrystallize with ethyl acetate to obtain yellow powder intermediate quaternary ammonium salt 17.1 (1.23 g, Y = 81%), which is used in the subsequent reaction without further purification.

[0265] Intermediate quaternary ammonium salt 17.2

[0266]

[0267] Dissolve 5-sulfonic acid-2,3,3-trimethyl-3H-indolium potassium salt (1 g, 2 eq) in 20 mL acetonitrile, then add 6-bromohexanoic acid (2.26 g, 3 eq). Stir the reaction system under nitrogen protection at 50°C, stop the reaction after 8 h of reaction. Remove most of the acetonitrile under reduced pressure, recrystallize with ethyl acetate to obtain yellow powder intermediate quaternary ammonium salt 17.2 (1.11 g, Y = 63%), which is used in the subsequent reaction without further purification.

[0268] Manufacture of Comparative Compound 2

[0269]

[0270] Dissolve 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) in 10 mL ethanol, add 0.1 g sodium acetate to catalyze the reaction, stop the reaction after stirring at 40°C for 12 h, and concentrate the solvent after the reaction solution is cooled to room temperature. The obtained crude product is purified by liquid chromatography to obtain blue solid Comparative 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 = 8.1 Hz, 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.

[0271] Manufacture of activated comparative compound 2

[0272]

[0273] Comparative compound 2 (0.05 g) was added to 3 mL of DMF solution, 2-(5- norbornene-2,3-dicarboximidyl)-1,1,3,3-tetramethylurea tetrafluoroborate (0.025 g) was added, followed by triethylamine (0.1 mL), and reacted at 25°C for 2 h, the reaction was stopped, the reaction solution was poured into ethyl acetate, centrifuged at 9000 rpm, the precipitate was collected, and purified by HPLC to obtain activated comparative compound 2 (0.053 g, Y = 87%). 1H NMR (500 MHz, DMSO-d6) δ 8.03 (d, J = 1.8 Hz, 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, 1.1 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), 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 C NMR (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, 24.24 ppm.

[0274] Example 18 Manufacture of Comparative Compound 3

[0275] Structure of Comparative Compound 3:

[0276]

[0277] Manufacture of Comparative Compound 3

[0278]

[0279] 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, and the reaction was stopped after stirring at 40 °C for 12 h. After the reaction solution was cooled to room temperature, the solvent was concentrated, and the crude product obtained after concentration was purified by liquid chromatography to obtain blue solid comparative compound 3 (0.05 g, Y = 30%).1 H NMR (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.

[0280] UV-Vis absorption spectrum and fluorescence spectrum determination of compound 5-6, compound 8-9 and comparative compound 1-2

[0281] The dye after vacuum drying was accurately weighed with a millionth scale, and a 2 mmol / L DMSO dye stock solution was prepared in a brown sample bottle, which was stored in a 4°C refrigerator for standby use.

[0282] When testing the UV-Vis absorption spectrum and fluorescence spectrum, 3 μL of the dye stock solution was taken with a micropipette gun and dissolved in a quartz cuvette containing 3 mL of dichloromethane solvent, mixed uniformly to obtain a dye concentration of 2.0 μmol / L for absorption spectrum and fluorescence emission spectrum testing. All tests were completed at 25°C.

[0283] Since the structures of compound 5, compound 6, compound 8 and compound 9 are close to comparative compound 1-2, the above-mentioned compounds are mainly selected for comparative study with comparative compound. As shown in Figure 1 The maximum absorption wavelength of the azaphthalocyanine dye (compound 5, compound 6, compound 8 and compound 9) with upconversion performance is significantly longer than that of the ordinary phthalocyanine dye (comparative compound 1-2). At the same time, as shown in Figure 2 The maximum emission wavelength of the azaphthalocyanine dye with upconversion performance is all above 700 nm, which is longer than that of the ordinary phthalocyanine dye. This performance can adapt to long-wavelength lasers and detectors in the imaging and photodynamic therapy process, which is beneficial to reduce the photobleaching rate of the dye and improve the tissue penetration depth.

[0284] Example 20 Upconversion fluorescence spectrum test of compounds 1-15, comparative examples 1-2

[0285] First, the tail emission wavelength of the dye was determined by fluorescence spectrum test, and then the fluorescence spectrum of the dye was tested at the excitation wavelength. The test results of the compounds involved in all examples are shown in Table 1 and Figure 3 a-o, i.e. compounds 1-15 of general formula I all have upconversion fluorescence, while the molecular structure of comparative compounds 1-3 is very similar to that of general formula I, but no upconversion fluorescence appears in the fluorescence spectrum test. In fact, it was predicted during the experimental design stage of this application that the molecular structure of comparative compounds 1 to 3 is similar to that of general formula I, which may exhibit similar fluorescence characteristics. However, the experimental results completely overturned the expectation, and these comparative compounds did not exhibit upconversion fluorescence. This unexpected discovery fully proves the non-predictability often existing in experimental disciplines.

[0286] Table 1:

[0287]

[0288] (Note: "+" means having upconversion performance; "-" means not having upconversion performance)

[0289] Example 21 Upconversion photodynamic effect test of compound 1, compound 5, compound 6 and comparative compound 1

[0290] Since the substitution of heavy atoms can promote the improvement of the photodynamic effect of the dye, the upconversion photodynamic effect of the heavy atom substituted compound 1 and the comparative compound 1 in the examples was studied. 1,3-Diphenyl isobenzofuran (DPBF) was used as a singlet oxygen trapping agent, and the singlet oxygen generation efficiency of the compound was detected by spectrophotometry.

[0291] Operation steps: 3 mL of dichloromethane was added to a 1x1 cm quartz cuvette, and 3 μL of dye molecule mother liquor was added to make the concentration of the dye 2 μM. After mixing, a 760 nm laser light source was used for irradiation, and the light power was uniformly kept at 20 mW / cm 2 The absorbance value of DPBF at 415 nm was determined at different time nodes, and the singlet oxygen production ability was judged by the rate of decrease of DPBF absorbance, and then the photodynamic effect was reflected.

[0292] As Figure 4As shown in a-d, compound 1, compound 5, compound 6 can generate singlet oxygen under 760 nm light. Thus resulting in the decrease of DPBF absorbance, while the contrast compound 1 does not have upconversion photodynamic effect, thus the photosensitizer designed by general formula I can generate a large amount of singlet oxygen under long-wavelength light excitation, thus playing a good photodynamic effect, having the potential for treating deep lesions.

[0293] Example 22 Fluorescent labeling of activated compound on lysine

[0294] Prepare 10 mmol / L L-lysine stock solution in PBS (pH = 7.4) in a 5 mL brown sample bottle, take 50 μL of 10 mmol / L L-lysine stock solution in a 0.5 mL centrifuge tube, and then add 100 mL of 5 mmol / L activated compound stock solution. After 6 h of reaction in a table constant temperature shaker, use methanol and ultrapure water as mobile phase, C18 packed reverse column as separation column, and perform liquid chromatography characterization of activated compound 1 and labeled L-lysine compound 1 at a wavelength of 254 nm.

[0295] For example, as shown in Figure 5 the retention time of activated compound 2 is 11.4 min, and after incubation with L-lysine, liquid chromatography characterization under the same conditions can find that the peak at 11.4 min is almost gone, and a new absorption peak with a retention time of 14.3 min appears, and the peak area of the new peak accounts for 91%, which shows that the activated compound 1 can well react with L-lysine and successfully connect L-lysine to the compound 2 dye. The labeling of the rest of the compounds is shown in Table 2, and the conventional dye control compound 2 can also be successfully labeled, and the nitrogen hetero dye after structural change can also be successfully labeled. This shows that the change of structure will not affect the ability of activated dye to label biomolecules.

[0296] Table 2:

[0297]

[0298] Example 23 Light stability test of compound 5

[0299] In order to verify that the dye with upconversion ability can have better light stability under long-wavelength irradiation, compound 1 is selected for verification.

[0300] Operation steps: add 3 mL of water to a 1 × 1 cm quartz cuvette, and add 3 μL of dye molecule stock solution to make the concentration of the dye 2 μM. After mixing, use 760 nm laser light source and 660 nm laser light source for irradiation, and the light power is uniformly kept at 20 mW / cm2 The absorption spectrum of compound 1 was determined at different time points, and the photobleaching rate of compound 1 was determined by the rate of decrease at the maximum absorption wavelength.

[0301] As shown in Figure 6 , the photobleaching rate of the compound under 760 nm laser irradiation is significantly lower than that under 660 nm laser irradiation, which indicates that the long-wavelength irradiation is beneficial to improve the light stability of the dye. Since both 660 nm and 760 nm lasers can make compound 5 produce bright fluorescence at 720 nm, compared with ordinary dyes, the compound according to the general formula I can have better stability in imaging and treatment applications.

[0302] The above is a further elaboration of the present application in combination with the specific preferred embodiments of the present application, and cannot be regarded as limiting the specific implementation of the present application to these descriptions. Any skilled person in the art can make some changes or modifications without departing from the technical scope of the present application, which shall be regarded as falling within the scope of the claims of the present application.

Claims

1. A dye having upconversion properties, characterized in that The dye is selected from compounds having the following structural formula: 。 2. Use of a dye having upconversion properties according to claim 1, characterized in that, The dye is applied to fluorescent labeling for non-disease diagnosis or treatment purposes, cell imaging, gene sequencing or biological macromolecule recognition.

3. Use according to claim 2, characterized in that, The dye emits fluorescence under the irradiation of light with a wavelength of 750-900 nm at room temperature.

4. Use according to claim 2, characterized in that, The dye is applied to cell imaging under up-conversion performance, gene sequencing under long-wavelength excitation and preparation of photosensitizers.

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