A compound with a pyridinium salt central ring structure, its preparation method and applications

Abstract

The present invention relates to a compound having a pyridinium salt central ring structure, a preparation method thereof, and an application thereof. The compound has a structure shown in the following general formula I. Compared with the near-infrared II region fluorescent probes in the prior art, the maximum absorption and emission wavelengths of the fluorescent probe of the general formula I compound of the present invention are both red-shifted. The fluorescent probe of the present invention can be specifically taken up by tumor cells and has the functions of early tumor diagnosis, targeted chemotherapy, surgical navigation, and photothermal therapy in one.

Description

Technical Field

[0001] The present invention belongs to the field of organic fluorescent probes, and particularly relates to a class of fluorescent probes, a preparation method thereof, and applications thereof. Background Art

[0002] Cancer is a major disease threatening human health. With the in-depth study of cancer, people have gradually realized the important role of mitochondria in the occurrence and development of tumors. Lipophilic delocalized cations (DLCs) are a class of compounds with mitochondrial targeting functions. Since the mitochondrial membrane potential of cancer cells is much higher than that of normal cells, DLCs accumulate more in the mitochondria of tumor cells, causing depolarization of the mitochondrial membrane of tumor cells and thus inducing apoptosis and necrosis. Rhodamine, MKT-077, and F16 are the most representative DLCs. Rhodamine is the first DLC found to have mitochondrial targeting function, and its anti-cancer activity has been confirmed in small animal experiments. MKT-077 is the first DLC to enter the clinical approval stage. Although it failed to pass clinical phase II due to limited anti-tumor activity and renal toxicity, it still laid the foundation for DLCs to become anti-cancer drugs.

[0003] Since DLCs have a conjugated structure with multiple aromatic rings, many DLCs are also natural fluorescent chromophores, so they have the potential to become integrated diagnosis and treatment probes. However, so far, the emission wavelengths of the developed DLCs are mostly in the traditional visible light-near infrared region I (400-900 nm), and the fluorescence penetration depth in this region is usually less than 3 mm, which limits their clinical applications.

[0004] In recent years, the development of near-infrared window two (NIR-II, 1000-1700 nm) fluorescent dyes has greatly expanded the clinical application value of fluorescence imaging. Compared with the traditional visible light-near infrared region I window, near-infrared region II fluorescence has the advantages of deep penetration depth, less tissue autofluorescence, high imaging signal-to-noise ratio, etc., and can perform high-resolution imaging on biological tissues with a depth of 1 cm, greatly exceeding the penetration depth of traditional fluorescence imaging.

[0005] However, so far, DLCs with NIR-II fluorescence are relatively rare. Therefore, it is of great significance to develop DLCs with NIR-II fluorescence and strong drug-likeness.

[0006] In 2020, the research group of Hong Xuechuan at Wuhan University developed the first NIR-II DLCs molecule with mitochondrial targeting function, H4-PEG-PT. H4-PEG-PT can not only image mitochondria at the subcellular level in osteosarcoma cells, but also efficiently convert light energy into heat energy to achieve mitochondrial-targeted photothermal cancer therapy (Nat. Commun. 2020, 11, 6183). However, the maximum emission wavelength of this probe is 1100 nm, and its IC50 for 143B is greater than 32 μM. This indicates that this probe still cannot reach an excellent level in terms of imaging tissue depth and chemotherapy activity.

[0007] Therefore, there is still a need to develop a new fluorescent probe with the four-in-one functions of early tumor diagnosis, targeted chemotherapy, surgical navigation, and photothermal therapy. Summary of the Invention

[0008] The technical objective of the present invention is to develop a class of fluorescent probes with NIR-II fluorescence by using the existing 6,7-bis(pyridinyl)-[1,2,5]thiadiazolo[3,4-g]quinoxaline as an electron acceptor.

[0009] Another technical objective of the present invention is to provide a preparation method for the fluorescent probe.

[0010] Another technical objective of the present invention is to provide the application of the fluorescent probe in NIR-II cell imaging, in vivo imaging, and tumor treatment.

[0011] On the one hand, the present invention provides a compound represented by the following general formula I:

[0012]

[0013] In general formula I:

[0014] X is S, O, Se or N;

[0015] C1 and C2 are each independently provided that the two are not simultaneously

[0016] R 1 is a straight bond, wherein, R 4 and R 5 are each independently selected from H, hydroxyl, amino, carboxyl, sulfonic acid group, halogen, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C8 alkoxy, substituted or unsubstituted C6-C 10 aryl or C5-C 10 heteroaryl; R 6 and R 7Each independently selected from H, hydroxyl, amino, carboxyl, sulfonic acid group, substituted or unsubstituted C1-C8 alkyl, C1-C8 alkoxy, halogen, or, R 6 and R 7 together with the adjacent carbon atom form a 5-10 membered N, P or O-containing heterocyclic group; Y is S, O, Se or N; where in the case of substitution, the substituents are selected from amino and hydroxyl;

[0017] R 2 is selected from substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C6-C 10 aryl or C5-C 10 heteroaryl, metal ions, the metal ions are selected from Mg 2+ 、Al 3+ 、Ca 2+ 、Mn 2+ 、Fe 2+ 、Cu 2+ 、Zn 2+ 、Ag + 、Au + and Hg 2+ ; where in the case of substitution, the substituents are selected from amino, hydroxyl, carboxyl, C1-C6 alkoxy and sulfonic acid group;

[0018] D is selected from the following groups:

[0019]

[0020] wherein, in the above formulas, R 8 -R 41 each independently selected from:

[0021] -H; amino; halogen; C1-C8 alkoxy; C1-C8 alkyl acyloxy; C1-C8 alkyl substituted or unsubstituted by silyl, hydroxyl, amino, alkynyl, azide, mercapto, halogen, carboxyl, trimethylsilyl C1-C4 alkoxycarbonyl;

[0022] --(CH2)n1-(OCH2CH2)n2-OR, wherein, n1 and n2 are each independently integers from 1 to 500, and R is selected from C1-C8 alkyl, amino, mercapto, halogen;

[0023] wherein, R', R" are C1-C8 alkylene, A is a cyclic peptide (such as c(RGDyk), c(RGDfk), c(RADyk)), prostate specific membrane antigen (PSMA), octreotide, monosaccharide or polysaccharide group, the monosaccharide can be selected from glucose, galactose, fructose, arabinose, rhamnose, ribose, lactose and maltose; B is a molecular group containing Fv segment, the molecule containing Fv segment can be selected from monoclonal antibody, bispecific antibody or single-chain Fv segment;

[0024]

[0025] Among them, M is selected from 64 Cu, 68 Ga, 177 Lu, Gd, 99m Tc and Al; R''' is C1-C 12 alkylene or -(CH2)n3-(OCH2CH2)n4-, where n3 is an integer from 0 to 100 and n4 is an integer from 1 to 100.

[0026] In the definitions of the above substituents, represents the bonding position.

[0027] In a specific embodiment,

[0028] In general formula I, one of C1 and C2 is the other is Preferably, one of C1 and C2 is the other is Preferably, both C1 and C2 are

[0029] R 1 is Y is S, O, Se or N; R 6 and R 7 are selected from amino-substituted C1-C4 alkyl, or R 6 and R 7 together with the adjacent carbon atom form a 5- to 10-membered oxygen-containing heterocyclic group. For example, R 1 is

[0030] R 2 is unsubstituted or substituted C1-C4 alkyl with hydroxyl, carboxyl or methoxy groups;

[0031] D is selected from

[0032] In a specific embodiment, the compound is selected from the following compounds:

[0033]

[0034]

[0035]

[0036] On the other hand, the present invention provides a method for preparing the compound shown in general formula I, and the reaction route is as follows:

[0037]

[0038] Wherein:

[0039] In the above reaction formula, Z is a borate group or a borate ester group, or D-Z is a stannous salt of the formula ;

[0040] X, R 1 、D、R 2 、C1 and C2 are defined as above respectively, and the method comprises the following steps:

[0041] a. Intermediate 3 is obtained by the nucleophilic substitution reaction of Compound 1 and Compound 2;

[0042] b. Intermediate 4 is obtained by the reduction reaction of Intermediate 3;

[0043] c. Compound 6 is obtained by the ring-closing reaction of Intermediate 4 and Compound 5;

[0044] d. Compound 6 and R 2 I undergoes an alkylation reaction to obtain a compound of general formula I.

[0045] On the other hand, the present invention provides a pharmaceutical composition comprising the compound of general formula I as described above and a pharmaceutically acceptable excipient.

[0046] In a specific embodiment, the pharmaceutical composition further comprises other tumor chemotherapeutic agents in addition to the compound of general formula I as described above.

[0047] On the other hand, the present invention provides the use of the compound of general formula I as described above or the pharmaceutical composition as described above in the preparation of a mitochondrial targeting agent, particularly a mitochondrial targeting anti-cancer agent.

[0048] On yet another hand, the present invention provides the use of the compound of general formula I as described above or the pharmaceutical composition as described above in the preparation of a fluorescent probe, a photothermal therapeutic agent or a tumor chemotherapeutic agent.

[0049] In a specific embodiment, the fluorescent probe can be used as a diagnostic agent or an imaging agent, such as a tumor diagnostic agent or a tumor imaging agent.

[0050] In a specific embodiment, the tumors include but are not limited to osteosarcoma, breast cancer, colorectal cancer, lung cancer, etc.

[0051] Beneficial effects

[0052] The maximum absorption and emission wavelengths of the compounds of the present invention are redshifted compared to existing near-infrared II region probes. The fluorescent probes of the present invention can be specifically taken up by tumor cells. Therefore, they have the four-in-one functions of early tumor diagnosis, targeted chemotherapy, surgical navigation, and photothermal therapy. Thus, they have great potential in clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Absorption spectra (A) and emission spectra (B) of TQP-1031, TQP-1196, and TQP-934 and TQP-1036 with the corresponding central ring of 6,7-bis(pyridyl)-[1,2,5]thiadiazolo[3,4-g]quinoxaline.

[0054] Figure 2 Near-infrared II region microscopic imaging diagrams of TQP-1031 and TQP-934, and TQP-1196 and TQP-1036.

[0055] Among them, the magnification is 20 times, the exposure time is 500 ms, and the laser power is 6 W.

[0056] Figure 3 Biodistribution diagrams of 1100 nm in the near-infrared II region at different time points (0, 2, 6, 24, 48 h) after intravenous injection of TQP-1031 into BALB / c mice.

[0057] Figure 4 Schematic diagram of surgical resection of tumors under the guidance of TQP-1031 fluorescence.

[0058] Figure 5 Shows the results of the cytotoxicity experiment of TQP-1031 on 143B and 3T3.

[0059] Figure 6 Heating curves of TQP-1031 and TQP-934, and TQP1196 and TQP-1036 under laser irradiation, where the laser power is 1 W / cm 2 .

[0060] Figure 7 Schematic diagram of the change in tumor volume size after intratumoral injection of TQP-1031 and PBS into nude mice, where IV is the initial tumor volume and FV is the tumor volume on the day of measurement. DETAILED DESCRIPTION OF THE INVENTION

[0061] The present invention will be further described below in conjunction with specific embodiments. These embodiments are only for the purpose of explanation and are not limited to the scope and essence of the present invention.

[0062] Embodiment

[0063] Example 1: Synthesis of Compound TQP-1031

[0064]

[0065] Take compound 1a (500 mg, 0.912 mmol) and compound 1b (1.64 g, 2.28 mmol) and dissolve them in toluene (10 mL). Pass nitrogen to displace the air in the reaction vessel and add potassium carbonate (315 mg, 2.28 mmol, dissolved in 2 mL of water) and Pd(PPh3)4 (52.7 mg, 0.046 mmol) under nitrogen protection. Heat and reflux with stirring at 110 °C for 12 h. After the reaction is completed, remove toluene by rotary evaporation, dilute the remaining reaction solution with ethyl acetate, and wash it with water several times. Combine the organic phases, dry them with anhydrous magnesium sulfate, filter, concentrate the filtrate by rotary evaporation, and pass it through a silica gel column to obtain reaction intermediate 1c (600 mg, yield 41.73%).

[0066] Take intermediate 1c (100 mg, 0.063 mmol) and dissolve it in a mixed solution of dichloromethane and methanol (40 mL of dichloromethane and 10 mL of methanol). Add zinc powder (497.71 mg, 7.61 mmol) and NH4Cl (135.73 mg, 2.54 mmol) under ice bath conditions. The reaction is carried out at room temperature for 2 hours. After the reaction is completed, filter, concentrate the filtrate by rotary evaporation, dissolve the remaining solid in EtOAc, and wash it with water repeatedly. Concentrate the organic phase by rotary evaporation.

[0067] Dissolve the obtained solid in acetic acid, pass nitrogen to displace the air in the reaction vessel and add 2,2'-pyridone (20 mg, 0.095 mmol) under nitrogen protection. Heat and reflux the reaction solution at 110 °C for 12 h. After the reaction is completed, cool the reaction solution to room temperature, dilute it with ethyl acetate, wash it with water several times, combine the organic phases, dry them with anhydrous magnesium sulfate, filter, concentrate the filtrate by rotary evaporation, and pass it through a silica gel column to obtain reaction intermediate 1e (27 mg, two-step yield 25%).

[0068] 11H NMR (400 MHz, CDCl3) δ 9.08 (d, J = 4.0 Hz, 2H), 8.68 (d, J = 7.6 Hz, 2H), 8.45–8.35 (m, 2H), 8.03 (t, J = 7.4 Hz, 2H), 7.81–7.70 (m, 8H), 7.62 (d, J = 4.1 Hz, 2H), 7.37 (d, J = 6.1 Hz, 8H), 4.12–4.03 (m, 8H), 2.08 (q, J = 12.1, 9.8 Hz, 16H), 1.46–1.37 (m, 8H), 1.18–1.09 (m, 8H), 0.95–0.88 (m, 8H), 0.71 (dq, J = 24.9, 7.3 Hz, 8H), -0.01 (s, 36H).

[0069] 13 13C NMR (126 MHz, CDCl3) δ 173.95, 156.96, 152.23, 152.10, 151.29, 150.62, 150.49, 148.42, 141.33, 140.80, 136.80, 135.57, 135.13, 134.67, 133.67, 127.52, 127.21, 125.13, 125.07, 123.58, 123.54, 123.00, 121.37, 120.39, 120.06, 120.00, 62.42, 55.20, 40.48, 34.52, 29.68, 24.75, 23.62, 17.41, -1.38.

[0070] Dissolve intermediate 1e (10 mg) in anhydrous DMF (1 mL), purge the air in the reaction vessel with nitrogen, and add an excess of CH3I (50 μL, 0.599 mmol) under nitrogen protection. Heat the mixture under reflux at 60 °C for 12 h. After the reaction is completed, cool the reaction mixture to room temperature, dilute it with ethyl acetate, and wash it several times with water. Dry the organic phase with anhydrous magnesium sulfate, filter, evaporate the filtrate to dryness, and obtain the final product TQP-1031 (8 mg, yield 79%) by passing through a silica gel column.

[0071] 11H NMR (600 MHz, CDCl3) δ 10.64 (d, J = 6.2 Hz, 1H), 9.13 (d, J = 4.1 Hz, 1H), 8.99 (dt, J = 8.0, 1.1 Hz, 1H), 8.92 (d, J = 4.1 Hz, 1H), 8.56 (td, J = 7.8, 1.4 Hz, 1H), 8.44–8.38 (m, 2H), 8.02 (td, J = 7.7, 1.7 Hz, 1H), 7.88–7.71 (m, 8H), 7.65–7.60 (m, 3H), 7.45 (ddd, J = 7.6, 4.7, 1.1 Hz, 1H), 7.42–7.33 (m, 6H), 4.67 (s, 3H), 4.08 (tt, J = 7.8, 1.5 Hz, 8H), 2.13–2.01 (m, 16H), 1.44–1.36 (m, 8H), 1.14 (t, J = 9.7 Hz, 8H), 0.94–0.89 (m, 9H), 0.79–0.64 (m, 8H), 0.00 (d, J = 1.7 Hz, 36H).

[0072] 13 13C NMR (126 MHz, CDCl3) δ 174.03, 173.93, 154.03, 152.86, 152.53, 152.41, 152.06, 151.68, 151.53, 151.35, 150.65, 149.86, 148.57, 147.73, 144.85, 143.40, 142.08, 141.98, 140.55, 140.54, 137.94, 136.40, 136.27, 135.13, 134.83, 134.55, 133.93, 133.10, 132.71, 128.01, 127.84, 127.74, 127.51, 127.32, 127.28, 125.97, 125.58, 125.36, 125.28, 124.27, 124.01, 123.06, 122.96, 122.62, 122.05, 120.79, 120.57, 120.26, 120.14, 120.05, 119.87, 62.46, 62.43, 55.27, 55.23, 47.26, 40.43, 40.32, 34.48, 34.46, 29.65, 29.60, 24.72, 23.64, 23.59, 17.40, -1.38.

[0073] MALDI-TOF / TOF Calculated: C 97 H 121 N6O8S3Si4 +[M]: 1705.75, measured [M]: 1705.713.

[0074] Example 2: Synthesis of Compound TQP-1196

[0075]

[0076] Take compound 1a (500 mg, 0.912 mmol) and compound 2b (1.64 g, 2.28 mmol) and dissolve them in toluene. Pass nitrogen to displace the air in the reaction vessel and add potassium carbonate (315 mg, 2.28 mmol, dissolved in 2 mL of water) and Pd(PPh3)4 (52.7 mg, 0.046 mmol) under nitrogen protection. Heat under reflux with stirring at 110 °C for 12 h. After the reaction is completed, rotary evaporate to remove toluene. Dilute the remaining reaction solution with ethyl acetate and wash it with water several times. Combine the organic phases and dry them over anhydrous magnesium sulfate, filter, rotary evaporate the filtrate, pass through a silica gel column to obtain the reaction intermediate 2c (470 mg, yield 32.90%).

[0077] Take intermediate 2c (100 mg, 0.063 mmol) and dissolve it in a mixed solution of dichloromethane and methanol (40 mL of dichloromethane, 10 mL of methanol). Add zinc powder (497.71 mg, 7.61 mmol) and NH4Cl (135.73 mg, 2.54 mmol) under ice bath conditions. The reaction is carried out at room temperature for 2 hours. After the reaction is completed, filter, rotary evaporate the filtrate. Dissolve the remaining solid in EtOAc and wash it with water repeatedly. Rotary evaporate the organic phase. Dissolve the obtained solid in acetic acid. Pass nitrogen to displace the air in the reaction vessel and add 2,2'-pyridone (20 mg, 0.095 mmol) under nitrogen protection. Heat the reaction solution under reflux at 110 °C for 12 h. After the reaction is completed, cool the reaction solution to room temperature and dilute it with ethyl acetate, wash it with water several times. Combine the organic phases and dry them over anhydrous magnesium sulfate, filter, rotary evaporate the filtrate, pass through a silica gel column to obtain the reaction intermediate 2e (69 mg, two-step yield 64.23%).

[0078] 11H NMR (400 MHz, CDCl3) δ 8.82 (d, J = 4.2 Hz, 2H), 8.48 (d, J = 7.8 Hz, 2H), 8.34 (d, J = 4.8 Hz, 2H), 7.91 (td, J = 7.8, 1.8 Hz, 2H), 7.51 (d, J = 8.3 Hz, 4H), 7.26 (q, J = 4.4 Hz, 4H), 7.13 (d, J = 8.1 Hz, 8H), 7.09–7.02 (m, 12H), 4.25–4.18 (m, 8H), 2.95 (t, J = 7.8 Hz, 8H), 2.64 (t, J = 7.8 Hz, 8H), 1.06–0.99 (m, 8H), 0.07 (s, 36H).

[0079] 13 13C NMR (101 MHz, CDCl3) δ 173.10, 156.56, 151.75, 151.20, 149.51, 147.87, 147.64, 145.64, 136.84, 135.63, 134.85, 134.14, 129.26, 128.23, 126.54, 125.24, 124.84, 123.40, 122.89, 122.48, 122.39, 120.84, 62.72, 36.12, 30.43, 17.39, -1.41.

[0080] Dissolve intermediate 2e (10 mg) in anhydrous DMF (1 mL), purge the air in the reaction vessel with nitrogen, and add an excess of CH3I (50 μL, 0.599 mmol) under nitrogen protection. Heat the mixture under reflux at 60 °C for 12 h. After the reaction is completed, cool the reaction mixture to room temperature, dilute it with ethyl acetate, and wash it several times with water. Dry the organic phase over anhydrous magnesium sulfate, filter, evaporate the filtrate to dryness, and pass it through a silica gel column to obtain the final product TQP-1196 (8 mg, yield 79%).

[0081] 11H NMR (400 MHz, CDCl3) δ 10.41 (s, 1H), 8.98 (d, J = 4.2 Hz, 1H), 8.80 (t, J = 7.0 Hz, 2H), 8.44 (t, J = 7.9 Hz, 1H), 8.31 (d, J = 4.7 Hz, 2H), 7.92 (t, J = 8.0 Hz, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.58 (d, J = 8.3 Hz, 2H), 7.48 - 7.29 (m, 5H), 7.17 - 6.98 (m, 20H), 4.51 (s, 3H), 4.25 - 4.13 (m, 8H), 2.92 (q, J = 7.8 Hz, 8H), 2.69 - 2.57 (m, 8H), 1.01 - 0.96 (m, 8H), 0.10 - 0.01 (m, 36H).

[0082] 13 13C NMR (126 MHz, CDCl3) δ 173.16, 153.83, 152.66, 152.44, 152.15, 151.43, 151.00, 149.77, 148.59, 148.51, 147.48, 145.45, 145.38, 144.76, 143.00, 137.98, 136.43, 136.20, 136.17, 134.91, 134.16, 133.82, 133.63, 129.48, 129.47, 126.85, 126.81, 126.76, 125.27, 125.25, 122.52, 122.35, 62.81, 36.20, 32.06, 30.53, 29.83, 22.82, 17.49, 14.25, -1.31.

[0083] ESI-LR calculation: C 95 H 107 N8O8S3Si4 + [M]: 1695.64, found [M]: 1696.4.

[0084] Example 3: Synthesis of Compound 3f

[0085]

[0086] Compound 3a (500 mg, 0.753 mmol) and compound 2b (1.35 g, 1.88 mmol) were dissolved in toluene (10 mL). Nitrogen was introduced to displace the air in the reaction vessel, and potassium carbonate (260 mg, 1.88 mmol, dissolved in 2 mL of water) and Pd(PPh3)4 (43.49 mg, 0.038 mmol) were added under nitrogen protection. The mixture was heated under reflux at 110 °C with stirring for 12 h. After the reaction was completed, toluene was removed by rotary evaporation. The remaining reaction solution was diluted with ethyl acetate and washed with water several times. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated by rotary evaporation, and passed through a silica gel column to obtain the reaction intermediate 3c (389 mg, yield 30.63%).

[0087] The intermediate 3c (100 mg, 0.059 mmol) was dissolved in a mixed solution of dichloromethane and methanol (40 mL of dichloromethane, 10 mL of methanol). Zinc powder (497.71 mg, 7.61 mmol) and NH4Cl (135.73 mg, 2.54 mmol) were added under ice bath conditions. The reaction was carried out at room temperature for 2 h. After the reaction was completed, the mixture was filtered, the filtrate was concentrated by rotary evaporation, the remaining solid was dissolved in EtOAc, and washed with water repeatedly. The organic phase was concentrated by rotary evaporation, the obtained solid was dissolved in acetic acid, nitrogen was introduced to displace the air in the reaction vessel, and 2,2'-pyridone (18.72 mg, 0.089 mmol) was added under nitrogen protection. The reaction solution was heated under reflux at 110 °C for 12 h. After the reaction was completed, the reaction solution was cooled to room temperature and diluted with ethyl acetate, washed with water several times. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated by rotary evaporation, and passed through a silica gel column to obtain the reaction intermediate 3e (70 mg, two-step yield 65.47%).

[0088] The intermediate 3e (10 mg) was dissolved in anhydrous DMF. Nitrogen was introduced to displace the air in the reaction vessel, and an excess of CH3I (50 μL, 0.599 mmol) was added under nitrogen protection. The mixture was heated under reflux at 60 °C for 12 h. After the reaction was completed, the reaction mixture was cooled to room temperature and diluted with ethyl acetate, washed with water several times. The organic phase was dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated by rotary evaporation, and passed through a silica gel column to obtain the final product 3f (8 mg, yield 79%). ESI-LR calculation: C 99 H 111 N8O 12 S3Si4 + [M]: 1811.66, found [M] +1 : 1812.3

[0089] Example 4: Synthesis of compound 4f

[0090]

[0091] Intermediate 1c (100 mg, 0.063 mmol) was dissolved in a mixed solution of dichloromethane and methanol (40 mL of dichloromethane, 10 mL of methanol). Zinc powder (497.71 mg, 7.61 mmol) and NH4Cl (135.73 mg, 2.54 mmol) were added under ice bath conditions. The reaction was carried out at room temperature for 2 hours. After the reaction was completed, the mixture was filtered, and the filtrate was concentrated by rotary evaporation. The remaining solid was dissolved in EtOAc and washed repeatedly with water. The organic phase was concentrated by rotary evaporation. The obtained solid was dissolved in acetic acid, and the air in the reaction vessel was displaced by nitrogen. Under nitrogen protection, 4d (20 mg, 0.095 mmol) was added. The reaction solution was heated to reflux at 110 °C for 12 h. After the reaction was completed, the reaction solution was cooled to room temperature and diluted with ethyl acetate, washed several times with water. The combined organic phases were dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated by rotary evaporation. The residue was purified by silica gel column chromatography to obtain reaction intermediate 4e (30 mg, two-step yield 28%).

[0092] Intermediate 4e (10 mg) was dissolved in anhydrous DMF (1 mL). The air in the reaction vessel was displaced by nitrogen. Under nitrogen protection, an excess of CH3I (50 μL, 0.599 mmol) was added. The mixture was heated to reflux at 60 °C for 12 h. After the reaction was completed, the reaction mixture was cooled to room temperature and diluted with ethyl acetate, washed several times with water. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated by rotary evaporation. The residue was purified by silica gel column chromatography to obtain the final product 4f (8 mg, yield 79%). MALDI-TOF / TOF calculation: C 97 H 121 N6O8S3Si4 + [M]: 1705.75, found [M]: 1705.713.

[0093] Example 5: Synthesis of Compound 5f

[0094]

[0095] Intermediate 1e (10 mg) was dissolved in anhydrous DMF (1 mL). The air in the reaction vessel was displaced by nitrogen. Under nitrogen protection, an excess of CH3I (50 μL, 0.599 mmol) was added. The mixture was heated to reflux at 60 °C for 7 days. After the reaction was completed, the reaction mixture was cooled to room temperature and diluted with ethyl acetate, washed several times with water. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated by rotary evaporation. The residue was purified by silica gel column chromatography to obtain the final product 5f (2 mg, yield 20%). ESI-LR calculation: C 98 H 126 N8O6S3Si4 2+ [M]: 1719.80, found [M] / 2: 860.5.

[0096] Example 6: Synthesis of Compound 6f

[0097]

[0098] Weigh intermediate 1e (10 mg) and dissolve it in anhydrous DMF (1 mL). Pass nitrogen gas to displace the air in the reaction vessel, and then add an excess of 1-iodobutane (50 μL) under nitrogen protection. Heat the mixture under reflux at 60 °C for 12 h. After the reaction is completed, cool the reaction mixture to room temperature, dilute it with ethyl acetate, and wash it with water several times. Dry the organic phase with anhydrous magnesium sulfate, filter, evaporate the filtrate to dryness, and pass it through a silica gel column to obtain the final product 6f (8 mg, yield 77%). MALDI-TOF / TOF calculation: C 100 H 127 N6O8S3Si4 + [M]: 1747.79, measured [M]: 1748.032.

[0099] Example 7: Synthesis of Compound 7f

[0100]

[0101] Weigh intermediate 1e (10 mg) and dissolve it in anhydrous DMF (1 mL). Pass nitrogen gas to displace the air in the reaction vessel, and then add an excess of 3-iodopropanol (50 μL) under nitrogen protection. Heat the mixture under reflux at 60 °C for 12 h. After the reaction is completed, cool the reaction mixture to room temperature, dilute it with ethyl acetate, and wash it with water several times. Dry the organic phase with anhydrous magnesium sulfate, filter, evaporate the filtrate to dryness, and pass it through a silica gel column to obtain the final product 7f (6 mg, yield 58%). MALDI-TOF / TOF calculation: C 99 H 125 N6O9S3Si4 + [M]: 1749.77, measured [M]: 1749.913.

[0102] Example 8: Synthesis of Compound 8f

[0103]

[0104] Weigh intermediate 1e (10 mg) and dissolve it in anhydrous DMF (1 mL). Pass nitrogen gas to displace the air in the reaction vessel, and then add an excess of 3-iodopropionic acid (100 mg) under nitrogen protection. Heat the mixture at 60 °C for 12 h. After the reaction is completed, cool the reaction mixture to room temperature, dilute it with ethyl acetate, and wash it with water several times. Dry the organic phase with anhydrous magnesium sulfate, filter, evaporate the filtrate to dryness, and pass it through a silica gel column to obtain the final product 7f (4 mg, yield 38%). MALDI-TOF / TOF calculation: C99 H 123 N6O 10 S3Si4 + [M]: 1763.75, measured [M]: 1763.806.

[0105] Example 9: Synthesis of Compound 9f

[0106]

[0107] Weigh the intermediate TQP-1031 (10 mg) and dissolve it in anhydrous DMF (1 mL). Purge the air in the reaction vessel with nitrogen and add an excess of 1-iodobutane (50 μL) under nitrogen protection. Heat the mixture under reflux at 60 °C for 7 days. After the reaction is completed, cool the reaction mixture to room temperature and dilute it with ethyl acetate, then wash it with water several times. Dry the organic phase with anhydrous magnesium sulfate, filter, evaporate the filtrate to dryness, and pass it through a silica gel column to obtain the final product 9f (2 mg, yield 20%). ESI-LR calculation: C 101 H 130 N6O8S3Si4 2+ [M]: 1762.82, measured [M] / 2: 882.3.

[0108] Example 10: Synthesis of Compound 10f

[0109]

[0110] Take Compound 1a (500 mg, 0.912 mmol) and Compound 10b (554 mg, 2.28 mmol) and dissolve them in toluene (10 mL). Purge the air in the reaction vessel with nitrogen and add potassium carbonate (315 mg, 2.28 mmol, dissolved in 2 mL of water) and Pd(PPh3)4 (52.7 mg, 0.046 mmol) under nitrogen protection. Heat and stir under reflux at 110 °C for 12 h. After the reaction is completed, evaporate toluene under reduced pressure, dilute the remaining reaction solution with ethyl acetate, and wash it with water several times. Combine the organic phases and dry them with anhydrous magnesium sulfate, filter, evaporate the filtrate to dryness, and pass it through a silica gel column to obtain the reaction intermediate 10c (260 mg, yield 45.93%).

[0111] Intermediate 10c (100 mg, 0.063 mmol) was dissolved in a mixed solution of dichloromethane and methanol (40 mL of dichloromethane, 10 mL of methanol). Zinc powder (497.71 mg, 7.61 mmol) and NH4Cl (135.73 mg, 2.54 mmol) were added under ice bath conditions. The reaction was carried out at room temperature for 2 hours. After the reaction, it was filtered, the filtrate was concentrated by rotary evaporation, the remaining solid was dissolved in EtOAc, and washed repeatedly with water. The organic phase was concentrated by rotary evaporation. The obtained solid was dissolved in acetic acid, the air in the reaction vessel was displaced by nitrogen, and 1d (20 mg, 0.095 mmol) was added under nitrogen protection. The reaction solution was heated to reflux at 110 °C for 12 h. After the reaction, the reaction solution was cooled to room temperature and diluted with ethyl acetate, washed several times with water, the combined organic phases were dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated by rotary evaporation, and passed through a silica gel column to obtain reaction intermediate 10e (33 mg, two-step yield 28%).

[0112] Intermediate 10e (10 mg) was dissolved in anhydrous DMF (1 mL). The air in the reaction vessel was displaced by nitrogen, and excess CH3I (50 μL, 0.599 mmol) was added under nitrogen protection. The mixture was heated to reflux at 60 °C for 12 h. After the reaction, the reaction mixture was cooled to room temperature and diluted with ethyl acetate, washed several times with water. The organic phase was dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated by rotary evaporation, and passed through a silica gel column to obtain the final product 10f (8 mg, yield 79%). MALDI-TOF / TOF calculation: C 43 H 27 N8S3 + [M]: 751.15, found [M]: 751.234.

[0113] Example 11: Synthesis of Compound 11f

[0114]

[0115] Compound 11a (500 mg, 0.755 mmol) and compound 1b (1.36 g, 1.89 mmol) were dissolved in toluene (10 mL). The air in the reaction vessel was displaced by nitrogen, and potassium carbonate (261 mg, 1.89 mmol, dissolved in 2 mL of water) and Pd(PPh3)4 (43.6 mg, 0.038 mmol) were added under nitrogen protection. It was heated to reflux and stirred at 110 °C for 12 h. After the reaction, toluene was removed by rotary evaporation, the remaining reaction solution was diluted with ethyl acetate, and washed several times with water. The combined organic phases were dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated by rotary evaporation, and passed through a silica gel column to obtain reaction intermediate 11c (510 mg, yield 40%).

[0116] Intermediate 11c (100 mg, 0.059 mmol) was dissolved in a mixed solution of dichloromethane and methanol (40 mL of dichloromethane, 10 mL of methanol). Zinc powder (465.69 mg, 7.13 mmol) and NH4Cl (127.18 mg, 2.38 mmol) were added under ice bath conditions. The reaction was carried out at room temperature for 2 hours. After the reaction was completed, it was filtered, the filtrate was concentrated by rotary evaporation, the remaining solid was dissolved in EtOAc, and washed repeatedly with water. The organic phase was concentrated by rotary evaporation. The obtained solid was dissolved in acetic acid, the air in the reaction vessel was displaced by passing nitrogen, and 1d (18.74 mg, 0.089 mmol) was added under nitrogen protection. The reaction solution was heated to reflux at 110 °C for 12 h. After the reaction was completed, the reaction solution was cooled to room temperature and diluted with ethyl acetate, washed several times with water, the combined organic phases were dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated by rotary evaporation, and passed through a silica gel column to obtain reaction intermediate 11e (36 mg, two-step yield 34%).

[0117] Intermediate 11e (10 mg) was dissolved in anhydrous DMF (1 mL). The air in the reaction vessel was displaced by passing nitrogen, and an excess of CH3I (50 μL, 0.599 mmol) was added under nitrogen protection. The mixture was heated to reflux at 60 °C for 12 h. After the reaction was completed, the reaction mixture was cooled to room temperature and diluted with ethyl acetate, washed several times with water. The organic phase was dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated by rotary evaporation, and passed through a silica gel column to obtain the final product 11f (8 mg, yield 79%). MALDI-TOF / TOF calculation: C 103 H 135 N8O8S3Si4 + [M]: 1819.86, measured [M]: 1820.271.

[0118] Example 12: Synthesis of Compound 12f

[0119]

[0120] Intermediate 1e (10 mg) was dissolved in anhydrous DMF (1 mL). The air in the reaction vessel was displaced by passing nitrogen, and an excess of 1-iodo-3-methoxypropane (118.18 mg, 0.591 mmol) was added under nitrogen protection. The mixture was heated to reflux at 60 °C for 12 h. After the reaction was completed, the reaction mixture was cooled to room temperature and diluted with ethyl acetate, washed several times with water. The organic phase was dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated by rotary evaporation, and passed through a silica gel column to obtain the final product 11f (4 mg, yield 38%). MALDI-TOF / TOF calculation: C 100 H 127 N6O9S3Si4 + [M]: 1763.79, measured [M]: 1763.925.

[0121] Example 13: Synthesis of Compound 13f

[0122]

[0123] Take Compound 1a (500 mg, 0.912 mmol) and Compound 13b (1.18 g, 2.28 mmol) and dissolve them in toluene (10 mL). Pass nitrogen gas to displace the air in the reaction vessel and add potassium carbonate (315 mg, 2.28 mmol, dissolved in 2 mL of water) and Pd(PPh3)4 (52.7 mg, 0.046 mmol) under the protection of nitrogen gas. Heat under reflux with stirring at 110 °C for 12 h. After the reaction is completed, remove toluene by rotary evaporation. Dilute the remaining reaction solution with ethyl acetate and wash it with water several times. Combine the organic phases and dry them over anhydrous magnesium sulfate, filter, evaporate the filtrate to dryness, and pass through a silica gel column to obtain the reaction intermediate 13c (505 mg, yield 48%).

[0124] Take intermediate 13c (100 mg, 0.085 mmol) and dissolve it in a methanol solution. Add zinc powder (670.59 mg, 10.27 mmol) and NH4Cl (183.14 mg, 3.43 mmol) under ice bath conditions. The reaction is carried out at room temperature for 2 hours. After the reaction is completed, filter, evaporate the filtrate to dryness, dissolve the remaining solid in EtOAc, and wash it repeatedly with water. Evaporate the organic phase to dryness. Dissolve the obtained solid in acetic acid, pass nitrogen gas to displace the air in the reaction vessel and add 1d (26.95 mg, 0.128 mmol) under the protection of nitrogen gas. Heat the reaction solution under reflux at 110 °C for 12 h. After the reaction is completed, cool the reaction solution to room temperature and dilute it with ethyl acetate, wash it with water several times. Combine the organic phases and dry them over anhydrous magnesium sulfate, filter, evaporate the filtrate to dryness, and pass through a silica gel column to obtain the reaction intermediate 13e (18 mg, two-step yield 16%).

[0125] Take intermediate 13e (10 mg) and dissolve it in anhydrous DMF (1 mL). Pass nitrogen gas to displace the air in the reaction vessel and add an excess of CH3I (50 μL, 0.599 mmol) under the protection of nitrogen gas. Heat the mixture under reflux at 60 °C for 12 h. After the reaction is completed, cool the reaction mixture to room temperature and dilute it with ethyl acetate, wash it with water several times. Dry the organic phase over anhydrous magnesium sulfate, filter, evaporate the filtrate to dryness, and pass through a silica gel column to obtain the final product 13f (5 mg, yield 49%). MALDI-TOF / TOF calculation: C 75 H 59 N8O8S3 + [M]: 1295.36, measured [M]: 1294.783

[0126] Example 14: Synthesis of Compound 14f

[0127]

[0128] Take Compound 1a (500 mg, 0.912 mmol) and Compound 14b (1.91 g, 2.28 mmol) and dissolve them in toluene (10 mL). Pass nitrogen gas to displace the air in the reaction vessel and add potassium carbonate (315 mg, 2.28 mmol, dissolved in 2 mL of water) and Pd(PPh3)4 (52.7 mg, 0.046 mmol) under nitrogen protection. Heat under reflux with stirring at 110 °C for 12 h. After the reaction is completed, remove toluene by rotary evaporation. Dilute the remaining reaction solution with ethyl acetate and wash it with water several times. Combine the organic phases, dry over anhydrous magnesium sulfate, filter, concentrate the filtrate by rotary evaporation, and pass through a silica gel column to obtain reaction intermediate 14c (734 mg, yield 44%).

[0129] Take intermediate 14c (100 mg, 0.055 mmol) and dissolve it in a methanol solution. Add zinc powder (432.64 mg, 6.63 mmol) and NH4Cl (118.15 mg, 2.21 mmol) under ice bath conditions. The reaction is carried out at room temperature for 2 h. After the reaction is completed, filter, concentrate the filtrate by rotary evaporation. Dissolve the remaining solid in EtOAc and wash it with water repeatedly. Concentrate the organic phase by rotary evaporation. Dissolve the obtained solid in acetic acid. Pass nitrogen gas to displace the air in the reaction vessel and add 1d (17.57 mg, 0.083 mmol) under nitrogen protection. Heat the reaction solution under reflux at 110 °C for 12 h. After the reaction is completed, cool the reaction solution to room temperature, dilute it with ethyl acetate, wash it with water several times. Combine the organic phases, dry over anhydrous magnesium sulfate, filter, concentrate the filtrate by rotary evaporation, and pass through a silica gel column to obtain reaction intermediate 14e (44 mg, two-step yield 42%).

[0130] Take intermediate 14e (10 mg) and dissolve it in anhydrous DMF (1 mL). Pass nitrogen gas to displace the air in the reaction vessel and add an excess of CH3I (50 μL, 0.599 mmol) under nitrogen protection. Heat the mixture under reflux at 60 °C for 12 h. After the reaction is completed, cool the reaction mixture to room temperature, dilute it with ethyl acetate, and wash it with water several times. Dry the organic phase over anhydrous magnesium sulfate, filter, concentrate the filtrate by rotary evaporation, and pass through a silica gel column to obtain the final product 14f (8 mg, yield 79%). MALDI-TOF / TOF calculation: C 99 H 101 N 10 O 26 S3 + [M]: 1941.60, measured [M]: 1941.873

[0131] Example 15: Synthesis of Compound 15f

[0132]

[0133] Compound 15a (543 mg, 0.912 mmol) and compound 1b (1.91 g, 2.28 mmol) were dissolved in toluene (10 mL). Nitrogen was introduced to displace the air in the reaction vessel, and potassium carbonate (315 mg, 2.28 mmol, dissolved in 2 mL of water) and Pd(PPh3)4 (52.7 mg, 0.046 mmol) were added under nitrogen protection. The mixture was heated under reflux at 110 °C with stirring for 12 h. After the reaction was completed, toluene was removed by rotary evaporation. The remaining reaction solution was diluted with ethyl acetate and washed with water several times. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated by rotary evaporation, and passed through a silica gel column to obtain reaction intermediate 15c (765 mg, yield 52%).

[0134] Intermediate 15c (89 mg, 0.055 mmol) was dissolved in a methanol solution. Zinc powder (432.64 mg, 6.63 mmol) and NH4Cl (118.15 mg, 2.21 mmol) were added under ice bath conditions. The reaction was carried out at room temperature for 2 h. After the reaction was completed, the mixture was filtered, and the filtrate was concentrated by rotary evaporation. The remaining solid was dissolved in EtOAc and washed with water repeatedly. The organic phase was concentrated by rotary evaporation. The obtained solid was dissolved in acetic acid. Nitrogen was introduced to displace the air in the reaction vessel, and 1d (17.57 mg, 0.083 mmol) was added under nitrogen protection. The reaction solution was heated under reflux at 110 °C for 12 h. After the reaction was completed, the reaction solution was cooled to room temperature, diluted with ethyl acetate, washed with water several times. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated by rotary evaporation, and passed through a silica gel column to obtain reaction intermediate 15e (43 mg, two-step yield 45%).

[0135] Intermediate 15e (10 mg) was dissolved in anhydrous DMF (1 mL). Nitrogen was introduced to displace the air in the reaction vessel, and an excess of CH3I (50 μL, 0.599 mmol) was added under nitrogen protection. The mixture was heated under reflux at 60 °C for 12 h. After the reaction was completed, the reaction mixture was cooled to room temperature, diluted with ethyl acetate, and washed with water several times. The organic phase was dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated by rotary evaporation, and passed through a silica gel column to obtain the final product 15f (8 mg, yield 79%). MALDI-TOF / TOF calculation: C 97 H 121 N6O8S2SeSi4 + [M]: 1753.69, found [M]: 1754.033.

[0136] Example 16: Synthesis of Compound 16f

[0137]

[0138] Compound 16a (500 mg, 1.3 mmol) and compound 2b (2.33 g, 3.26 mmol) were dissolved in toluene (10 mL). Nitrogen was introduced to displace the air in the reaction vessel, and potassium carbonate (450 mg, 3.26 mmol, dissolved in 2 mL of water) and Pd(PPh3)4 (75.24 mg, 0.065 mmol) were added under nitrogen protection. The mixture was heated under reflux at 110 °C and stirred for 12 h. After the reaction was completed, toluene was removed by rotary evaporation. The remaining reaction solution was diluted with ethyl acetate and washed several times with water. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated by rotary evaporation, and passed through a silica gel column to obtain reaction intermediate 16c (1.1 g, yield 60%).

[0139] Intermediate 16c (100 mg, 0.071 mmol) was dissolved in methanol solution. Zinc powder (558.11 mg, 8.55 mmol) and NH4Cl (152.41 mg, 2.85 mmol) were added under ice bath conditions. The reaction was carried out at room temperature for 2 h. After the reaction was completed, the mixture was filtered, the filtrate was concentrated by rotary evaporation, the remaining solid was dissolved in EtOAc, and washed repeatedly with water. The organic phase was concentrated by rotary evaporation. The obtained solid was dissolved in acetic acid. Nitrogen was introduced to displace the air in the reaction vessel, and 1d (22.70 mg, 0.107 mmol) was added under nitrogen protection. The reaction solution was heated under reflux at 110 °C for 12 h. After the reaction was completed, the reaction solution was cooled to room temperature, diluted with ethyl acetate, and washed several times with water. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated by rotary evaporation, and passed through a silica gel column to obtain reaction intermediate 16e (50 mg, two-step yield 46%).

[0140] Intermediate 16e (10 mg) was dissolved in anhydrous DMF (1 mL). Nitrogen was introduced to displace the air in the reaction vessel, and an excess of CH3I (50 μL, 0.599 mmol) was added under nitrogen protection. The mixture was heated under reflux at 60 °C for 12 h. After the reaction was completed, the reaction mixture was cooled to room temperature, diluted with ethyl acetate, and washed several times with water. The organic phase was dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated by rotary evaporation, and passed through a silica gel column to obtain the final product 16f (8 mg, yield 79%). MALDI-TOF / TOF calculation: C 87 H 103 N8O8SSi4 + [M]: 1531.67, found [M]: 1531.777.

[0141] Test examples:

[0142] Test example 1: Infrared absorption and emission spectra of the synthesized fluorescent probe

[0143] The measured probe was dissolved in dichloroethane to prepare a dichloroethane solution with a concentration of 50 μM, which was placed in a 1-cm-thick quartz cuvette. The absorption spectrum of the probe was measured using a UV-visible-infrared spectrophotometer (UV-2600, Shimadzu Corporation, Japan), and the recorded wavelength range was 500 - 1200 nm. The fluorescence emission spectra of the probe in different solvents were measured on a fluorescence spectrometer (iHR320, HORIBA, Japan) with an excitation wavelength set at 808 nm, and the recorded wavelength range was 820 - 1500 nm. The structures of the reference compounds TQP-934 and TQP-1036 are shown in the following figure:

[0144]

[0145] The test results are as Figure 1 shown. It can be seen from the figure that compared with TQP-934 and TQP-1036 with a 6,7-bis(pyridyl)-[1,2,5]thiadiazolo[3,4-g]quinoxaline central ring, the absorption and emission of the compounds TQP-1031 and TQP-1196 with a pyridinium salt central ring structure in this application have undergone a red shift.

[0146] Test Example 2: Near-infrared II region microscopic imaging diagrams of TQP-1031 and TQP1196

[0147] Experimental method:

[0148] The cell lines selected for this experiment were 143B and 3T3. Among them, 143B is a human osteosarcoma cell, and 3T3 is a mouse embryonic fibroblast. The above cells were all purchased from ATCC. The subcultured 3T3 and 143B cells were inoculated into a 3.5-mm-sized culture dish. After the cells were cultured to a good state, the culture medium was changed. The fluorescent probes TQP-1031 and TQP-1196 were added to make the final concentration of the probes approximately 10 mM; incubated for about 1 h; after incubation, the culture medium was aspirated; washed three times with PBS buffer to wash away the unlabeled probes, and new culture medium was added. A near-infrared deep-cooled liquid nitrogen fluorescence microscope was used to observe the intensity of the near-infrared second-region fluorescent probe signal in the range of 400 - 1700 nm. The near-infrared second-region high-magnification microscopic imaging instrument used an InGaAs camera with a pixel of 640×512, and the imaging started after the camera was cooled to -190 °C by a liquid nitrogen cooling system. The excitation laser in the imaging instrument was generated by an 808-nm laser diode. The filter used was a 1100-nm LP. The imaging software used was Lightfield, and the post-image processing software was Image J. The microscopic magnification was 20 times.

[0149] Experimental results:

[0150] As Figure 2As shown, TQP-1031 binds most strongly to 143B with the strongest fluorescence signal; while its binding to 3T3 is less, indicating that TQP-1031 has the ability to selectively bind to tumor cells. On the other hand, it was found that the binding ability of TQP-934 to 143B was weak, and the fluorescence signal could be ignored. On the other hand, due to the low fluorescence quantum yield of TQP-1196, the fluorescence microscopic signals were relatively weak. However, it can also be seen that TQP-1196 binds to cells, while its control probe TQP-1036 has no signal, which indicates that the positive charges carried by TQP-1031 and TQP-1196 play a key role in the tumor cell targeting ability of this molecule.

[0151] Test Example 3: Distribution experiment of TQP-1031 in BALB / c mice in the second near-infrared region at 1100 nm at different time points Experimental method:

[0152] In vivo imaging of mice was performed using a second near-infrared in vivo imaging instrument (MARS, Hengguang Zhiying). The second near-infrared in vivo imaging instrument uses a 640×512 pixel InGaAs camera, which starts working after being cooled to -80 °C by a water cooling system. The excitation laser in the imaging instrument is generated by an 808 nm laser diode. The filter used for imaging is 1100 nm LP. The imaging software used is Lightfield, and the post-image processing software is Image J. The imaging mice are BALB / c mice, which need to be depilated first. Calculate the mass of the fluorescent probe required according to a body weight of 12 mg / kg. The mice need to be anesthetized during imaging. Dissolve the fluorescent probe in 200 μL of PBS (pH = 7.4, containing 10 μL of DMSO) and inject it via the tail vein. Then immediately perform imaging, and then repeat sampling at the target data acquisition time points.

[0153] Experimental results:

[0154] As Figure 3 shown, through the whole-body images of mice in the supine position selected at different time points (0 h, 2 h, 6 h, 24 h, 48 h), it can be found that when the probe is just injected into the mice, the probe enters the liver through the vascular circulatory system and then accumulates in the liver. After 24 h, fluorescent signals appear in the intestine and spleen.

[0155] Test Example 4: Use of fluorescent probe for surgical navigation

[0156] Experimental method:

[0157] The imaging mice were 4T1 tumor-bearing mice. The mice needed to be anesthetized before imaging. The fluorescent probe was dissolved in 10 μL PBS (pH = 7.4, containing 1 μL DMSO), and directly injected into the tumor. Wait for 2 h to allow the probe to fully diffuse in the tumor site, and then perform surgical resection under fluorescence guidance. The exposure time was 50 ms, and all other imaging methods were the same as in Test Example 3.

[0158] Experimental results:

[0159] Successfully performed as Figure 4 shown, successfully performed 4T1 subcutaneous tumor resection and lymph node dissection under NIR-II fluorescence guidance.

[0160] Test Example 5: Cytotoxicity of the fluorescent probe

[0161] Experimental method:

[0162] Add 200 μL of cell suspension to a 96-well plate, place it in a constant temperature and humidity incubator for 12 h to allow it to adhere to the wall, and then replace it with 200 μL of drug solutions with different concentration gradients prepared with basal medium. Set up a blank control group and a PBS control group; after adding the drug for 24 h, aspirate the supernatant in the 96-well plate, and add 20 μL of 5 mg mL -1 MTT solution to each well, and let it stand for 4 h; then aspirate the MTT solution with a pipette, and add 150 μL of DMSO solution to each well. Detect with an enzyme-labeled instrument. Set the oscillation time to 10 min, and then measure the absorbance value at 490 nm. Calculate the cell viability according to the following formula:

[0163]

[0164] Among them, A sample refers to the absorbance of the experimental group with different concentrations of the probe added, while A control is the absorbance of the control group without the probe, and A blank is the absorbance of the blank group without the probe and cells. At least three replicate experiments were set for each concentration gradient to achieve data credibility.

[0165] Experimental results:

[0166] As Figure 5 shown, TQP-1031 has certain growth inhibitory activity against 143B cells, while it has no cell inhibitory activity against 3T3 cells in the concentration range of 1 - 50 μM.

[0167] Test Example 6: Study on the photothermal properties of the fluorescent probe

[0168] Experimental method:

[0169] Dissolve TQP-934, TQP-1031, TQP-1036, and TQP-1196 in dimethyl sulfoxide to prepare a 50 μM solution. Take 50 μL and place it in an EP tube. Place the EP tube in the MARS imaging system. Continuously irradiate it with an 808 nm laser for 300 s, and the laser power is 1 W / cm 2 , and the temperature curve is accurately recorded by a FLIR E95 thermal imaging camera.

[0170] Experimental results:

[0171] As Figure 6 shown, the temperatures of the TQP-1031 and TQP-934 solutions can both reach above 40 °C after irradiation for 300 s, and the temperatures of the TQP-1196 and TQP-1036 solutions can both reach above 50 °C after irradiation for 300 s. Therefore, they all have the potential to be used as photothermal therapy agents.

[0172] Test Example 7: Tumor suppression activity of fluorescent probes

[0173] Experimental method:

[0174] Digest 143B cells with trypsin and prepare a cell suspension of about 5×10 6 mL -1 . Take female nude mice and subcutaneously inoculate 150 μL of the cell suspension into the forelimbs, and observe the tumor formation. When the tumor diameter reaches about 3 mm, inject TQP-1031 (dissolved in 50 μL of PBS containing 5 μL of DMSO) into the tumors of tumor-bearing mice at a dose of 3 mg / Kg on the 2nd, 4th, 6th, 8th, 10th, 12th, 14th, 16th, 18th, and 20th days. As a control, untreated tumor-bearing mice are intravenously injected with the same dose of PBS solution on the same days as the treated mice. After each intravenous injection, measure the length and width of the oval tumor with a vernier caliper. Calculate the tumor growth according to the ratio of the final volume (FV) minus the initial volume (IV) to the initial volume (IV). Each group has 3 mice. The volume is calculated according to the following formula:

[0175]

[0176] where l is the length of the tumor and w is the width of the tumor.

[0177] Experimental results:

[0178] As Figure 7 shown, TQP-1031 has chemotherapeutic activity against 143B subcutaneous tumors. Therefore, it can be developed into an antitumor chemotherapy drug.

Claims

1. A compound represented by the following general formula I: In general formula I: X is S, O or Se; C1 and C2 are each independently provided that they are not both R 1 is a straight key, wherein, R 4 and R 5 each independently selected from H, hydroxy, amino, carboxyl, sulfonic acid group, halogen, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C8 alkoxy, substituted or unsubstituted C6-C 10 aryl or C5-C 10 heteroaryl; R 6 and R 7 each independently selected from H, hydroxy, amino, carboxyl, sulfonic acid group, substituted or unsubstituted C1-C8 alkyl, C1-C8 alkoxy, halogen, or R 6 and R 7 together with the adjacent carbon atom form a 5-10 membered N, P or O-containing heterocyclic group; Y is S, O, Se or N; wherein in the case of substitution, the substituent is selected from amino and hydroxy; R 2 selected from substituted or unsubstituted C1-C8 alkyl groups, wherein in the case of substitution, the substituents are selected from amino, hydroxy, carboxy, C1-C6 alkoxy and sulfo groups; D is selected from the following groups: wherein, in the above formulas, R 8 -R 41 each independently selected from: -H; amino; halogen; C1-C8 alkoxy; C1-C8 alkyl acyloxy; C1-C8 alkyl substituted or unsubstituted with silyl, hydroxy, amino, alkynyl, azide, mercapto, halogen, carboxyl, trimethylsilyl C1-C4 alkoxycarbonyl; --(CH2)n1-(OCH2CH2)n2-OR, wherein n1 and n2 are each independently an integer from 1 to 500, and R is selected from C1-C8 alkyl, amino, mercapto, halogen; - wherein R' and R" are C1-C8 alkylene groups A is a cyclic peptide, prostate-specific membrane antigen, octreotide, monosaccharide or polysaccharide group, and the monosaccharide is selected from glucose, galactose, fructose, arabinose, rhamnose, ribose, lactose and maltose; B is a molecular group containing an Fv segment, and the molecule containing an Fv segment is selected from monoclonal antibody, bispecific antibody or single-chain Fv segment; - Among them, M is selected from 64 Cu, 68 Ga, 177 Lu, Gd, 99m Tc and Al; R''' is C1-C 12 alkylene or -(CH2)n3-(OCH2CH2)n4-, where n3 is an integer from 0 to 100 and n4 is an integer from 1 to 100. In the definitions of the above substituents, represents the bonding position.

2. The compound according to claim 1, wherein, A is a cyclic peptide, and the cyclic peptide is selected from c(RGDyk), c(RGDfk) and c(RADyk).

3. The compound according to claim 1, wherein, In General Formula I, one of C1 and C2 is the other is R 1 is Y is S, O, Se or N; R 6 and R 7 are selected from H and amino-substituted C1-C4 alkyl, or R 6 and R 7 together with the adjacent carbon atom form a 5-10 membered oxygen-containing heterocyclic group; R 2 is a C1-C4 alkyl group which is unsubstituted or substituted by a hydroxyl group, a carboxyl group or a methoxy group; D is selected from 4. The compound according to claim 1, wherein, One of C1 and C2 is The other is Or both C1 and C2 are And / or R 1 For 5. The compound according to claim 1, wherein The compound is selected from the following compounds:

6. A method for preparing the compound according to any one of claims 1 to 4, and the reaction route is as follows: In the above reaction formula, Z is a boronic acid group or a boronic acid ester group, or D-Z is a stannous salt of the formula ; X, R 1 , D, R 2 , C1 and C2 are defined in the same way as any one of claims 1-4 respectively, The method comprises the following steps: a. Compound 1 and compound 2 undergo a nucleophilic substitution reaction to obtain intermediate 3; b. Intermediate 3 undergoes a reduction reaction to obtain intermediate 4; c. Intermediate 4 and compound 5 undergo ring closure to obtain compound 6; d, Compound 6 and R 2 Compound I is obtained by alkylation of I to give a compound of general formula I.

7. A pharmaceutical composition comprising the compound according to any one of claims 1-5 and a pharmaceutically acceptable excipient.

8. Use of the compound according to any one of claims 1-5 or the pharmaceutical composition according to claim 7 in the preparation of a mitochondrial targeting preparation.

9. The use according to claim 8, wherein, The mitochondrial targeting preparation is a mitochondrial targeting anti-cancer preparation.

10. Use of the compound according to any one of claims 1-5 or the pharmaceutical composition according to claim 7 in the preparation of a fluorescent probe, a photothermal therapeutic agent or an anti-tumor chemotherapeutic agent.

11. The use according to claim 10, wherein, The fluorescent probe is used as a diagnostic agent or an imaging agent.

12. According to the use described in claim 11, wherein The diagnostic agent or imaging agent is a tumor diagnostic agent or a tumor imaging agent.

13. The use according to claim 10, wherein, The tumor is selected from osteosarcoma, breast cancer, colorectal cancer, lung cancer.

Images