Preparation method and application of liver cancer targeted diagnosis and treatment probe IR-BTOGP-GPC3 compound

Abstract

The invention belongs to the technical field of hepatocellular carcinoma targeting molecules, and particularly relates to a preparation method and application of a hepatocellular carcinoma targeting diagnosis and treatment probe IR-BTOGP-GPC3 compound. The IR-BTOGP-GPC3 compound provided by the invention has a structure as shown in a formula 1, wherein n in the formula 1 is 10-60. The IR-BTOGP-GPC3 compound can be used for accurately identifying and diagnosing hepatocellular carcinoma, and has excellent phototherapy performance; while the intramolecular energy dissipation can be effectively reduced, good key molecular characteristics, including tissue permeation, photo-thermal efficiency and fluorescence quantum yield, are achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of hepatocellular carcinoma targeted molecules, and specifically relates to a preparation method and application of a liver cancer targeted diagnostic and therapeutic probe IR-BTOGP-GPC3 compound. Background Art

[0002] In the near-infrared II (NIR-II) spectral region (1000-1700 nm), phototherapy molecules based on NIR-II have excellent tissue penetration depth, minimal autofluorescence interference, and high spatial resolution. These remarkable optical properties make them very suitable for addressing challenging clinical scenarios, such as deep-seated and refractory tumors. Currently, in the field of precise tumor diagnosis and treatment, the development of integrated phototherapy probes that combine NIR-II fluorescence imaging with photothermal therapy (PTT) / photodynamic therapy (PDT) is a research hotspot. In the existing technology, there is no phototherapy probe that can accurately identify and diagnose hepatocellular carcinoma. Summary of the Invention

[0003] The present invention aims to provide a method for preparing and applying the IR-BTOGP-GPC3 compound, a targeted diagnostic and therapeutic probe for liver cancer. The IR-BTOGP-GPC3 compound provided by the present invention can accurately identify and diagnose hepatocellular carcinoma and exhibits excellent phototherapeutic properties. It can effectively reduce intramolecular energy dissipation while also possessing excellent key molecular properties, including tissue penetration, photothermal efficiency, and fluorescence quantum yield.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] An IR-BTOGP-GPC3 compound having the structure shown in Formula 1:

[0006]

[0007] In formula 1, n is 10 to 60.

[0008] The present invention provides a method for preparing the IR-BTOGP-GPC3 compound described in the above technical solution, comprising the following steps:

[0009] The IR-BTOG compound, sodium azide and an organic solvent are mixed to perform an azide reaction to obtain an IR-BTOGP compound;

[0010] The IR-BTOGP compound, copper (I)-thiophene-2-carboxylate, alkynone-polyethylene glycol-GPC3, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine and an organic solvent are mixed to perform a superposition-alkyl click cyclization reaction to obtain the IR-BTOGP-GPC3 compound with the structure represented by Formula 1;

[0011] The IR-BTOG compound has a structure shown in Formula 2, and the IR-BTOGP compound has a structure shown in Formula 3;

[0012]

[0013] Preferably, the preparation method of the IR-BTOG compound comprises the following steps:

[0014] Compound 7, n-butyl lithium solution, tributyltin chloride and an organic solvent are mixed to undergo a nucleophilic reaction to obtain compound 8;

[0015] The compound 8, 4,7-dibromobenzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole), bistriphenylphosphine palladium dichloride and an organic solvent are mixed to carry out a first reaction to obtain the IR-BTOG compound;

[0016] The compound 7 has a structure shown in Formula 4; the compound 8 has a structure shown in Formula 5;

[0017]

[0018] Preferably, the molar ratio of the IR-BTOG compound to sodium azide is 0.058:0.72.

[0019] Preferably, the mass ratio of the IR-BTOGP compound to alkynone-polyethylene glycol-GPC3 is 85-87:159; the mass ratio of the IR-BTOGP compound to copper (I)-thiophene-2-carboxylate is 85-87:10; the mass ratio of the IR-BTOGP compound to tri[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine is 85-87:5.

[0020] The present invention provides the use of the IR-BTOGP-GPC3 compound described in the above technical solution or the IR-BTOGP-GPC3 compound obtained by the preparation method in the preparation of a preparation for specific targeted identification and diagnosis of hepatocellular carcinoma.

[0021] The present invention provides the use of the IR-BTOGP-GPC3 compound described in the above technical solution or the IR-BTOGP-GPC3 compound obtained by the preparation method in the preparation of anti-hepatocellular carcinoma targeted drugs.

[0022] The present invention provides an anti-hepatocellular carcinoma targeted drug, comprising the IR-BTOGP-GPC3 compound described in the above technical solution or the IR-BTOGP-GPC3 compound obtained by the preparation method.

[0023] The present invention provides the use of the IR-BTOGP-GPC3 compound described in the above technical solution or the IR-BTOGP-GPC3 compound obtained by the preparation method in the preparation of near-infrared II light diagnostic and therapeutic probes.

[0024] The present invention provides a near-infrared II phototherapy probe, comprising the IR-BTOGP-GPC3 compound described in the above technical solution or the IR-BTOGP-GPC3 compound obtained by the preparation method.

[0025] The present invention provides an IR-BTOGP-GPC3 compound having the structure shown in Formula 1, where n is 10 to 60. The present invention couples the IR-BTOG compound with a GPC3 peptide to obtain an IR-BTOGP-GPC3 compound, wherein the IR-BTOG compound has excellent long-wavelength penetrance and fluorescence properties, and the GPC3 peptide is a specific targeting molecule. The IR-BTOGP-GPC3 compound provided by the present invention has excellent tissue penetration depth, excellent photothermal conversion efficiency, and a NIR-II fluorophore with high fluorescence quantum yield, and can accurately identify and diagnose hepatocellular carcinoma, with excellent targeted diagnostic and therapeutic effects for hepatocellular carcinoma.

[0026] At the same time, the excellent phototherapeutic properties of the IR-BTOGP-GPC3 compound provided by the present invention can provide new opportunities for the application of precision phototherapy in hepatocellular carcinoma and provide an important structural design basis for the future development of NIR-II phototherapy preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Flow chart for preparing IR-BTOGP-GPC3 compound for example;

[0028] Figure 2 The H NMR spectrum of compound 4 prepared in Example ( 1 H NMR) spectra;

[0029] Figure 3 The C NMR spectrum of compound 4 prepared in Example ( 13 C NMR) spectra;

[0030] Figure 4 This is a high resolution mass spectrum (HRMS) of compound 4 prepared in Example;

[0031] Figure 5 The H NMR spectrum of compound 7 prepared in Example ( 1 H NMR) spectra;

[0032] Figure 6 The C NMR spectrum of compound 7 prepared in Example ( 13 C NMR) spectra;

[0033] Figure 7 This is a high resolution mass spectrum (HRMS) of compound 7 prepared in Example;

[0034] Figure 8 The H NMR spectrum of the IR-BTOGP compound prepared in Example ( 1 H NMR) spectra;

[0035] Figure 9 The C NMR spectrum of the IR-BTOGP compound prepared in this example ( 13 C NMR) spectra;

[0036] Figure 10 High resolution mass spectrometry (HRMS) of the IR-BTOGP compound prepared in Example;

[0037] Figure 11 The H NMR spectrum of the IR-BTOGP-GPC3 compound prepared in Example ( 1 H NMR) spectra;

[0038] Figure 12 The C NMR spectrum of the IR-BTOGP-GPC3 compound prepared in Example ( 13 C NMR) spectra;

[0039] Figure 13 Transmission electron microscopy (TEM) image of the IR-BTOGP-GPC3 compound prepared in Example;

[0040] Figure 14 The dynamic light scattering (DLS) graph of the IR-BTOGP-GPC3 compound prepared in Example 1 and the relationship between size and PDI over time;

[0041] Figure 15 This is the spectrum of the IR-BTOGP-GPC3 compound prepared in Example;

[0042] Figure 16 Graph showing the evaluation of intracellular phototherapy effects of the compounds prepared in Examples and Comparative Examples;

[0043] Figure 17 The effect diagram of in vivo NIR-II fluorescence imaging of the compounds prepared in Examples and Comparative Examples;

[0044] Figure 18 The figures show the in vivo anti-tumor efficacy of the compounds prepared in Examples and Comparative Examples. DETAILED DESCRIPTION

[0045] The present invention provides an IR-BTOGP-GPC3 compound having the structure shown in Formula 1:

[0046]

[0047] In Formula 1, n can be 10 to 60. In a specific embodiment of the present invention, n is 24.

[0048] The present invention provides a method for preparing the IR-BTOGP-GPC3 compound described in the above technical solution, comprising the following steps:

[0049] The IR-BTOG compound, sodium azide and an organic solvent are mixed to perform an azide reaction to obtain an IR-BTOGP compound;

[0050] The IR-BTOGP compound, copper (I)-thiophene-2-carboxylate, alkynone-polyethylene glycol-GPC3, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine and an organic solvent are mixed to perform a superposition-alkyl click cyclization reaction to obtain the IR-BTOGP-GPC3 compound with the structure represented by Formula 1;

[0051] The IR-BTOG compound has a structure shown in Formula 2, and the IR-BTOGP compound has a structure shown in Formula 3;

[0052]

[0053]

[0054] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0055] In the present invention, an IR-BTOG compound, sodium azide, and an organic solvent are mixed to perform an azide reaction to obtain an IR-BTOGP compound. In one embodiment of the present invention, the molar ratio of the IR-BTOG compound to sodium azide can be 0.058:0.72, and the organic solvent can be DMF. The present invention has no particular requirements for the amount of the organic solvent used, as long as the azide reaction proceeds smoothly.

[0056] In one embodiment of the present invention, the temperature of the azide reaction can be 65-75°C, specifically 70°C, and the reaction time can be 4-6 hours, specifically 5 hours. After the azide reaction, an azide reaction solution is obtained. In the present invention, the azide reaction solution is preferably post-treated to obtain the IR-BTOGP compound. The post-treatment preferably includes: diluting the azide reaction solution with water, extracting the resulting diluted reaction solution with ethyl acetate to obtain an organic phase; drying the organic phase with a desiccant and removing the solvent to obtain a crude product; and purifying the crude product by column chromatography to obtain the IR-BTOGP compound. In the present invention, the extraction can be performed twice, and the organic phases from each extraction are combined and dried. The desiccant used for drying can be magnesium sulfate. The solvent removal method can be vacuum evaporation. The column chromatography purification can be flash chromatography, and the eluting solvents can be dichloromethane (DCM) and methanol (MeOH), with the volume ratio of DCM to MeOH being 5:1.

[0057] After obtaining the IR-BTOGP compound, the present invention mixes the IR-BTOGP compound, copper (I)-thiophene-2-carboxylate, alkynone-polyethylene glycol-GPC3, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) and an organic solvent, and performs a superposition-alkyl click cyclization reaction to obtain the IR-BTOGP-GPC3 compound having the structure shown in Formula 1. As one embodiment of the present invention, the alkynone-polyethylene glycol-GPC3 can be alkynone-polyethylene glycol 1000-GPC3 (Alkyne-PEG1000-GPC3), and the alkynone-polyethylene glycol 1000-GPC3 is purchased from Sichuan Ruixi Biological Co., Ltd. The copper (I) -thiophene-2-carboxylate may be copper (I) thiophene-2-carboxylate (CuTc); the mass ratio of the IR-BTOGP compound to alkynone-polyethylene glycol-GPC3 may be 85 to 87:159, specifically 86:159; the mass ratio of the IR-BTOGP compound to copper (I) -thiophene-2-carboxylate may be 85 to 87:10, specifically 86:10; the mass ratio of the IR-BTOGP compound to tris [(1-benzyl-1H-1,2,3-triazol-4-yl)methyl] amine may be 85 to 87:5, specifically 86:5; the organic solvent may be THF. The present invention has no special requirements for the amount of the organic solvent, as long as the superposition-alkyl click cyclization reaction is carried out smoothly.

[0058] In one embodiment of the present invention, the temperature of the superposition-alkyl click cyclization reaction can be 15-30°C, the time can be 0.5 hours, and the superposition-alkyl click cyclization reaction is carried out under stirring. In the present invention, the superposition-alkyl click cyclization reaction produces a cyclization reaction solution. The cyclization reaction solution is preferably post-treated to obtain the IR-BTOGP-GPC3 compound with the structure represented by Formula 1. The post-treatment preferably includes: filtering the cyclization reaction solution through diatomaceous earth to obtain a liquid product; removing the solvent from the liquid product to obtain a crude product; purifying the crude product by column chromatography to obtain a primary purified product; and recrystallizing the primary purified product from methyl tert-butyl ether to obtain the IR-BTOGP-GPC3 compound with the structure represented by Formula 1. In the present invention, the solvent removal method can be vacuum evaporation. The elution solvents used in the column chromatography can be DCM and MeOH, and the volume ratio of DCM to MeOH can be 5:1. The present invention has no particular requirements for the specific method of recrystallization.

[0059] As an embodiment of the present invention, the preparation method of the IR-BTOG compound comprises the following steps:

[0060] Compound 7, n-butyl lithium solution, tributyltin chloride and an organic solvent are mixed to undergo a nucleophilic reaction to obtain compound 8;

[0061] The compound 8, 4,7-dibromobenzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole), bistriphenylphosphine palladium dichloride and an organic solvent are mixed to carry out a first reaction to obtain the IR-BTOG compound;

[0062] The compound 7 has a structure shown in Formula 4; the compound 8 has a structure shown in Formula 5;

[0063]

[0064] The present invention mixes compound 7, an n-butyllithium (n-BuLi) solution, tributyltin chloride, and an organic solvent to perform a nucleophilic reaction to obtain compound 8. As one embodiment of the present invention, the n-butyllithium solution is obtained by mixing n-butyllithium and hexane, and the concentration of the n-butyllithium solution is 1.6M; the molar ratio of compound 7, n-butyllithium, and tributyltin chloride is 1.048:1.29:1.29; and the organic solvent can be THF.

[0065] As one embodiment of the present invention, mixing compound 7, n-butyllithium solution, tributyltin chloride, and an organic solvent may include: adding the n-butyllithium solution dropwise to a THF solution of compound 7 at -78°C under nitrogen to carry out a first reaction; then adding tributyltin chloride to carry out a second reaction; the first reaction is carried out with stirring at a temperature of -78°C for 2 hours; and the second reaction is carried out with stirring at a temperature of 15-30°C for 1 hour. As one embodiment of the present invention, after the nucleophilic reaction, a nucleophilic reaction mixture is obtained. In the present invention, the nucleophilic reaction mixture is preferably post-treated to obtain compound 8. The post-treatment preferably includes: pouring the nucleophilic reaction mixture into water, extracting the resulting reaction solution with ethyl acetate to obtain an organic phase; drying the organic phase with a desiccant and removing the solvent to obtain compound 8. In the present invention, the extraction may be performed twice, and the organic phases from each extraction are combined and dried. The desiccant used for drying may be magnesium sulfate, and the solvent removal method may be vacuum evaporation.

[0066] After obtaining compound 8, the present invention mixes compound 8, 4,7-dibromobenzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole) (BBTD), bistriphenylphosphine palladium dichloride (Pd(PPh3)2Cl2), and an organic solvent to perform a first reaction to obtain the IR-BTOG compound. As one embodiment of the present invention, the molar ratio of compound 8, 4,7-dibromobenzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole), and bistriphenylphosphine palladium dichloride can be 0.81:0.27:0.048; and the organic solvent can be xylene.

[0067] As one embodiment of the present invention, the temperature of the first reaction can be 130°C, and the holding time can be 10 hours. The first reaction is carried out under stirring. After the first reaction, a first reaction mixture is obtained. In the present invention, the first reaction mixture is preferably post-treated to obtain the IR-BTOG compound. The post-treatment preferably includes: cooling the first reaction mixture to room temperature, pouring it into water, extracting the resulting reaction solution with ethyl acetate to obtain an organic phase; drying the organic phase with a desiccant and removing the solvent to obtain a crude product; and purifying the crude product by column chromatography to obtain pure IR-BTOG. In the present invention, the extraction can be performed twice, and the organic phases from each extraction are combined and dried. The desiccant used for drying can be magnesium sulfate. The solvent removal method can be vacuum evaporation. The column chromatography purification can be silica gel column chromatography. The eluents for the column chromatography can be DCM and ethyl acetate (EA), and the volume ratio of DCM to EA can be 10:1.

[0068] The present invention provides the use of the IR-BTOGP-GPC3 compound described in the above technical solution or the IR-BTOGP-GPC3 compound obtained by the preparation method in the preparation of a preparation for specific targeted identification and diagnosis of hepatocellular carcinoma.

[0069] The present invention provides the use of the IR-BTOGP-GPC3 compound described in the above technical solution or the IR-BTOGP-GPC3 compound obtained by the preparation method in the preparation of anti-hepatocellular carcinoma targeted drugs.

[0070] The present invention provides an anti-hepatocellular carcinoma targeted drug, comprising the IR-BTOGP-GPC3 compound described in the above technical solution or the IR-BTOGP-GPC3 compound obtained by the preparation method.

[0071] The present invention provides the use of the IR-BTOGP-GPC3 compound described in the above technical solution or the IR-BTOGP-GPC3 compound obtained by the preparation method in the preparation of near-infrared II light diagnostic and therapeutic probes.

[0072] The present invention provides a near-infrared II phototherapy probe, comprising the IR-BTOGP-GPC3 compound described in the above technical solution or the IR-BTOGP-GPC3 compound obtained by the preparation method.

[0073] Figure 1 Flow chart of the preparation of IR-BTOGP-GPC3 compound in the embodiment of the present invention. In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present invention.

[0074] Example 1

[0075] (1) Preparation of Compound 2:

[0076] 2-Bromo-1,3-di((6-bromohexyl)oxy)benzene (Compound 2) was synthesized according to the literature “Wan, H.; Yue, J.; Zhu, S.; Uno, T.; Zhang, X.; Yang, Q.; Yu, K.; Hong, G.; Wang, J.; Li, L.; et al. A bright organic NIR-II nanofluorophore for three-dimensional imaging into biological tissues. Nat. Commun. 2018, 9, 1171”.

[0077] (2) Preparation of compound 4: Under nitrogen protection, Pd(PPh3)4 (269.2 mg, 0.233 mmol) was added to a xylene (15 mL) solution containing compound 2 (1.2 g, 2.35 mmol) and tributyl(2-thienyl)tin (compound 3) (878 mg, 2.35 mmol), and the mixture was stirred at 130°C for 3 h. The obtained product system was cooled to room temperature and poured into water, extracted twice with ethyl acetate, the organic phase was collected, dried over MgSO4, filtered, and the obtained filtrate was vacuum evaporated to remove the solvent. The obtained crude product was chromatographed on a silica gel column using PE and EA as the eluting solvents, with the volume ratio of PE to EA being 5:1. Compound 4 was obtained as a white solid (1.08 g, 88.6%). Compound 4 is 2-(2,6-bis((6-bromohexyl)oxy)phenyl)thiophene. H NMR spectrum of compound 4 ( 1 HNMR) images Figure 2 As shown; NMR carbon spectrum ( 13 C NMR) Figure 3 As shown; High resolution mass spectrometry (HRMS) is shown Figure 4 shown.

[0078] 1 H NMR (500MHz, CDCl3) δ7.40(d,J=3.5Hz,1H),7.29(d,J=5.1Hz,1H),7.10(s,1H),7.02(dd,J=13.3,9.3Hz,1H),6.54(d,J=8.3Hz, 1H),6.45(s,1H),3.95-3.92(m,4H),3.34(d,J=8.0Hz,4H),1.80(dd,J=14.3,7.1Hz,8H),1.48-1.46(m,4H),1.39-1.37(m,4H).

[0079] 13 C NMR (500MHz, CDCl3) δ157.12,156.73,133.98,128.70,128.46,128.04,125.63,125.05,113.02,105.77,105.34,102.17,77. 29,77.04,76.79,74.15,69.06,68.79,68.58,33.90,33.87,32.70,32.66,28.97,28.95,27.87,27.79,25.32,25.27,13.63.

[0080] HRMS(ESI)calcd for C 22 H 31 O2Br2S2+ ,([M+H + ])519.0385,Found 519.0372

[0081] (3) Preparation of compound 7: Under nitrogen protection, 1-bromopyrrolidine-2,5-dione (383.8 mg, 2.16 mmol) was added to a DMF (15 mL) solution containing compound 4 (1.12 g, 2.16 mmol). The mixture was stirred at -8°C for 1 h, and then heated to room temperature and stirred for 4 h. The resulting reaction system was poured into water and extracted twice with ethyl acetate. The organic phase was collected, dried over MgSO4, and filtered. The filtrate was evaporated in vacuo to remove the solvent. The crude product was chromatographed on a silica gel column using DCM and MeOH as the eluting solvents. The volume ratio of DCM to MeOH was 5:1. 2-(2,6-bis((6-bromohexyl)oxy)phenyl)-5-bromothiophene (compound 5) was obtained as a light yellow oil (1.01 g, 78.9%). Under nitrogen protection, compound 5 (1 g, 1.67 mmol) and tributyl (3-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)thiophen-2-yl)stannane (compound 6) (895 mg, 1.67 mmol) were dissolved in xylene (15 mL), and Pd (PPh 3 ) 4 (193.49 mg, 0.167 mmol) was added to the reaction system. The reaction was stirred at 130 ° C for 3 h. The resulting product system was cooled to room temperature and poured into water. The reaction mixture was dried with ethyl acetate. Extract twice, collect the organic phase, dry it over MgSO4, filter, and vacuum evaporate the filtrate to remove the solvent. The crude product is chromatographed on a silica gel column using DCM and MeOH as the eluting solvents in a volume ratio of 5:1. Compound 7 is obtained as a brown-black oil (0.81 g, 63.9%). Compound 7 is 5'-(2,6-bis((6-bromohexyl)oxy)phenyl)-3-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2,2'-dithiophene. The H NMR spectrum of compound 7 ( 1 HNMR) images Figure 5 As shown; NMR carbon spectrum ( 13 CNMR) Figure Figure 6 As shown; High resolution mass spectrometry (HRMS) is shown Figure 7 shown.

[0082] 1H NMR(500MHz,CDCl3)δ7.34-7.20(m,2H),7.08(t,J=8.3Hz,1H),6.99-6.92(m,1H),6.80(t,J=4.8Hz,1H),6.53(d,J=3.7Hz,1H),4.21-4.17(m,2H),3.97-3.87(m,4H),3.82-3.78(m,2H),3.65(dd,J=5.8,3.7Hz,2H),3.58-3.53(m,4H),3.45-3.42(m,2H),3.30-3.25(m,7H),1.74(dd,J=8.8,7.0Hz,6H),1.39(dd,J=9.3,6.0Hz,6H),1.21-1.17(m,4H)。

[0083] 13 C NMR(126MHz,CDCl3)δ157.10,157.01,156.61,154.44,152.24,152.17,151.93,136.56,135.93,134.55,133.62,133.21,132.96,132.31,131.77,130.98,130.03,129.81,129.39,129.06,128.58,128.49,128.48,126.42,125.16,124.49,124.01,122.51,122.19,122.05,121.49,121.06,119.86,119.74,118.80,118.33,118.10,117.07,116.43,113.68,113.15,109.49,109.31,105.47,105.30,105.25,77.38,77.13,76.87,72.86,71.95,71.92,71.77,70.97,70.90,70.82,70.73,70.69,70.67,70.63,70.57,70.05,69.97,69.84,69.10,68.91,60.43,59.04,34.00,33.96,33.90,33.88,32.71,32.66,32.53,31.96,31.47,30.21,29.89,29.73,29.05,28.90,27.88,27.81,26.60,25.55,25.44,25.34,25.10,22.73,21.10,14.24,14.18。

[0084] HRMS(ESI)calcd for C 33 H 47 O6Br2S2 + ,([M+H + ])763.1154,Found 763.1145.

[0085] (4) Preparation of IR-BTOG compound:

[0086] Compound 7 (800 mg, 1.048 mmol) was added to THF (15.00 mL), and n-BuLi solution (solvent: hexane, concentration: 1.6 M, volume: 806 μL, 1.29 mmol) was added dropwise at -78 ° C under nitrogen protection; after stirring at -78 ° C for 2 h, tributyltin chloride (420 mg, 1.29 mmol) was added, and the reaction system was heated to room temperature and stirred for 1 h. The resulting reaction system was poured into water and extracted twice with ethyl acetate. The organic phase was collected, dried over MgSO4, filtered, and the filtrate was evaporated in vacuo to remove the solvent to obtain compound 8. Under nitrogen protection, Pd(PPh3)2Cl2 (56.04 mg, 0.048 mmol) was added to a xylene (10 mL) solution containing 4,7-dibromobenzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole) (93.68 mg, 0.27 mmol) and compound 8 (944.5 mg, 0.81 mmol). The mixture was stirred at 130°C for 10 h. The resulting product system was cooled to room temperature and poured into water. The mixture was extracted twice with ethyl acetate. The organic phase was collected, dried over MgSO4, filtered, and the filtrate was evaporated in vacuo. The crude product was chromatographed on a silica gel column using DCM and MeOH as the eluents in a volume ratio of 10:1. IR-BTOG was obtained as a brown solid (258.6 mg, 56.0%). H NMR spectrum of IR-BTOG compound ( 1 HNMR) images Figure 8 As shown; NMR carbon spectrum ( 13 CNMR) Figure Figure 9 As shown; High resolution mass spectrometry (HRMS) is shown Figure 10 shown.

[0087] 1H NMR(500MHz, CDCl3) δ7.69(dd,J=12.0,7.1Hz,2H),7.58(dd,J=13.7,5.0Hz,2H),7.50(dd,J=7.7,2.7Hz,2H),7.2 1(dd,J=8.3,3.5Hz,3H),6.65(dd,J=11.4,8.4Hz,3H),4.55(s,1H),4.33(s,1H),4.03(ddd,J=47.7,27.1,18.6).

[0088] (5) Preparation of IR-BTOGP-GPC3 compound:

[0089] The IR-BTOG compound (100 mg, 0.058 mmol) and sodium azide (47 mg, 0.72 mmol) were dissolved in DMF (10 mL) and heated at 70 ° C for 5 h. Water was added to the reaction system and stirred until the solid was dissolved. The mixture was extracted twice with ethyl acetate, and the organic phase was collected and dried over MgSO4. The filtered solution was evaporated in vacuo to remove the solvent. The crude product was subjected to column chromatography on silica gel. The elution solvents used were dichloromethane (DCM) and methanol (MeOH). The volume ratio of DCM to MeOH was 5: 1 to give a dark brown solid as the IR-BTOGP compound (86 mg, 0.055 mmol). The IR-BTOGP compound was dissolved in THF (5 mL), and copper (I)-thiophene-2-carboxylate (CuTc) (10 mg), alkyne-PEG1000-GPC3 (159 mg) and tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) (5 mg) were added. The mixture was stirred at room temperature for 0.5 h. The resulting reaction system was filtered through celite, and the resulting filtrate was evaporated in vacuo to remove the solvent. The resulting crude product was chromatographed on a silica gel column using dichloromethane (DCM) and methanol (MeOH) as the eluting solvents. The volume ratio of DCM to MeOH was 5:1. The substance obtained by column chromatography was recrystallized from methyl tert-butyl ether to obtain the IR-BTOGP-GPC3 compound as a dark brown solid (167.6 mg, 85.5%). The H NMR spectrum of the IR-BTOGP-GPC3 compound ( 1 HNMR) images Figure 11 As shown; NMR carbon spectrum ( 13 CNMR) Figure Figure 12 shown.

[0090] 1H NMR (500MHz, CDCl3) δ7.60(dd,J=11.9,7.6Hz,2H),7.54-7.44(m,3H),7.40(t,J=6.4Hz,4H),7.11(s,3H),6.56(dd,J=11.0,8.7Hz, 4H), 4.35 (d, J = 110.4Hz, 3H), 4.02-3.23 (m, 168H), 1.81 (dd, J = 25.6, 19.2Hz, 26H), 1.47-1.14 (m, 34H), 0.78 (dd, J = 9.0, 4.8Hz, 8H).

[0091] 13 C NMR (126MHz, CDCl3) δ132.15,131.13,128.48,115.00,105.47,89.64,77. 31,77.06,76.80,71.92,70.57,59.05,33.99,32.71,29.03,27.87,25.45.

[0092] Optical parameters in water: Absorption coefficient ε(10 3 L / mol·cm, 1064nm): 45.4, absorption wavelength λexmax = 1092nm, emission wavelength λemmax = 1378nm, quantum yield 0.00736%; (QY data were quantified using IR-26, with a quantum yield of 0.05%).

[0093] Comparative Example 1

[0094] A PBS blank control was set up.

[0095] Test Example 1

[0096] IR-BTOGP-GPC3 compound can be abbreviated as BTOGP-GPC3 NPs. Figure 13 Transmission electron microscopy (TEM) image of BTOGP-GPC3 NPs prepared in Example; Figure 14 Figure a is a dynamic light scattering (DLS) graph of BTOGP-GPC3 NPs prepared in Example. Figure 14 (b) is a graph showing the relationship between the size and PDI of BTOGP-GPC3 NPs prepared in Example 1 over time; Figure 15 This is the spectrum of BTOGP-GPC3 NPs prepared in Example.

[0097] The morphology and size distribution of the samples were analyzed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The results showed that BTOGP-GPC3 NPs were spherical with an average diameter of approximately 90.68±9.08nm ( Figure 13The hydrodynamic diameter of BTOGP-GPC3 NPs measured by DLS was 91.94±7.45nm, which was consistent with the TEM results ( Figure 14 (a) Figure 14 Figure b shows that the size and polydispersity index (PDI) of BTOGP-GPC3 NPs remained stable in PBS for 14 days. In addition, the spectrum of BTOGP-GPC3 NPs showed a high absorption peak in the range of 1100-1300 nm, proving that they have excellent excitation light penetration depth. A broad emission peak in the range of 1200-1700 nm indicates that BTOGP-GPC3 NPs have important potential in bioimaging ( Figure 15 ).

[0098] Test Example 2: Evaluation of Intracellular Phototherapy Effects

[0099] Figure 16 Graph showing the evaluation of intracellular phototherapy effects of the compounds prepared in Examples and Comparative Examples; Figure 16 Figure a is the relationship between the fluorescence signals of BTOGPNPs and BTOGP-GPC3 NPs and time. Figure 16 Figure b is the cleaved caspase-3 staining images of BTOGP-GPC3 NPs prepared in the laser irradiation example and the comparative example PBS under different conditions. Figure 16 Figure c is a live cell / dead cell staining diagram of the compound of BTOGP-GPC3 NPs prepared in the laser irradiation example and PBS in the comparative example. Figure 16 Figure d shows the change of the average fluorescence intensity of BTOGP NPs and BTOGP-GPC3 NPs over time. Figure 16 Figure e is a pie chart showing the survival and mortality rates of tumor cells to which BTOGP-GPC3 NPs prepared in the example and PBS were added, respectively. Figure 16 Figure f is a schematic diagram of the fluorescence signals of BTOGP-GPC3 NPs in LX-2 cells, HepG-2 cells without pre-blocking, and HepG-2 cells with pre-blocking.

[0100] Using HepG-2 cells as an in vitro liver cancer model, the liver cancer targeting ability of BTOGP-GPC3 NPs was evaluated through a culture experimental system. The results showed that compared with the BTOGPNPs group, the fluorescence signal of the BTOGP-GPC3 NPs group was significantly enhanced after 60 minutes of incubation ( Figure 16 The average fluorescence intensity of the BTOGP-GPC3 NPs group increased nearly 8 times from the initial value, while that of the BTOGPNPs group increased only 2.5 times ( Figure 16(d) This significant difference indicates that the internalization efficiency of BTOGP-GPC3 NPs is significantly improved due to the active targeting effect of GPC3 peptide. At the same time, reverse evaluation was performed through GPC3 peptide pre-blocking experiment, further confirming the above targeting effect. Figure 16 As shown in Figure f, there is a significant difference in the fluorescence accumulation of HepG-2 cells after PBS washing compared with normal liver cells LX-2 cells. HepG-2 cells showed obvious fluorescence enrichment without pre-blocking. However, after pre-blocking with a 10-fold excess of free GPC3 peptide, the fluorescence signal of BTOGP-GPC3NPs in the cells was significantly weakened. The results clearly show that the tumor targeting effect of BTOGP-GPC3 NPs mainly depends on the specific binding affinity of GPC3 peptide. In summary, the results of the cell targeting experiment clearly show that the change of GPC3 peptide significantly enhances the molecular targeting of BTOGP-GPC3 NPs, providing solid supporting evidence for its application prospects in tumor targeted detection and treatment.

[0101] Immunofluorescence staining of cleaved caspase-3 was used to show cell apoptosis under different treatments. Figure 16 Figure b shows that laser irradiation did not affect the cleaved caspase staining in the PBS group. The level of cleaved caspase-3 in BTOGP-GPC3 NPs+L was higher than that in the control PBS group. In addition, live cell / dead cell staining analysis confirmed the above data. Figure 16 As can be seen in Figures c and e, the BTOGP-GPC3 NPs+L group has significant cytotoxicity against tumor cells, which is significantly better than the control molecule. The above results indicate that BTOGP-GPC3 NPs have special anti-tumor activity, which is mainly due to the significant changes in the photothermal properties of BTOGP-GPC3 NPs after optimization of SO conformation lock and alkoxy engineering strategy.

[0102] In summary, BTOGP-GPC3 NPs optimized by SoCLs-assisted alkoxy engineering strategy selectively targeted tumor cells through GPC3 peptide and showed significant anti-HCC effect in HepG-2 cells under light triggering.

[0103] Test Example 3: In vivo NIR-II fluorescence imaging

[0104] Figure 17 The effect diagram of in vivo NIR-II fluorescence imaging of the compounds prepared in Examples and Comparative Examples; Figure 17 Figure a is the relationship between the fluorescence signals of BTOGPNPs and BTOGP-GPC3 NPs at the tumor site and time. Figure 17 Figure b is a schematic diagram of the aggregation ability of BTOGPNPs at the tumor site. Figure 17Figure c is a schematic diagram of the aggregation ability of BTOGP-GPC3 NPs at the tumor site. Figure 17 The d in the middle is the normalized intensity and signal-to-noise ratio signal diagram at 1300nm. Figure 17 The middle e is the imaging result of the mouse upper abdomen at 1300nm. Figure 16 The middle f is the imaging result of the mouse upper abdomen and legs at 1300nm. Figure 17 The image g in the middle is the imaging result of the mouse leg at 1300nm. Figure 17 The h in the middle is the resolution and image quality diagram of 1300nm filter imaging. Figure 17 The i in the figure is the relationship between the tumor and normal tissue signal ratios of BTOGP-GPC3 NPs and BTOGPNPs at different times. Figure 17 In the figure, j is the normalized intensity and signal-to-noise ratio signal at 1500 nm. Figure 17 The image in the middle shows the imaging result of the mouse upper abdomen at 1500nm. Figure 17 The image (1) in the middle shows the imaging results of the mouse upper abdomen and legs at 1500nm. Figure 17 The image of the mouse leg is shown in Figure 1500nm. Figure 17 In the figure, n is the resolution and image quality of imaging with a 1500nm filter. Figure 17 The middle o is the relationship diagram of the tumor and normal tissue signal ratio of BTOGP-GPC3NPs and BTOGPNPs, Figure 17 p in the figure is the signal fitting curve of BTOGP-GPC3NPs for experimental groups with different lipid layer thicknesses. Figure 17 The q in the middle is the change of the content of BTOGP-GPC3 NPs in blood samples within 48 hours. Figure 17 The graph r in the figure shows the change of BTOGP-GPC3 NPs content in fecal samples within 48 hours. Figure 17 The graph in the middle is the change of BTOGP-GPC3NPs content in blood over time. Figure 17 The figure t in the middle shows the change of BTOGP-GPC3 NPs content in feces over time.

[0105] Inspired by the good phototherapy performance of BTOGP-GPC3 NPs in vitro, this test case further studied its potential for phototherapy in vivo. First, the NIR-II fluorescence imaging performance of BTOGP-GPC3 NPs was evaluated using the HepG-2 mouse orthotopic tumor model. Figure 17 As shown in Figure a, compared with the BTOGPNPs group, the fluorescence signal at the tumor site in the BTOGP-GPC3 NPs group significantly accumulated over time and stabilized after 8 hours. To further verify its distribution in vivo, fluorescence images of isolated organs were obtained by dissecting euthanized mice. Figure 17As can be seen in Figures b and c, the BTOGP-GPC3 NPs group exhibited stronger aggregation ability at the tumor site compared with the BTOGPNPs group, indicating that the GPC3-modified fluorescent molecules had a stronger targeting effect in vivo.

[0106] In addition, spleen and liver act as the mononuclear phagocytic system (MPS) and BTOGP and BTOGP-GPC3 NPs showed specific amount of accumulation in these major distribution organs, e.g. Figure 17 As shown in Figures i and o. Notably, BTOGP-GPC3 NPs more clearly outline HCC than BTOGP NPs, achieving a high image tumor-to-normal tissue (T / N) signal ratio from 4.26 to 4.10. This result indicates that BTOGP-GPC3 NPs significantly improve tumor imaging clarity, laying a solid foundation for further tumor detection and treatment applications.

[0107] Subsequently, BTOGP-GPC3 NPs (200 μM, 200 μL) were intravenously infused into BALB / c mice bearing orthotopic tumors. Under the stimulation of a 1064 nm laser, BTOGP-GPC3 NPs effectively achieved rapid imaging of major arteries. Imaging results of the upper abdomen and legs were further analyzed using long-pass filters at 1300 nm and 1500 nm. Figure 17 f, l, e, g, k, and m). In the 1300 nm filter imaging mode, the two major arteries adjacent to the HCC appeared blurred at 1.8 mm, with a signal-to-noise ratio (SBR) of 2.23 and a full width at half maximum (FWHM) of 260 μm and 800 μm, respectively. In contrast, using a 1500 nm filter, the vascular contours around the HCC were more clearly depicted, with intervascular distances of 1.01 mm and 1.25 mm, respectively. The SNR improved to 10.87, and the full width at half maximum (FWHM) was 300 μm, 360 μm, and 190 μm, respectively. Figure 17 In addition, in leg imaging, NIR-II imaging can identify four consecutive veins. Compared with 1300 nm filter imaging (SBR: 4.8, FWHM: 520 μm), 1500 nm filter imaging (SBR: 24.75, FWHM: 190 μm) has better resolution and image quality ( Figure 17 h and n).

[0108] Based on the strong performance of the optimized BTOGP-GPC3 NPs in the long-wave spectrum, the imaging penetration of BTOGP-GPC3 NPs in the NIR-II window (1500nm filter) was evaluated by filling capillaries and depositing 1% lipid layers of different thicknesses. The signal fitting curve analysis of the experimental groups with different lipid layer thicknesses showed that BTOGP-GPC3 NPs had excellent imaging penetration ability, achieving an optical penetration depth of 8 to 10 mm ( Figure 17 The results indicate that BTOGP-GPC3 NPs have important application potential in deep tissue bioimaging.

[0109] To study the in vivo pharmacokinetics of BTOGP-GPC3 NPs, blood and fecal samples were collected at different times for monitoring. The blood circulation and fecal excretion of BTOGP-GPC3 NPs were observed within 48 hours after intravenous injection. Figure 17 As shown in the q and s, the circulation half-life of BTOGP-GPC3 NPs is about 54 minutes. In addition, the fecal excretion data showed that about 66.4% of BTOGP-GPC3 NPs were excreted through feces within 48 hours after intravenous injection, indicating that the hepatic excretion process is slow ( Figure 17 r and t).

[0110] Test Example 4: In vivo anti-tumor efficacy

[0111] Figure 18 The figures are effect diagrams of the in vivo anti-tumor efficacy of the compounds prepared in Examples and Comparative Examples; Figure 18 Middle a is the bioluminescent signal image of the luciferase-labeled tumor site observed by imaging in mice. Figure 18 Middle b is the graph showing the changes in tumor luminescence signal over time after mice were injected with BTOGP-GPC3 NPs and PBS, Figure 18 The c in the figure is the curve of the related tumor signals of BTOGP-GPC3 NPs and PBS changing with time. Figure 18 Middle d is the weight curve of mice injected with BTOGP-GPC3 NPs and PBS respectively within 14 days. Figure 18 Middle e is a schematic diagram of the liver tumor area of ​​mice injected with BTOGP-GPC3 NPs and PBS respectively. Figure 18 Middle f is the histogram of tumor volume of BTOGP-GPC3 NPs and PBS within 14 days after initial tumor elimination. Figure 18 (g) shows the tumor apoptosis detected by HE, KI and TUNEL staining of BTOGP-GPC3 NPs and PBS, respectively.

[0112] Based on the above experimental results, in vivo experiments further demonstrated the effectiveness of optical diagnosis and treatment under the optimized strategy. To obtain non-invasive visualization of tumor size, HCC cells were first pre-transfected with luciferase. At intervals of 14 days, the bioluminescent signal of the luciferase-labeled tumor site was observed by an in vivo imaging system ( Figure 18 (a) and the tumor volume was assessed based on the luminescence intensity. Figure 18 As can be seen in Figures b and c, the tumor growth rates of the BTOGP-GPC3 NPs and PBS groups were significantly enhanced, while the tumor growth of the BTOGP-GPC3 NPs group was inhibited. The liver cancer inhibition rate was 99.8% in the BTOGP-GPC3 NPs group. The relative body weight of each group was not affected during the treatment, indicating that these molecular phototherapy agents are non-toxic ( Figure 18 (d) Figure 18 As shown in Figure 5 (f), the BTOGP-GPC3 NPs-treated group exhibited sustained tumor suppression after initial tumor elimination, with no signs of tumor recurrence observed during the 14-day observation period. Consequently, the survival rate of this group remained at 100%. In contrast, the control group exhibited varying degrees of tumor progression and mortality, indicating a significantly reduced therapeutic effect compared to BTOGP-GPC3 NPs-treated patients.

[0113] Under the experimental protocol, mouse liver tissues were surgically removed and subsequently analyzed under different conditions. Figure 18 Middle e shows that the liver of mice in the BTOGP-GPC3 NPs group was bright red and the tumor area was extremely small, which was consistent with the results of bioluminescence imaging ( Figure 18 Middle b). HE staining to detect tumor apoptosis ( Figure 18 (g) A significant increase in apoptotic cells and elevated TUNEL expression were observed in tumor sections. Significant downregulation of the proliferation marker KI-67 indicated significant inhibition of tumor cell growth and increased apoptosis.

[0114] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. An IR-BTOGP-GPC3 compound having the structure shown in Formula 1: In formula 1, n is 10 to 60.

2. The method for preparing the IR-BTOGP-GPC3 compound according to claim 1, comprising the following steps: Mixing the IR-BTOG compound, sodium azide, and an organic solvent to perform an azide reaction to obtain the IR-BTOG-N3 compound; The IR-BTOG-N3 compound, copper (I)-thiophene-2-carboxylate, alkynone-polyethylene glycol-GPC3, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine and an organic solvent are mixed to perform a superposition-alkyl click cyclization reaction to obtain the IR-BTOGP-GPC3 compound with the structure represented by Formula 1; The IR-BTOG compound has a structure shown in Formula 2, and the IR-BTOG-N3 compound has a structure shown in Formula 3; 3. The preparation method according to claim 2, characterized in that The preparation method of the IR-BTOG compound comprises the following steps: Compound 7, n-butyl lithium solution, tributyltin chloride and an organic solvent are mixed to undergo a nucleophilic reaction to obtain compound 8; The compound 8, 4,7-dibromobenzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole), bistriphenylphosphine palladium dichloride and an organic solvent are mixed to carry out a first reaction to obtain the IR-BTOG compound; The compound 7 has a structure shown in Formula 4; the compound 8 has a structure shown in Formula 5; 4. The preparation method according to claim 2, characterized in that The molar ratio of the IR-BTOG compound to sodium azide is 0.058:0.

72.

5. The preparation method according to claim 2, characterized in that The mass ratio of the IR-BTOGP compound to acetyl ketone-polyethylene glycol-GPC3 is 85-87:159; the mass ratio of the IR-BTOGP compound to copper (I)-thiophene-2-carboxylate is 85-87:10; the mass ratio of the IR-BTOGP compound to tri[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine is 85-87:

5.

6. Use of the IR-BTOGP-GPC3 compound according to claim 1 or the IR-BTOGP-GPC3 compound obtained by the preparation method according to any one of claims 2 to 5 in the preparation of a preparation for specific targeted recognition and diagnosis of hepatocellular carcinoma.

7. Use of the IR-BTOGP-GPC3 compound according to claim 1 or the IR-BTOGP-GPC3 compound obtained by the preparation method according to any one of claims 2 to 5 in the preparation of an anti-hepatocellular carcinoma targeted drug.

8. An anti-hepatocellular carcinoma targeted drug comprising the IR-BTOGP-GPC3 compound according to claim 1 or the IR-BTOGP-GPC3 compound obtained by the preparation method according to any one of claims 2 to 5.

9. Use of the IR-BTOGP-GPC3 compound according to claim 1 or the IR-BTOGP-GPC3 compound obtained by the preparation method according to any one of claims 2 to 5 in the preparation of near-infrared II photodiagnostic probes.

10. A near-infrared II phototherapy probe, comprising the IR-BTOGP-GPC3 compound according to claim 1 or the IR-BTOGP-GPC3 compound obtained by the preparation method according to any one of claims 2 to 5.

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