Preparation method of tumor dual-targeting magnetic resonance / fluorescence imaging dual-mode contrast agent

Abstract

The invention discloses a preparation method of a tumor dual-targeting magnetic resonance / fluorescent dual-mode imaging contrast agent, which comprises the following steps: by taking a triphenylamine derivative as a fluorescent parent nucleus, coupling a manganese group, a biotin unit and the triphenylamine derivative through a conjugation effect to obtain the tumor dual-targeting magnetic resonance / fluorescent dual-mode imaging contrast agent. The magnetic resonance and fluorescence bimodal contrast agent TPA-BM with dual targeting of biotin receptors on the surfaces of tumor cells and intracellular mitochondria is prepared. The contrast agent TPA-BM is efficiently enriched in tumor cells by utilizing the dual active targeting property of the contrast agent TPA-BM, and precise positioning of tumor tissues is realized by virtue of the advantages of high tissue penetrability of T1 magnetic resonance and high sensitivity and real-time visualization of fluorescence of the contrast agent TPA-BM. The contrast agent TPA-BM prepared by the invention has the advantages of low toxicity, stable signal and the like, can realize preoperative diagnosis and intraoperative navigation of tumors, and has important theoretical and practical significance in clinical application of early diagnosis and precise surgical resection of tumors.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to a method for preparing a tumor dual-targeting magnetic resonance / fluorescence imaging dual-modality contrast agent. Background Art

[0002] As the disease with the highest mortality rate, cancer is an important health problem that humans need to face. How to achieve accurate diagnosis and effective treatment of tumors has become a frontier issue that urgently needs to be solved. At present, cancer is mostly treated with comprehensive means, including a combination of surgery and radiotherapy and chemotherapy. In addition, targeted therapy, immunotherapy, photothermal therapy, and photodynamic therapy have gradually become popular research directions in tumor treatment in recent years. It is worth noting that surgical resection and drug therapy are still the preferred methods for the treatment of malignant tumors. However, surgery mainly distinguishes normal tissue from tumor tissue by inspection and palpation, and conventional objective methods may lead to the remnant of tiny tumor tissue or unnecessary resection of normal tissue. Therefore, precise positioning of tumor tissue and its edges is a scientific and technological technology that modern medicine urgently needs to develop.

[0003] Studies have shown that the introduction of image-guided surgery (IGS) is expected to solve the above problems. It increases the contrast of tumor imaging and accurately distinguishes tumor tissue from normal tissue during surgery. At present, imaging methods such as computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT), ultrasound (US) and optical imaging have been successfully used in IGS. As a non-invasive clinical imaging method, MRI is widely used in clinical diagnosis due to its unlimited tissue penetration depth and high-resolution anatomical images. However, its low sensitivity and complex and expensive equipment limit the application of magnetic resonance imaging technology in intraoperative navigation. The high sensitivity, high spatial resolution, low cost and simple equipment of fluorescence imaging (FL) enable it to perform not only conventional tumor imaging, but also real-time imaging, thus becoming one of the most promising tools for guiding the precise positioning of tumors during surgery. It can be said that the combination of magnetic resonance imaging and fluorescence imaging can achieve complementary advantages in tumor imaging: excellent magnetic resonance and fluorescence multimodal imaging technology can provide more accurate anatomical information of tumors, while increasing intraoperative control and evaluation of image-guided treatment, which is expected to provide a better imaging strategy for future surgical navigation.

[0004] Targeted drug delivery systems enable contrast agents to accurately reach specific physiological sites, organs, tissues or cells to exert therapeutic effects, thereby enhancing contrast agent accumulation at the tumor site, enhancing the signal-to-noise ratio, and reducing side effects on normal tissues. Therefore, finding suitable targeting ligands to make contrast agents more targeted is a hot topic in contrast agent research. At present, biotin has been widely studied in the field of targeted tumor therapy due to its relatively low molecular weight, relatively simple chemical structure, low toxicity and high tumor specificity. Studies have shown that biotin regulates its intracellular uptake by participating in the binding of intracellular vitamin transporters, and rapidly proliferating tumor cells have a higher demand for biotin than normal tissues. Based on this, the coupling of biotin with contrast agent molecular groups can not only specifically target tumor tissues, but also achieve accurate qualitative and localization of tumor tissues, achieving the purpose of preoperative diagnosis and intraoperative navigation, which has important theoretical and practical significance. However, due to the heterogeneity of tumors, the types or expression levels of receptors expressed by tumor tissues vary greatly, and single target molecular probes may have false positive or false negative diagnostic results in application. Therefore, constructing multimodal molecular probes with cell membrane molecular receptors and organelles of different biological significance as dual targets is of great significance for enhancing the selectivity and affinity of the probes, improving the uptake of the probes in tumor tissues and the image quality of tumor imaging. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a dual-modality contrast agent for dual-targeting magnetic resonance / fluorescence imaging of tumors. By conjugation coupling between a manganese group, a biotin unit and a triphenylamine derivative, a contrast agent TPA-BM with dual-targeting functions of tumor cell biotin receptor targeting and mitochondria is prepared, so as to realize dual-modality magnetic resonance / fluorescence imaging of tumor tissues or cells, and is expected to realize accurate qualitative identification and positioning of tumor tissues, and achieve the purpose of preoperative diagnosis and intraoperative navigation, which has important theoretical and practical significance.

[0006] To achieve the above object, the technical solution adopted by the present invention is: a method for preparing a tumor dual-targeting magnetic resonance / fluorescence imaging dual-modality contrast agent, comprising the following steps:

[0007] (1) dissolving 3-bromo-1-propanol, D-biotin, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine in N,N-dimethylformamide in sequence and stirring at room temperature for 18 to 36 hours; the molar ratio of 3-bromo-1-propanol, D-biotin, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine is 1:(1.0 to 2.0):(1.0 to 2.0):(1.0 to 2.0); after the reaction is completed, slowly dripping the reaction solution into ultrapure water until a white solid precipitates, then filtering with suction, and redissolving the filter cake in dichloromethane and purifying it by silica gel column chromatography to obtain a white powder compound 1;

[0008] (2) Under nitrogen protection, 5-iodo-2-thiopheneacetonitrile, tetrakis(triphenylphosphine)palladium, anhydrous potassium carbonate and pyridine-4-boric acid are dissolved in a mixture of tetrahydrofuran and water in sequence, and stirred at 50-150° C. for 5-15 hours; the molar ratio of 5-iodo-2-thiopheneacetonitrile, tetrakis(triphenylphosphine)palladium, anhydrous potassium carbonate and pyridine-4-boric acid is 1:(0.05-0.15):(1-2):(1.0-2.0); after the reaction is completed, the reaction solution is diluted with ethyl acetate to separate the organic layer; then, the organic layer is washed with brine and dried over anhydrous sodium sulfate; finally, the organic layer is purified by silica gel column chromatography to obtain white crystalline compound 2;

[0009] (3) Under nitrogen protection, 4-boric acid triphenylamine, 4-bromo-2-hydroxybenzaldehyde, tetrakis(triphenylphosphine)palladium, anhydrous potassium carbonate and tetrabutylammonium bromide are dissolved in tetrahydrofuran, and stirred at 50-150° C. for 5-25 hours to obtain a reaction solution; the molar ratio of the 4-boric acid triphenylamine, 4-bromo-2-hydroxybenzaldehyde, tetrakis(triphenylphosphine)palladium, anhydrous potassium carbonate and tetrabutylammonium bromide is 1:(1.2-2.4):(0.05-0.15):(1.2-2.4):(0.5-1); after the reaction solution is cooled to room temperature, the reaction solution is diluted with dichloromethane to separate an organic layer; then, the organic layer is washed with brine and dried over anhydrous sodium sulfate; finally, the organic layer is purified by silica gel column chromatography to obtain a yellow powder compound 3;

[0010] (4) Compound 3, 3-(tert-butoxycarbonylamino)propyl bromide and anhydrous potassium carbonate are dissolved in N,N-dimethylformamide in sequence and stirred at 75-95° C. for 5-8 hours; the molar ratio of compound 3, 3-(tert-butoxycarbonylamino)propyl bromide and anhydrous potassium carbonate is 1:(1.25-2.5):(1.25-2.5); after the reaction liquid temperature drops to room temperature, the reaction liquid is poured into ultrapure water and continued to be stirred at room temperature for 0.3-0.7 hours until a light yellow solid is precipitated, and then filtered, and the filter cake is redissolved in dichloromethane and purified by silica gel column chromatography to obtain a yellow powder compound 4;

[0011] (5) Compound 2, Compound 4 and potassium tert-butoxide are dissolved in anhydrous ethanol and stirred at 75-95° C. for 2-3 hours; the molar ratio of Compound 2, Compound 4 and potassium tert-butoxide is 1:(1.0-2.0):(1.0-2.0); after the temperature of the reaction solution drops to room temperature, anhydrous ethanol is added to the reaction solution and stirring is continued for 0.3-0.7 hours; after solids are precipitated from the solution, the solution is filtered, the filter cake is redissolved in dichloromethane and purified by silica gel column chromatography to obtain an orange powdered compound 5;

[0012] (6) Under nitrogen protection, compound 5 is dissolved in anhydrous dichloromethane and stirred at 20-35° C. for 0.3-0.7 hours; then, trifluoroacetic acid is added to the reaction solution and stirring is continued for 8-16 hours; the molar ratio between compound 5 and trifluoroacetic acid is 1:(1.0-2.0); after the reaction is completed, the pH is adjusted to 6-8 with sodium hydroxide solution, and the reaction solution is diluted with dichloromethane to separate the organic layer; finally, the organic layer is washed with brine and dried with anhydrous sodium sulfate to obtain orange powder compound 6;

[0013] (7) After dissolving compound 6 in anhydrous dichloromethane, N,N-diisopropylethylamine was slowly added dropwise thereto; then, benzoyl isothiocyanate was slowly added to the solution and stirred at 20-35° C. for 0.3-0.7 hours; the molar ratio of compound 6 to benzoyl isothiocyanate was 1:(1.5-3.0); after the reaction was completed, the reaction solution was diluted with dichloromethane to separate the organic layer; then, the organic layer was washed with brine and dried over anhydrous sodium sulfate; finally, the organic layer was purified by silica gel column chromatography to obtain orange powder compound 7;

[0014] (8) Compound 7 and Compound 1 were dissolved in o-dichlorobenzene and stirred at 100-130° C. for 16-24 hours; the molar ratio of Compound 1 to Compound 7 was 1:(1.0-1.5); after the reaction was completed and the reaction solution cooled to room temperature, ethyl acetate was added to the reaction solution and the stirring was continued for 0.3-0.7 hours; after a red solid precipitated from the solution, the solution was filtered and the filter cake was redissolved in dichloromethane and the organic layer was purified by silica gel column chromatography to obtain a dark red powdered Compound 8 (TPA-B);

[0015] (9) Compound 8 (TPA-B) and anhydrous manganese dichloride were dissolved in methanol and stirred at 20-35° C. for 8-16 hours; the molar ratio of compound 8 (TPA-B) to anhydrous manganese chloride was 1:(1.0-2.0); after the reaction was completed, the reaction solution was diluted with dichloromethane to separate the organic layer; finally, the organic layer was washed with brine and dried with anhydrous sodium sulfate to obtain a dark red powder compound TPA-BM.

[0016] Preferably, in step (1), the molar volume ratio between 3-bromo-1-propanol and N,N-dimethylformamide is 1 mol:(4-8) L; and the eluent used for purification is a mixture of dichloromethane and methanol in a volume ratio of 30 / 1-50 / 1.

[0017] Preferably, in the step (2), the molar volume ratio of 5-iodo-2-thiopheneacetonitrile to tetrahydrofuran and water is 1 mol: (2.5-12.5) mL: (0.5-2.5) mL; and the eluent used for purification is a mixture of petroleum ether and ethyl acetate in a volume ratio of 1 / 1-1 / 10.

[0018] Preferably, in step (3), the molar volume ratio between 4-boric acid triphenylamine and tetrahydrofuran is 1 mol: (2-6) L; and the eluent used for purification is a mixture of petroleum ether and dichloromethane in a volume ratio of 6 / 1-8 / 1.

[0019] Preferably, in step (4), the molar volume ratio between compound 3 and N,N-dimethylformamide is 1 mol:(2.5-12.5) L; and the eluent used for purification is a mixture of petroleum ether and ethyl acetate in a volume ratio of 4 / 1-8 / 1.

[0020] Preferably, in step (5), the molar volume ratio between compound 4 and anhydrous ethanol is 1 mol: (5-10) L; and the eluent used for purification is a mixture of petroleum ether and ethyl acetate in a volume ratio of 1 / 1-4 / 1.

[0021] Preferably, in step (6), the molar volume ratio between compound 5 and anhydrous dichloromethane is 1 mol:(7-14.5)L.

[0022] Preferably, in step (7), the molar volume ratio between compound 6 and anhydrous dichloromethane is 1 mol:(5-10) L; and the eluent used for purification is a mixture of petroleum ether and ethyl acetate in a volume ratio of 1 / 1-1 / 10.

[0023] Preferably, in step (8), the molar volume ratio between compound 7 and o-dichlorobenzene is 1 mol:(1-3) L; and the eluent used for purification is a mixture of dichloromethane and ethyl acetate in a volume ratio of 20 / 1-100 / 1.

[0024] Preferably, in step (9), the molar volume ratio between compound 8 (TPA-B) and methanol is 1 mol: (5-10) L.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention uses triphenylamine derivatives as fluorescent mother nuclei, and couples manganese groups, biotin units and triphenylamine derivatives through conjugation to prepare magnetic resonance and fluorescence dual-modality contrast agents TPA-BM with dual targeting of biotin receptors on the surface of tumor cells and mitochondria in cells. The contrast agent TPA-BM utilizes its dual active targeting to efficiently enrich in tumor cells, and achieves precise positioning of tumor tissues by virtue of its high tissue penetration of T1 magnetic resonance and high sensitivity and real-time visualization of fluorescence. The contrast agent TPA-BM prepared by the present invention has the advantages of low toxicity and stable signals, and can realize preoperative diagnosis and intraoperative navigation of tumors, and has important theoretical and practical significance in the clinical application of early diagnosis and precise surgical resection of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the 1H NMR spectrum of compound 1;

[0028] Figure 2 is the 1H NMR spectrum of compound 2;

[0029] Figure 3 is the 1H NMR spectrum of compound 3;

[0030] Figure 4 is the 1H NMR spectrum of compound 4;

[0031] Figure 5 is the 1H NMR spectrum of compound 5;

[0032] Figure 6 is the 1H NMR spectrum of compound 6;

[0033] Figure 7 is the 1H NMR spectrum of compound 7;

[0034] Figure 8 is the 1H NMR spectrum of compound 8 (TPA-B);

[0035] Fig. 9 is the 13C NMR spectrum of compound 8 (TPA-B);

[0036] Fig.10 is the FT-IR graph of compound TPA-BM and compound 8 (TPA-B);

[0037] Fig.11 This is the full scan spectrum of XPS of compound TPA-BM;

[0038] Fig.12 is the Mn 2p pattern of XPS of compound TPA-BM;

[0039] Fig.13 It is the UV-visible absorption spectrum of compound TPA-BM;

[0040] Fig.14 This is the fluorescence emission spectrum of compound TPA-BM at 690nm when the excitation wavelength is 460nm; the concentration is 10μM;

[0041] Fig.15 It is the fluorescence emission spectrum of compound TPA-BM at 690nm under different ratios of toluene / DMSO, when the excitation wavelength is 460nm; the concentration is 10μM;

[0042] Fig.16 It is the fluorescence emission spectra of compound TPA-BM and ICG under PBS with different pH;

[0043] Fig.17 It is the fluorescence emission spectra of compound TPA-BM and ICG at different concentrations;

[0044] Fig.18 is the fluorescence emission spectrum of compound TPA-BM and ICG after incubation at 37°C for 7 days;

[0045] Fig.19 The compound TPA-BM and ICG were subjected to 25mW / cm 2 Fluorescence emission spectra after illumination;

[0046] Fig. 20 It is a statistical graph of cell survival rate after 4T1 cells were incubated with compounds TPA-BM and compound 8 (TPA-B) at different concentration gradients for 24 hours;

[0047] Fig.21It is a statistical graph of cell survival rate after NIH 3T3 cells were incubated with different concentration gradients of compounds TPA-BM and compound 8 (TPA-B) for 24 hours;

[0048] Fig. 22 It is a laser confocal colocalization image;

[0049] Fig.23 are laser confocal images of NIH 3T3 cells and 4T1 cells incubated with the same concentration of TPA-BM for different time periods (scale bar = 30 μm);

[0050] Fig.24 Magnetic resonance images of NIH 3T3 cells and 4T1 cells incubated with different concentrations of TPA-BM for 2 hours;

[0051] Fig.25 These are the magnetic resonance images of tumor-bearing mice at 0 to 24 hours after tail vein injection of TPA-BM;

[0052] Fig.26 These are fluorescent images of tumor-bearing mice injected with TPA-BM from the tail vein at 0 to 48 hours. DETAILED DESCRIPTION

[0053] The technical solutions in the embodiments of the present invention will be further described below in conjunction with the embodiments and drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0054] All raw materials and reagents in the examples of the present application were purchased on the market or prepared according to conventional methods well known to those skilled in the art.

[0055] Example

[0056] A preparation method of a contrast agent having dual-modality imaging capability and capable of specifically targeting tumor tissue comprises the following steps:

[0057] (1) Preparation of Compound 1: 3-bromo-1-propanol (1.38 g, 10 mmol), D-biotin (4.88 g, 20 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (3.82 g, 20 mmol) and 4-dimethylaminopyridine (2.44 g, 20 mmol) were dissolved in 80 mL of N,N-dimethylformamide in sequence and stirred at room temperature for 18 hours. After the reaction was completed, the reaction solution was slowly dripped into ultrapure water until a white solid precipitated. The mixed solution was filtered and the filter cake was redissolved in 50 mL of dichloromethane. Finally, the organic layer was purified by silica gel column chromatography using a mixture of dichloromethane and methanol (v / v=50 / 1) to obtain 2.26 g of white powdered Compound 1. 1 HNMR images Figure 1 As shown: 1 H NMR (400MHz, CDCl3) δ4.51(q,J=4.0Hz,1H),4.31(q,J=4.0Hz,1H),4.21(dd,J=12.0,4.0Hz,2H),3.61(t,J=4.0Hz,2H),3.46(t,J=8.0Hz,1H),3.13 -3.18(m,1H),2.91(dd,J=12.0,8.0Hz,1H),2.73(d,J=12.0Hz,1H),2.32( t,J=8.0Hz,2H),2.20-2.06(m,2H),1.77-1.61(m,4H),1.52-1.38(m,2H).

[0058] The synthetic route of compound 1 is:

[0059]

[0060] (2) Preparation of Compound 2: Under nitrogen protection, 5-iodo-2-thiopheneacetonitrile (0.99 g, 4 mmol), tetrakis(triphenylphosphine)palladium (684 mg, 0.6 mmol), anhydrous potassium carbonate (1.1 g, 8 mmol) and pyridine-4-boric acid (1.2 g, 8 mmol) were dissolved in a mixture of 50 mL of tetrahydrofuran and 10 mL of water, and stirred at 80° C. for 5 hours. After the reaction was completed, the reaction solution was diluted with 100 mL of ethyl acetate to separate the organic layer. Then, the organic layer was washed with brine and dried over anhydrous sodium sulfate. Finally, the organic layer was purified by silica gel column chromatography using a mixture of petroleum ether and ethyl acetate (v / v=1 / 1) to obtain 480 mg of white crystalline Compound 2. 1 H NMR images Figure 2 As shown: 1H NMR (400MHz, CDCl3) δ8.62(d,J=4.0Hz,2H),7.43(d,J=4.0Hz,2H),7.38(d,J=4.0Hz,1H),7.10(dt,J=4.0,4.0Hz,1H),3.96(d,J=4.0Hz,2H).

[0061] The synthetic route of compound 2 is:

[0062]

[0063] (3) Preparation of Compound 3: Under nitrogen protection, 4-boric acid triphenylamine (1.44 g, 5 mmol), 4-bromo-2-hydroxybenzaldehyde (2.4 g, 12 mmol), tetrakis(triphenylphosphine)palladium (855 mg, 0.75 mmol), anhydrous potassium carbonate (1.64 g, 12 mmol) and tetrabutylammonium bromide (1.6 g, 5 mmol) were dissolved in 30 mL of tetrahydrofuran and stirred at 80° C. for 15 hours. After the solution cooled to room temperature, the mixture was diluted with 100 mL of dichloromethane to separate the organic layer. Then, the organic layer was washed with brine and dried over anhydrous sodium sulfate. Finally, the organic layer was purified by silica gel column chromatography using a mixture of petroleum ether and dichloromethane (v / v=8 / 1) to obtain 1.46 g of yellow powdered Compound 3. 1 H NMR images Figure 3 As shown: 1 H NMR (400MHz, CDCl3) δ11.1(s,1H),9.88(s,1H),7.57(d,J=8.0Hz,1H),7.50(dt,J= 8.0,4.0Hz,2H),7.30(dt,J=8.0,4.0Hz,2H),7.27-7.21(m,3H),7.18-7.05(m,8H).

[0064] The synthetic route of compound 3 is:

[0065]

[0066] (4) Preparation of compound 4: Compound 3 (1.46 g, 4 mmol), N-Boc-3-aminopropyl bromide (2.38 g, 10 mmol) and anhydrous potassium carbonate (1.38 g, 10 mmol) were dissolved in 10 mL of N,N-dimethylformamide in sequence and stirred at 75°C for 8 hours. After the solution temperature dropped to room temperature, the reaction solution was poured into ultrapure water and continued to be stirred at room temperature for 0.5 hour until a light yellow solid precipitated from the solution. Then, the mixed solution was filtered and the filter cake was redissolved in 50 mL of dichloromethane. Finally, the organic layer was purified by silica gel column chromatography using a mixture of petroleum ether and ethyl acetate (v / v=8 / 1) to obtain 1.88 g of yellow powdered compound 4. 1 H NMR images Figure 4 As shown: 1 HNMR (400MHz, CDCl3) δ10.44(s,1H),7.86(d,J=4.0Hz,1H),7.47(d,J=8.0Hz,2H),7.31-7.22(m,6H),7.14(d,J=12. 0Hz, 6H), 7.06 (t, J = 8.0Hz, 3H), 4.85 (s, 1H), 4.21 (t, J = 8.0Hz, 2H), 3.37 (s, 2H), 2.08 (t, J = 8.0Hz, 2H), 1.43 (s, 9H).

[0067] The synthetic route of compound 4 is:

[0068]

[0069] (5) Preparation of Compound 5: Compound 2 (200 mg, 1 mmol), Compound 4 (1.04 g, 2 mmol) and potassium tert-butoxide (224 mg, 2 mmol) were dissolved in 15 mL of anhydrous ethanol and stirred at 75°C for 3 hours; after the solution temperature dropped to room temperature, 5 mL of anhydrous ethanol was added to the mixed solution and the stirring was continued for 0.5 hour; after the solid precipitated from the solution, the mixed solution was filtered and the filter cake was redissolved in 10 mL of dichloromethane; finally, the organic layer was purified by silica gel column chromatography using a mixture of petroleum ether and ethyl acetate (v / v=4 / 1) to obtain 492 mg of orange powdered Compound 5. 1 H NMR images Figure 5 As shown: 1H NMR (400MHz, DMSO-d6) δ8.59(s,2H),8.09(d,J=8.0Hz,1H),7.94(s,1H),7.85(s,1H),7.69(s,4H),7.47(s,1H),7.38 -7.31(m,6H),7.06(q,J=12.0Hz,6H),7.01(s,1H),6.93(s,1H),4.20(s,2H),3.05(s,1H),1.91(s,1H),1.33(s,9H).

[0070] The synthetic route of compound 5 is:

[0071]

[0072] (6) Preparation of Compound 6: Under nitrogen protection, Compound 5 (492 mg, 0.7 mmol) was dissolved in 5 mL of anhydrous dichloromethane and stirred at 35° C. for 0.5 hour; then, trifluoroacetic acid (320 mg, 1.4 mmol) was added to the solution and stirred for 8 hours; after the reaction was completed, the pH was adjusted to 6-8 with 10 mL of sodium hydroxide solution, and the mixture was diluted with 100 mL of dichloromethane to separate the organic layer; finally, the organic layer was washed with brine and dried over anhydrous sodium sulfate to obtain an orange powdered compound 6, which 1 H NMR images Figure 6 As shown: 1 H NMR (400MHz, DMSO-d6) δ8.62 (s, 2H), 8.10 (s, 1H), 7.91 (d, J = 20.2Hz, 2H), 7.77 (s,5H),7.49-7.34(m,8H),7.10(s,9H),4.30(s,2H),2.85(s,2H),1.96(s,2H).

[0073] The synthetic route of compound 6 is:

[0074]

[0075] (7) Preparation of compound 7: Compound 6 (0.6 g, 1 mmol) was dissolved in 10 mL of anhydrous dichloromethane, and 0.5 mL of N,N-diisopropylethylamine was slowly added dropwise thereto; then, benzoyl isothiocyanate (488 mg, 3 mmol) was slowly added to the solution, and stirred at 30°C for 0.5 hour; after the reaction was completed, the mixture was diluted with 50 mL of dichloromethane to separate the organic layer; then, the organic layer was washed with brine and dried over anhydrous sodium sulfate; finally, the organic layer was purified by silica gel column chromatography using a mixture of petroleum ether and ethyl acetate (v / v=1 / 1) to obtain 600 mg of orange powdered compound 7, whose 1H NMR spectrum is shown as follows: Figure 7 As shown: 1H NMR (400MHz, DMSO-d6) δ8.56 (d, J = 24.0, 2H), 8.12 (d, J = 4.0 Hz, 1H), 8.01 (s, 1H), 7.86 (t, J = 8.0 Hz, 3H), 7.60 (s, 1H), 7.43 (t, J = 8.0 Hz, 5H), 7.35 (s, 4H), 7.12-7.02 (m, 8H), 4.43 (s, 2H), 3.93 (s, 2H), 2.23 (s, 2H).

[0076] The synthetic route of compound 7 is:

[0077]

[0078] (8) Preparation of Compound 8 (TPA-B): Compound 7 (740 mg, 1 mmol) and Compound 1 (873 mg, 1.5 mmol) were dissolved in 1 mL of o-dichlorobenzene and stirred at 100°C for 24 hours. After the reaction was completed and the solution cooled to room temperature, 10 mL of ethyl acetate was added to the mixture and the mixture was stirred for 0.5 hours. After a red solid precipitated from the solution, the mixture was filtered and the filter cake was redissolved in 20 mL of dichloromethane. Finally, the organic layer was purified by silica gel column chromatography using a mixture of dichloromethane and methanol (v / v=50 / 1) to obtain 500 mg of dark red powdered Compound 8 (TPA-B). 1 H NMR images Figure 8Shown: 1H NMR (400MHz, DMSO-d6) δ11.23(s,1H),11.0(s,1H),9.03(d,J=4.0Hz,2H),8.42(d,J=4.0Hz,2H),8.29(s,1H),8.16(d,J=12.0Hz,2H),7.85(d,J=4. 0Hz,2H),7.75(d,J=8.0Hz,2H),7.65(s,1H),7.60(t,J=8.0Hz,2H),7.45 (t,J=8.0Hz,4H),7.35(t,J=8.0Hz,4H),7.10(t,J=12.0Hz,6H),7.03(d,J =8.0Hz,2H),6.39(d,J=16.0Hz,2H),4.62(s,2H),4.35(s,2H),4.27(s,1H),4.13(s,3H),3.92(s,1H),3.02(s,1H),2.78(d,J=12.0Hz,1H),2.56(s,1H),2.28-2.23(m,3H),2.16(t,J=8.0Hz,2H),1.54(s,1H),1.41(d,J=16.0Hz,3H),1.24(s,2H),0.95(d,J=4.0Hz,1H),0.84(d,J=8.0Hz,1H), and its 13C NMR spectrum is shown as follows Fig. 9 As shown, 13 C NMR(101MHz,DMSO-D6)δ180.78,173.13,168.29,163.26,158.45,148.32,147 .68,147.30,146.52,145.55,145.20,137.67,137.22,133.97,133.48,132.7 3,130.25,128.82,125.11,124.23,122.89,120.61,119.17,117.21,110.63,67.26,61.62,59.73,58.16,55.97,42.93,33.74,29.99,28.59,27.96,24.92.

[0079] (9) Preparation of compound TPA-BM: Compound 8 (TPA-B) (113 mg, 0.1 mmol) and anhydrous manganese dichloride (50 mg, 0.2 mmol) were dissolved in 1 mL of methanol and stirred at 35 °C for 16 hours. After the reaction was completed, the mixture was diluted with 50 mL of dichloromethane to separate the organic layer. Then, the organic layer was washed with brine and dried over anhydrous sodium sulfate to obtain a dark red powder compound TPA-BM.

[0080] The synthetic routes of the compounds 8 (TPA-B) and TPA-BM are:

[0081]

[0082] Fourier transform infrared spectroscopy shows ( Fig.10 ), 3286cm in the spectrum of TPA-BM -1 The typical NH stretching disappears. In addition, the two peaks at the Mn2p orbital in the XPS spectrum of TPA-BM, Mn2p 3 / 2 The characteristic peaks appearing at the main peak and at Mn2p 1 / 2 The main peak has a characteristic peak ( Fig.11 and Fig.12 ), all of which indicated the successful preparation of TPA-BM.

[0083] In addition, the in vitro optical properties of the contrast agent TPA-BM were systematically analyzed using UV-visible absorption spectroscopy and fluorescence emission spectroscopy. Figure 13-14 As shown. The ultraviolet absorption peak of TPA-BM is around 450nm, and its maximum emission wavelength in PBS is at 690nm, and the fluorescence emission wavelength range covers the near-infrared light. Moreover, Fig.15 The fluorescence intensity of the compound TPA-BM increased with the increase of water content, indicating that TPA-BM also has AIE effect. The fluorescence emission spectrum of TPA-BM in vitro showed stability at different pH values. Fig.16 ). It is worth noting that the fluorescence intensity of TPA-BM gradually increased with increasing concentration, while ICG (indocyanine green, the only cyanine dye drug approved by the US Food and Drug Administration for surgical navigation) showed an opposite growth trend ( Fig.17 ). Unlike ICG, the fluorescence intensity of the contrast agent TPA-BM did not change significantly after incubation at 37°C for 7 days ( Fig.18 In addition, the fluorescence intensity of the contrast agent TPA-BM did not change significantly after 20 minutes of illumination, while the fluorescence of ICG decreased significantly, which indicates that the contrast agent TPA-BM prepared by the present invention exhibits more stable imaging performance in vitro than the commercially available contrast agent ICG ( Fig.19 ).

[0084] After investigating the biocompatibility of TPA-BM, it was found that TPA-BM showed almost no toxicity to normal NIH 3T3 cells, while tumor cells, due to the presence of hydrogen peroxide, would produce a Fenton-like reaction with divalent manganese ions, thus causing certain damage to tumor cells ( Fig. 20 and Fig.21). Since the structure of TPA-BM not only contains mitochondrial targeting groups, but also cleverly introduces biotin targeting receptor groups, biotin, it can more efficiently target tumor cells. Fig. 22 As shown in Figure 2, the red fluorescence of the contrast agent TPA-BM can overlap with the green fluorescence of the mitochondrial fluorescent probe, indicating that TPA-BM can efficiently and specifically target the mitochondria of tumor cells. Finally, the enhancement effect of TPA-BM on T1-weighted magnetic resonance imaging was further evaluated at the cellular level. The results are shown in Figure 2. Fig.23 and Fig.24 As shown in Figure 3, the signal intensity of T1-weighted images and pseudo-color images of 4T1 cells gradually increased with the increase of TPA-BM concentration, while the magnetic resonance signal of NIH 3T3 cells did not show obvious signal enhancement at the highest concentration, indicating that TPA-BM is expected to become a targeted contrast agent for tumor T1-weighted magnetic resonance imaging.

[0085] In order to further verify the ability of compound TPA-BM to image tumor tissues in magnetic resonance and fluorescence, a mouse breast cancer model was first established on the upper thigh of BalB / c mice, TPA-BM was injected through the tail vein, and T1-weighted images and pseudo-color images of tumor-bearing mice at different time points were collected on a 3.0T magnetic resonance scanner. Fig.25 As shown in the figure, after injection of TPA-BM, the magnetic resonance signal at the tumor site was significantly enhanced, and the signal reached a peak at 3 hours after injection; then, the signal weakened over time. The in vivo fluorescence signal was continuously observed using a small animal in vivo imaging device, such as Fig.26 As shown, TPA-BM can effectively accumulate in the tumor site, reaching a peak 3 hours after injection, and tumor signals can still be observed at 48 hours after injection, which is consistent with the results of magnetic resonance imaging. This suggests that TPA-BM has good and stable biological imaging capabilities, and its tumor targeting and retention properties are expected to become a targeted contrast agent for tumor magnetic resonance / fluorescence imaging.

Claims

1. A method for preparing a dual-modality contrast agent for tumor dual-targeting magnetic resonance / fluorescence imaging, characterized in that The steps include: (1) dissolving 3-bromo-1-propanol, D-biotin, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine in N,N-dimethylformamide in sequence and stirring at room temperature for 18 to 36 hours; the molar ratio of 3-bromo-1-propanol, D-biotin, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine is 1:(1.0 to 2.0):(1.0 to 2.0):(1.0 to 2.0); after the reaction is completed, slowly dripping the reaction solution into ultrapure water until a white solid precipitates, then filtering with suction, and redissolving the filter cake in dichloromethane and purifying it by silica gel column chromatography to obtain a white powder compound 1; (2) Under nitrogen protection, 5-iodo-2-thiopheneacetonitrile, tetrakis(triphenylphosphine)palladium, anhydrous potassium carbonate and pyridine-4-boric acid are dissolved in a mixture of tetrahydrofuran and water in sequence, and stirred at 50-150° C. for 5-15 hours; the molar ratio of 5-iodo-2-thiopheneacetonitrile, tetrakis(triphenylphosphine)palladium, anhydrous potassium carbonate and pyridine-4-boric acid is 1:(0.05-0.15):(1-2):(1.0-2.0); after the reaction is completed, the reaction solution is diluted with ethyl acetate to separate the organic layer; then, the organic layer is washed with brine and dried over anhydrous sodium sulfate; finally, the organic layer is purified by silica gel column chromatography to obtain white crystalline compound 2; (3) Under nitrogen protection, 4-boric acid triphenylamine, 4-bromo-2-hydroxybenzaldehyde, tetrakis(triphenylphosphine)palladium, anhydrous potassium carbonate and tetrabutylammonium bromide are dissolved in tetrahydrofuran, and stirred at 50-150° C. for 5-25 hours to obtain a reaction solution; the molar ratio of the 4-boric acid triphenylamine, 4-bromo-2-hydroxybenzaldehyde, tetrakis(triphenylphosphine)palladium, anhydrous potassium carbonate and tetrabutylammonium bromide is 1:(1.2-2.4):(0.05-0.15):(1.2-2.4):(0.5-1); after the reaction solution is cooled to room temperature, the reaction solution is diluted with dichloromethane to separate an organic layer; then, the organic layer is washed with brine and dried over anhydrous sodium sulfate; finally, the organic layer is purified by silica gel column chromatography to obtain a yellow powder compound 3; (4) Compound 3, 3-(tert-butoxycarbonylamino)propyl bromide and anhydrous potassium carbonate are dissolved in N,N-dimethylformamide in sequence and stirred at 75-95° C. for 5-8 hours; the molar ratio of compound 3, 3-(tert-butoxycarbonylamino)propyl bromide and anhydrous potassium carbonate is 1:(1.25-2.5):(1.25-2.5); after the reaction liquid temperature drops to room temperature, the reaction liquid is poured into ultrapure water and continued to be stirred at room temperature for 0.3-0.7 hours until a light yellow solid is precipitated, and then filtered, and the filter cake is redissolved in dichloromethane and purified by silica gel column chromatography to obtain a yellow powder compound 4; (5) Compound 2, Compound 4 and potassium tert-butoxide are dissolved in anhydrous ethanol and stirred at 75-95° C. for 2-3 hours; the molar ratio of Compound 2, Compound 4 and potassium tert-butoxide is 1:(1.0-2.0):(1.0-2.0); after the temperature of the reaction solution drops to room temperature, anhydrous ethanol is added to the reaction solution and stirring is continued for 0.3-0.7 hours; after solids are precipitated from the solution, the solution is filtered, the filter cake is redissolved in dichloromethane and purified by silica gel column chromatography to obtain an orange powdered compound 5; (6) Under nitrogen protection, compound 5 is dissolved in anhydrous dichloromethane and stirred at 20-35° C. for 0.3-0.7 hours; then, trifluoroacetic acid is added to the reaction solution and stirring is continued for 8-16 hours; the molar ratio between compound 5 and trifluoroacetic acid is 1:(1.0-2.0); after the reaction is completed, the pH is adjusted to 6-8 with sodium hydroxide solution, and the reaction solution is diluted with dichloromethane to separate the organic layer; finally, the organic layer is washed with brine and dried with anhydrous sodium sulfate to obtain orange powder compound 6; (7) After dissolving compound 6 in anhydrous dichloromethane, N,N-diisopropylethylamine was slowly added dropwise thereto; then, benzoyl isothiocyanate was slowly added to the solution and stirred at 20-35° C. for 0.3-0.7 hours; the molar ratio of compound 6 to benzoyl isothiocyanate was 1:(1.5-3.0); after the reaction was completed, the reaction solution was diluted with dichloromethane to separate the organic layer; then, the organic layer was washed with brine and dried over anhydrous sodium sulfate; finally, the organic layer was purified by silica gel column chromatography to obtain orange powder compound 7; (8) Compound 7 and Compound 1 were dissolved in o-dichlorobenzene and stirred at 100-130° C. for 16-24 hours; the molar ratio of Compound 1 to Compound 7 was 1:(1.0-1.5); after the reaction was completed and the reaction solution cooled to room temperature, ethyl acetate was added to the reaction solution and the stirring was continued for 0.3-0.7 hours; after a red solid precipitated from the solution, the solution was filtered and the filter cake was redissolved in dichloromethane and the organic layer was purified by silica gel column chromatography to obtain a dark red powdered Compound 8 (TPA-B); (9) Compound 8 (TPA-B) and anhydrous manganese dichloride were dissolved in methanol and stirred at 20-35° C. for 8-16 hours; the molar ratio of compound 8 (TPA-B) to anhydrous manganese chloride was 1:(1.0-2.0); after the reaction was completed, the reaction solution was diluted with dichloromethane to separate the organic layer; finally, the organic layer was washed with brine and dried with anhydrous sodium sulfate to obtain a dark red powder compound TPA-BM.

2. The method for preparing a tumor dual-targeting magnetic resonance / fluorescence imaging dual-modality contrast agent as claimed in claim 1, characterized in that: In the step (1), the molar volume ratio between 3-bromo-1-propanol and N,N-dimethylformamide is 1 mol:(4-8) L; the eluent used for purification is a mixture of dichloromethane and methanol in a volume ratio of 30 / 1-50 / 1.

3. The method for preparing a tumor dual-targeting magnetic resonance / fluorescence imaging dual-modality contrast agent as claimed in claim 1, characterized in that: In the step (2), the molar volume ratio of 5-iodo-2-thiopheneacetonitrile to tetrahydrofuran and water is 1 mol: (2.5-12.5) mL: (0.5-2.5) mL; the eluent used for purification is a mixture of petroleum ether and ethyl acetate in a volume ratio of 1 / 1-1 / 10.

4. The method for preparing a tumor dual-targeting magnetic resonance / fluorescence imaging dual-modality contrast agent as claimed in claim 1, characterized in that: In the step (3), the molar volume ratio between 4-boric acid triphenylamine and tetrahydrofuran is 1 mol: (2-6) L; the eluent used for purification is a mixture of petroleum ether and dichloromethane in a volume ratio of 6 / 1-8 / 1.

5. The method for preparing a tumor dual-targeting magnetic resonance / fluorescence imaging dual-modality contrast agent as claimed in claim 1, characterized in that: In the step (4), the molar volume ratio between compound 3 and N,N-dimethylformamide is 1 mol: (2.5-12.5) L; the eluent used for purification is a mixture of petroleum ether and ethyl acetate in a volume ratio of 4 / 1-8 / 1.

6. The method for preparing a tumor dual-targeting magnetic resonance / fluorescence imaging dual-modality contrast agent as claimed in claim 1, characterized in that: In the step (5), the molar volume ratio between compound 4 and anhydrous ethanol is 1 mol: (5-10) L; the eluent used for purification is a mixture of petroleum ether and ethyl acetate in a volume ratio of 1 / 1-4 / 1.

7. The method for preparing a tumor dual-targeting magnetic resonance / fluorescence imaging dual-modality contrast agent as claimed in claim 1, characterized in that: The molar volume ratio between compound 5 and anhydrous dichloromethane in step (6) is 1 mol: (7-14.5) L.

8. The method for preparing a tumor dual-targeting magnetic resonance / fluorescence imaging dual-modality contrast agent as claimed in claim 1, characterized in that: In the step (7), the molar volume ratio between compound 6 and anhydrous dichloromethane is 1 mol: (5-10) L; the eluent used for purification is a mixture of petroleum ether and ethyl acetate in a volume ratio of 1 / 1-1 / 10.

9. The method for preparing a tumor dual-targeting magnetic resonance / fluorescence imaging dual-modality contrast agent as claimed in claim 1, characterized in that: In the step (8), the molar volume ratio of compound 7 to o-dichlorobenzene is 1 mol:(1-3) L; the eluent used for purification is a mixture of dichloromethane and ethyl acetate in a volume ratio of 20 / 1-100 / 1.

10. The method for preparing a tumor dual-targeting magnetic resonance / fluorescence imaging dual-modality contrast agent according to claim 1, characterized in that: The molar volume ratio between compound 8 (TPA-B) and methanol in step (9) is 1 mol: (5-10) L.

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