A new method for the construction of meso aryl substituted heptamethine cyanine dyes

Abstract

The present application belongs to the technical field of fluorescent probe, and particularly relates to a new method for constructing a meso-aryl-substituted heptamethine cyanine dye. The present application uses cyanine ketone as a raw material, aryl lithium as a reaction reagent, and a meso-aryl-substituted heptamethine cyanine (ArCy7) dye is obtained through a one-pot reaction with a high yield. The ArCy7 dye prepared by the method of the present application effectively improves the water solubility, chemical / photo stability and anti-aggregation ability of the Cy7 dye, thereby promoting the application of the dye in constructing a fluorescent probe, protein labeling or tumor diagnosis and treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of fluorescent probe, and particularly relates to a new method for constructing meso-aryl substituted heptamethine cyanine dye. BACKGROUND

[0002] Heptamethine cyanine dye (Cy7) is one of the widely used fluorescent dyes in the field of fluorescence technology. It has been widely used in various fields of fluorescence technology, including protein labeling, biosensing, surgical navigation, super-resolution imaging, photodynamic therapy, etc. In addition to the near-infrared excitation and emission wavelength, another feature of Cy7 is the relatively large molar extinction coefficient (ε: usually greater than 2×10 5 M -1 cm -1 ), much larger than most other fluorescent dyes (ε: generally less than 1×10 5 M -1 cm -1 ), thus to some extent making up for the impact of low fluorescence quantum yield (Φ) of near-infrared fluorescent dyes on fluorescence brightness (ε×Φ). However, compared with many visible region fluorescent dyes, the traditional Cy7 not only has low fluorescence quantum yield, but also has weak light stability, easy aggregation, lack of functionalization sites and other shortcomings. Although in recent years the introduction of polycyclic systems has effectively inhibited the non-radiative transition caused by C=C bond isomerization and significantly improved the fluorescence quantum yield of Cy5, this strategy has little effect on the improvement of Cy7 fluorescence quantum yield. Given the wide application prospect of Cy7 in biomedical imaging field, how to overcome the above shortcomings through structural modification and further improve the application effect of this kind of dye in biological system has always been one of the important challenges in the field of fluorescence technology.

[0003] Studies have shown that the introduction of a cyclohexene structure and a meso-aryl substituent into the polymethine conjugated chain of Cy7 can improve the dye's fluorescence quantum yield and photochemical stability. The introduction of a steric shielding group on the meso-aryl group can further enhance the dye's photostability and reduce its aggregation in aqueous solution, thus providing guidance for the future development of Cy7 dyes. Three main methods have been reported to date for the synthesis of meso-aryl-substituted heptamethine cyanine (ArCy7) dyes. One method, first reported in 2006 by the Achilefu group at the University of Washington, involves the preparation of ArCy7 dyes via a Pd-catalyzed Suzuki coupling reaction using Cl-Cy7 and phenylboronic acid. However, the meso-C-Cl functional group of Cl-Cy7 is a weak Suzuki coupling substrate, resulting in low yields in most reported syntheses of ArCy7 and hindering the synthesis of Cy7 dyes with meso-sterically hindered aryl groups. Another method uses phenyl-substituted cyclohexene as a starting material, undergoing formylation-condensation to generate a Schiff base intermediate, which is then reacted with indolinium quaternary ammonium to obtain the product. However, this method is not universally applicable, requiring the synthesis of different phenyl-substituted cyclohexene starting materials to prepare different ArCy7 derivatives, and therefore has not received widespread attention. A third method, reported by Smith's group at the University of Notre Dame, borrows from the recently reported "Zincke salt (N-2,4-dinitrophenylpyridinium salt)" Cy7 dye synthesis method by Stacko's group at Masaryk University in the Czech Republic. Although this method can synthesize the meso-sterically hindered phenyl-substituted Cy7 dye s775z, the synthesis of the "Zincke salt" involves multiple reaction steps, making it difficult to obtain a variety of meso-sterically hindered ArCy7 dyes. Given the potential for improved performance and broad application prospects of ArCy7 dyes, the development of more efficient synthetic methods would substantially advance their application in fluorescence technology. Summary of the Invention

[0004] To address the technical problems in the above-mentioned prior art, the present invention provides a new synthetic route, namely the cyanine ketone method, for preparing ArCy7 dyes. This route uses cyanine ketone (Cyanine-Keto, CyAK) as a raw material (which can be conveniently prepared from ClCy7) and aryl lithium as a reaction reagent. ArCy7 dyes can be obtained in high yields through a "one-pot reaction". Moreover, due to the extremely strong nucleophilicity of aryl lithium, various ArCy7 dyes with large steric hindrance can also be prepared by this method. Using this route, the present invention provides a series of representative ArCy7 dyes. Compared with the classic Cy7 dyes, these dyes all have higher fluorescence brightness and stronger chemical and light stability.

[0005] The technical solutions of the present invention are as follows:

[0006] A novel method for constructing a meso-aryl-substituted heptamethine cyanine dye comprises the following steps:

[0007] At -78°C under N2, a solution of anhydrous THF containing cyanone was added dropwise to a reaction solution of anhydrous THF containing an aryl lithium reagent, and the reaction mixture was then heated to ambient temperature and stirred for reaction. After the reaction, HCl was added to quench the reaction, and the resulting mixture was extracted with CH2Cl2. The organic layer was dried, concentrated, and purified by column chromatography to obtain a meso-aryl-substituted heptamethine cyanine dye.

[0008] Wherein, the structural formula of the cyanine ketone is selected from Formula 1a or Formula 1b:

[0009]

[0010] The structural formula of the aryl lithium reagent is selected from at least one of Formulas 2a-2o:

[0011]

[0012] Preferably, the molar ratio of the cyanine ketone to the aryl lithium reagent is 1:5.

[0013] Preferably, the synthesis steps of the cyanine ketone 1a are as follows: Cl-Cy7 and sodium acetate are dissolved in DMF solution, stirred at 100°C for 3 hours, cooled to room temperature, added with water, stirred, filtered, and purified by column chromatography to obtain 1a.

[0014] Preferably, the molar ratio of Cl-Cy7 to sodium acetate is 1:3.

[0015] Preferably, the synthesis steps of the cyanine ketone 1b are as follows: PEG-Cl-Cy7, N-hydroxysuccinimide and NaH are dissolved in DMF, stirred at room temperature under N2 environment for 12 hours, and concentrated and purified by column chromatography to obtain 1b.

[0016] Preferably, the molar ratio of PEG-Cl-Cy7, N-hydroxysuccinimide and NaH is 1:5:5.

[0017] Preferably, the synthesis steps of the aryl lithium reagents 2a-2i and 2m-2o are as follows: n-BuLi is added dropwise to anhydrous THF containing the aryl bromide derivatives corresponding to 2a-2i and 2m-2o at -78°C under N2 environment, and stirred for 30 minutes to obtain 2a-2i and 2m-2o.

[0018] Preferably, the synthesis steps of the aryl lithium reagents 2j-2l are as follows: n-BuLi is added dropwise to a THF solution of 1,3-dimethoxybenzene, 1,3-diethoxybenzene or 1,3-diphenoxybenzene at 0°C under N2 environment, and stirred for 1 hour to obtain 2j-2l.

[0019] A medical photosensitizer is synthesized using the novel method for constructing a meso-aryl-substituted heptamethine cyanine dye as described above, and its structural formula is as follows:

[0020]

[0021] The invention relates to an application of the above-mentioned medical photosensitizer as a preparation of an anti-tumor photodynamic therapy drug.

[0022] The beneficial effects of the present invention are as follows:

[0023] The "cyanone method" provided by the present invention is simple to synthesize, and can prepare various Cy7 fluorescent dyes substituted with meso-sterically hindered aromatic groups in high yield, thus overcoming the shortcomings of traditional dyes of this type, such as low fluorescence quantum yield, poor light / chemical stability, and easy aggregation. At the same time, the present invention confirms that the introduction of two methoxy groups at the two ortho positions of the meso-aryl group can greatly improve the anti-aggregation ability of the dye in water; in addition, by introducing a large sterically hindered PEG chain into the central aromatic group, not only can the aggregation of the dye in water be inhibited, but the water solubility of the dye can also be greatly improved, thereby promoting the application of such dyes in protein labeling. In addition, the compound 3f provided by the present invention is a heavy atom-free photodynamic photosensitizer, which has the advantages of an excitation wavelength in the near-infrared region, a long triplet lifetime, and low dark toxicity. Experiments have confirmed that 3f can produce a large amount of 1 O2 has a strong killing ability against cancer cells / tumors, so it has potential biological application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 For compound 1a 1 H NMR spectrum;

[0025] Figure 2 For compound 1a 13 C NMR spectrum;

[0026] Figure 3 is the HRMS pattern of compound 1a;

[0027] Figure 4 Compound 1b 1 H NMR spectrum;

[0028] Figure 5 Compound 1b 13 C NMR spectrum;

[0029] Figure 6 is the HRMS pattern of compound 1b;

[0030] Figure 7 For compound 3a 1H NMR spectrum;

[0031] Figure 8 For compound 3a 13 C NMR spectrum;

[0032] Figure 9 is the HRMS pattern of compound 3a;

[0033] Figure 10 Absorption spectra of ICG and 3i-3p in PBS (10 mM, pH = 7.4) as the concentrations change (2-40 μM);

[0034] Figure 11 Normalized absorption and emission spectra of 3i-3p in (A) PBS, (B) CH3CN, and (C) CH2Cl2, respectively;

[0035] Figure 12 The absorption spectrum of 3i-3p (1 μM) in PBS containing 1 mM GSH (0-30 min);

[0036] Figure 13 The graph shows the change of absorbance values ​​of 3a-3p, Cy7 and Cl-Cy7 at the maximum absorption wavelength in CH3CN over time under continuous 750nm laser irradiation;

[0037] Figure 14 For 750nm laser (30mW / cm 2 ) The UV-visible absorption spectra of DPBF / PS-AN (A) or DPBF / ICG (B) in DMEM under irradiation change over time, recorded every 50 seconds;

[0038] Figure 15 The cytotoxicity experiment of photosensitizer PS-AN. In the figure, (dark) indicates that A549 cells were treated with different concentrations of PS-AN for 1 hour and then incubated for 24 hours; (light) indicates that A549 cells were first treated with different concentrations of PS-AN for 1 hour and then irradiated with 750nm laser (30mW / cm 2 , 30 min) and continued incubation for 24 h after irradiation;

[0039] Figure 16 The cells loaded with PS-AN / DCFH-DA (A) or PS-AN / MCR-DMA (B) were not irradiated or irradiated (750 nm, 30 mW / cm 2Confocal imaging after 30 min); for DCFH-DA, the collection wavelength was 500 nm-750 nm (λex = 488 nm), and the scale bar was 100 μm; for MCR-DMA, the collection wavelength was 640 nm-750 nm (λex = 633 nm), and the scale bar was 50 μm;

[0040] Figure 17 The cells pre-loaded with PS-AN were not irradiated or irradiated (750 nm, 30 mW / cm 2 , 30 min), and then confocal imaging after Calcein AM / PI co-staining; for Calcein AM, the collection wavelength was 505 nm-535 nm (λex = 488 nm); for PI, the collection wavelength was 590 nm-700 nm (λex = 561 nm), and the scale bar is 100 μm;

[0041] Figure 18 (A) Tumor-bearing nude mice were injected with PS-AN (50 μM, 300 μL) in situ and then irradiated with 750 nm laser (50 mW / cm 2 (A) are photos of the tumors of the tumor-bearing mice in different treatment groups after 19 minutes of light irradiation. (B) are photos of the tumors of the tumor-bearing mice in different treatment groups after 19 minutes of light irradiation. (C) and (D) are the curves of the changes in tumor volume and body weight of the tumor-bearing mice in different treatment groups, respectively. DETAILED DESCRIPTION

[0042] In order to further illustrate the technical solution of the present invention, the present invention is further described below through examples.

[0043] Example 1 Preparation of Aryl-Substituted Heptamethine Cyanine Dye

[0044] (1) Synthesis of cyanine ketone 1a. The synthetic route is as follows:

[0045]

[0046] Cl-Cy7 (200 mg, 0.313 mmol) and sodium acetate (77 mg, 0.939 mmol) were dissolved in 20 mL of dry DMF solution and stirred at 100°C for 3 h. After cooling to room temperature, water was added and the mixture was treated with a magnetic stirrer for 10 min. A large amount of red solid precipitated. The crude product was collected by filtration and separated by column chromatography (petroleum ether / ethyl acetate = 50 / 1) to obtain a red solid 1a (0.313 g, yield 82.8%). 1H NMR (600MHz, CDCl3) δ8.21(d,J=12.6Hz,2H),7.21(t,J=6.6Hz,4H),6.93(t,J=7.2Hz,2H),6.71(d,J=7.8Hz,2H ),5.50(d,J=13.2Hz,2H),3.77(m,4H),2.64(t,J=5.4Hz,4H),1.89(m,2H),1.69(s,12H),1.30(t,J=7.2Hz,6H); 13 C NMR (150MHz, CDCl3) δ186.34,161.81,143.75,139.85,132.80,127.64,126.55,121 .81,120.43,106.39,92.15,46.56,37.05,28.71,25.87,22.59,11.13; ESI-MS[M+H] + :calcdfor 493.3219,Found 493.3221.

[0047] (2) Synthesis of cyanine ketone 1b. The synthetic route is as follows:

[0048]

[0049] PEGylated meso-chlorine-substituted PEG-Cl-Cy7 (1.50 g, 1.812 mmol), N-hydroxysuccinimide (1.04 g, 9.058 mmol), and NaH (217 mg, 9.058 mmol) were dissolved in DMF and stirred at room temperature under N2 for 12 h. The solution was concentrated by rotary evaporation to obtain a red oil, which was then purified by column chromatography (petroleum ether / ethyl acetate = 1 / 1) to obtain a red liquid 1b. 1 H NMR(600MHz, CDCl3)δ8.19(s,2H),7.19(t,J=6.6Hz,4H),6.93(t,J=6.6Hz, 2H),6.71(d,J=6.6Hz,2H),5.56(d,J=12.6Hz,2H),3.93(s,4H),3.76(t,J=6 Hz,4H),3.66(d,J=6.6Hz,4H),3.63(m,4H),3.61(t,J=5.4Hz,4H),3.52(t,J =4.8Hz,4H),3.37(s,6H),2.62(t,J=5.4Hz,4H),1.88(m,2H),1.68(s,12H); 13C NMR (150MHz, CDCl3) δ186.70,162.41,144.31,139.48,132.76,127.96,127.61,121.69,120.64,1 07.13,92.74,71.92,71.08,70.65,67.24,59.02,46.58,42.85,29.69,28.78,25.87; ESI-MS[M+H] + :calcd for 729.4474,Found 729.4471.

[0050] (3) Synthesis of aryl lithium reagents 2a-2i and 2m-2o. The synthesis steps are as follows:

[0051] At -78°C under N2, n-BuLi (1.6 M in hexane) was injected dropwise into anhydrous THF containing the aryl bromide derivatives corresponding to 2a-2i and 2m-2o, respectively, and stirred for 30 min to obtain the aryl lithium reagents 2a-2i and 2m-2o.

[0052]

[0053] (4) Synthesis of aryl lithium reagents 2j-2l. The synthesis steps are as follows:

[0054] At 0°C under N2, n-BuLi (1.6 M in hexane) was slowly added dropwise to a THF solution of 1,3-dimethoxybenzene, 1,3-diethoxybenzene, or 1,3-diphenoxybenzene and stirred for 1 h to obtain aryl lithium reagents 2j-2l.

[0055]

[0056] (5) Synthesis of compounds 3a-3p. The synthetic routes are as follows:

[0057]

[0058] At −78°C under N₂, a solution of cyanone 1a or 1b (0.2 mmol, 1 eq.) in anhydrous THF (5 mL) was carefully added dropwise to a reaction solution of aryl lithium reagents 2a-2o (5 eq.) in anhydrous THF (6 mL). The reaction mixture was then warmed to ambient temperature and stirred for 12 h. After completion of the reaction, 1N HCl was added to quench the reaction, and the resulting mixture was extracted with CH₂Cl₂. The organic layer was dried over Na₂SO₄ and concentrated on a rotary evaporator to afford a black solid, which was then purified by column chromatography (CH₂Cl₂ / CH₃OH = 30 / 1-50 / 1) to afford the final products 3a-3p.

[0059] 7.25(d,J=7.2Hz,2H),7.17(d,J=12.6Hz,2H),7.12(d,J=13.8Hz,2H),6.22(d,J=14.4Hz,2H),4.15(q,J=6.6Hz,4H),2.69(t,J=5.4Hz,4H),1.95(s,2H),1.23(t,J=7.2Hz,6H),1.11(s,12H); 13 C NMR(150MHz,d-DMSO)δ171.21,161.78,147.69,142.15,141.15,139.11,131.05,129.58,129.09,128.35,126.26,125.03,122.95,111.20,100.16,61.27,48.67,27.39,24.56,21.33,12.51;ESI-MS[M] + :calcd for 553.3578,Found553.3551.

[0060] Hz,2H),7.11(d,J=7.8Hz,1H),7.08(d,J=13.8Hz,2H),6.23(m,2H),4.15(m,4H),2.71(d,J=1.8Hz,4H),2.11(s,3H),1.99(m,2H),1.24(t,J=7.2Hz,6H),1.11(s,6H),1.07(s,6H); 13 C NMR(150MHz,d-DMSO)δ171.24,161.09,146.58,142.16,141.23,138.37,135.80,130.61,130.38,129.54,128.97,128.88,126.68,125.03,122.96,111.22,100.23,60.22,48.69,27.58,24.48,21.43,18.91,12.52;ESI-MS[M] + :calcd for 567.3734,Found567.3745.

[0061] J=7.2Hz,2H),7.10(d,J=14.4Hz,2H),6.25(d,J=14.4Hz,2H),4.15(m,4H),2.74(t,J=6.0Hz,4H),2.08(s,6H),1.98(m,2H),1.25(t,J=7.2Hz,6H),1.09(s,12H); 13 C NMR(150MHz,d-DMSO)δ171.26,160.41,145.10,142.15,141.29,137.65,135.76,129.53,128.97,128.65,128.07,125.02,122.97,111.23,100.28,60.24,48.70,27.51,24.44,21.47,19.32,12.53;ESI-MS[M] + :calcd for 581.3891,Found 581.3900.

[0062] Hz,2H),4.16(s,4H),2.69(s,4H),1.94(s,2H),1.22(s,6H),1.12(s,12H); 13 C NMR(150MHz,d-DMSO)δ171.43,157.98,150.52,147.77,146.77,142.10,141.19,130.24,129.01,125.24,124.86,122.98,111.40,100.55,48.74,39.20,27.38,24.46,21.15,12.57;ESI-MS[M] + :calcd for 554.3530,Found 554.3535.

[0063] 7.60(t,J=7.2Hz,1H),7.50(d,J=4.2Hz,1H),7.37(d,J=7.8Hz,2H),7.31(s,4H),7.12(s,2H),6.86(d,J=13.8Hz,2H),6.24(d,J=14.4Hz,2H),4.12(d,J=6.6Hz,4H),2.80(s,4H),2.12(m,1H),2.02(m,1H),1.19(t,J=6.6Hz,6H),0.94(s,6H),0.49(s,6H); 13C NMR(150 MHz,d-DMSO)δ171.23,156.11,150.83,148.32,146.57,146.03,141.98,141.16,130.78,130.68,129.97,128.94,128.30,127.31,125.95,125.21,122.92,122.49,111.36,100.60,48.57,27.09,26.87,24.55,21.27,12.55;ESI-MS[M] + :calcd for 604.3687,Found 604.3705.

[0064] J=8.4Hz,2H),7.54(m,4H),7.26(m,6H),7.04(t,J=7.2Hz,2H),6,78(d,J=13.8Hz,2H),6.20(d,J=14.4Hz,2H),4.05(d,J=6.6Hz,4H),2.92(t,J=4.8Hz,4H),2.21(t,J=5.4Hz,2H),1.15(t,J=6.6Hz,6H),0.34(s,12H); 13 C NMR(150MHz,d-DMSO)δ171.01,158.45,146.95,141.93,141.14,132.63,132.04,131.29,130.02,128.87,128.80,127.76,127.37,126.12,126.07,124.98,122.77,111.14,100.43,48.32,39.07,26.64,24.84,21.59,12.45;ESI-MS[M] + :calcd for 653.3891,Found 653.3898.

[0065] 6H),7.05(t,J=7.2Hz,2H),6.80(d,J=14.4Hz,2H),6.20(d,J=13.8Hz,2H),4.23(s,3H),4.06(d,J=6.6Hz,4H),2.90(s,4H),2.21(t,J=4.8Hz,2H),1.15(t,J=7.2Hz,6H),0.36(s,12H); 13C NMR (150 MHz, d-DMSO) δ 170.97, 158.44, 153.02, 147.08, 141.92, 141.12, 132.13, 130.78, 128.82, 128.48, 127.55, 126.54, 126.21, 124.95, 124.01, 122.80, 122.55, 111.16, 100.44, 64.35, 60.22, 48.35, 28.14, 26.62, 24.77, 12.49; ESI-MS [M] + : calcd for 683.3996, Found 683.3997.

[0066] (t, J = 6 Hz, 2H), 6.84 (m, 3H), 6.32 (d, J = 13.8 Hz, 2H), 6.09 (d, J = 7.2 Hz, 2H), 4.22 (d, J = 6.6 Hz, 4H), 3.35 (s, 4H), 2.73 (m, 6H), 2.18 (s, 3H), 1.99 (s, 2H), 1.29 (m, 12H); 13 C NMR (150 MHz, d-DMSO) δ 170.97, 158.44, 153.02, 147.08, 141.92, 141.12, 132.13, 130.78, 128.82, 128.48, 127.55, 126.54, 126.21, 124.95, 124.01, 122.80, 122.55, 111.16, 100.44, 64.35, 60.22, 48.35, 28.14, 26.62, 24.77, 12.49; ESI-MS [M] + : calcd for 748.4262, Found 748.4271.

[0067] J = 10.2 Hz, 4H), 3.70 (s, 3H), 2.68 (m, 4H), 1.99 (s, 1H), 1.66 (s, 1H), 1.24 (t, J = 10.8 Hz, 6H), 1.15 (s, 6H), 1.10 (s, 6H); 13C NMR(150MHz,d-DMSO)δ170.94,159.34,156.44,146.74,142.20,141.16,130.88,130.69,130.54,128.95,127.04,124.93,122.93,121.27,111.89,111.12,100.11,56.13,48.62,27.42,27.33,24.52,21.29,12.49;ESI-MS[M] + :calcd for583.3683,Found 583.3730.

[0068] 7.26(d,J=13.8Hz,2H),7.16(t,J=7.2Hz,2H),6.94(d,J=9Hz,2H),6.15(d,J=13.8Hz,2H),4.12(q,J=6.6Hz,4H),3.66(s,6H),2.65(t,J=5.4Hz,4H),1.90(t,J=5.4Hz,2H),1.24(t,J=7.2Hz,6H),1.13(s,12H); 13 C NMR(150MHz,d-DMSO)δ170.71,157.16,156.87,145.50,142.26,141.14,130.96,130.45,128.92,124.80,122.90,114.78,111.01,104.71,100.04,60.21,56.51,48.54,27.36,24.54,21.25,12.46;ESI-MS[M] + :calcd for613.3789,Found 613.3797.

[0069] 2H),6.87(s,2H),6.17(m,2H),4.11(s,4H),3.96(s,4H),2.49(s,4H),1.89(s,2H),1.22(s,12H),1.14(s,12H); 13C NMR(150MHz,d-DMSO)δ170.62,157.50,156.56,145.53,142.28,141.11,131.27,130.74,128.91,124.73,122.86,110.97,105.90,99.88,99.87,64.38,48.56,38.93,27.42,24.69,15.13,12.45;ESI-MS[M] + :calcd for 641.4102,Found641.4105.

[0070] (m,2H),7.25-7.19(m,5H),7.11(d,J=7.6Hz,2H),7.05(m,1H),6.98(m,1H),6.92(d,J=7.6Hz,1H),6.83(d,J=6.4Hz,1H),6.72(m,1H),6.61(m,1H),6.22(m,2H),4.18(m,4H),2.62(s,4H),1.41(s,2H),1.29-1.1.23(m,12H),1.16(d,J=4.2Hz,6H); 13 C NMR(150MHz,d-DMSO)δ171.08,158.48,157.61,156.22,154.04,146.33,142.15,141.14,131.53,130.77,130.46,128.98,125.05,124.29,122.92,119.36,114.40,114.13,111.22,109.22,100.39,48.73,27.54,27.40,24.58,27.17,12.52;ESI-MS[M] + :calcd for 737.4102,Found737.4156.

[0071] J=6.3Hz,4H),4.02(s,4H),3.47(s,4H),3.15(s,6H),2.65(s,4H),1.91(s,2H),1.24(s,6H),1.16(s,12H); 13C NMR(150MHz,d-DMSO)δ170.66,157.09,156.73,145.68,142.26,141.13,130.81,130.73,128.91,124.75,122.89,116.05,110.96,106.34,99.86,70.74,69.26,58.85,48.57,38.91,27.49,24.73,21.31,12.44;ESI-MS[M] + :calcd for701.4313,Found 701.4355.

[0072] 8.4Hz,2H),6.17(d,J=14Hz,2H),4.13(t,J=7.2Hz,4H),4.01(t,J=4Hz,4H),3.56(t,J=4.4Hz,4H),3.44(t,J=4.4Hz,4H),3.28(t,J=5.2Hz,4H),3.12(s,6H),2.64(t,J=4.8Hz,4H),1.92(m,2H),1.24(t,J=7.2Hz,6H),1.16(s,12H); 13 CNMR(150MHz,d-DMSO)δ170.70,157.09,156.60,145.71,142.23,141.14,130.88,130.67,128.92,124.78,122.89,115.65,110.96,105.95,99.84,71.83,70.51,69.63,69.12,58.49,48.58,38.91,27.50,24.70,21.30,12.42;ESI-MS[M] + :calcd for789.4838,Found 789.4883.

[0073] 6.73(s,1H),6.17(d,J=14Hz,2H),4.11(t,J=6.8Hz,4H),4.01(t,J=4Hz,4H),3.57(t,J=3.6Hz,4H),3.44(t,J=4.4Hz,4H),3.36(m,8H),3.28(t,J=5.2Hz,4H),3.10(s,6H),2.65(m,4H),1.92(m,2H),1.24(m,6H),1.16(s,12H); 13C NMR (150MHz, d-DMSO) δ170.72,157.10,156.60,146.01,142.25,141.16,130.88,130.70,128.91,124.77,122.92,115.66,11 0.95,105.96,99.82,71.66,70.67,70.40,70.02,69.68,69.12,58.43,48.59,38.91,27.49,24.62,21.34,12.45; ESI-MS[M] + :calcd for 877.5362, Found 877.5414.

[0074] 6.26(d,J=14Hz,2H),4.31(t,J=4.4Hz,4H),4.01(t,J=3.6Hz,4H),3.75(t,J=4.8Hz,4H),3.57(t,J=4.4Hz ,4H),3.52(m,6H),3.43-3.28(m,28H),3.19(s,6H),3.12(s,4H),2.63(s,4H),1.92(m,2H),1.16(s,12H); 13 C NMR (150MHz, CDCl3) δ171.48,157.36,156.51,145.82,142.87,140.38,131.77,130.48,128.48,124.50,121.75,115.74,111.07,10 5.47,100.34,71.84,70.97,70.92,70.64,70.55,70.46,69.38,68.92,67.98,59.03,58.96,48.47,45.01,27.77,24.92; ESI-MS[M] + :calcd for1113.6622,Found 1113.6698.

[0075] Example 2 Water solubility and anti-aggregation ability test

[0076] Studies have shown that by introducing water-soluble, large steric groups at the two ortho positions of the aryl group in the cyanine dye, the water solubility and resistance to self-aggregation of the dye can be effectively improved. The "cyanone method" can easily construct a heptamethine cyanine dye substituted with a sterically shielded aryl group at the middle position. First, the present invention uses the "cyanone method" to synthesize a heptamethine cyanine dye (3i) substituted with a 2-dimethoxyphenyl group at the middle position, a heptamethine cyanine dye (3j) substituted with a 2,6-dimethoxyphenyl group at the middle position, a heptamethine cyanine dye (3k) substituted with a 2,6-diethoxyphenyl group at the middle position, and a heptamethine cyanine dye (3l) substituted with a 2,6-diphenoxyphenyl group at the middle position. Among them, 3j exhibited the best water solubility (octanol / water partition coefficient: LogP = 1.005) and the strongest anti-aggregation ability in PBS (10 mM, pH = 7.4). In contrast, ICG and 3i exhibited distinct H aggregation absorption peaks, while 3k and 3l had low water solubility, which was attributed to the hydrophobicity of the 2,6-diethoxyphenyl and 2,6-diphenoxyphenyl groups. These results indicate that the meso-2,6-dimethoxyphenyl group is sufficiently effective in preventing the self-aggregation of heptamethine indocyanines. To further improve the water solubility of the dye, the present invention subsequently synthesized a series of polyethylene glycol-functionalized meso-aryl-substituted heptamethine cyanine dyes, including meso-2,6-bis(ethylene glycol monomethyl ether)phenyl-substituted heptamethine cyanine dye (3m) (LogP=0.849), meso-2,6-bis(dipolyethylene glycol monomethyl ether)phenyl-substituted heptamethine cyanine dye (3n) (LogP=0.431), and meso-2,6-bis(tripolyethylene glycol monomethyl ether)phenyl-substituted heptamethine cyanine dye (3o) (LogP=0.403). The water solubility of this series of dyes is not only greatly improved, but also, due to the large steric hindrance of the meso-aryl group, they all exhibit strong anti-aggregation ability in PBS. To further improve the water solubility of the dye, the present invention synthesized polyethylene glycol-functionalized cyanine ketone 1b, and on this basis, synthesized heptamethine cyanine dye 3p with four PEG3 chains. As expected, this molecule showed the highest water solubility (LogP = 0.092) and no aggregation peak was observed in PBS (see Figure 10 In general, the cyanine ketone method can be used to conveniently construct various heptamethine cyanine dyes substituted with meso-aryl groups. It is also confirmed that the introduction of methoxy groups or polyethylene glycol chains at the two ortho positions of the meso-aryl group can greatly improve the water solubility and anti-aggregation ability of the dye in water.

[0077] Example 3 Photophysical property test

[0078] The photophysical properties of dyes 3a-3p were evaluated in dichloromethane (CH2Cl2), acetonitrile (MeCN), and PBS (10 mM, pH = 7.4, containing 30% MeCN) and compared with the traditional heptamethine cyanine dye (Cy7) (Table 1). Figure 11As shown, 3a-3p all exhibit near-infrared absorption / emission wavelengths, with maxima in the 757-778 nm / 784-811 nm range, respectively, which are longer than those of Cy7 (740-756 nm / 774-790 nm). This is likely due to the stronger electron-withdrawing ability of the meso-aryl group compared to the meso-H atom. 3h has a low fluorescence quantum yield (Φ: 0.05 in CH2Cl2; Φ: 0.07 in CH3CN; Φ: 0.07 in PBS) due to the a-PeT interaction from the phenoxazine group to the dye matrix. However, the fluorescence quantum yields of 3a-3p (Φ: 0.26-0.41 in CH2Cl2; Φ: 0.18-0.32 in CH3CN; Φ: 0.12-0.24 in PBS) are comparable to or higher than those of Cy7. It is worth noting that compared with Cy7 dye (ε: 2.00-2.48×10 5 M -1 cm -1 ), these dyes all exhibit large molar extinction coefficients (ε: 2.00-3.95×10 5 M -1 cm -1 ), so their fluorescence brightness (ε×Φ) (except 3h) is significantly higher than that of Cy7 dye, suggesting that this series of dyes has potential in improving resolution.

[0079] Table 1 Photophysical properties of 3a-3p and Cy7 dyes in dichloromethane, acetonitrile and PBS

[0080]

[0081]

[0082]

[0083] Example 4 Chemical / Light Stability Test

[0084] Studies have shown that the phenolic groups in the heptamethine cyanine dyes substituted with phenolic groups at the mesoposition, such as CW800 and ZW800-1, can be rapidly replaced by biothiols in aqueous solutions. In contrast, 3a-3p exhibit strong stability to biothiols due to the stability of the central C-aryl bond. When 1 mM GSH was added to PBS containing dyes 3a-3p, their absorption spectra did not change (see Figure 12). In addition, studies have shown that electron-rich heptamethine indole cyanine dyes substituted with meso-phenoloxy or meso-alkoxy groups, such as UL766, CW800, and ZW800-1, are susceptible to attack by singlet oxygen, leading to photobleaching. However, compared with Cy7 and Cl-Cy7 dyes, meso-aryl substituted heptamethine cyanine dyes 3a-3p all exhibit higher photostability (see Figure 13 ).

[0085] Example 5 Application of Compound 3f as a Photodynamic Photosensitizer

[0086] Photodynamic therapy (PDT) is a new cancer treatment method developed and clinically applied in recent years. Its basic principle is to use the light energy absorbed by photosensitizer (PS) to convert oxygen ( 3 O2) into highly active reactive oxygen species (ROS), thereby killing cancer cells. Most of the currently reported photosensitizers of this type have excitation wavelengths in the visible region, which is not conducive to further photodynamic therapy of in vivo tumors. Compound 3f, constructed based on the "cyanone method" of this invention, is a heavy atom-free triplet photosensitizer. This photosensitizer uses a heptamethine cyanine dye as the parent chromophore and electron acceptor, and anthracene as the electron donor, and is designed based on the SOCT-ISC mechanism.

[0087] In order to better simulate the physiological environment, the present invention first evaluated the generation of singlet oxygen by 3f in the cell culture medium DMEM under light, and used 1,3-diphenylbenzisofuran (DPBF) as a singlet oxygen scavenger and ICG as a reference compound at 750nm (30mW / cm 2 ) as the excitation light. Figure 14 As shown, when using 750nm (30mW / cm 2 When the laser irradiated DPBF / 3f continuously, the absorbance value of DPBF at 416nm decreased rapidly, indicating that 3f produced a large amount of 1 O2; in contrast, no O2 was observed when DPBF / ICG was illuminated. 1 O2 is generated.

[0088] In order to further verify the biological application effect of photosensitizer 3f, the present invention tested the biological toxicity and photodynamic therapy effect of the photosensitizer through CCK8 experiment. A549 cells were incubated with different concentrations of 3f for 1 hour, the control group was placed in a cell culture incubator and cultured for 24 hours, and the illumination group was irradiated with 750nm laser (30mW / cm 2 After irradiation (30 minutes), the cells were placed in a cell culture incubator and cultured for 24 hours. Figure 15 As shown in the figure, 3f showed low dark toxicity in the concentration range of 0-2 μM, and its phototoxicity gradually increased with the increase of concentration. The half lethal concentration (EC 50) is 0.8 μM. When A549 cells are pre-incubated with 3f and commercial reactive oxygen species probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and then irradiated, the cells emit strong green fluorescence signals, indicating that 3f generates a large amount of ROS under irradiation Figure 16 A); when A549 cells are pre-incubated with 3f and singlet oxygen fluorescent probe (MCR-DMA) developed by the inventors, and then irradiated, the cells emit strong red fluorescence signals, indicating that the ROS generated by 3f in the cells after irradiation is 1 O2( Figure 16 B). To further confirm the PDT effect of 3f, the inventors also performed a Calcein AM (green, live cells) / propidium iodide (PI, red, dead cells) double staining experiment. As shown in Figure 17 , cells pre-incubated with 3f but not irradiated show strong green fluorescence signals, indicating that 3f itself does not cause cell death; in contrast, when cells pre-incubated with 3f are irradiated with 750 nm (30 mW / cm 2 ) laser for 30 minutes, the cells show strong red fluorescence signals, indicating that 3f can kill cancer cells by generating 1 O2under irradiation.

[0089] Finally, the inventors evaluated the PDT effect of 3f in tumor-bearing nude mice, which were prepared by subcutaneously injecting A549 cells into BALB / c mice. The tumor-bearing nude mice were divided into PBS, PBS / irradiation, 3f, and 3f / irradiation groups, and the tumor sites of the tumor-bearing nude mice were injected with PBS (300 μL) and 3f (50 μM, 300 μL) in situ, respectively, and then treated with non-irradiation or irradiation (750 nm, 50 mW / cm 2 , 60 min). As shown in Figure 18 , the tumors of the tumor-bearing mice in the 3f / irradiation group gradually ablated over time and basically disappeared after 19 days of irradiation, which is mainly due to the near-infrared excitation wavelength and high 1 O2generation ability of 3f; however, the tumors of the tumor-bearing mice in the other groups significantly increased over time. In addition, the body weight of all tumor-bearing nude mice did not significantly decrease during the photodynamic therapy, indicating that 3f has good biocompatibility and negligible side effects.

[0090] In summary, the present invention provides a new method for constructing a Cy7 fluorescent dye substituted with a mesosterically hindered aromatic group—the cyanone method. Using this method, a series of meso-aryl-substituted cyanine dyes 3a-3p were constructed in high yield. The present invention has confirmed that the meso-small sterically hindered 2,6-dimethoxyphenyl substituent can effectively prevent the self-aggregation of Cy7 dyes; importantly, given that aryl lithium has extremely strong nucleophilicity, the water-soluble, highly hindered PEG3 chain can be easily introduced into the ArCy7 dye by this method. This type of dye not only has strong water solubility, but also effectively inhibits the self-aggregation of the dye in water, and therefore has potential application value in protein labeling. In addition, the photosensitizer 3f provided by the present invention has the characteristics of an absorption wavelength in the near-infrared region, good biocompatibility, low dark toxicity, and high singlet oxygen generation efficiency. Therefore, it can produce reactive oxygen species under near-infrared light irradiation, thereby killing cancer cells, and has potential biological application value.

[0091] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for constructing a meso-aryl-substituted heptamethine cyanine dye, characterized in that: The following steps are involved: At −78°C under N₂, a solution of cyanone dissolved in anhydrous THF was added dropwise to a reaction solution of anhydrous THF containing an aryl lithium reagent. The reaction mixture was then warmed to ambient temperature and stirred. After completion of the reaction, HCl was added to quench the reaction. The resulting mixture was extracted with CH₂Cl₂, and the organic layer was dried, concentrated, and purified by column chromatography to obtain the meso-aryl-substituted heptamethine cyanine dye. Wherein, the structural formula of the cyanine ketone is selected from Formula 1a or Formula 1b: ； The structural formula of the aryl lithium reagent is selected from at least one of Formulas 2a-2o: 。 2. The method for constructing a meso-aryl-substituted heptamethine cyanine dye according to claim 1, wherein The molar ratio of the cyanine ketone to the aryl lithium reagent is 1:

5.

3. The method for constructing a meso-aryl-substituted heptamethine cyanine dye according to claim 1, wherein The synthesis steps of the cyanine ketone 1a are as follows: Cl-Cy7 and sodium acetate are dissolved in DMF solution, stirred at 100°C for 3 hours, cooled to room temperature, added with water, stirred, filtered, and purified by column chromatography to obtain 1a.

4. The method for constructing a meso-aryl-substituted heptamethine cyanine dye according to claim 3, wherein The molar ratio of Cl-Cy7 to sodium acetate is 1:

3.

5. The method for constructing a meso-aryl-substituted heptamethine cyanine dye according to claim 1, wherein The synthesis steps of the cyanine ketone 1b are as follows: PEG-Cl-Cy7, N-hydroxysuccinimide and NaH are dissolved in DMF, stirred at room temperature under N2 environment for 12 hours, and concentrated and purified by column chromatography to obtain 1b.

6. The method for constructing a meso-aryl-substituted heptamethine cyanine dye according to claim 5, wherein The molar ratio of PEG-Cl-Cy7, N-hydroxysuccinimide and NaH is 1:5:

5.

7. The method for constructing a meso-aryl-substituted heptamethine cyanine dye according to claim 1, wherein The synthesis steps of the aryl lithium reagents 2a-2i and 2m-2o are as follows: n-BuLi was added dropwise to anhydrous THF containing the corresponding aryl bromide derivatives of 2a-2i and 2m-2o at −78°C under N2 environment and stirred for 30 minutes to obtain 2a-2i and 2m-2o.

8. The method for constructing a meso-aryl-substituted heptamethine cyanine dye according to claim 1, wherein The synthesis steps of the aryl lithium reagents 2j-2l are as follows: n-BuLi is added dropwise to a THF solution of 1,3-dimethoxybenzene, 1,3-diethoxybenzene, or 1,3-diphenoxybenzene at 0°C under N2 environment and stirred for 1 hour to obtain 2j-2l.

9. A medical photosensitizer, characterized in that: The medical photosensitizer is synthesized by the method for constructing a meso-aryl-substituted heptamethine cyanine dye according to any one of claims 1 to 8, and its structural formula is as follows: 。 10. Use of the medical photosensitizer according to claim 9 as a drug for preparing anti-tumor photodynamic therapy.

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