Phthalocyanine rare earth complex as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of fluorescent dyes, and particularly relates to a phthalocyanine rare earth complex as well as a preparation method and application thereof. The phthalocyanine rare earth complex has a narrow absorption band, the half-peak width is 20-47nm, and the overlapping risk can be reduced; the maximum absorption wavelength is 673-772 nm and is convenient to adjust, the emission wavelengths are all located at 1534 nm, that is, the emission wavelengths are all located in a near-infrared II region, and the high-capacity in-vivo excitation spectrum imaging device is suitable for being used for high-capacity in-vivo excitation spectrum imaging. The phthalocyanine rare earth complex can realize almost 100% of photon collection efficiency in a near-infrared II region in spectral imaging. And meanwhile, the narrow absorption band also allows a small amount of excitation scanning to realize spectrum reconstruction, so that the spectral imaging speed is improved, and the requirement of rapid decision making in an operation is met.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent dyes, and in particular relates to a phthalocyanine rare earth complex and a preparation method and application thereof. Background Art

[0002] Fluorescence-guided surgical navigation uses fluorescent dyes to mark tumors or key anatomical structures (such as nerves and blood vessels), and displays areas that are difficult to observe with the naked eye in real time during surgery, thereby improving surgical precision. Compared with traditional visual / palpation, the core role of fluorescence-guided surgical navigation is to simultaneously obtain anatomical information (such as ureteral position) and functional information (such as vascular perfusion status or bleeding leakage) of the tissue coverage area in a single imaging, thereby assisting surgeons in optimizing intraoperative decision-making, improving surgical integrity and reducing the risk of accidental injury. However, such as indocyanine green (ICG), it can only be marked in single color and cannot display multiple types of information at the same time. In addition, the emission spectrum of traditional fluorophores (such as ICG) is relatively wide, and the signals crosstalk with each other during multi-color marking, resulting in blurred imaging; for example, when marking tumors and blood vessels at the same time, the spectra of the two fluorescent signals partially overlap and are difficult to distinguish. Doctors cannot obtain multiple information (such as tumor boundary + vascular perfusion + nerve position) through a single imaging.

[0003] The main principle of hyperspectral imaging technology is to scan and image the excitation or emission spectra to obtain a three-dimensional dataset (x, y, λ). Using the spectral information obtained from this 3D dataset, different unmixing algorithms are applied to unmix overlapping spectra, thereby distinguishing different dye markers. Currently, hyperspectral imaging technology has been used to identify dozens of markers in bacteria, cells, and transparent animals such as zebrafish. In theory, hyperspectral imaging can simultaneously reveal multiple structural and functional information, enhancing intraoperative decision-making. However, in living mammals, the emission of conventional fluorescent probes primarily falls within the 400-1000nm wavelength range, which is susceptible to tissue scattering, autofluorescence, and absorption. This 400-1000nm wavelength range can distort data during in vivo imaging and affect unmixing accuracy. Mammalian tissues have varying thickness and density, and complex light paths further distort the spectrum. Respiration and heartbeats cause image motion artifacts, affecting data stability. Existing spectral unmixing methods rely on manual intervention or are only effective in simple cell experiments. The complexity and dynamic nature of in vivo data make automated analysis difficult.

[0004] Therefore, developing a new imaging system based on narrow spectrum probes in the near-infrared II region (1000-1700nm) and constructing an automated spectral unmixing algorithm for the living environment have become the key to breaking through the real-time feedback of multiple parameters during surgery. Summary of the Invention

[0005] (1) Technical issues to be resolved

[0006] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a phthalocyanine rare earth complex and its preparation method and application, which solves the technical problems in the prior art that the spectra partially overlap and are difficult to distinguish, and doctors cannot obtain multiple information through a single imaging.

[0007] (2) Technical solution

[0008] In a first aspect, the present invention provides a phthalocyanine rare earth complex having a structure shown in general formula A:

[0009]

[0010] Wherein, the rare earth ion Ln is selected from at least one of Ce, Pm, Eu, Gd, Tb, Pr, Nd, Sm, Dy, Ho, Er, Tm and Yb, and R1, R2, R3 and R4 are each independently selected from any one of H, C1-C5 alkoxy and the groups represented by formula I-IV.

[0011]

[0012] Optionally, the phthalocyanine rare earth complex has a maximum absorption wavelength between 670-772 nm, an emission wavelength at 1534 nm, and a molecular weight between 1000-2500 Da.

[0013] Optionally, the complex represented by the general formula A has any of the following specific structures:

[0014]

[0015] In a second aspect, the present invention provides a method for preparing the phthalocyanine rare earth complex described in the first aspect, comprising the following steps:

[0016] S1, cyclizing a phthalonitrile derivative to obtain phthalocyanine;

[0017] S2, introducing a rare earth metal into the center of the phthalocyanine through a coordination reaction to obtain an intermediate;

[0018] S3, the intermediate and the tripod ligand NaL OM e reaction to obtain a phthalocyanine rare earth complex with a structure shown in general formula A.

[0019] Optionally, the preparation method includes the following reactions:

[0020]

[0021] Under the protection of a non-oxidizing inert atmosphere, compound (1) reacts with lithium block in a first organic solvent at 100-140° C. to generate compound (2);

[0022] Under the protection of a non-oxidizing inert atmosphere, compound (2) reacts with a rare earth salt of acetylacetonate in a second organic solvent at 200-220° C. to generate a reaction intermediate;

[0023] The intermediate and the tripod ligand NaL OM e. reacting in a third organic solvent to obtain a rare earth phthalocyanine complex (3) having a structure represented by general formula A.

[0024] Optionally, the general structural formula of the acetylacetonate rare earth salt is Ln(acac)3, wherein the rare earth ion Ln is at least one of Ce, Pm, Eu, Gd, Tb, Pr, Nd, Sm, Dy, Ho, Er, Tm and Yb.

[0025] Optionally, the non-oxidizing inert atmosphere is selected from at least one of nitrogen, argon and helium;

[0026] The first organic solvent is selected from at least one of n-butanol, n-pentanol and n-heptanol;

[0027] The second organic solvent is selected from at least one of trichlorobenzene, dimethyl sulfoxide and toluene;

[0028] The third organic solvent is selected from at least one of chloroform, tetrahydrofuran and methanol.

[0029] Optionally, the compound (2) has any of the following specific structures:

[0030]

[0031] In a third aspect, the present invention provides use of the phthalocyanine rare earth complex described in the first aspect and the phthalocyanine rare earth complex obtained by the preparation method described in the second aspect in preparing a contrast agent.

[0032] Optionally, the phthalocyanine rare earth complex is used to prepare a mesenteric lymph node contrast agent.

[0033] (3) Beneficial effects

[0034] The phthalocyanine rare earth complexes of the present invention have a narrow absorption band with a half-width of 20-47 nm, which reduces the risk of overlap. Their maximum absorption wavelength is between 673 and 772 nm, making them easily adjustable. Their emission wavelengths are all located at 1534 nm, falling uniformly within the near-infrared II region, making them suitable for high-volume in vivo excitation spectral imaging. The phthalocyanine rare earth complexes can achieve nearly 100% photon collection efficiency in the near-infrared II region for spectral imaging. The narrow absorption band also allows for spectral reconstruction using fewer excitation scans, increasing the speed of spectral imaging and meeting the need for rapid decision-making during surgery.

[0035] The narrow absorption band (half-maximum width 20-47nm) effectively reduces spectral crosstalk when multiple probes coexist. Using fluorescent materials with emission wavelengths in the near-infrared II region (1000-1700nm), the tissue's absorption / scattering of 1000-1700nm photons is extremely low, and autofluorescence is almost zero, significantly improving the signal-to-noise ratio and reducing tissue interference with the fluorescence signal. This provides high-fidelity spectral information in in vivo imaging, laying the foundation for subsequent real-time automated multispectral unmixing.

[0036] The phthalocyanine rare earth complex fluorescent material provided by the present invention can achieve high-capacity in vivo spectral imaging, reveal key anatomical structures, primary tumors and metastatic tumors during surgery, and provide surgeons with precise resection guidance during the operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The chemical structural formula of the phthalocyanine rare earth complex (1b-9b) of the present invention and its absorption spectrum and emission spectrum in dichloromethane are shown.

[0038] Figure 2 The absorption spectra (a, b) and excitation spectrum (c) of the phthalocyanine rare earth complex (1b-9b) of the present invention in tetrahydrofuran are shown.

[0039] Figure 3 The phthalocyanine rare earth complexes (7b and 9b) of the present invention are used for hyperspectral imaging of blood vessels and gastrointestinal tract of living normal mice; wherein, Figure 3 a is a schematic diagram of the multispectral imaging scheme of a normal mouse model; Figure 3 b is the result collected in the 1500-1700 nm band; Figure 3 c is the result collected in the 850-1000 nm band; Figure 3 d is the vascular contrast; Figure 3 e Above is the relative residual of all pixels.

[0040] Figure 4 Schematic diagram of fluorescent surgical navigation using phthalocyanine rare earth complex fluorescent dyes (2b, 4b, 6b, 7b, 9b) for colorectal tumor resection in mice; Figure 4 a is a schematic diagram of the anatomical structure labeling scheme for five-color imaging of mouse colorectal cancer model; Figure 4 b The right side is the five-color unmixed image, and the left side is the five-color superimposed image; Figure 4 c is Figure 4 Intensity cross-section at the dotted line position in b; Figure 4 d is Figure 4 b Fluorescence signal-to-noise ratio of the resected primary tumor and metastasis in the tumor channel; Figure 4 e is Figure 4Average intensity measurements of ROI1 and ROI2 in b.

[0041] Figure 5 A diagram of a device for using phthalocyanine rare earth complex fluorescent dyes (2b, 4b, 6b, 7b, 9b) for surgical fluorescence navigation.

[0042] Figure 6 Flowchart for automating image analysis and spectral extraction for deep learning neural network models.

[0043] Figure 7 An interactive interface for an automated in vivo spectral imaging program integrated with a deep learning neural network. DETAILED DESCRIPTION

[0044] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation methods in conjunction with the accompanying drawings.

[0045] Preparation Example 1

[0046]

[0047] (1) Compound 1a was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0048] (2) Synthesis of dye 1b

[0049] Compound 1a, DBU, and Er(acac)3 were mixed in a molar ratio of 1:4:5 and dissolved in 5 mL of 1,2,4-trichlorobenzene. The reaction system was degassed and exchanged with a nitrogen atmosphere for three cycles, then heated to 210°C for 0.5 h. After cooling to room temperature, the high-boiling point solvent was removed by column chromatography under reduced pressure using petroleum ether as the eluent, and then eluted with a mixed solvent of dichloromethane and methanol in a volume ratio of 10:1. The eluate was collected. After concentration, an equal molar amount of the ligand NaL was added to the phthalocyanine. OM e (dissolved in a small amount of methanol) and refluxed for 4 h. After the reaction solution was cooled, the solvent was removed by rotary evaporation and the product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1) to obtain dye 1b (see the above reaction formula).

[0050] Characterization data of dye 1b are as follows:

[0051] 1 H NMR (600 MHz, CDCl 3 ) chemical shift (δ): 35.60 (s, 8H), 29.23 (s, 18H), 21.32 (s, 8H), −49.00 (s, 5H).

[0052] MALDI-TOF-MS: Molecular formula: C 43 H 39CoErN8O9P3, theoretical molecular weight [M]+1131.0714, measured molecular weight 1131.6823.

[0053] Preparation Example 2

[0054]

[0055] (1) Compound 2a was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0056] (2) Synthesis of dye 2b

[0057] Compound 2a, DBU, and Er(acac)3 were mixed in a molar ratio of 1:4:5 and dissolved in 5 mL of 1,2,4-trichlorobenzene. The reaction mixture was degassed and replaced with a nitrogen atmosphere three times, then heated to 210°C for 0.5 h. After cooling to room temperature, the high-boiling solvent was removed by column chromatography under reduced pressure using petroleum ether as the eluent. The eluate was then eluted with a 10:1 volume ratio of dichloromethane and methanol, and the eluate was collected. After concentration, an equimolar amount of the ligand NaLOMe (dissolved in a small amount of methanol) was added to 2a, and the mixture was refluxed for 4 h. After cooling, the reaction solution was rotary evaporated to remove the solvent, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1) to yield dye 2b (see the above reaction formula).

[0058] Characterization data of dye 2b are as follows:

[0059] 1 H NMR (600 MHz, CDCl 3 ) chemical shift (δ): 37.35-33.95 (m, 8H), 29.06 (s, 18H), 21.93-20.49 (m, 4H), 10.40-9.69 (m, 36H), -48.36 (s, 5H).

[0060] Maldi-TOF-MS: Molecular formula: C 59 H 71 CoErN8O9P3, theoretical molecular weight [M]+1355.3221, measured molecular weight 1355.0419.

[0061] Preparation Example 3

[0062]

[0063] (1) Synthesis of compound 3a

[0064] Compound 1 and lithium block were mixed in a molar ratio of 1:2 and dissolved in 8 mL of n-pentanol. The reaction mixture was degassed and replaced with a nitrogen atmosphere three times. The reaction system was heated to 100°C for 4 hours. After the reaction was completed, the reaction was cooled to room temperature and the solvent was removed by rotary evaporation to obtain a crude product. The crude product was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 5:1) to obtain compound 3a.

[0065] The characterization data of compound 3a are as follows:

[0066] 1 H NMR (400 MHz, CDCl3), chemical shift (δ): 6.48 (dd, coupling constant J = 119.6, 48.5 Hz, 8H); 3.75 (m, 8H); 1.88 (m, 8H); 1.68-1.47 (m, 16H); 1.12 (t, J = 5.8 Hz, 12H); -5.84 (s, 2H).

[0067] Maldi-TOF-MS molecular formula: C 52 H 58 N8O4, theoretical molecular weight [M] + :858.4581, measured molecular weight [M] + :858.9194.

[0068] (2) Synthesis of dye 3b

[0069] Compound 3a, DBU and Er(acac)3 were mixed in a molar ratio of 1:4:5 and dissolved in 5 mL of 1,2,4-trichlorobenzene. The reaction mixture was degassed and exchanged with a nitrogen atmosphere, repeated three times, and then heated to 210°C for 0.5 h. After cooling to room temperature, petroleum ether was used as the eluent, and the high-boiling point solvent was removed by column chromatography under reduced pressure. Then, the mixture was eluted with a mixed solvent of dichloromethane and methanol in a volume ratio of 10:1, and the eluate was collected. After concentrating it, an equal molar amount of ligand NaL was added to compound 3a. OM e (dissolved in a small amount of methanol) and refluxed for 4 h. After the reaction solution was cooled, the solvent was removed by rotary evaporation and separated by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1) to obtain dye 3b (see the above reaction formula).

[0070] Characterization data of dye 3b are as follows:

[0071] 1H NMR (600 MHz, CDCl3) chemical shift (δ): 35.01 (m, 8H), 29.12 (s, 18H), 21.19-19.72 (m, 4H), 16.11-13.21 (m, 8H), 8.18 (m, 8H), 7.05 (d, 8H), 5.62 (s, 9H), 4.24 (d, 12H), -47.93 (s, 5H).

[0072] Maldi-TOF-MS: Molecular formula: C 63 H 81 CoErN8O 13 P3, theoretical molecular weight [M]+1476.3646; measured molecular weight 1476.1287.

[0073] Preparation Example 4

[0074]

[0075] (1) Synthesis of compound 4a

[0076] Compound 2 and lithium block were mixed in a 1:2 molar ratio and dissolved in 8 ml of n-pentanol. The reaction mixture was degassed and then replaced with a nitrogen atmosphere three times. The reaction system was then heated to 100°C for 4 hours. After the reaction, the mixture was cooled to room temperature and the solvent was removed by rotary evaporation to obtain a crude product. The product was then separated by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1) to afford compound 4a.

[0077] The characterization data of compound 4a are as follows:

[0078] 1 H NMR (400 MHz, CDCl₃), chemical shift (δ): 8.54 (d, J = 7.2 Hz, 4H), 7.79 (t, J = 7.6 Hz, 4H), 7.35 (d, J = 7.9 Hz, 4H), 4.54 (t, J = 6.3 Hz, 7H), 2.42-2.32 (m, 12H), 2.02 dt, J = 15.2, 7.4 Hz, 10H), 1.71 (dt, J = 14.4, 7.4 Hz, 14H), 1.58-1.46 (m, 24H), 1.01 (t, J = 6.8 Hz, 13H), -1.23 (d, J = 142.0 Hz, 3H), -1.41 (s, 2H).

[0079] Maldi-TOF-MS: Molecular formula: C 60 H 74 N8O4, theoretical molecular weight [M] + :971.5833; Measured molecular weight [M] + :972.0654.

[0080] (2) Synthesis of dye 4b

[0081] Compound 4a, DBU and Er(acac)3 were mixed in a molar ratio of 1:4:5 and dissolved in 5 mL of 1,2,4-trichlorobenzene. The reaction mixture was degassed and exchanged with a nitrogen atmosphere, repeated three times, and then heated to 210°C for 0.5 h. After cooling to room temperature, petroleum ether was used as the eluent, and the high-boiling point solvent was removed by column chromatography under reduced pressure. Then, the mixture was eluted with a mixed solvent of dichloromethane and methanol in a volume ratio of 10:1, and the eluate was collected. After concentration, an equal molar amount of ligand NaL was added to compound 4a. OM e (dissolved in a small amount of methanol) and refluxed for 4 h. After the reaction solution was cooled, the solvent was removed by rotary evaporation and separated by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1) to obtain dye 4b (see the above reaction formula).

[0082] Characterization data of dye 4b are as follows:

[0083] 1 H NMR (400 MHz, CDCl3) chemical shift (δ): 36.25 (s, 2H), 29.28 (s, 18H), 20.77 (d, 10H), 14.17 (m, 24H), 9.42 (d, 8H), 5.75 (d, 8H), 3.78 (s, 12H), -52.01 (s, 5H).

[0084] Maldi-TOF-MS: Molecular formula: C 71 H 95 CoErN8O 13 The theoretical molecular weight of P3 [M]+ is 1587.4900; the measured molecular weight is 1587.3079.

[0085] Preparation Example 5

[0086]

[0087] (1) Synthesis of compound 5a

[0088] Compound 3 and lithium block were mixed in a 1:2 molar ratio and dissolved in 8 ml of n-pentanol. The reaction mixture was degassed and replaced with a nitrogen atmosphere three times, then heated to 100°C for 4 hours. After the reaction was completed, the mixture was cooled to room temperature and the solvent was removed by rotary evaporation to obtain a crude product. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 8:1) to obtain compound 5a.

[0089] The characterization data of compound 5a are as follows:

[0090] 1H NMR (400 MHz, CDCl3), chemical shift (δ): 8.87 (d, J = 7.5 Hz, 4H), 7.93 (t, J = 7.6 Hz, 4H), 7.72 (d, J = 7.7 Hz, 4H), 7.38 (d, J = 9.0 Hz, 16H), 6.79 (d, J = 9.0 Hz, 16H), 3.68 (s, 24H), -0.24 (s, 2H).

[0091] Maldi-TOF-MS: molecular formula C 88 H 70 N 12 Theoretical molecular weight of O8 [M+H]+ is 1424.5440; the measured molecular weight is 1424.2120.

[0092] (2) Synthesis of dye 5b

[0093] Compound 5a, DBU and Er(acac)3 were mixed in a molar ratio of 1:4:5 and dissolved in 5 mL of 1,2,4-trichlorobenzene. The reaction mixture was degassed and exchanged with a nitrogen atmosphere, repeated three times, and then heated to 210°C for 0.5 h. After cooling to room temperature, a large amount of petroleum ether was added for precipitation, and the filter cake was collected. It was then eluted with a mixed solvent of dichloromethane and methanol in a volume ratio of 10:1, and the eluate was collected. After concentrating it, an equal molar amount of ligand NaL was added to compound 5a. OM e (dissolved in a small amount of methanol) and refluxed for 4 h. After the reaction solution was cooled, the solvent was removed by rotary evaporation, and the residue was redissolved in chloroform and separated by silica gel column chromatography (petroleum ether:ethyl acetate = 8:1) to obtain dye 5b (see the above reaction formula).

[0094] Characterization data of dye 5b are as follows:

[0095] 1 H NMR (600 MHz, CDCl3) chemical shift (δ): 30.95 (s, 4H), 30.15 (d, 18H), 27.11 (s, 8H), 22.71-13.89 (m, 16H), 8.89 (s, 24H), -45.67 (s, 5H).

[0096] Maldi-TOF-MS: Molecular formula: C 99 H 91 CoE 12 O 17 Theoretical molecular weight of P3: 2039.4510; measured molecular weight: 2040.5001.

[0097] Preparation Example 6

[0098]

[0099] (1) Synthesis of compound 6a

[0100] Compound 4 and lithium block were mixed in a 1:2 molar ratio and dissolved in 8 ml of n-pentanol. The reaction mixture was degassed and then replaced with a nitrogen atmosphere three times. The reaction system was then heated to 100°C for 4 hours. After the reaction was completed, the mixture was cooled to room temperature and the solvent was removed by rotary evaporation to obtain a crude product. The crude product was purified by silica gel column chromatography (eluent: petroleum ether:ethyl acetate = 10:1) to obtain compound 6a.

[0101] The characterization data of compound 6a are as follows:

[0102] 1 H NMR (400 MHz, CDCl3), chemical shift (δ): 4.95-4.79 (m, 16H), 4.57-4.41 (m, 16H), 2.19 (dt, J = 13.4, 6.7 Hz, 16H), 2.10-2.02 (m, 16H), 1.69 (dt, J = 15.2, 7.3 Hz, 16H), 1.54 (dq, J = 14.6, 7.2 Hz, 32H), 1.43 (dq, J = 14.2, 7.0 Hz, 16H), 1.05 (t, J = 7.3 Hz, 24H), 0.93 (t, J = 7.3 Hz, 24H).

[0103] Maldi-TOF-MS: Molecular formula: C 112 H 178 N8O 16 , theoretical molecular weight [M+H] + 1893.3394; measured molecular weight [M] + :1893.3137.

[0104] (2) Synthesis of dye 6b

[0105] Compound 6a, DBU and Er(acac)3 were mixed in a molar ratio of 1:4:5 and dissolved in 5 mL of 1,2,4-trichlorobenzene. The reaction mixture was degassed and exchanged with a nitrogen atmosphere, repeated three times, and then heated to 210°C for 0.5 h. After cooling to room temperature, petroleum ether was used as the eluent, and the high-boiling point solvent was removed by column chromatography under reduced pressure. Then, the mixture was eluted with a mixed solvent of dichloromethane and methanol in a volume ratio of 10:1, and the eluate was collected. After concentration, an equal molar amount of ligand NaL was added to compound 6a. OM e (dissolved in a small amount of methanol) and refluxed for 4 h. After the reaction solution was cooled, the solvent was removed by rotary evaporation and separated by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1) to obtain dye 6b (see the above reaction formula).

[0106] Characterization data of dye 6b are as follows:

[0107] 1 H NMR (600 MHz, CDCl3) chemical shift (δ): 27.73 (s, 18H), 15.54-11.95 (m, 32H), 10.16-7.85 (m, 32H), 7.87-6.35 (m, 32H), 5.52 (m, 32H), 4.28 (m, 48H), -46.06 (s, 5H). Maldi-TOF-MS: Molecular formula: C 123 H 199 CoErN8O 25 P3, theoretical molecular weight [M+H]+2509.2436, measured molecular weight 2509.2421.

[0108] Preparation Example 7

[0109]

[0110] (1) Synthesis of compound 7a

[0111] Compound 5 and lithium block were mixed in a 1:2 molar ratio and dissolved in 8 ml of n-pentanol. The reaction mixture was degassed and then replaced with a nitrogen atmosphere three times. The mixture was then heated to 100°C for 4 hours. After the reaction was completed, the mixture was cooled to room temperature and the solvent was removed by rotary evaporation to obtain a crude product. The crude product was purified by silica gel column chromatography (eluent: petroleum ether:ethyl acetate = 5:1) to obtain compound 7a.

[0112] The characterization data of compound 7a are as follows:

[0113] 1 H NMR (400 MHz, CDCl3), chemical shift (δ): 9.06 (d, J = 7.3 Hz, 4H), 8.07 (t, J = 7.6 Hz, 4H), 7.69 (d, J = 7.9 Hz, 4H), 4.43-4.24 (m, 8H), 3.70 (s, 12H), 2.06-1.87 (m, 8H), 1.44-1.28 (m, 16H), 0.81 (t, J = 7.1 Hz, 12H).

[0114] Maldi-TOF-MS molecular formula: C 56 H 70 N 12 , theoretical molecular weight [M+H]+911.5846; measured molecular weight 912.0332.

[0115] (2) Synthesis of dye 7b

[0116] Compound 7a, DBU and Er(acac)3 were mixed in a molar ratio of 1:4:5 and dissolved in 5 mL of 1,2,4-trichlorobenzene. The reaction mixture was degassed and exchanged with a nitrogen atmosphere, repeated three times, and then heated to 210°C for 0.5 h. After cooling to room temperature, petroleum ether was used as the eluent, and the high-boiling point solvent was removed by column chromatography under reduced pressure. Then, the mixture was eluted with a mixed solvent of dichloromethane and methanol in a volume ratio of 10:1, and the eluate was collected. After concentration, an equal molar amount of ligand NaL was added to compound 7a. OM e (dissolved in a small amount of methanol) and refluxed for 4 h. After the reaction solution was cooled, the solvent was removed by rotary evaporation and separated by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1) to obtain dye 7b (see the above reaction formula).

[0117] Characterization data of dye 7b are as follows:

[0118] 1 H NMR (400 MHz, CDCl 3 ) chemical shift (δ): 34.98 (s, 4H), 29.12 (s, 9H), 27.76 (s, 18H), 21.99 (s, 12H), 20.50 (d, 8H), 14.28 (d, 8H), 12.00 (d, 8H), 7.20 (d, 8H), 4.45 (s, 12H), −46.59 (s, 5H).

[0119] Maldi-TOF-MS: Molecular formula: C 67 H 91 CoE 12 O9P3, theoretical molecular weight [M]+1527.4911; measured molecular weight 1527.2397.

[0120] Preparation Example 8

[0121]

[0122] (1) Synthesis of compound 8a

[0123] Compound 6 and lithium block were mixed in a 1:2 molar ratio and dissolved in 8 ml of n-pentanol. The reaction mixture was degassed and then replaced with a nitrogen atmosphere three times. The mixture was then heated to 100°C for 4 hours. After cooling to room temperature, a large amount of methanol was added to the reaction mixture and stirred for 5 minutes. The mixture was then filtered and the filter cake was collected. The filter cake was recrystallized from a dichloromethane and methanol solution to yield compound 8a.

[0124] The characterization data of compound 8a are as follows:

[0125] 1H NMR (400 MHz, CDCl 3 ), chemical shift (δ): 8.65 (d, J=6.2 Hz, 4H), 7.83 (t, J=7.3 Hz, 4H), 7.06 (d, J=7.4 Hz, 4H), 4.78 (s, 16H), 2.68-2.59 (m, 8H).

[0126] Maldi-TOF-MS molecular formula: C 44 H 38 N 12 , theoretical molecular weight [M]+734.3342, measured molecular weight: 34.6919.

[0127] (2) Synthesis of dye 8b

[0128] Compound 8a, DBU and Er(acac)3 were mixed in a molar ratio of 1:4:5 and dissolved in 5 mL of 1,2,4-trichlorobenzene. The reaction mixture was degassed and exchanged with a nitrogen atmosphere, repeated three times, and then heated to 210°C for 0.5 h. After cooling to room temperature, a large amount of petroleum ether was added for precipitation, and the filter cake was collected. It was then eluted with a mixed solvent of dichloromethane and methanol in a volume ratio of 10:1, and the eluate was collected. After it was concentrated, an equal molar amount of ligand NaL was added to compound 8a. OM e (dissolved in a small amount of methanol) and refluxed for 4 h. After the reaction solution was cooled, the solvent was removed by rotary evaporation and separated by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain dye 8b (see the above reaction formula).

[0129] The characterization data of compound 8b are as follows:

[0130] 1 H NMR (400 MHz, CDCl3) chemical shift (δ): 38.34-31.26 (m, 4H), 28.33 (s, 18H), 25.99 (s, 8H), 23.51-17.83 (m, 16H), 15.68-11.90 (m, 8H), -46.94 (t, 5H).

[0131] Maldi-TOF-MS: Molecular formula: C 55 H 59 CoE 12 O9P3, theoretical molecular weight [M]+1351.2404; measured molecular weight 1351.9495.

[0132] Preparation Example 9

[0133]

[0134] (1) Synthesis of compound 9a

[0135] Compound 7 and lithium block were mixed in a molar ratio of 1:2 and dissolved in 8 ml of n-pentanol. The reaction mixture was degassed and then exchanged with a nitrogen atmosphere, repeated three times. The reaction system was then heated to 140°C and stirred for 8 hours until the reaction was complete. The reaction mixture was rotary evaporated to remove the solvent and extracted with dichloromethane. The resulting organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to remove the solvent. The resulting mixture was separated by chromatography on a neutral alumina column using a volume ratio of 1:1 dichloromethane:tetrahydrofuran as the eluent to obtain a crude product. The crude product was then recrystallized from a dichloromethane and methanol system to obtain compound 9a.

[0136] The characterization data of compound 9a are as follows:

[0137] Maldi-TOF-MS: Molecular formula: C 48 H 54 N 12 , theoretical molecular weight [M]+798.4594; measured molecular weight 798.8345.

[0138] (2) Synthesis of dye 9b

[0139] Compound 9a, DBU and Er(acac)3 were mixed in a molar ratio of 1:4:5 and dissolved in 5 mL of 1,2,4-trichlorobenzene. The reaction mixture was degassed and exchanged with a nitrogen atmosphere, repeated three times, and then heated to 210 ° C for 0.5 h. After cooling to room temperature, a large amount of petroleum ether was added to precipitate and the filter cake was collected. It was then eluted with a mixed solvent of dichloromethane and methanol in a volume ratio of 10:1, and the eluate was collected. After concentrating it, an equal molar amount of ligand NaL was added to compound 9a. OM e (dissolved in a small amount of methanol) and refluxed for 4 h. After the reaction solution was cooled, the solvent was removed by rotary evaporation and separated by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1) to obtain dye 9b (see the above reaction formula).

[0140] Characterization data of dye 9b are as follows:

[0141] 1 H NMR (600 MHz, CDCl 3 ) chemical shift (δ): 39.70 (s, 1H), 28.50 (s, 18H), 19.69 (d, 8H), 16.59 (s, 4H), 13.64 (d, 8H), 12.27 (d, 8H), 7.85 (s, 12H), −50.26 (s, 5H).

[0142] MALDI-TOF MS: Molecular formula: C 59 H 75 CoE 12The theoretical molecular weight of O9P3 [M+H]+ is 1416.3697; the measured molecular weight is 1416.0651.

[0143] Example 10

[0144] The spectral characterization method of the fluorescent dyes 1b-9b of the present invention comprises the following steps: firstly, the dyes 1b-9b are dissolved in dichloromethane to prepare a solution with a concentration of 10 μM, and then UV-visible absorption spectrum, fluorescence emission spectrum and fluorescence absolute quantum yield test are performed. Figure 1 , showing the chemical structure of the phthalocyanine rare earth complex (1b-9b) of the present invention and the absorption and emission spectra in dichloromethane; this series of dyes exhibits narrow and finely controllable dispersed absorption characteristics within a limited spectral range, indicating that they have the potential to be used as high-capacity multiple fluorescence surgical navigation contrast agents.

[0145] Dyes 1b-9b were dissolved in tetrahydrofuran and loaded into a capillary tube. A femtosecond optical parametric oscillator laser was used as an adjustable excitation source (spectral resolution of 1 nm). The excitation light was transmitted through a 450 μm core diameter metal-clad multimode optical fiber, and incremental scanning was performed in 5 nm steps within the wavelength range of 670-790 nm. The excitation light was collimated and directed to the sample plane through a dichroic mirror. The emitted fluorescence was filtered out of stray light by a coaxial dichroic mirror and a 1500 nm long-pass filter. The signal intensity was then collected by an electrically cooled near-infrared camera. The excitation spectrum was drawn after correcting the laser power at different excitation wavelengths. Figure 2 , showing the absorption spectra (a, b) and excitation spectra (c) of the phthalocyanine rare earth complexes (1b-9b) of the present invention in tetrahydrofuran. The excitation spectra of this series of dyes are highly consistent with the absorption spectra, verifying their suitability for excitation spectrum imaging technology and providing a precise wavelength control basis for multimodal fluorescence surgical navigation.

[0146] Example 11

[0147] The preparation method of the F127 hydrophilically modified dye of the present invention comprises the steps of:

[0148] A phthalocyanine rare earth complex and a polyoxyethylene polyoxypropylene ether triblock copolymer (polyoxyethylene polyoxypropylene (F127)) are dissolved in chloroform, stirred for 5-10 minutes, and the solvent is removed by rotary evaporation. The mixture is dried in vacuo, heated to 60-90°C, dissolved in a 60-90°C phosphate buffered saline (PBS), sonicated, cooled to room temperature, and then concentrated by ultrafiltration through a 30KD ultrafiltration tube to obtain a final contrast agent (such as F127 / 2b). The mass ratio of the phthalocyanine rare earth complex fluorescent dye to the polyoxyethylene polyoxypropylene is 1:(200-50), and the final contrast agent concentration is 200-500 μM.

[0149] The preparation method of the bovine serum albumin (BSA) modified dye of the present invention comprises the steps of:

[0150] Under intense ultrasound, a solution of a phthalocyanine rare earth complex dissolved in dimethyl sulfoxide is added dropwise to a phosphate-buffered saline solution (PBS) containing bovine serum albumin (BSA). The solution is then concentrated by ultrafiltration through a 30 kDa ultrafiltration tube to obtain the final contrast agent (e.g., BSA / 2b). The molar ratio of the phthalocyanine rare earth complex to bovine serum albumin (BSA) is 1:(1-5), and the final contrast agent concentration is 100-200 μM.

[0151] The present invention further verifies the in vivo spectral fidelity of the dye through in vivo hyperspectral imaging in the near-infrared II region. The specific implementation steps are as follows: 150 μL of 500 μmol F127 / 2b micelle solution was gavaged to the experimental mice 2 hours before imaging, and 200 μL of 500 μmol F127 / 7b micelle solution was injected through the tail vein 5 minutes before imaging; to eliminate in vivo metabolic interference, the mice were euthanized after injection to avoid spectral artifacts. A wide-field imaging system equipped with a femtosecond optical parametric oscillator laser was used for high-resolution spectral acquisition to obtain an in vivo near-infrared II region hyperspectral data set. Figure 3 , showing the phthalocyanine rare earth complexes (7b and 9b) of the present invention used for hyperspectral imaging of blood vessels and gastrointestinal tract of living normal mice; wherein, Figure 3 a is a schematic diagram of the multispectral imaging scheme of the normal mouse model. Figure 3 b is the result collected in the 1500-1700nm band. The upper left is a vector diagram of hyperspectral imaging, showing two signal sources EP737 (cyan) and EP772 (red). The upper right is their spectral diagram, and the lower figure is the unmixing result of blood vessels (EP737) and intestines (EP772). Figure 3 c is the result collected in the 850-1000nm band. The upper left is a vector diagram of hyperspectral imaging, showing three inclusions: EP737 (cyan), EP772 (red) and autofluorescence (AF, magenta). The upper right is their spectral diagram, and the lower figure is the unmixing result of blood vessels (EP737) and intestines (EP772), removing the autofluorescence signal. Figure 3 d is the vascular contrast, which quantifies the imaging quality of the 1500-1700nm and 850-1000nm data sets; the results show that near-infrared II region emission can effectively improve the imaging quality of in vivo hyperspectral images. Figure 3The relative residuals for all pixels are shown above, and the residual map is shown below. The unmixing accuracy of the 1500-1700 nm and 850-1000 nm datasets is quantified. As can be seen, the imaging results accurately reconstruct the anatomical structure of the intestine and blood vessels. The data demonstrate that erbium phthalocyanine complexes, represented by 7b and 9b, maintain spectral characteristics in living tissue that are highly consistent with in vitro testing, confirming the method's excellent spectral fidelity in complex biological environments. This technological breakthrough provides reliable technical support for achieving real-time, precise multimodal fluorescence navigation during surgical procedures, demonstrating significant clinical application prospects.

[0152] Example 12

[0153] The present invention implements dynamic compressed in vivo spectral imaging technology based on the erbium phthalocyanine complex as follows: The dyes 2b, 4b, 6b, 7b, and 9b utilize near-infrared II emission with narrowly separated excitation spectra, combined with differentiated surface modification strategies, to achieve multi-target simultaneous navigation. Among them, the F127 hydrophilic-modified dye (such as F127 / 4b) specifically accumulates in situ colorectal tumors and metastases with abnormal blood vessels due to its long circulation properties; while the bovine serum albumin (BSA)-modified BSA / 9b selectively accumulates in mesenteric lymph nodes due to its rapid clearance effect.

[0154] Specifically, in CT-26 orthotopic colon cancer model mice, 200 μL of 500 μM BSA / 9b solution was injected intravenously 73 hours before imaging to label mesenteric nodes. One hour later, 200 μL of 500 μM F127 / 6b micelles were injected to label orthotopic colorectal tumors. 2.5 hours and 0.5 hours before imaging, 60 μL each of 500 μM F127 / 7b and F127 / 2b were gavage-administered. Before imaging, 200 μL of 500 μM F127 / 4b micelles were additionally injected intravenously to construct a multi-temporal targeting system. After anesthesia, hair removal, and laparotomy to expose the intestine, a dynamic compressed spectral imaging system was used to acquire time-lapse hyperspectral data: 671, 690, 730, and 760 (100 mW cm) were integrated through a five-in-one metal-coated multimode fiber (200 μm input / 400 μm output). - 2) and 785nm (125mW cm - 2) Five semiconductor lasers are homogenized by liquid core fiber to form a uniform excitation field; the customized LightField software module precisely controls the 50ms laser pulse and 10ms delay trigger, combined with a 12.5kHz analog-to-digital conversion rate and a high-gain InGaAs camera (1500nm long-pass filter) to achieve synchronous exposure and high-fidelity data acquisition. Figure 4 , showing a schematic diagram of fluorescent surgical navigation using phthalocyanine rare earth complex fluorescent dyes (2b, 4b, 6b, 7b, 9b) for colorectal tumor resection in mice. Figure 4a is a schematic diagram of the anatomical structure labeling scheme for five-color imaging of the mouse colorectal cancer model. Figure 4 b The right side is a five-color unmixed image, and the left side is a five-color overlay image, which emphasizes the specific anatomical structures labeled with different fluorophores. The scale bar is 5 mm. Figure 4 c is Figure 4 The intensity cross-section at the dotted line position in b shows that the overlapping signals of the colon capillaries and the colon can also be well separated in imaging. Figure 4 d is Figure 4 b Fluorescence signal-to-noise ratio of the resected primary tumor and metastasis in the tumor channel. Figure 4 e is Figure 4 The average intensity measurements of ROI1 and ROI2 in b reveal different movement patterns and frequencies within the gastrointestinal tract, including segmentation and peristalsis. It can be seen that the linear unmixing algorithm can accurately separate anatomical structures such as the cecum, small intestine, and mesenteric lymph nodes, and simultaneously visualize in situ tumors, metastases, and intestinal blood supply. Dynamic monitoring further reveals two movement patterns: intestinal peristalsis (12 times / minute) and phasic contractions (6 times / minute), reflecting the physiological process of chyme delivery and mixing. Compared with traditional single fluorophore imaging, this method significantly improves the efficiency of intraoperative multi-target identification through the excitation spectrum separation characteristics and compressed sampling technology, providing real-time, multi-dimensional navigation information for precise tumor resection, identification of necrotic intestinal segments, and optimization of surgical decisions, and has important value for clinical translation.

[0155] Example 13

[0156] The real-time automated intraoperative high-capacity spectral imaging system of the present invention combines intelligent algorithms with fluorescent dye technology. The specific process is as follows:

[0157] First, a deep learning model was constructed based on 32 sets of mouse in vivo hyperspectral datasets (see Example 8), of which 19 sets were used for training, 3 sets for validation, and 10 sets for testing. The automated spectral recognition network EndmemberNet was trained. The network adopts a two-stage architecture:

[0158] Target detection: The YOLOv5 model (with the CSPDarknet53 feature extraction network at its core) is used in conjunction with multi-scale feature fusion technology (FPN+PAN architecture) to quickly identify the locations of key areas in the image, such as tumors and lymph nodes, and annotate their bounding boxes.

[0159] Fine segmentation stage: The DeepLabv3+ model (based on ResNet-50 and combined with multi-scale dilated convolution technology) is used to generate pixel-level segmentation masks for the detected targets to accurately distinguish normal tissue from diseased areas.

[0160] To enable real-time intraoperative analysis, a software system integrated with EndmemberNet was developed (written in Python and supports both GPU and CPU execution). When accelerated by a graphics card such as the GeForce RTX 3060, the system can simultaneously complete the following tasks:

[0161] Image acquisition: Trigger five laser groups in a preset sequence (see Example 8) to automatically capture synchronized images at different excitation wavelengths;

[0162] Data integration: Pack multi-band images into three-dimensional data blocks (spatial coordinates x, y + spectral dimension λ) and store them in user-specified paths;

[0163] Intelligent analysis: EndmemberNet is called in real time to first identify the target spectral features in the image, and then separate the mixed signals through a linear unmixing algorithm (for example, distinguishing the overlapping spectra of tumors from surrounding normal tissues);

[0164] The linear unmixing algorithm used includes: A linear unmixing algorithm is used to decompose the multispectral data into the contributions of individual fluorophores. The observed spectrum of each pixel in the multispectral dataset and the fluorophore reference spectrum extracted from the same data cube are modeled as a system of linear equations:

[0165] Y=AX+m

[0166] Here, Y represents the observed pixel spectrum, A is the extracted fluorophore reference spectrum, and m is the noise present in the observed spectrum. The vector X corresponds to the relative abundance of the fluorophore at each pixel. Given Y and A, matrix inversion or regularization techniques are used to obtain the solution for X to prevent potential overfitting and noise amplification:

[0167] X=(A T A) -1 A T Y

[0168] This approach assumes that the spectra in A are linearly independent and accurately represent the fluorophores present in the sample.

[0169] Dynamic display: The unmixed multi-target imaging results (such as marking tumor boundaries and lymph node locations with different colors) are displayed in real time on the operation interface, and the original data and analysis results are automatically saved.

[0170] This system has achieved a breakthrough in increasing the speed of hyperspectral imaging to a level that is synchronized with surgical operations, allowing researchers to obtain analysis results instantly during experiments or operations, and providing an efficient automated tool for accurately locating lesions and evaluating surgical outcomes.

[0171] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A phthalocyanine rare earth complex, characterized in that: It has the structure shown in formula A: Wherein, the rare earth ion Ln is selected from at least one of Ce, Pm, Eu, Gd, Tb, Pr, Nd, Sm, Dy, Ho, Er, Tm and Yb, and R1, R2, R3 and R4 are each independently selected from any one of H, C1-C5 alkoxy and the groups represented by formula I-IV.

2. The phthalocyanine rare earth complex according to claim 1, characterized in that: The complex represented by the general formula A has any of the following specific structures:

3. The method for preparing the phthalocyanine rare earth complex according to claim 1 or 2, characterized in that: The following steps are involved: S1, cyclizing a phthalonitrile derivative to obtain phthalocyanine; S2, introducing a rare earth metal into the center of the phthalocyanine through a coordination reaction to obtain an intermediate; S3, the intermediate and the tripod ligand NaL OMe The reaction is carried out to obtain a phthalocyanine rare earth complex having a structure shown in general formula A.

4. The preparation method according to claim 3, characterized in that The preparation method comprises the following reactions: Under the protection of a non-oxidizing inert atmosphere, compound (1) reacts with lithium block in a first organic solvent at 100-140° C. to generate compound (2); Under the protection of a non-oxidizing inert atmosphere, compound (2) reacts with a rare earth salt of acetylacetonate in a second organic solvent at 200-220° C. to generate a reaction intermediate; The intermediate and the tripod ligand NaL OMe The reaction is carried out in a third organic solvent to obtain a rare earth phthalocyanine complex (3) with a structure represented by general formula A.

5. The preparation method according to claim 4, characterized in that The general structural formula of the acetylacetonate rare earth salt is Ln(acac)3, wherein the rare earth ion Ln is at least one of Ce, Pm, Eu, Gd, Tb, Pr, Nd, Sm, Dy, Ho, Er, Tm and Yb.

6. The preparation method according to claim 4, characterized in that The non-oxidizing inert atmosphere is selected from at least one of nitrogen, argon and helium; The first organic solvent is selected from at least one of n-butanol, n-pentanol and n-heptanol; The second organic solvent is selected from at least one of trichlorobenzene, dimethyl sulfoxide and toluene; The third organic solvent is selected from at least one of chloroform, tetrahydrofuran and methanol.

7. The preparation method according to claim 4, characterized in that The compound (2) has any of the following specific structures:

8. Use of the phthalocyanine rare earth complex according to any one of claims 1 to 2 and the phthalocyanine rare earth complex prepared by the preparation method according to any one of claims 3 to 7 in the preparation of contrast agents.

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