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

Abstract

This invention belongs to the field of targeted molecular technology for hepatocellular carcinoma, specifically relating to the preparation method and application of the hepatocellular carcinoma targeted diagnostic probe compound IR-BTOGP-GPC3. The IR-BTOGP-GPC3 compound provided by this invention has the structure shown in Formula 1, where n is 10–60. The IR-BTOGP-GPC3 compound can accurately identify and diagnose hepatocellular carcinoma and exhibits excellent phototherapy performance; while effectively reducing intramolecular energy dissipation, it possesses good key molecular properties, including tissue penetration, photothermal efficiency, and fluorescence quantum yield.

Description

Technical Field

[0001] This invention belongs to the field of hepatocellular carcinoma targeted molecular technology, specifically relating to the preparation method and application of the hepatocellular carcinoma targeted diagnostic and therapeutic probe compound IR-BTOGP-GPC3. Background Technology

[0002] With superior tissue penetration depth, minimal autofluorescence interference, and high spatial resolution in the near-infrared II (NIR-II) spectral region (1000–1700 nm), NIR-II-based phototherapy molecules possess remarkable optical properties that make them ideal for addressing challenging clinical scenarios, such as deep and refractory tumors. Currently, in the field of precision tumor diagnosis and treatment, the development of integrated phototherapy probes combining NIR-II fluorescence imaging with photothermal therapy (PTT) / photodynamic therapy (PDT) is a research hotspot. However, existing technologies lack phototherapy probes capable of accurately identifying and diagnosing hepatocellular carcinoma. Summary of the Invention

[0003] The purpose of this invention is to provide a method for preparing and applying the IR-BTOGP-GPC3 compound, a targeted diagnostic probe for liver cancer. The IR-BTOGP-GPC3 compound provided by this invention can accurately identify and diagnose hepatocellular carcinoma, exhibits excellent phototherapy performance, and effectively reduces intramolecular energy dissipation while possessing favorable key molecular properties, including tissue penetration, photothermal efficiency, and fluorescence quantum yield.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

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

[0006]

[0007] In Equation 1, n ranges from 10 to 60.

[0008] This invention provides a method for preparing the IR-BTOGP-GPC3 compound described above, comprising the following steps:

[0009] The IR-BTOG compound, sodium azide, and organic solvent were mixed and subjected to an azide reaction to obtain the IR-BTOGP compound.

[0010] The IR-BTOGP compound, copper(I)-thiophene-2-carboxylate, acetylene-polyethylene glycol-GPC3, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine and organic solvent were mixed and subjected to a superimposed-alkyl click cyclization reaction to obtain the IR-BTOGP-GPC3 compound with the structure shown in Formula 1.

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

[0012]

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

[0014] Compound 7, n-butyllithium solution, tributyltin chloride and organic solvent were mixed and subjected to 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 were mixed and subjected to a first reaction to obtain the IR-BTOG compound;

[0016] Compound 7 has the structure shown in Formula 4; compound 8 has the 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 acetylacetonate-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; and the mass ratio of the IR-BTOGP compound to tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine is 85-87:5.

[0020] This invention provides the application of the IR-BTOGP-GPC3 compound described in the above technical solution or the IR-BTOGP-GPC3 compound obtained by the above preparation method in the preparation of agents for specific targeted recognition and diagnosis of hepatocellular carcinoma.

[0021] This invention provides the application of the IR-BTOGP-GPC3 compound described in the above technical solution or the IR-BTOGP-GPC3 compound obtained by the above preparation method in the preparation of targeted drugs for hepatocellular carcinoma.

[0022] This invention provides a targeted drug for treating hepatocellular carcinoma, comprising the IR-BTOGP-GPC3 compound described in the above technical solution or the IR-BTOGP-GPC3 compound obtained by the preparation method described above.

[0023] This invention provides the application of the IR-BTOGP-GPC3 compound described in the above technical solution or the IR-BTOGP-GPC3 compound obtained by the above preparation method in the preparation of near-infrared II phototherapy probes.

[0024] This 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 described above.

[0025] This invention provides an IR-BTOGP-GPC3 compound having the structure shown in Formula 1, where n is 10–60. This invention couples an IR-BTOG compound with a GPC3 peptide to obtain the IR-BTOGP-GPC3 compound. The IR-BTOG compound exhibits superior long-wavelength penetration and fluorescence properties, while the GPC3 peptide is a specific targeting molecule. The IR-BTOGP-GPC3 compound provided by this invention possesses superior tissue penetration depth, superior photothermal conversion efficiency, and a high fluorescence quantum yield NIR-II fluorophore, enabling precise identification and diagnosis of hepatocellular carcinoma, and demonstrating excellent targeted diagnostic and therapeutic effects for hepatocellular carcinoma.

[0026] Meanwhile, the excellent phototherapy properties of the IR-BTOGP-GPC3 compound provided by this 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 formulations. Attached Figure Description

[0027] Figure 1 The flowchart for preparing the IR-BTOGP-GPC3 compound is shown in the example.

[0028] Figure 2 The proton nuclear magnetic resonance spectrum of compound 4 prepared for example ( 1 H NMR spectrum;

[0029] Figure 3 The carbon NMR spectrum of compound 4 prepared for example ( 13 C NMR spectrum;

[0030] Figure 4 High-resolution mass spectra (HRMS) of compound 4 were prepared for the example;

[0031] Figure 5 The proton nuclear magnetic resonance spectrum of compound 7 prepared for example ( 1 H NMR spectrum;

[0032] Figure 6 The carbon NMR spectrum of compound 7 prepared for example ( 13 C NMR spectrum;

[0033] Figure 7 High-resolution mass spectra (HRMS) of compound 7 were prepared for the example;

[0034] Figure 8 The proton nuclear magnetic resonance spectrum of the IR-BTOGP compound prepared for the example ( 1 H NMR spectrum;

[0035] Figure 9 Carbon NMR spectra of IR-BTOGP compounds prepared for examples ( 13 C NMR spectrum;

[0036] Figure 10 High-resolution mass spectra (HRMS) of IR-BTOGP compounds were prepared for this example.

[0037] Figure 11 The proton nuclear magnetic resonance spectrum of the IR-BTOGP-GPC3 compound prepared for the example ( 1 H NMR spectrum;

[0038] Figure 12 Carbon NMR spectra of the IR-BTOGP-GPC3 compound prepared for the example ( 13 C NMR spectrum;

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

[0040] Figure 14 Dynamic light scattering (DLS) plot and time-varying size-PDI relationship plot of the IR-BTOGP-GPC3 compound prepared for the example;

[0041] Figure 15 The spectrum of the IR-BTOGP-GPC3 compound prepared for the example;

[0042] Figure 16 Effect diagrams of intracellular phototherapy efficacy evaluation of the compounds prepared for the examples and comparative examples;

[0043] Figure 17 The images show the in vivo NIR-II fluorescence imaging effects of the compounds prepared in the examples and comparative examples;

[0044] Figure 18 The graph shows the in vivo antitumor efficacy of the compounds prepared in the examples and comparative examples. Detailed Implementation

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

[0046]

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

[0048] This invention provides a method for preparing the IR-BTOGP-GPC3 compound described above, comprising the following steps:

[0049] The IR-BTOG compound, sodium azide, and organic solvent were mixed and subjected to an azide reaction to obtain the IR-BTOGP compound.

[0050] The IR-BTOGP compound, copper(I)-thiophene-2-carboxylate, acetylene-polyethylene glycol-GPC3, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine and organic solvent were mixed and subjected to a superimposed-alkyl click cyclization reaction to obtain the IR-BTOGP-GPC3 compound with the structure shown in Formula 1.

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

[0052]

[0053]

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

[0055] This invention involves mixing an IR-BTOG compound, sodium azide, and an organic solvent to perform an azide reaction, thereby obtaining the IR-BTOGP compound. In one embodiment of this 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. This invention does not have specific requirements regarding the amount of 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 time can be 4-6 hours, specifically 5 hours. After the azide reaction, an azide reaction solution is obtained. Preferably, the azide reaction solution is post-treated to obtain the IR-BTOGP compound. The post-treatment preferably includes: diluting the azide reaction solution with water, extracting the 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; purifying the crude product by column chromatography to obtain the IR-BTOGP compound. In this 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 column chromatography, and the elution solvent can be dichloromethane (DCM) and methanol (MeOH), with a volume ratio of DCM to MeOH of 5:1.

[0057] After obtaining the IR-BTOGP compound, the present invention mixes the IR-BTOGP compound, copper(I)-thiophene-2-carboxylate, acetylacetonate-polyethylene glycol-GPC3, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), and an organic solvent to perform a superimposed-alkyl click cyclization reaction to obtain the IR-BTOGP-GPC3 compound with the structure shown in Formula 1. As one embodiment of the present invention, the acetylacetonate-polyethylene glycol-GPC3 can be acetylacetonate-polyethylene glycol 1000-GPC3 (Alkyne-PEG1000-GPC3), which was purchased from Sichuan Ruixi Biotechnology Co., Ltd. The copper(I)-thiophene-2-carboxylate can be cuprous(I)thiophene-2-carboxylate (CuTc); the mass ratio of the IR-BTOGP compound to acetylacetonate-polyethylene glycol-GPC3 can be 85-87:159, specifically 86:159; the mass ratio of the IR-BTOGP compound to copper(I)-thiophene-2-carboxylate can be 85-87:10, specifically 86:10; the mass ratio of the IR-BTOGP compound to tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine can be 85-87:5, specifically 86:5; the organic solvent can be THF. This invention does not have special requirements for the amount of the organic solvent used, as long as the superimposed-alkyl click cyclization reaction proceeds smoothly.

[0058] In one embodiment of the present invention, the temperature of the superimposed-alkyl click cyclization reaction can be 15–30°C, and the time can be 0.5 h. The superimposed-alkyl click cyclization reaction is carried out under stirring. In the present invention, after the superimposed-alkyl click cyclization reaction, a cyclization reaction solution is obtained. Preferably, the cyclization reaction solution is post-treated to obtain the IR-BTOGP-GPC3 compound with the structure shown in Formula 1. The post-treatment preferably includes: filtering the cyclization reaction solution with 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 first-purified product; and recrystallizing the first-purified product with methyl tert-butyl ether to obtain the IR-BTOGP-GPC3 compound with the structure shown in Formula 1. In the present invention, the solvent removal method can be vacuum evaporation. The elution solvent used for column chromatography can be DCM and MeOH, and the volume ratio of DCM to MeOH can be 5:1. The present invention does not have special requirements for the specific implementation of the recrystallization.

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

[0060] Compound 7, n-butyllithium solution, tributyltin chloride and organic solvent were mixed and subjected to 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 were mixed and subjected to a first reaction to obtain the IR-BTOG compound;

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

[0063]

[0064] In this invention, compound 7, n-butyllithium (n-BuLi) solution, tributyltin chloride, and an organic solvent are mixed and subjected to a nucleophilic reaction to obtain compound 8. As one embodiment of this invention, the n-butyllithium solution is obtained by mixing n-butyllithium with hexane, and the concentration of the n-butyllithium solution is 1.6 M; 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] In one embodiment of the present invention, the mixing of compound 7, n-butyllithium solution, tributyltin chloride, and organic solvent may include: adding n-butyllithium solution dropwise to the THF solution of compound 7 under nitrogen atmosphere at -78°C to carry out a first reaction; then adding tributyltin chloride to carry out a second reaction; the first reaction is carried out under stirring conditions at -78°C for 2 hours; the second reaction is carried out under stirring conditions at 15–30°C for 1 hour. In another embodiment of the present invention, a nucleophilic reaction mixture is obtained after the nucleophilic reaction. Preferably, the nucleophilic reaction mixture is 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), bis(triphenylphosphine)palladium dichloride (Pd(PPh3)2Cl2), and an organic solvent to carry out 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 bis(triphenylphosphine)palladium dichloride can be 0.81:0.27:0.048; the organic solvent can be xylene.

[0067] In 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 conditions. After the first reaction, a first reaction mixture is obtained. Preferably, the first reaction mixture is 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; 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, and the column chromatography purification can be silica gel column chromatography. The eluent for the column chromatography is DCM and ethyl acetate (EA), and the volume ratio of DCM to EA can be 10:1.

[0068] This invention provides the application of the IR-BTOGP-GPC3 compound described in the above technical solution or the IR-BTOGP-GPC3 compound obtained by the above preparation method in the preparation of agents for specific targeted recognition and diagnosis of hepatocellular carcinoma.

[0069] This invention provides the application of the IR-BTOGP-GPC3 compound described in the above technical solution or the IR-BTOGP-GPC3 compound obtained by the above preparation method in the preparation of targeted drugs for hepatocellular carcinoma.

[0070] This invention provides a targeted drug for treating hepatocellular carcinoma, comprising the IR-BTOGP-GPC3 compound described in the above technical solution or the IR-BTOGP-GPC3 compound obtained by the preparation method described above.

[0071] This invention provides the application of the IR-BTOGP-GPC3 compound described in the above technical solution or the IR-BTOGP-GPC3 compound obtained by the above preparation method in the preparation of near-infrared II phototherapy probes.

[0072] This 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 described above.

[0073] Figure 1 This is a flowchart illustrating the preparation of the IR-BTOGP-GPC3 compound according to an embodiment of the present invention. To further illustrate the present invention, the technical solutions provided by the present invention are described in detail below with reference to embodiments, but these should not be construed 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-thiophene)tin (Compound 3) (878 mg, 2.35 mmol). The mixture was stirred at 130 °C for 3 h. The resulting product system was cooled to room temperature and poured into water. It was extracted twice with ethyl acetate. The organic phase was collected, dried over MgSO4, filtered, and the filtrate was vacuum evaporated to remove the solvent. The crude product was subjected to silica gel column chromatography. The eluting solvents were PE and EA, with a volume ratio of PE to EA of 5:1. Compound 4 was obtained as a white solid (1.08 g, 88.6%). Compound 4 is 2-(2,6-di((6-bromohexyl)oxy)phenyl)thiophene. The nuclear magnetic resonance (NMR) spectrum of Compound 4 was obtained. 1 HNMR (HNMR) image as follows Figure 2 As shown; Carbon NMR spectrum ( 13 (C NMR) image as follows Figure 3 As shown; High-resolution mass spectrometry (HRMS) image as shown. Figure 4 As 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 stirred at room temperature for another 4 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 vacuum evaporated to remove the solvent. The crude product was subjected to silica gel column chromatography with DCM and MeOH as the elution solvents, in a volume ratio of 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)thiophen-2-yl)stanane (compound 6) (895 mg, 1.67 mmol) were dissolved in xylene (15 mL). Pd(PPh3)4 (193.49 mg, 0.167 mmol) was added to the reaction system, and the mixture was stirred at 130 °C for 3 h. After cooling the resulting product system to room temperature, it was poured into water and ethyl acetate was used. Extraction was performed twice. The organic phase was collected, dried over MgSO4, filtered, and the filtrate was vacuum evaporated to remove the solvent. The crude product was subjected to silica gel column chromatography using DCM and MeOH in a volume ratio of 5:1. Compound 7 was obtained as a brownish-black oil (0.81 g, 63.9%). Compound 7 is 5'-(2,6-di((6-bromohexyl)oxy)phenyl)-3-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2,2'-dithiophene. The 1H NMR spectrum of compound 7 was... 1 HNMR (HNMR) image as follows Figure 5 As shown; Carbon NMR spectrum ( 13 CNMR) diagram as follows Figure 6 As shown; High-resolution mass spectrometry (HRMS) image as shown. Figure 7 As 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 compounds:

[0086] Compound 7 (800 mg, 1.048 mmol) was added to THF (15.00 mL), and n-BuLi solution (hexane, 1.6 M, 806 μL, 1.29 mmol) was added dropwise under nitrogen protection at -78 °C. 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, extracted twice with ethyl acetate, and the organic phase was collected, dried over MgSO4, filtered, and the filtrate was vacuum evaporated to remove the solvent, yielding 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. After cooling the resulting product system to room temperature, it was poured into water and extracted twice with ethyl acetate. The organic phase was collected, dried over MgSO4, filtered, and the filtrate was evaporated under vacuum. The crude product was subjected to silica gel column chromatography using DCM and MeOH in a volume ratio of 10:1. The IR-BTOG compound was obtained as a brown solid (258.6 mg, 56.0%). The 1H NMR spectrum of the IR-BTOG compound was... 1 HNMR (HNMR) image as follows Figure 8 As shown; Carbon NMR spectrum ( 13 CNMR) diagram as follows Figure 9 As shown; High-resolution mass spectrometry (HRMS) image as shown. Figure 10 As 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 the mixture was stirred until the solid dissolved. The mixture was extracted twice with ethyl acetate. The organic phase was collected, dried over MgSO4, and filtered. The filtrate was evaporated under vacuum to remove the solvent. The crude product was subjected to column chromatography on silica gel. The elution solvents were dichloromethane (DCM) and methanol (MeOH) in a volume ratio of 5:1. The resulting dark brown solid was 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 diatomaceous earth, and the filtrate was evaporated under vacuum to remove the solvent. The crude product was subjected to silica gel column chromatography, using dichloromethane (DCM) and methanol (MeOH) as the eluent, with a DCM to MeOH volume ratio of 5:1. The substance obtained from the column chromatography was recrystallized from methyl tert-butyl ether to give the IR-BTOGP-GPC3 compound as a dark brown solid (167.6 mg, 85.5%). The 1H NMR spectrum of the IR-BTOGP-GPC3 compound was... 1 HNMR (HNMR) image as follows Figure 11 As shown; Carbon NMR spectrum ( 13 CNMR) diagram as follows Figure 12 As 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, quantum yield 0.05%).

[0093] Comparative Example 1

[0094] Set up a PBS blank control.

[0095] Test Example 1

[0096] The IR-BTOGP-GPC3 compound can be abbreviated as BTOGP-GPC3 NPs. Figure 13 Transmission electron microscopy (TEM) image of BTOGP-GPC3 NPs prepared for the example; Figure 14 Image a shows the dynamic light scattering (DLS) pattern of the BTOGP-GPC3 NPs prepared in the example. Figure 14 Figure b shows the relationship between the size and PDI of BTOGP-GPC3 NPs prepared in the example over time; Figure 15 The spectrum of BTOGP-GPC3 NPs prepared for the example.

[0097] The morphology and size distribution of the samples were analyzed using transmission electron microscopy (TEM) and dynamic light scattering (DLS). The results showed that the BTOGP-GPC3 NPs were spherical with an average diameter of approximately 90.68 ± 9.08 nm. Figure 13The hydrodynamic diameter of BTOGP-GPC3 NPs measured by DLS was 91.94 ± 7.45 nm, 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 over 14 days. Furthermore, the spectrum of BTOGP-GPC3 NPs exhibited a high absorption peak in the 1100–1300 nm range, demonstrating excellent excitation light penetration depth. A broad emission peak was also observed in the 1200–1700 nm range, indicating that BTOGP-GPC3 NPs have significant potential in bioimaging. Figure 15 ).

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

[0099] Figure 16 Effect diagrams of intracellular phototherapy efficacy evaluation of the compounds prepared for the examples and comparative examples; Figure 16 Figure 'a' shows the relationship between the fluorescence signals of BTOGP NPs and BTOGP-GPC3 NPs and time. Figure 16 In the middle b, the images show cleaved caspase-3 staining of BTOGP-GPC3 NPs prepared in the laser irradiation example and PBS in the comparative example under different conditions. Figure 16 In the middle (c), there are live / dead cell staining images of BTOGP-GPC3 NPs prepared in the laser irradiation example and PBS in the comparative example. Figure 16 Figure d shows the change in average fluorescence intensity of BTOGP NPs and BTOGP-GPC3 NPs over time. Figure 16 Figure e shows a pie chart of tumor cell survival and mortality rates after adding BTOGP-GPC3 NPs prepared in the examples and the control PBS, respectively. Figure 16 The diagram in Figure f shows 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 hepatocellular carcinoma model, the liver cancer targeting ability of BTOGP-GPC3 NPs was evaluated through a culture experiment. The results showed that, compared with the BTOGP NPs group, the fluorescence signal in the BTOGP-GPC3 NPs group was significantly enhanced after 60 min of incubation. Figure 16 (a) The average fluorescence intensity of the BTOGP-GPC3 NPs group increased nearly 8-fold from the initial value, while that of the BTOGPNPs group increased only 2.5-fold. 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 the GPC3 peptide. Furthermore, a reverse evaluation using a GPC3 peptide pre-blockade experiment further confirms this targeting effect. Figure 16 As shown in Figure f, the fluorescence accumulation of HepG-2 cells after PBS washing was significantly different compared to normal LX-2 hepatocytes. HepG-2 cells exhibited significant fluorescence enrichment without pre-blockage. However, after pre-blockage with a 10-fold excess of free GPC3 peptide, the fluorescence signal of intracellular BTOGP-GPC3NPs was significantly weakened. These results clearly indicate that the tumor-targeting effect of BTOGP-GPC3 NPs mainly depends on the specific binding affinity of GPC3 peptides. In conclusion, the cell-targeting experiment results clearly demonstrate that alterations to GPC3 peptides significantly enhance the molecular targeting of BTOGP-GPC3 NPs, providing solid supporting evidence for their application in tumor-targeted detection and therapy.

[0101] Apoptosis was observed under different treatments by immunofluorescence staining of lysed caspase-3. Figure 16 As shown in Figure b, laser irradiation had no effect on 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. Furthermore, live / dead cell staining analysis confirmed these data. Figure 16 As shown in c and e, the BTOGP-GPC3 NPs+L group exhibited significant cytotoxicity against tumor cells, significantly superior to the control molecule. These results indicate that BTOGP-GPC3 NPs possess unique antitumor activity, primarily due to the substantial changes in their photothermal properties resulting from SO conformation locking and alkoxy group engineering optimization strategies.

[0102] In summary, BTOGP-GPC3 NPs optimized by a synergistic alkoxy group engineering strategy with SOCLs exhibited a significant anti-HCC effect in HepG-2 cells under light-triggered conditions by selectively targeting tumor cells with GPC3 peptides.

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

[0104] Figure 17 The images show the in vivo NIR-II fluorescence imaging effects of the compounds prepared in the examples and comparative examples; Figure 17 Figure 'a' shows the relationship between the fluorescence signal of BTOGP NPs and BTOGP-GPC3 NPs at the tumor site and time. Figure 17 Figure b shows the aggregation ability of BTOGPNPs at the tumor site. Figure 17In the middle, c is a schematic diagram illustrating the aggregation ability of BTOGP-GPC3 NPs at the tumor site. Figure 17 The image shows the normalized intensity and signal-to-noise ratio of a signal with d = 1300 nm. Figure 17 Image e in the image shows the imaging results of the upper abdomen of a mouse at 1300 nm. Figure 16 Image f in the middle shows the imaging results of the upper abdomen and legs of a mouse at 1300 nm. Figure 17 Image g shows the imaging results of a mouse leg at 1300nm. Figure 17 In the middle, h represents the resolution and image quality of imaging with a 1300nm filter. Figure 17 In the figure, i represents the signal ratio between tumor and normal tissue for BTOGP-GPC3 NPs and BTOGPNPs at different time points. Figure 17 The image shows the normalized intensity and signal-to-noise ratio of signal j at 1500 nm. Figure 17 The image in the middle (k) shows the imaging results of the upper abdomen of a mouse at 1500 nm. Figure 17 Image l in the middle shows the imaging results of the upper abdomen and legs of a mouse at 1500nm. Figure 17 The image in the middle (m) shows the imaging results of a mouse leg at 1500 nm. Figure 17 In the image, n represents the resolution and image quality of an image formed using a 1500nm filter. Figure 17 The image in the middle represents the signal ratio between tumor and normal tissues for BTOGP-GPC3NPs and BTOGPNPs. Figure 17 In the figure, p represents the signal fitting curve of BTOGP-GPC3NPs for experimental groups with different lipid layer thicknesses. Figure 17 The graph in Figure q shows the change in the content of BTOGP-GPC3 NPs in a blood sample over 48 hours. Figure 17 The graph in Figure r shows the change in the content of BTOGP-GPC3 NPs in fecal samples over 48 hours. Figure 17 The graph in 's' represents the change in the concentration of BTOGP-GPC3NPs in blood over time. Figure 17 The graph in t represents the change in the content of BTOGP-GPC3 NPs in feces over time.

[0105] Inspired by the excellent phototherapy performance of BTOGP-GPC3 NPs in vitro, this test case further investigated its potential for in vivo phototherapy. First, the NIR-II fluorescence imaging performance of BTOGP-GPC3 NPs was evaluated using a HepG-2 mouse orthotopic tumor model. For example... 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, stabilizing 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 from Figures b and c, compared with the BTOGPNPs group, the BTOGP-GPC3 NPs group showed a stronger aggregation ability at the tumor site, indicating that the GPC3-modified fluorescent molecules have a stronger targeting effect in vivo.

[0106] Furthermore, the spleen and liver, as major organs of distribution of the mononuclear phagocytic system (MPS) BTOGP and BTOGP-GPC3 NPs, exhibit specific accumulations, such as... Figure 17 As shown in i and o. Notably, BTOGP-GPC3 NPs delineated HCC more clearly than BTOGPNPs, achieving a high image tumor-to-normal tissue (T / N) signal ratio, from 4.26 to 4.10. These results demonstrate that BTOGP-GPC3 NPs significantly improve tumor imaging clarity, laying a solid foundation for further applications in tumor detection and treatment.

[0107] Subsequently, BTOGP-GPC3 NPs (200 μM, 200 μL) were intravenously infused into BALB / c mice with orthotopic tumors. Under stimulation with a 1064 nm laser, BTOGP-GPC3 NPs effectively achieved rapid imaging of the 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 1300nm filter imaging mode, the two adjacent major arteries around HCC appear blurred at 1.8mm, with a signal-to-noise ratio (SBR) of 2.23 and full width at half maximum (FWHM) of 260μm and 800μm, respectively. Conversely, using the 1500nm filter, the contours of the vessels around HCC are more clearly depicted, with inter-vessel distances of 1.01mm and 1.25mm, respectively. The SBR improves to 10.87, and the FWHMs are 300μm, 360μm, and 190μm, respectively. Figure 17 (d and j). Furthermore, in leg imaging, NIR-II imaging can identify four consecutive veins. Compared to 1300nm filter imaging (SBR: 4.8, FWHM: 520μm), 1500nm filter imaging (SBR: 24.75, FWHM: 190μm) offers better resolution and image quality. Figure 17 (h and n).

[0108] Based on the superior performance of optimized BTOGP-GPC3 NPs in the long-wavelength spectrum, the imaging penetration of BTOGP-GPC3 NPs within the NIR-II window (1500 nm filter) was evaluated by filling capillaries and depositing lipid layers of varying thicknesses (1%). Signal fitting curve analysis of the experimental groups with different lipid layer thicknesses showed that BTOGP-GPC3 NPs possess excellent imaging penetration capabilities, achieving optical penetration depths of 8–10 mm. Figure 17 (p). The results indicate that BTOGP-GPC3 NPs have significant application potential in deep tissue bioimaging.

[0109] To investigate the in vivo pharmacokinetics of BTOGP-GPC3 NPs, blood and fecal samples were collected at different time points for monitoring. Blood circulation and fecal excretion of BTOGP-GPC3 NPs were observed within 48 hours after intravenous injection. Figure 17 As shown in q and s, the circulating half-life of BTOGP-GPC3 NPs is approximately 54 min. Furthermore, fecal excretion data indicate that approximately 66.4% of BTOGP-GPC3 NPs are excreted in feces within 48 hours after intravenous injection, suggesting a slow hepatic excretion process. Figure 17 (r and t).

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

[0111] Figure 18 The in vivo antitumor efficacy of the compounds prepared in the examples and comparative examples is illustrated in graphs. Figure 18 Image a shows the bioluminescence signal observed in mice using luciferase-labeled tumor sites via in vivo imaging. Figure 18 Figure b shows the changes in tumor luminescence signal over time in mice after injection of BTOGP-GPC3 NPs and PBS, respectively. Figure 18 In the figure, 'c' represents the time-dependent curves of tumor signals related to BTOGP-GPC3 NPs and PBS. Figure 18 Figure d shows the body weight curves of mice injected with BTOGP-GPC3 NPs and PBS, respectively, over 14 days. Figure 18 Image e shows a schematic diagram of the liver tumor region in mice injected with BTOGP-GPC3 NPs and PBS, respectively. Figure 18 The graph in f is a bar chart of BTOGP-GPC3 NPs and PBS tumor volume within 14 days after initial tumor elimination. Figure 18 The figure in the middle shows the tumor apoptosis detection using 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 photodiagnosis and treatment under the optimized strategy. To obtain non-invasive visualization of tumor size, HCC cells were first pre-transfected with luciferase. At 14-day intervals, the bioluminescent signals at the luciferase-labeled tumor sites were observed using an in vivo imaging system. Figure 18 (a) and assess tumor volume based on luminescence intensity. From Figure 18 Figures b and c show that tumor growth was significantly enhanced in the BTOGP-GPC3 NPs and PBS groups, while tumor growth was inhibited in the BTOGP-GPC3 NPs group. The liver cancer inhibition rate was 99.8% in the BTOGP-GPC3 NPs group. Relative body weight was not affected in any group during treatment, indicating that these molecular phototherapy agents are non-toxic. Figure 18 (d). For example Figure 18 As shown in Figure f, the BTOGP-GPC3 NPs treatment group exhibited sustained tumor suppression after initial tumor elimination, with no signs of tumor recurrence observed during the 14-day observation period. Therefore, the survival rate in this group remained at 100%. In contrast, the control group showed varying degrees of tumor progression and mortality, indicating a significantly reduced therapeutic effect compared to BTOGP-GPC3 NPs treatment.

[0113] In the experimental protocol, mouse liver tissue was surgically removed and subsequently analyzed under different conditions. Figure 18 The results showed that the livers of mice in the BTOGP-GPC3 NPs group were bright red, with extremely small tumor areas, consistent with the bioluminescence imaging results. Figure 18 (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. The substantial downregulation of the proliferation marker KI-67 indicates substantial inhibition of tumor cell growth, coupled with increased apoptosis.

[0114] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

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

2. The method for preparing the IR-BTOGP-GPC3 compound according to claim 1, comprising the following steps: The IR-BTOG compound, sodium azide, and organic solvent were mixed and subjected to an azide reaction to obtain the IR-BTOG-N3 compound. The IR-BTOG-N3 compound, copper(I)-thiophene-2-carboxylate, acetylene-polyethylene glycol-GPC3, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine and organic solvent were mixed and subjected to a superimposed-alkyl click cyclization reaction to obtain the IR-BTOGP-GPC3 compound with the structure shown in Formula 1. The IR-BTOG compound has the structure shown in Formula 2, and the IR-BTOG-N3 compound has the structure shown in Formula 3; 3. The preparation method according to claim 2, characterized in that, The preparation method of the IR-BTOG compound includes the following steps: Compound 7, n-butyllithium solution, tributyltin chloride and organic solvent were mixed and subjected to 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 were mixed and subjected to a first reaction to obtain the IR-BTOG compound; Compound 7 has the structure shown in Formula 4; compound 8 has the 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 acetylacetonate-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; and the mass ratio of the IR-BTOGP compound to tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine is 85-87:

5.

6. The use of the IR-BTOGP-GPC3 compound of claim 1 or the IR-BTOGP-GPC3 compound obtained by any one of claims 2 to 5 in the preparation of agents for specific targeting and diagnosis of hepatocellular carcinoma.

7. The use of the IR-BTOGP-GPC3 compound of claim 1 or the IR-BTOGP-GPC3 compound obtained by the preparation method of any one of claims 2 to 5 in the preparation of a targeted drug for hepatocellular carcinoma.

8. A targeted drug for hepatocellular carcinoma, comprising the IR-BTOGP-GPC3 compound of claim 1 or the IR-BTOGP-GPC3 compound obtained by any one of the preparation methods of claims 2 to 5.

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

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

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