Hemicyanine fluorescent molecule for detecting bacterial infection and bacterial metabolism as well as preparation and application of hemicyanine fluorescent molecule

Abstract

The invention discloses a hemicyanine fluorescent molecule for detecting bacterial infection and bacterial metabolism as well as preparation and application thereof, relates to a hemicyanine-based molecule as well as a preparation method and application thereof, and belongs to the field of detection. The compound can specifically detect bacteria, even drug-resistant bacteria and the fatty acid beta-oxidation physiological process of the bacteria. Through the enhancement of the fluorescence signal, the bacteria type or metabolism condition can be visually judged. By introducing carbon chains with different lengths, the sensitivity of the probe to bacteria is improved, and the accuracy of bacteria detection is promoted. The compound AC5 realizes the detection of 100 mu M of drug resistance of pseudomonas aeruginosa / ceftazidime. The probe provides a promising novel method for early detection of in-vivo deep bacterial infection and research of bacterial oxidative metabolism. Furthermore, a mannose part is introduced, so that the compound D1 shows targeting on the cellular level, and the compound D1 can be used for researching the oxidative metabolism condition of macrophages. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of detection of bacterial infections and bacterial metabolism. Specifically, it relates to a class of hemicyanine fluorescent probes, a preparation method of the near-infrared hemicyanine fluorescent probes, an application thereof in detecting the activity of bacterial infections and metabolism, and a detection kit therefor. Background Art

[0002] Bacterial infections have gradually become a major threat to global human health. As early as 2019, bacterial infections became the second leading cause of death globally after ischemic heart disease. 1 At the same time, bacterial infections have certain similar symptoms to other physiological or pathological processes, such as inflammation, tumors, etc. 2 It is necessary to accurately diagnose bacterial infections at an early stage and distinguish the types of bacterial infections. Clinically, the commonly used diagnostic method for bacterial infections is blood routine, and indirect diagnosis of bacterial infections is carried out through indicators such as the number of white blood cells, the concentration of procalcitonin, and C-reactive protein. 3 Or microbiological tests are performed, which involve stages such as inoculation, staining microscopy, culture, and staining of specimens. In particular, Gram staining is a traditional general method for judging the types of bacterial infections. 4 For the above methods, one is an indirect method, which is prone to misjudgment because non-bacterial infection pathological processes can also cause changes in the above indicators. Therefore, multiple detection means often need to be used for assistance to determine the symptoms belonging to bacterial infections; the second method is direct in nature, but involves multiple operation processes and is relatively cumbersome, which is not conducive to rapid diagnosis.

[0003] Currently, fluorescent molecular probes are a generally recognized effective tool for accurately observing the physiological activities of organisms. 5 And the hemicyanine fluorophore provides favorable conditions for the design of activatable fluorescent probes because its emission reaches the near-infrared region, with deeper tissue penetration and reduced interference from background autofluorescence. 6 There have been many reports on hemicyanine fluorescent probes for detecting enzyme activity. Such probes exhibit advantages such as high sensitivity and large changes in fluorescence intensity, which are easy to detect. Therefore, it is feasible and necessary to design and develop hemicyanine fluorescent probes based on organic small molecules for specific detection of bacterial infections and their activities. Moreover, so far, there have been no reports on fluorescent probes for detecting bacterial metabolism at home and abroad.

[0004] On the one hand, the present invention designs and synthesizes a class of hemicyanine fluorescent probe molecules for detecting bacterial infection types and bacterial metabolic activities. The synthetic raw materials involved in this route are inexpensive, the synthetic route is short (the shortest requires six steps), and the synthetic operation is simple. Therefore, such fluorescent probes can be effectively, quickly and massively synthesized. On the second hand, the present invention also relates to a method for detecting bacteria and metabolic activities of the fluorescent probe. Through fluorescence imaging, bacteria can be visually located, and the bacterial infection situation can be directly reflected by the fluorescence intensity, and the metabolic activities in bacteria that were difficult to detect in the past can be detected. Furthermore, it can be used for comparative evaluation of the metabolic levels between different bacterial species, exploration of compounds that promote or inhibit bacterial metabolic activities, or basic research on related enzyme groups, etc. On the third hand, since the present invention also involves the introduction of mannose 7 , at the cellular level, targeting of macrophages can be achieved. Therefore, it can be further used to study the fatty acid metabolism of macrophages, and then to study the relationship between macrophage metabolism and the development of different diseases. Summary of the Invention

[0005] The object of the present invention is to overcome the above-mentioned defects in the prior art, and provide a near-infrared fluorescent probe for detecting bacterial infection or bacterial metabolism with low synthesis cost, short synthetic route (only 6 steps are required at least to obtain), simple synthetic operation (no dangerous reagents are involved), and at the same time having high selectivity, stable properties and simple operation, and its preparation method and application.

[0006] To solve the technical problems of the present invention, the present invention provides the following technical solutions:

[0007] In the first aspect, the present invention provides a compound or a pharmaceutically acceptable salt thereof:

[0008]

[0009] R1 is independently selected from a hydrogen atom, R2 is independently selected from a hydrogen atom, fluorine, chlorine, bromine, iodine, aldehyde group or difluoromethyl;

[0010] R3 is independently selected from a hydrogen atom, a normal alkyl group or wherein, m takes 2, 3, 4, 5, 6, 7, 8, 9, 10

[0011] X1 is independently selected from O, S,

[0012] n is selected from 1-20, preferably 4-18.

[0013] Furthermore, the compound described in the present invention is selected from:

[0014]

[0015] The second aspect of the technical solution of the present invention is to provide a preparation method of the compound described in the first aspect or a pharmaceutically acceptable salt thereof, which is characterized by including the following steps:

[0016]

[0017]

[0018] Wherein: the definitions of R1, R2, and X1 are the same as any one of claims 1-4;

[0019] (1) Add methyl bromoacetate, bromoalkanoic acid, base and solvent to a round-bottom flask, react under the conditions of 0-100 °C and stirring. After the reaction is completed, directly concentrate and purify the reaction solution under reduced pressure to obtain I or bromoalkanoic acid, tert-butyl trichloroacetimidate, boron trifluoride diethyl ether solution and solvent, react under the conditions of 0-100 °C and stirring. After the reaction is completed, directly concentrate and purify the reaction solution under reduced pressure to obtain I;

[0020] (2) Add resorcinol or a derivative of resorcinol or 3-hydroxybenzenethiol or a derivative of 3-hydroxybenzenethiol, compound II, base and solvent to a round-bottom flask, react under the conditions of 0-100 °C and stirring. After the reaction is completed, extract the reaction solution, concentrate and purify it under reduced pressure to obtain III;

[0021] (3) Add III prepared in step (2), a solvent, I prepared in step (1), and a base into a round-bottom flask, react at room temperature under anhydrous conditions with argon protection. After the reaction is completed, extract the reaction solution, concentrate it under reduced pressure and purify to obtain IV or further react with a dichloro solution of trifluoroacetic acid, concentrate it under reduced pressure and purify to obtain IV or (3) Add III prepared in step (2), a solvent, I prepared in step (1), and a base into a round-bottom flask, react at room temperature under anhydrous conditions with argon protection. After the reaction is completed, extract the reaction solution, concentrate it under reduced pressure and purify to obtain IV or further react with a dichloro solution of trifluoroacetic acid, concentrate it under reduced pressure and purify to obtain IV or and (3αS,4S,6R,6αS)-6-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-ol, a solvent, and a condensing agent to react. After purification, react with I prepared in step (1) and a base at room temperature under anhydrous conditions with argon protection. After the reaction is completed, extract the reaction solution, concentrate it under reduced pressure and purify, then react with an aqueous solution of trifluoroacetic acid at a certain concentration at 0 - 100 °C. After the reaction is completed, extract, concentrate under reduced pressure and dry, and further react with N,N'-disuccinimidyl carbonate, a base and a solvent at 0 - 100 °C with stirring. After the reaction is completed, extract the reaction solution, concentrate it under reduced pressure and dry. Finally, react with mannosyl hydrochloride, a base and a solvent at 0 - 100 °C with stirring. After the reaction is completed, extract the reaction solution, concentrate it under reduced pressure and purify to obtain compound IV.

[0022] The third aspect of the technical solution of the present invention is to provide the use of the compound described in the first aspect or a pharmaceutically acceptable salt thereof in the preparation of fluorescence imaging of bacteria. In one embodiment, it can be used as a fluorescent probe to stain bacteria, so as to detect the presence of bacteria under a confocal laser scanning microscope or a fluorescence microscope. Among them, in one embodiment, the pathogenic microorganism is Gram-negative bacteria 19606 (Acinetobacter baumannii), 25922 (Escherichia coli), 27853 (Pseudomonas aeruginosa), 700603 (Klebsiella pneumoniae); Gram-positive bacteria 29213 (Staphylococcus aureus) and 700221 (Enterococcus faecium).

[0023] The fourth aspect of the technical solution of the present invention is to provide the use of the compound described in the first aspect or a pharmaceutically acceptable salt thereof in the preparation of an infection diagnostic reagent for bacterial metabolism research.

[0024] The fifth aspect of the technical solution of the present invention is to provide the use of the compound described in the first aspect or a pharmaceutically acceptable salt thereof in the preparation of immunocyte metabolism research.

[0025] The sixth aspect of the technical solution of the present invention is to provide the use of the compound described in the first aspect or its pharmaceutically acceptable salt in the preparation of a fluorescent probe. In one embodiment, it can be used as a fluorescent probe to stain Pseudomonas aeruginosa / ceftazidime-resistant at 100 μM, and in combination with other commercial nuclear dyes, to detect whether Pseudomonas aeruginosa / ceftazidime-resistant at 100 μM is viable under a confocal laser scanning microscope. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings are used to provide a further understanding of the present invention and are a part of the specification, and together with the following specific embodiments are used to explain the present invention, but do not constitute a limitation to the present invention. In the drawings:

[0027] Figure 1 Schematic diagram of the probe detecting the physiological process of fatty acid β-oxidation (FAO) of bacteria.

[0028] Figure 2 Showing the absorption and emission spectra of the compound.

[0029] Figure 3 Showing the cytotoxicity experiment of the compound.

[0030] Figure 4 Showing the confocal imaging experiment of the compound and drug-resistant bacteria.

[0031] Figure 5 Showing the confocal imaging experiment of the compound and different bacteria. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0032] The following provides a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0033] On the one hand, the present invention provides a class of near-infrared hemicyanine fluorescent probes, and the near-infrared hemicyanine fluorescent probe has the structure shown in Formula IV;

[0034]

[0035] Among them, in the near-infrared hemicyanine fluorescent probe shown in Formula IV, the recognition group of the aliphatic side chain is used, and mannose is used as the targeting group.

[0036] Preparation steps of Compound AC1 in Example 1:

[0037]

[0038] Preparation Example 1, Preparation of Compound 1

[0039] 6-Bromohexanoic acid (1.000 g, 5.12 mmol) was dissolved in ultradry acetonitrile (5.0 mL). After slowly adding bromomethyl acetate (1.00 mL, 10.25 mmol) under argon protection at room temperature, N,N-diisopropylethylamine (1.78 mL, 10.25 mmol) was added to the above system, and the reaction system was stirred at 50 °C for 15 minutes. After the reaction was completed, silica gel was directly added to the reaction system and purified by medium-pressure column chromatography (petroleum ether:ethyl acetate = 99:1), obtaining 0.844 g of white solid with a yield of 61.6%. 1 H NMR (400 MHz, Chloroform-d) δ 5.77 (s, 2H), 3.44 (t, J = 6.7 Hz, 2H), 2.42 (t, J = 7.4 Hz, 2H), 2.15 (s, 3H), 1.91 (p, J = 7.0 Hz, 2H), 1.71 (p, J = 7.5 Hz, 2H), 1.52 (p, J = 7.8, 7.2 Hz, 2H). 13 C NMR (100 MHz, Chloroform-d) δ 172.22, 169.77, 79.24, 33.76, 33.46, 32.38, 27.57, 23.77, 20.83.

[0040] Preparation Example 2, Preparation of Compound AC1 Compound 1 (0.167 g, 0.627 mmol) and Compound 2 6 (0.100 g, 0.21 mmol) were dissolved in ultradry N,N-dimethylformamide (2.0 mL). After adding N,N-diisopropylethylamine (145 μL, 0.836 mmol) to the above system, the reaction system was stirred overnight at 65 °C. The reaction system was diluted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered by suction, concentrated under reduced pressure, and the residue was purified by medium-pressure column chromatography (dichloromethane:methanol = 97:3) to obtain Compound AC1 as a blue solid, 0.019 g, with a yield of 12.9%. 11H NMR (400 MHz, CD3OD-d4) δ 8.05 (dd, J = 14.2, 8.5 Hz, 2H), 7.40 - 7.36 (m, 4H), 7.25 (dd, J = 8.0, 4.7 Hz, 2H), 7.21 (q, J = 7.1 Hz, 2H), 6.90 (d, J = 8.6 Hz, 2H), 6.76 (d, J = 8.7 Hz, 2H), 6.13 (dd, J = 14.2, 7.7 Hz, 2H), 4.13 (q, J = 7.2 Hz, 2H), 4.09 (t, J = 7.5 Hz, 2H), 2.73 (t, J = 6.3 Hz, 4H), 2.30 (t, J = 7.3 Hz, 2H), 2.05 - 2.01 (m, 2H), 1.83 - 1.79 (m, 2H), 1.71 - 1.66 (m, 2H), 1.50 - 1.44 (m, 2H), 1.39 (s, 12H), 1.36 (t, J = 7.3 Hz, 3H). 13 13C NMR (176 MHz, CD3OD-d4) δ 177.24, 172.27, 169.73, 162.80, 161.79, 154.54, 145.73, 142.23, 141.13, 133.92, 128.85, 128.74, 127.05, 126.96, 122.43, 115.75, 114.27, 113.47, 112.21, 102.89, 100.72, 79.04, 68.38, 50.58, 39.96, 33.15, 28.69, 28.41, 26.83, 25.12, 24.01, 23.71, 20.27, 19.16, 11.45.

[0041] Preparation steps of compound AC2 in Example 2:

[0042]

[0043] Preparation Example 3, Preparation of Compound 3

[0044] Dissolve 9-bromononanoic acid (0.500 g, 2.108 mmol) in ultradry acetonitrile (3.0 mL). After slowly adding bromomethyl acetate (413 μL, 4.22 mmol) under argon protection at room temperature, add N,N-diisopropylethylamine (734 μL, 4.22 mmol) to the above system. The reaction system is stirred at 50 °C for 1 hour. After the reaction is completed, silica gel is directly added to the reaction system and purified by medium-pressure column chromatography (petroleum ether:ethyl acetate = 99:1) to obtain 0.553 g of colorless transparent oil, with a yield of 84.5%. 11H NMR (400 MHz, Chloroform-d) δ 5.73 (s, 2H), 3.40 (t, J = 6.8 Hz, 2H), 2.35 (t, J = 7.5 Hz, 2H), 2.11 (s, 3H), 1.84 (p, J = 6.9 Hz, 2H), 1.62 (q, J = 7.3 Hz, 2H), 1.42 (t, J = 7.4 Hz, 2H), 1.31 (s, 6H). 13 13C NMR (100 MHz, Chloroform-d) δ 172.46, 169.70, 79.13, 33.94, 33.89, 32.74, 28.99, 28.85, 28.53, 28.06, 24.49, 20.75

[0045] Preparation Example 4, Preparation of Compound AC2

[0046] Dissolve Compound 3 (0.193 g, 0.63 mmol) and Compound 2 6 (0.100 g, 0.21 mmol) in ultradry N,N-dimethylformamide (2.0 mL). After adding N,N-diisopropylethylamine (145 μL, 0.63 mmol) to the above system, the reaction system was stirred overnight at 65 °C. The reaction system was diluted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered by suction, concentrated under reduced pressure, and the residue was purified by medium-pressure column chromatography (dichloromethane:methanol = 98:2) to obtain Compound AC2 as a blue solid, 0.029 g, with a yield of 18%. 1 1H NMR (400 MHz, CD3OD-d4). δ 8.75 (d, J = 14.8 Hz, 1H), 7.66 (d, J = 7.1 Hz, 1H), 7.59–7.51 (m, 2H), 7.48–7.41 (m, 2H), 7.38 (s, 1H), 7.03–6.91 (m, 2H), 6.49 (d, J = 14.9 Hz, 1H), 5.71 (s, 2H), 4.40 (q, J = 7.2 Hz, 2H), 4.12 (t, J = 6.4 Hz, 2H), 2.75 (dt, J = 17.0, 5.8 Hz, 4H), 2.36 (t, J = 7.4 Hz, 2H), 2.06 (s, 3H), 1.94 (q, J = 6.1 Hz, 2H), 1.82 (s, 8H), 1.67–1.58 (m, 2H), 1.49 (q, J = 7.1 Hz, 5H), 1.37 (s, 5H). 1313C NMR (126 MHz, CD3OD-d4) δ 177.25, 172.46, 169.79, 162.93, 161.82, 161.81, 154.59, 145.74, 142.29, 141.20, 134.00, 128.93, 128.84, 127.06, 127.03, 122.54, 115.77, 114.37, 113.55, 112.33, 103.03, 100.79, 79.08, 68.73, 50.65, 40.16, 33.32, 28.93, 28.86, 28.77, 28.60, 27.00, 25.69, 24.34, 23.88, 20.32, 19.26, 11.62.

[0047] Preparation steps of compound AC3 in Example 3:

[0048]

[0049] Preparation Example 5, Preparation of Compound 4

[0050] Dissolve 11-bromoundecanoic acid (1.000 g, 3.77 mmol) in ultradry acetonitrile (6.0 mL). After slowly adding bromomethyl acetate (1.313 mL, 7.54 mmol) under argon protection at room temperature, add N,N-diisopropylethylamine (740 μL, 7.54 mmol) to the above system. The reaction system was stirred at 50 °C for 1 hour. After the reaction was completed, silica gel was directly added to the reaction system and purified by medium-pressure column chromatography (petroleum ether:ethyl acetate = 99:1) to obtain 1.001 g of colorless transparent oil, with a yield of 78.7%. 1 1H NMR (400 MHz, Chloroform-d) δ 5.73 (s, 1H), 3.40 (t, J = 6.8 Hz, 2H), 2.35 (t, J = 7.5 Hz, 2H), 2.10 (s, 3H), 1.84 (p, J = 7.0 Hz, 2H), 1.62 (p, J = 7.7 Hz, 2H), 1.40 (q, J = 7.1 Hz, 2H), 1.28 (s, 10H). 13 13C NMR (101 MHz, CDCl3) δ 172.52, 169.70, 79.12, 34.02, 33.93, 32.81, 29.33, 29.28, 29.14, 28.95, 28.71, 28.14, 24.55, 20.75.

[0051] Preparation Example 6, Preparation of Compound AC3

[0052] Compound 3 (0.211 g, 0.63 mmol), Compound 2 6(0.100 g, 0.21 mmol) was dissolved in ultradry N,N-dimethylformamide (2.0 mL). After adding N,N-diisopropylethylamine (145 μL, 0.63 mmol) to the above system, the reaction system was stirred overnight at 65 °C. The reaction system was diluted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered by suction, concentrated under reduced pressure, and the residue was purified by medium-pressure column chromatography (dichloromethane:methanol = 98:2) to obtain compound AC3 as a blue solid, 0.038 g, with a yield of 23.1%. 1 H NMR (400 MHz, Methanol-d4) δ 8.78 (d, J = 14.8 Hz, 1H), 7.69 (d, J = 7.4 Hz, 1H), 7.57 (qd, J = 8.0, 1.4 Hz, 2H), 7.51–7.44 (m, 2H), 7.42 (s, 1H), 7.08–6.95 (m, 2H), 6.53 (d, J = 14.8 Hz, 1H), 5.74 (s, 2H), 4.43 (q, J = 7.2 Hz, 2H), 4.15 (t, J = 6.3 Hz, 2H), 2.78 (dt, J = 19.5, 6.2 Hz, 4H), 2.38 (t, J = 7.4 Hz, 2H), 2.09 (s, 3H), 2.00–1.93 (m, 2H), 1.86 (s, 8H), 1.63 (t, J = 7.0 Hz, 2H), 1.51 (t, J = 7.2 Hz, 5H), 1.35 (s, 10H). 13 C NMR (126 MHz, CD3OD-d4) δ 177.18, 172.37, 169.69, 162.86, 161.71, 154.51, 145.64, 142.22, 141.13, 133.92, 128.88, 128.78, 127.00, 126.98, 122.46, 115.70, 114.29, 113.46, 112.29, 112.27, 102.97, 100.74, 100.72, 78.99, 68.65, 50.59, 40.07, 33.60, 33.26, 29.21, 29.10, 29.05, 28.94, 28.85, 28.71, 28.63, 26.93, 26.91, 25.70, 24.38, 24.32, 23.77, 20.27, 19.21, 11.55.

[0053] Procedure for the preparation of compound AC4 in Example 4:

[0054]

[0055] Preparation Example 7, Preparation of Compound 5

[0056] 12-Bromododecanoic acid (0.500 g, 1.79 mmol) was dissolved in ultradry acetonitrile (2.5 mL). After slowly adding bromomethyl acetate (351 μL, 3.58 mmol) under argon protection at room temperature, N,N-diisopropylethylamine (627 μL, 3.58 mmol) was added to the above system, and the reaction system was stirred at 50 °C for 1 hour. After the reaction was completed, silica gel was directly added to the reaction system and purified by medium-pressure column chromatography (petroleum ether:ethyl acetate = 99:1) to obtain 0.523 g of a colorless transparent oil, with a yield of 57.3%. 1 H NMR (400 MHz, Chloroform-d) δ 5.77 (s, 2H), 3.44 (t, J = 6.9 Hz, 2H), 2.39 (t, J = 7.5 Hz, 2H), 2.15 (s, 3H), 1.88 (p, J = 7.0 Hz, 2H), 1.67 (p, J = 7.3 Hz, 2H), 1.45 (p, J = 6.9 Hz, 2H), 1.31 (s, 12H). 13 C NMR (100 MHz, Chloroform-d) δ 172.62, 169.79, 79.19, 34.13, 34.02, 32.90, 29.50, 29.46, 29.43, 29.25, 29.05, 28.82, 28.24, 24.64, 20.83.

[0057] Preparation Example 8, Preparation of Compound AC4

[0058] Compound 3 (0.193 g, 0.63 mmol) and Compound 2 6 (0.100 g, 0.21 mmol) were dissolved in ultradry N,N-dimethylformamide (2.0 mL). After adding N,N-diisopropylethylamine (145 μL, 0.63 mmol) to the above system, the reaction system was stirred overnight at 65 °C. The reaction system was diluted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered by suction, concentrated under reduced pressure, and the residue was purified by medium-pressure column chromatography (dichloromethane:methanol = 98:2) to obtain 0.029 g of Compound AC2 as a blue solid, with a yield of 17.2%. 11H NMR (400 MHz, Methanol-d4) δ 8.82 (d, J = 14.8 Hz, 1H), 7.69 (d, J = 7.4 Hz, 1H), 7.61–7.54 (m, 2H), 7.53–7.46 (m, 2H), 7.44 (s, 1H), 7.06 (d, J = 2.4 Hz, 1H), 7.01 (dd, J = 8.6, 2.3 Hz, 1H), 6.54 (d, J = 14.8 Hz, 1H), 5.74 (s, 2H), 4.43 (q, J = 7.3 Hz, 2H), 4.18 (t, J = 6.4 Hz, 2H), 2.82 (t, J = 6.2 Hz, 2H), 2.77 (t, J = 6.2 Hz, 2H), 2.38 (t, J = 7.4 Hz, 2H), 2.09 (s, 3H), 1.98 (t, J = 6.2 Hz, 2H), 1.86 (s, 8H), 1.62 (m, J = 10.5 Hz, 2H), 1.53 (d, J = 7.3 Hz, 5H), 1.35 (s, 12H). 13 13C NMR (175 MHz, Methanol-d4) δ 177.21, 172.40, 169.70, 162.91, 161.84, 154.57, 145.72, 142.22, 141.14, 133.98, 128.85, 128.75, 127.01, 126.94, 122.43, 115.71, 114.28, 113.48, 112.21, 102.87, 100.71, 78.97, 68.61, 50.57, 39.99, 33.24, 29.24, 29.17, 29.01, 28.94, 28.81, 28.69, 28.62, 26.85, 25.67, 24.30, 23.75, 20.27, 19.15, 11.46.

[0059] Preparation steps of compound AC5 in Example 5:

[0060]

[0061] Preparation Example 9, Preparation of Compound C4

[0062] tert-Butyl 18-bromooctadecanoate (0.94 g, 0.225 mmol), Compound 2 6(0.60 g, 0.151 mmol) was dissolved in ultradry N,N-dimethylformamide (2.0 mL). After N,N-diisopropylethylamine (105 μL, 0.60 mmol) was added to the above system, the reaction system was stirred overnight at 50 °C. The reaction system was diluted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered by suction, concentrated under reduced pressure. The residue was purified by medium-pressure column chromatography (dichloromethane:methanol = 97:3) and then dissolved in ultradry dichloromethane (2.0 mL). Trifluoroacetic acid (2 mL) was added in an ice-water bath, and the reaction system was reacted at 0 °C for 30 minutes. It was concentrated under reduced pressure, and the residue was purified by medium-pressure column chromatography (dichloromethane:methanol = 96:4) to obtain compound C4 as a blue solid, 0.012 g, with a yield of 88.2%. 1 H NMR (400 MHz, Methanol-d4) δ 8.77 (d, J = 14.8 Hz, 1H), 7.65 (d, J = 7.4 Hz, 1H), 7.56–7.50 (m, 2H), 7.49–7.42 (m, 2H), 7.40 (s, 1H), 7.01 (d, J = 2.5 Hz, 1H), 6.97 (dd, J = 8.6, 2.4 Hz, 1H), 6.50 (d, J = 14.8 Hz, 1H), 4.39 (q, J = 7.3 Hz, 2H), 4.14 (t, J = 6.4 Hz, 2H), 2.79 (t, J = 6.3 Hz, 2H), 2.73 (t, J = 6.2 Hz, 2H), 2.26 (s, 2H), 1.94 (p, J = 6.3 Hz, 2H), 1.82 (s, 8H), 1.65–1.50 (m, 4H), 1.48 (t, J = 7.3 Hz, 3H), 1.27 (s, 24H). 13 C NMR (151 MHz, Methanol-d4) δ 178.62, 177.80, 164.33, 163.26, 155.97, 147.13, 143.60, 142.53, 135.39, 130.25, 130.14, 128.40, 128.34, 123.80, 117.12, 115.67, 114.89, 113.59, 104.23, 102.11, 69.99, 54.79, 51.96, 41.34, 35.05, 30.74, 30.70, 30.61, 30.43, 30.37, 30.26, 30.18, 30.08, 28.23, 27.04, 26.15, 25.10, 21.67, 12.83. Preparation Example 10, Preparation of Compound AC5

[0063] Dissolve bromomethyl acetate (0.008 g, 0.054 mmol) and compound C4 (0.012 g, 0.018 mmol) in ultradry N,N-dimethylformamide (2.0 mL). After adding N,N-diisopropylethylamine (9.45 μL, 0.054 mmol) to the above system, the reaction system was stirred overnight at 65 °C. The reaction system was diluted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered by suction, concentrated under reduced pressure, and the residue was purified by medium-pressure column chromatography (dichloromethane:methanol = 97:3) to obtain compound AC5 as a blue solid, 0.012 g, with a yield of 88.6%. 1 H NMR (400 MHz, Methanol-d4) δ 8.70 (d, J = 15.0 Hz, 1H), 7.55 (d, J = 7.3 Hz, 1H), 7.44 (dd, J = 4.7, 1.6 Hz, 2H), 7.40–7.34 (m, 2H), 7.32 (s, 1H), 6.93 (d, J = 2.7 Hz, 1H), 6.88 (dd, J = 8.6, 2.4 Hz, 1H), 6.41 (d, J = 14.8 Hz, 1H), 5.61 (s, 2H), 4.30 (q, J = 7.2 Hz, 2H), 4.05 (t, J = 6.4 Hz, 2H), 2.73–2.67 (m, 2H), 2.64 (t, J = 6.5 Hz, 2H), 2.24 (t, J = 7.4 Hz, 2H), 1.96 (s, 3H), 1.86 (d, J = 5.6 Hz, 2H), 1.73 (s, 8H), 1.50 (t, J = 7.3 Hz, 4H), 1.39 (s, 3H), 1.19 (s, 24H). 13 C NMR (151 MHz, Methanol-d4) δ 177.19, 172.35, 162.90, 161.84, 154.53, 145.70, 142.17, 141.10, 133.97, 128.80, 128.69, 126.96, 126.89, 122.36, 115.68, 114.22, 113.44, 112.14, 102.78, 100.68, 78.94, 68.55, 50.52, 39.90, 33.19, 29.28, 29.23, 29.22, 29.17, 29.15, 29.11, 29.02, 28.92, 28.89, 28.73, 28.64, 28.57, 26.78, 25.59, 24.25, 22.28, 20.23, 11.38.

[0064] Procedure for the preparation of compound C1 in Example 6:

[0065]

[0066] Preparation Example 11

[0067] Dissolve Compound 6 (0.110 g, 0.375 mmol) and Compound 2 6 (0.50 g, 0.125 mmol) in ultradry N,N-dimethylformamide (2.0 mL). After adding N,N-diisopropylethylamine (88 μL, 0.5 mmol) to the above system, the reaction system is stirred overnight at 50 °C. The reaction system is diluted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered by suction, concentrated under reduced pressure, and the residue is purified by medium-pressure column chromatography (dichloromethane:methanol = 97:3) to obtain Compound 7 as a blue solid, 0.015 g, with a yield of 19.5%. 1 H NMR (400 MHz, Methanol-d4) δ 8.80 (d, J = 14.8 Hz, 1H), 7.67 (d, J = 7.4 Hz, 1H), 7.57–7.53 (m, 2H), 7.51–7.45 (m, 2H), 7.42 (s, 1H), 7.04 (d, J = 3.2 Hz, 1H), 6.99 (dd, J = 8.6, 2.4 Hz, 1H), 6.52 (d, J = 14.8 Hz, 1H), 4.41 (q, J = 7.3 Hz, 2H), 4.17 (t, J = 6.3 Hz, 2H), 2.81 (t, J = 6.9 Hz, 2H), 2.75 (t, J = 6.0 Hz, 2H), 2.30 (t, J = 7.3 Hz, 2H), 1.96 (p, J = 6.3 Hz, 2H), 1.92–1.86 (m, 2H), 1.85 (s, 6H), 1.74–1.66 (m, 2H), 1.61–1.54 (m, 2H), 1.50 (t, J = 7.3 Hz, 3H), 1.47 (s, 9H). 13 C NMR (151 MHz, Chloroform-d) δ 176.88, 173.43, 162.62, 161.54, 154.57, 145.94, 142.03, 141.38, 133.49, 129.46, 128.87, 127.68, 127.33, 122.53, 115.89, 115.61, 113.09, 112.92, 104.80, 101.65, 80.14, 69.06, 60.36, 50.65, 41.78, 35.69, 34.47, 29.84, 29.51, 29.37, 29.32, 29.22, 29.15, 28.40, 28.27, 26.12, 25.18, 20.55, 14.42, 14.27, 13.25.

[0068] Preparation Example 12

[0069] Compound 7 (0.015 g, 0.024 mmol) was dissolved in ultradry dichloromethane (2.0 mL), and trifluoroacetic acid (2 mL) was added in an ice-water bath. The reaction system was reacted at 0 °C for 30 minutes, concentrated under reduced pressure, and the residue was purified by medium-pressure column chromatography (dichloromethane:methanol = 96:4) to obtain Compound C1 as a blue solid, 0.012 g, with a yield of 89.6%. 1 H NMR (400 MHz, Methanol-d4) δ 8.80 (d, J = 14.9 Hz, 1H), 7.67 (d, J = 7.6 Hz, 1H), 7.55 (dd, J = 5.6, 1.6 Hz, 2H), 7.50–7.45 (m, 2H), 7.43 (s, 1H), 7.05 (d, J = 2.3 Hz, 1H), 6.99 (dd, J = 8.6, 2.4 Hz, 1H), 6.52 (d, J = 14.8 Hz, 1H), 4.40 (q, J = 7.1 Hz, 2H), 4.16 (t, J = 6.3 Hz, 2H), 2.80 (t, J = 6.2 Hz, 2H), 2.75 (t, J = 6.1 Hz, 2H), 2.30 (t, J = 7.4 Hz, 2H), 1.96 (t, J = 6.1 Hz, 2H), 1.84 (s, 9H), 1.62 (d, J = 7.1 Hz, 2H), 1.49 (t, J = 7.3 Hz, 4H), 1.43–1.37 (s, 6H). 13 C NMR (151 MHz, Methanol-d4) δ 178.64, 177.66, 164.34, 163.28, 155.98, 147.15, 143.63, 142.55, 135.40, 130.24, 130.14, 128.41, 128.34, 123.82, 117.12, 115.66, 114.88, 113.57, 104.21, 102.11, 69.99, 51.97, 41.33, 34.92, 30.34, 30.27, 30.18, 30.15, 30.09, 28.21, 27.03, 26.06, 25.11, 21.69, 12.82. Preparation steps of Compound C2 in Example 7

[0070]

[0071] Preparation Example 13

[0072] Compound 8 (0.120 g, 0.375 mmol) and Compound 2 6(0.100 g, 0.251 mmol) was dissolved in ultradry N,N-dimethylformamide (2.0 mL). After adding N,N-diisopropylethylamine (175 μL, 1.00 mmol) to the above system, the reaction system was stirred overnight at 50 °C. The reaction system was diluted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered by suction, concentrated under reduced pressure, and the residue was purified by medium-pressure column chromatography (dichloromethane:methanol = 97:3) to obtain Compound 9 as a blue solid, 0.020 g, with a yield of 12.5%. 1 H NMR (500 MHz, Methanol-d4) δ 8.78 (d, J = 14.7 Hz, 1H), 7.66 (d, J = 7.3 Hz, 1H), 7.54 (q, J = 8.6, 8.0 Hz, 2H), 7.46 (q, J = 7.8, 7.0 Hz, 2H), 7.41 (s, 1H), 7.02 (s, 1H), 6.97 (d, J = 11.0 Hz, 1H), 6.51 (d, J = 14.8 Hz, 1H), 4.40 (q, J = 7.3 Hz, 2H), 4.14 (t, J = 6.4 Hz, 2H), 2.79 (t, J = 6.1 Hz, 2H), 2.74 (t, J = 6.0 Hz, 2H), 2.21 (t, J = 7.4 Hz, 2H), 1.95 (p, J = 6.0 Hz, 2H), 1.83 (s, 8H), 1.59–1.52 (m, 4H), 1.49 (t, J = 7.3 Hz, 3H), 1.44 (s, 9H), 1.33 (s, J = 4.6 Hz, 10H). 13 C NMR (126 MHz, Methanol-d4) δ 178.59, 175.13, 164.30, 163.22, 155.96, 147.11, 143.61, 142.54, 135.36, 130.24, 130.13, 128.40, 128.33, 123.83, 117.10, 115.68, 114.89, 113.62, 104.29, 102.09, 81.31, 70.03, 51.97, 41.41, 36.43, 30.61, 30.50, 30.42, 30.36, 30.22, 30.11, 28.36, 28.27, 27.09, 26.22, 25.17, 21.67, 12.89.

[0073] Preparation Example 14

[0074] Compound 9 (0.020 g, 0.031 mmol) was dissolved in ultradry dichloromethane (2.0 mL), trifluoroacetic acid (2 mL) was added in an ice-water bath, and the reaction system was reacted at 0 °C for 30 minutes. It was concentrated under reduced pressure, and the residue was purified by medium-pressure column chromatography (dichloromethane:methanol = 96:4) to obtain Compound C2 as a blue solid, 0.016 g, with a yield of 88.9%. 1 H NMR (500 MHz, Methanol-d4) δ 8.78 (d, J = 15.0 Hz, 1H), 7.65 (d, J = 7.4 Hz, 1H), 7.53 (d, J = 7.5 Hz, 2H), 7.49–7.42 (m, 2H), 7.41 (s, 1H), 7.03 (d, J = 2.4 Hz, 1H), 6.97 (dd, J = 8.6, 2.3 Hz, 1H), 6.50 (d, J = 14.9 Hz, 1H), 4.39 (q, J = 7.2 Hz, 2H), 4.14 (t, J = 6.4 Hz, 2H), 2.79 (t, J = 6.0 Hz, 2H), 2.73 (t, J = 6.0 Hz, 2H), 2.27 (t, J = 7.4 Hz, 2H), 1.95 (q, J = 6.2 Hz, 2H), 1.83 (s, 8H), 1.59 (t, J = 7.4 Hz, 2H), 1.53 (t, J = 7.5 Hz, 2H), 1.48 (t, J = 7.3 Hz, 3H), 1.34 (s, 10H). 13 C NMR (151 MHz, Methanol-d4) δ 177.19, 176.45, 162.89, 161.83, 154.53, 145.70, 142.17, 141.09, 133.94, 128.79, 128.69, 126.96, 126.89, 122.37, 115.67, 114.22, 113.45, 112.12, 102.77, 100.65, 68.55, 50.51, 47.97, 47.83, 47.69, 47.55, 47.41, 47.26, 47.12, 39.88, 33.66, 29.14, 29.04, 28.94, 28.80, 28.75, 28.63, 26.77, 25.61, 24.70, 23.66, 20.22, 11.36.

[0075] Preparation steps of Compound C3 in Example 8

[0076]

[0077] Preparation Example 15

[0078] Compound 10 (0.189 g, 0.565 mmol), Compound 26 (0.150 g, 0.376 mmol) was dissolved in ultradry N,N-dimethylformamide (2.0 mL). After N,N-diisopropylethylamine (263 μL, 1.5 mmol) was added to the above system, the reaction system was stirred overnight at 50 °C. The reaction system was diluted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered by suction, concentrated under reduced pressure, and the residue was purified by medium-pressure column chromatography (dichloromethane:methanol = 97:3) to obtain Compound 11 as a blue solid, 0.030 g, with a yield of 12.2%. 1 H NMR (400 MHz, Methanol-d4) δ 8.77 (d, J = 15.0 Hz, 1H), 7.66 (dt, J = 7.4, 1.0 Hz, 1H), 7.54 (dd, J = 6.4, 1.2 Hz, 2H), 7.49–7.42 (m, 2H), 7.40 (s, 1H), 7.01 (d, J = 2.9 Hz, 1H), 6.97 (dd, J = 8.5, 2.3 Hz, 1H), 6.50 (d, J = 14.8 Hz, 1H), 4.40 (q, J = 7.4 Hz, 2H), 4.14 (t, J = 6.3 Hz, 2H), 2.79 (t, J = 5.8 Hz, 2H), 2.74 (t, J = 6.2 Hz, 2H), 2.20 (t, J = 7.4 Hz, 2H), 1.98–1.91 (m, 2H), 1.83 (s, 8H), 1.56 (dt, J = 14.0, 6.9 Hz, 4H), 1.49 (d, J = 7.3 Hz, 3H), 1.43 (s, 9H), 1.32 (s, 12H). 13 C NMR (151 MHz, Methanol-d4) δ 178.58, 175.15, 164.28, 163.20, 155.94, 147.10, 143.59, 142.52, 135.36, 130.24, 130.14, 128.39, 128.34, 123.82, 117.10, 115.68, 114.88, 113.61, 104.28, 102.10, 81.33, 70.04, 51.96, 41.42, 36.44, 30.64, 30.58, 30.55, 30.43, 30.36, 30.21, 30.10, 28.36, 28.29, 27.07, 26.21, 25.17, 21.66, 12.89.

[0079] Preparation Example 16

[0080] Compound 11 (0.030 g, 0.046 mmol) was dissolved in ultradry dichloromethane (2.0 mL), and trifluoroacetic acid (2.0 mL) was added in an ice-water bath. The reaction system was reacted at 0 °C for 30 minutes, concentrated under reduced pressure, and the residue was purified by medium-pressure column chromatography (dichloromethane:methanol = 96:4) to obtain Compound C3 as a blue solid, 0.025 g, with a yield of 91.2%. 1 H NMR (400 MHz, Methanol-d4) δ 8.79 (d, J = 14.8 Hz, 1H), 7.65 (d, J = 7.4 Hz, 1H), 7.57–7.51 (m, 2H), 7.50–7.43 (m, 2H), 7.41 (s, 1H), 7.03 (d, J = 2.3 Hz, 1H), 6.98 (dd, J = 8.7, 2.3 Hz, 1H), 6.51 (d, J = 14.8 Hz, 1H), 4.39 (q, J = 7.2 Hz, 2H), 4.15 (t, J = 6.4 Hz, 2H), 2.79 (t, J = 5.6 Hz, 2H), 2.74 (t, J = 6.0 Hz, 2H), 2.27 (t, J = 7.4 Hz, 2H), 1.95 (p, J = 6.6 Hz, 2H), 1.83 (s, 8H), 1.58 (dd, J = 15.7, 8.6 Hz, 4H), 1.49 (t, J = 7.3 Hz, 3H), 1.33 (s, 12H). 13 C NMR (151 MHz, CD3OD) δ 178.64, 177.70, 164.34, 163.28, 155.98, 147.15, 143.62, 142.55, 135.40, 130.25, 130.14, 128.41, 128.35, 123.82, 117.13, 115.67, 114.89, 113.58, 104.22, 102.12, 70.00, 51.97, 41.34, 34.97, 30.64, 30.59, 30.57, 30.42, 30.41, 30.25, 30.20, 30.09, 28.22, 27.06, 26.11, 25.11, 21.68, 12.82.

[0081] Preparation steps of Compound D1 in Example 9

[0082]

[0083] Preparation Example 17

[0084] Compound 12 8(0.500 g, 1.03 mmol), (3αS,4S,6R,6αS)-6-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-ol (0.537 g, 2.06 mmol), DMAP (0.189 g, 1.54 mmol), EDCI (0.306 g, 1.54 mmol) and a total of 12 mL of ultradry dichloromethane and ultradry N,N-diisopropylethylamine in a ratio of 5:1 were added to the above system, and the mixture was stirred at room temperature for about 1 hour. After the reaction was completed, the reaction system was diluted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered by suction, concentrated under reduced pressure, and the residue was purified by medium-pressure column chromatography (dichloromethane:methanol = 96:4) to obtain 0.214 g of compound 13 as a blue solid, with a yield of 27.3%. 1 H NMR (500 MHz, Methanol-d4) δ 8.65 (d, J = 14.6 Hz, 1H), 7.60 (d, J = 7.5 Hz, 1H), 7.52–7.34 (m, 5H), 6.82 (dd, J = 8.6, 2.2 Hz, 1H), 6.77 (d, J = 2.3 Hz, 1H), 6.34 (d, J = 14.7 Hz, 1H), 6.01 (s, 1H), 4.82 (dd, J = 5.8, 3.5 Hz, 2H), 4.68 (d, J = 5.8 Hz, 1H), 4.33 (q, J = 6.0 Hz, 1H), 4.26 (t, J = 7.4 Hz, 2H), 4.07 (dd, J = 6.4, 3.5 Hz, 1H), 4.04–3.99 (m, 1H), 3.89 (dd, J = 8.6, 5.5 Hz, 1H), 2.76 (t, J = 5.7 Hz, 2H), 2.70 (t, J = 6.0 Hz, 2H), 2.38 (t, J = 7.2 Hz, 2H), 1.91 (dt, J = 21.9, 6.8 Hz, 4H), 1.79 (s, 6H), 1.71 (p, J = 7.2 Hz, 2H), 1.54–1.46 (m, 2H), 1.41 (s, 3H), 1.35 (s, 3H), 1.29 (d, J = 2.9 Hz, 6H). 1313C NMR (101 MHz, Methanol-d4) δ 176.64, 172.09, 164.18, 162.76, 155.04, 144.80, 141.77, 141.69, 135.40, 129.15, 128.79, 126.52, 125.80, 122.38, 115.07, 114.33, 112.72, 112.07, 108.69, 102.22, 101.65, 100.63, 84.86, 82.13, 79.25, 72.95, 65.99, 50.24, 44.31, 33.16, 28.53, 27.14, 26.89, 25.74, 25.59, 24.81, 23.99, 23.85, 23.72, 23.29, 20.35.

[0085] Preparation Example 18

[0086] Compound 13 (0.100 g, 0.137 mmol), Compound 3 (0.139 g, 0.411 mmol), N,N-diisopropylethylamine (96 μL, 0.548 mmol). After adding 2 mL of ultra-dry N,N-diisopropylethylamine to the above system, the mixture was heated to 60 °C and stirred overnight. After the reaction was completed, the reaction system was diluted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered by suction, concentrated under reduced pressure, and the residue was purified by medium-pressure column chromatography (dichloromethane:methanol = 95:5) to obtain Compound 14 as a blue solid, 0.037 g, with a yield of 28.5%. 11H NMR (400 MHz, Methanol-d4) δ 8.79 (d, J = 14.8 Hz, 1H), 7.68 (d, J = 7.4 Hz, 1H), 7.57 (d, J = 6.0 Hz, 2H), 7.48 (td, J = 8.4, 4.2 Hz, 2H), 7.44 (s, 1H), 7.08–6.96 (m, 2H), 6.53 (d, J = 14.9 Hz, 1H), 6.05 (s, 1H), 5.74 (s, 2H), 4.87–4.84 (m, 1H), 4.71 (d, J = 5.9 Hz, 1H), 4.38 (p, J = 6.5 Hz, 3H), 4.17 (t, J = 6.3 Hz, 2H), 4.11 (dd, J = 6.4, 3.5 Hz, 1H), 4.05 (dd, J = 8.5, 6.4 Hz, 1H), 3.92 (dd, J = 8.6, 5.5 Hz, 1H), 2.79 (dt, J = 20.6, 6.0 Hz, 4H), 2.40 (dt, J = 16.7, 7.2 Hz, 5H), 2.09 (s, 3H), 1.98 (t, J = 7.2 Hz, 4H), 1.86 (s, 8H), 1.75 (t, J = 7.2 Hz, 2H), 1.65–1.60 (m, 2H), 1.58–1.53 (m, 5H), 1.44 (s, 3H), 1.39 (s, 3H), 1.36 (s, 9H), 1.32 (d, J = 3.4 Hz, 6H). 13 13C NMR (176 MHz, Methanol-d4) δ 177.59, 172.37, 172.06, 169.69, 162.93, 161.90, 154.59, 145.53, 142.09, 141.54, 134.15, 128.85, 128.78, 127.05, 126.94, 122.42, 115.75, 114.40, 113.58, 112.70, 112.45, 108.68, 103.22, 100.74, 100.62, 86.80, 84.85, 82.12, 79.24, 78.99, 72.95, 68.64, 65.99, 50.56, 44.54, 33.26, 33.14, 29.20, 29.09, 29.03, 28.94, 28.83, 28.70, 28.63, 27.01, 25.72, 25.69, 25.60, 24.82, 24.31, 24.00, 23.84, 23.29, 20.28, 19.19.

[0087] Preparation Example 19

[0088] Compound 14 was added to a 1 mL system of frozen trifluoroacetic acid:water = 9:1. After reacting for about 10 minutes, it was diluted with dichloromethane, water was added, and after extraction, it was dried by suction. Then, N,N-disuccinimidyl carbonate (0.010 g, 0.037 mmol), 2.0 mL of ultra-dry dichloromethane, and triethylamine (10 μL) were added. After reacting for 10 minutes, TLC was used to determine that the raw materials had completely reacted. After rotary evaporation, a mixed solution of ethyl acetate and petroleum ether was used to wash away the residual raw materials, and then it was dried by suction. Next, mannose hydrochloride (0.040 g, 0.188 mmol), 2 mL of ultra-dry N,N-diisopropylethylamine, and triethylamine (15 μL) were added. The mixture was stirred at room temperature for about 45 minutes. After TLC was used to determine that the raw materials had completely reacted, the reaction system was diluted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered by suction, concentrated under reduced pressure, and the residue was purified by medium-pressure column chromatography (dichloromethane:methanol = 98:2) to obtain Compound D1 as a blue solid, 0.009 g, with a yield of 26.3%. 1 H NMR(700MHz,Methanol-d4)δ8.77(d,J=14.8Hz,1H),7.65(d,J=7.5Hz,1H),7.54(dt,J=11.2,7.5Hz,2H),7.49–7.40(m,3H),7.03(d,J=2.4Hz,1H),7.00–6.97(m,1H),6.50(d,J=14.8Hz,1H),5.71(s,2H),4.35(q,J=7.5Hz,2H),4.28(dd,J=4.6,1.6Hz,1H),4.14(t,J=6.3Hz,2H),4.00(dd,J=9.7,4.7Hz,2H),3.89–3.81(m,1H),3.78–3.73(m,2H),3.65(dt,J=9.4,4.7Hz,1H),3.57(t,J=9.8Hz,1H),2.79(t,J=6.0Hz,2H),2.73(t,J=6.2Hz,2H),2.35(t,J=7.4Hz,3H),2.34–2.25(m,2H),2.06(s,3H),1.93(dq,J=24.3,7.6,6.9Hz,4H),1.83(s,8H),1.73(dq,J=15.0,7.4Hz,2H),1.61(t,J=7.2Hz,2H),1.53(ddt,J=12.1,7.6,2.5Hz,3H),1.44–1.39(m,2H),1.33(s,9H). 1313C NMR(176MHz, Methanol-d4) δ 177.54, 175.03, 172.41, 169.72, 162.93, 161.91, 154.59, 145.57, 142.08, 141.57, 134.07, 128.87, 128.76, 127.07, 126.92, 122.37, 115.75, 115.73, 114.39, 113.51, 112.49, 103.20, 100.72, 93.63, 78.99, 72.01, 69.23, 68.61, 67.05, 60.73, 53.59, 50.54, 44.56, 35.03, 33.24, 29.18, 29.06, 29.00, 28.91, 28.81, 28.69, 28.60, 27.10, 26.97, 25.95, 25.67, 24.98, 24.30, 23.75, 20.29, 19.16.

[0089] Pharmacological experiments

[0090] Experimental Example 1: Absorption and Emission Spectra Determination Experiment of Compounds AC6 and D1

[0091] Dissolve compounds AC6 and D1 in dimethyl sulfoxide, configure with physiological saline, and then use a microplate reader to measure the absorption and normal emission spectra of the compounds. See Figure 2 .

[0092] Experimental Example 2: Cytotoxicity Determination Experiment of Compounds AC6 and D1

[0093] The CCK-8 method was used to measure cell viability. HepG-2 cells (5×10 4 cells / mL, 100 μL) were seeded in 96-well plates and cultured at 37 °C and 5% CO2 for 24 hours. Then, they were treated with compounds AC5 and D1 at concentrations of 0 μM, 0.078 μM, 0.156 μM, 0.312 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5.0 μM, 10.0 μM, and 20.0 μM, respectively, and cultured at 37 °C and 5% CO2 for 24 hours. Then, 20 μL of CCK8 was added to each well, and then cultured at 37 °C and 5% CO2 for 1 hour. The absorption OD value of each group was measured on a microplate reader at a wavelength of 450 nm. According to the OD values of each well, the cell viability was calculated using the following formula: Cell viability (%) = (OD of experimental group / OD of control group) × 100%. See Figure 3 .

[0094] Experimental Example 3: Confocal Imaging Experiment of Compound AC5 after Acting with Pseudomonas aeruginosa / Ceftazidime Resistance at 100 μM

[0095] Pseudomonas aeruginosa / ceftazidime-resistant (100 μM) was cultured overnight in a medium at 37 °C on a shaker at 180 rpm for about 12 h. The activated bacterial solution cultured overnight was collected by centrifugation and rinsed twice with physiological saline. The collected bacteria were resuspended in physiological saline to obtain a bacterial suspension with an OD 600 value between 2.0 and 2.5. The bacteria and 10.0 μM probe were co-incubated at 37 °C on a shaker at 180 rpm for 5 h. Then the bacteria were rinsed with physiological saline to wash away the unbound probe, and then stained with 0.2% Hoechst 33258 at 37 °C on a shaker at 180 rpm for 30 min. Finally, 10 μL of the suspension was dropped onto an 8-well chamber cover glass (specially for laser confocal microscopy), and an agarose gel pad was covered on it (to prevent the movement of bacteria from affecting the imaging effect), and confocal imaging was performed. All images were collected using a ZEISS LSM 710 laser confocal scanning microscope (63× oil immersion lens). See Figure 4 .

[0096] Experimental Example 4: Confocal Imaging Experiment of Compound D1 on Different Bacteria

[0097] 19606 (Acinetobacter baumannii), 29213 (Staphylococcus aureus), 25922 (Escherichia coli), 700221 (Enterococcus faecalis), 27853 (Pseudomonas aeruginosa), 700603 (Klebsiella pneumoniae), 27853-cdz100 (Pseudomonas aeruginosa / ceftazidime-resistant 100 μM) were cultured overnight in a medium at 37 °C on a shaker at 180 rpm for about 12 h. The activated bacterial solution cultured overnight was collected by centrifugation and rinsed twice with physiological saline. The collected bacteria were resuspended in physiological saline to obtain a bacterial suspension with an OD 600 value between 2.0 and 2.5. The bacteria and 10.0 μM probe were co-incubated at 37 °C on a shaker at 180 rpm for 5 h. Then the bacteria were rinsed with physiological saline to wash away the unbound probe, and then stained with 0.2% Hoechst 33258 at 37 °C on a shaker at 180 rpm for 30 min. Finally, 10 μL of the suspension was dropped onto an 8-well chamber cover glass (specially for laser confocal microscopy), and an agarose gel pad was covered on it (to prevent the movement of bacteria from affecting the imaging effect), and confocal imaging was performed. All images were collected using a ZEISS LSM 710 laser confocal scanning microscope (63× oil immersion lens). See Figure 5 .

[0098] References

[0099] 1. C.J.L. Murray, K.S. Ikuta and F. Sharara, The Lancet, 2022, 399, 629 - 655.

[0100] 2. L.-L. Wu, Q. Wang, Y. Wang, N. Zhang, Q. Zhang and H.-Y. Hu, Chemical Science, 2020, 11, 3141 - 3145.

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Claims

1. A compound represented by the formula IV structure or a pharmaceutically acceptable salt thereof: R1 is independently selected from a hydrogen atom, R2 is independently selected from a hydrogen atom, fluorine, chlorine, bromine, iodine, aldehyde group or difluoromethyl; R3 is independently selected from a hydrogen atom, a normal alkyl group or wherein, m is 2, 3, 4, 5, 6, 7, 8, 9, 10; X1 is independently selected from O, S; n is selected from 1 - 20, preferably 4 - 18.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, The compound is selected from:

3. Use of the compound according to any one of claims 1 - 2 or a pharmaceutically acceptable salt thereof in the preparation of a fluorescent imaging agent for bacteria.

4. Use of the compound according to any one of claims 1 - 2 or a pharmaceutically acceptable salt thereof in the preparation of an infection diagnostic reagent for bacterial metabolism research.

5. Use of the compound according to any one of claims 1 - 2 or a pharmaceutically acceptable salt thereof in the preparation of immunocyte metabolism research.

6. Use of the compound according to any one of claims 1 - 2 or a pharmaceutically acceptable salt thereof in the preparation of a fluorescent probe.

7. A process for preparing the compound according to any one of claims 1-2 or a pharmaceutically acceptable salt thereof, characterized in that, The preparation method thereof is as follows: Wherein: R1, R2, R 3， X1 and n are defined as in any one of claims 1-2; (1) Methyl bromoacetate, bromoalkanoic acid, a base and a solvent are reacted under the conditions of 0 - 100 °C and stirring to obtain I, or bromoalkanoic acid, tert - butyl trichloroacetimidate, boron trifluoride diethyl etherate solution and a solvent are reacted under the conditions of 0 - 100 °C and stirring to obtain I; (2) Resorcinol or a derivative of resorcinol or 3 - hydroxybenzenethiol or a derivative of 3 - hydroxybenzenethiol, compound II, a base and a solvent are reacted under the conditions of 0 - 100 °C and stirring to obtain III; (3) III prepared in step (2), a solvent, I prepared in step (1) and a base are reacted together to obtain IV, or further reacted with an aqueous solution of trifluoroacetic acid, concentrated under reduced pressure and purified to obtain IV, or reacted with (3αS,4S,6R,6αS) - 6 - ((R) - 2,2 - dimethyl - 1,3 - dioxolan - 4 - yl) - 2,2 - dimethyltetrahydrofuro[3,4 - d][1,3]dioxol - 4 - ol, a solvent, a condensing agent, purified and then reacted with I prepared in step (1) and a base. After the reaction is completed, it is purified, acid is added, and after the reaction is completed at 0 - 100 °C, it is extracted, concentrated under reduced pressure and dried, and further reacted with a condensing agent, a base and a solvent under the conditions of 0 - 100 °C and stirring. After the reaction is completed, the obtained compound is finally reacted with mannose hydrochloride, a base and a solvent under the conditions of 0 - 100 °C and stirring to obtain compound IV.

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