Synthesis and application of a pH-activated photosensitizer

Abstract

The present invention belongs to the field of biological detection and biomedicine technology, and specifically relates to the synthesis and application of a pH-activated photosensitizer. In order to construct a NIR-Ⅱ activated photosensitizer, the present invention uses rhodamine derivatives as raw materials to modify and synthesize a pH-activated photosensitizer. This NIR-Ⅱ region activated photosensitizer itself does not have the ability to emit fluorescence, and needs to be activated by light after H+ stimulation, thereby generating a large amount of reactive oxygen and fluorescence emission. Among them, the reactive oxygen generated will destroy the protein, DNA, and lipids in the cell, leading to cell apoptosis, thereby killing tumor cells; the fluorescence emission can produce a strong fluorescent signal in the NIR-Ⅱ region, and the cancerous site can be imaged in vivo by NIR-Ⅱ. It can be seen that the pH-activated photosensitizer of the present invention can provide new ideas for the design of activated photosensitizers, which has important reference significance for realizing the integration of diagnosis and treatment and overcoming the problem of cancer.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection and biomedicine, and particularly relates to the synthesis and application of a pH-activated photosensitizer. Background Art

[0002] With the continuous improvement of the quality of life, people are paying more and more attention to health issues. In recent years, the cancer problem has become a focus issue in medical and health care and has attracted much attention. The commonly used methods for treating cancer are radiotherapy, chemotherapy and surgical resection. However, these treatment methods have more or less defects, resulting in unsatisfactory treatment results. Photodynamic therapy (PDT) is a non-invasive diagnosis and treatment method. Due to its many advantages such as minimal surgical trauma, excellent selectivity, few side effects, ability to coordinate surgical treatment, and ability to eliminate hidden lesions, it is widely used in cancer treatment and has attracted much attention from scholars.

[0003] PDT is a new disease treatment method based on the interaction between light, photosensitizers, and oxygen. A photosensitizer is one of the key elements of PDT treatment. It is a photoactive substance that can absorb light energy of a specific wavelength and convert it into chemical energy, thereby triggering a series of chemical reactions. Among them, activated photosensitizers can only produce fluorescence and reactive oxygen species when stimulated by specific biomarkers in tumor cells. By selectively activating photosensitizers to control the production of reactive oxygen species in tumor cells, tumor cells can be directly killed and toxic side effects on normal tissues can be reduced. This allows for more accurate detection and more precise treatment of tumor cells. Therefore, photosensitizers that can be specifically activated by biomarkers in tumors have received widespread attention.

[0004] Near-infrared light has a wavelength between visible light and mid-infrared light, ranging from 700 to 2500 nm. Near-infrared light is subdivided into different optical windows based on wavelength. For example, the first near-infrared (NIR-I) region, which has been studied earlier, has a wavelength range of 700 to 900 nm, while the second near-infrared (NIR-II) region, which has been studied more in recent years, has a wavelength range of 900 to 1700 nm. Compared with visible light and NIR-I imaging, NIR-II imaging offers many advantages, including fewer adverse reactions, faster imaging speed, and low autofluorescence background. Because light scattering intensity decreases exponentially with increasing wavelength, NIR-II imaging has superior tissue penetration and spatial resolution compared to visible light and NIR-I imaging. Due to the complexity of the tumor microenvironment, the adjuvant effect of single-use photodynamic therapy on tumor elimination and immunotherapy is often weakened. Therefore, if the advantages of NIR-Ⅱ imaging and activated photosensitizers can be combined to develop NIR-Ⅱ activated photosensitizers, it is expected to further enhance the diagnostic and therapeutic effects of photodynamic therapy.

[0005] In addition, since tumor tissue is more acidic than normal tissue, it can be used as a target for pH-activated photosensitizers. + As a biomarker in cancer cells to activate photosensitizers, it will surely have broad application prospects. Summary of the Invention

[0006] In order to overcome the above-mentioned deficiencies of the prior art, the present invention uses rhodamine derivatives as raw materials to construct a pH-activated photosensitizer. The pH-activated photosensitizer is an integrated diagnosis and treatment dosage form, which can provide a new strategy for the integrated diagnosis and treatment of cancer treatment and has broad development prospects.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] The first aspect of the present invention provides a pH-activated photosensitizer, the structural formula of which is shown below:

[0009]

[0010] The second aspect of the present invention provides a method for preparing the pH-activated photosensitizer according to the first aspect. According to the following reaction formula, the preparation method comprises the following steps:

[0011]

[0012] S1. Dissolve compound 2b in acetic acid, add 2,2':5',2"-terthiophene-5-carboxaldehyde, and reflux at 100-120°C for 8-15 hours. After the reaction, cool under reduced pressure, concentrate, and purify by column chromatography to obtain compound 2b+3t.

[0013] S2. Dissolve compound 2b+3t in dichloromethane, add NHS and EDC, and reflux with stirring at 35°C-50°C for 4-7 hours. Then add adamantane and triethylamine, and continue to reflux with stirring at 35°C-50°C for 8-15 hours. After the reaction, concentrate under reduced pressure and purify by column chromatography to obtain a pH-activated photosensitizer.

[0014] Only produced when specific biomarkers are present 1O2-activated photosensitizers can minimize damage to normal tissue. Among them, photosensitizers that do not contain heavy metal atoms have the advantages of low toxicity, long triplet lifetime, good photostability, and low cost. When the fluorescence emission range of the photosensitizer is in the near-infrared region II (NIR-II) (900-1700 nm), it can be used for in vivo biological imaging. Compared with visible light and near-infrared region I imaging, NIR-II imaging has many advantages, including fewer adverse reactions, faster imaging speed, deep tissue penetration, high image contrast, and low autofluorescence background. The present invention synthesized a photosensitizer that does not contain heavy metal atoms by modifying a rhodamine derivative and adding a thiophene structure to its structure. Experimental testing showed that the synthesized photosensitizer exhibited good singlet oxygen generation ability. Based on this, the photosensitizer was further modified to obtain a pH-activated photosensitizer. By monitoring the changes in the fluorescence emission spectrum of this activated photosensitizer, it was found that this activated photosensitizer has good fluorescence emission ability in the NIR-II region. These research results provide new ideas for integrated cancer diagnosis and treatment.

[0015] Preferably, in S1, the molar ratio of compound 2b to 2,2':5',2"-terthiophene-5-carboxaldehyde is 0.8-1.0:2.0-3.0.

[0016] Preferably, the column chromatography purification in S1 uses dichloromethane:methanol=200:1-100; v / v (more preferably 11 v / v) as the elution solvent.

[0017] Preferably, in S2, the molar ratio of the compound 2b+3t, NHS, EDC and amantadine is 0.06-0.09:0.1-0.2:0.06-0.09:0.2-0.3.

[0018] Preferably, in S2, the concentration of the compound 2b+3t in dichloromethane is 40-60 mg / 20 mL.

[0019] Preferably, in S2, NHS and EDC are added under light-proof conditions.

[0020] Preferably, the column chromatography purification in S2 uses dichloromethane:ethanol = 100:1-100:v / v (more preferably 5v / v) as the elution solvent.

[0021] The third aspect of the present invention provides the use of the pH-activated photosensitizer described in the second aspect in the preparation of an integrated tumor diagnosis and treatment preparation.

[0022] The activated photosensitizer synthesized by the method of the present invention is +After stimulation, light exposure can generate a large amount of reactive oxygen species and fluorescence emission, thereby killing tumor cells. It can also produce a strong fluorescence signal in the NIR-Ⅱ region and perform NIR-Ⅱ in vivo imaging of cancerous sites. It is an integrated dosage form for tumor diagnosis and treatment with broad application prospects.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention uses rhodamine derivatives as raw materials to construct a pH-activated photosensitizer. This NIR-II region activated photosensitizer itself does not have the ability to emit fluorescence and needs to be exposed to H + After stimulation, it can be activated by light, thereby generating a large amount of reactive oxygen species and fluorescence emission. Among them, the reactive oxygen species generated will destroy the proteins, DNA, and lipids in the cells, leading to cell apoptosis, thereby killing tumor cells; the fluorescence emission can produce a strong fluorescence signal in the NIR-Ⅱ region, which can be used for NIR-Ⅱ in vivo imaging of the cancerous site. It can be seen that the pH-activated photosensitizer of the present invention is an integrated diagnosis and treatment dosage form, which can provide new ideas for the design of activated photosensitizers and has important reference significance for realizing integrated diagnosis and treatment and overcoming the problem of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 2b+t 1 HNMR spectrum.

[0026] Figure 2 2b+2t 1 HNMR spectrum.

[0027] Figure 3 2b+3t 1 HNMR spectrum.

[0028] Figure 4 2b+t 13 C NMR spectrum.

[0029] Figure 5 2b+2t 13 C NMR spectrum.

[0030] Figure 6 2b+3t 13 C NMR spectrum.

[0031] Figure 7 This is the HRMS diagram of 2b+t.

[0032] Figure 8 This is the HRMS diagram of 2b+2t.

[0033] Figure 9 This is the HRMS diagram of 2b+3t.

[0034] Figure 10 The UV absorption spectrum of 10 μM photosensitizer in 10% DMSO / H2O;

[0035] Figure 11 (a) Fluorescence emission spectra of DCFH in the solution at different illumination times after adding 2b+t; (b) Fluorescence emission spectra of DCFH in the solution at different illumination times after adding 2b+2t; (c) Fluorescence emission spectra of DCFH in the solution at different illumination times after adding 2b+3t; (d) Comparison of the fluorescence emission intensity of DCFH at 525 nm over time.

[0036] Figure 12 (a) UV absorption spectra of DPBF in the solution at different illumination times after adding 2b+t; (b) UV absorption spectra of DPBF in the solution at different illumination times after adding 2b+2t; (c) UV absorption spectra of DPBF in the solution at different illumination times after adding 2b+3t; (d) comparison of the absorption intensity of DPBF at 410nm as a function of illumination time.

[0037] Figure 13 pH-activated photosensitizer 1 HNMR spectrum.

[0038] Figure 14 pH-activated photosensitizer 13 C NMR spectrum.

[0039] Figure 15 HRMS diagram of pH-activated photosensitizer.

[0040] Figure 16 (a) pH-activated photosensitizer plus H + The fluorescence intensity changes of DCFH in the pre-solution under different illumination times; (b) the pH-activated photosensitizer plus H + The fluorescence intensity changes of DCFH in the solution at different illumination times; (c) The fluorescence intensity changes of DCFH in the solution at different illumination times; (d) The fluorescence intensity changes of DCFH in the solution at different illumination times; (e) The fluorescence intensity changes of DCFH in the solution at different illumination times; (f) The fluorescence intensity changes of DCFH in the solution at different illumination times; (g) The fluorescence intensity changes of DCFH in the solution at different illumination times; (h) The fluorescence intensity changes of DCFH in the solution at different + Comparison of the fluorescence emission intensity of DCFH at 525 nm in the solution before and after treatment over time; the numbers in (a) and (b) are pH values.

[0041] Figure 17 pH-activated photosensitizers at different H + Fluorescence emission spectra at the concentration. DETAILED DESCRIPTION

[0042] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0043] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0044] Example 1: Synthesis and characterization of photosensitizers

[0045] 1. Synthesis of photosensitizer

[0046] The synthetic reaction formula of the photosensitizer is as follows:

[0047]

[0048] (1) Synthesis of compound 2b+t:

[0049] Compound 2b (199.9 mg, 0.42 mmol) was added to a 100 mL round-bottom flask and dissolved in 10 mL of acetic acid. Thiophene aldehyde (58 mg, 0.52 mmol) was then added to the flask and heated at 110°C for 12 h. After completion of the reaction, the mixture was cooled and concentrated under reduced pressure. The resulting product was then purified by column chromatography (dichloromethane:methanol = 400:1-100; 5 v / v) to obtain the purple desired product (yield 156 mg, 65.16%). The NMR and mass spectra of the product are as follows:

[0050] 1H NMR(600MHz,Chloroform-d)δ7.96(d,J=7.7Hz,1H),7.63(td,J=7.5,1.1Hz,1H),7.57-7.52(m,2H),7.37(d,J=5.1H z,1H),7.24-7.19(m,2H),7.10(dd,J=5.1,3.6Hz,1H),6.49(d,J=8.9Hz,1H),6.44(d,J=2.5Hz,1H),6.35(dd,J=8.9 ,2.6Hz,1H),3.37(q,J=7.1Hz,4H),2.92(dddd,J=15.7,6.7,4.7,1.6Hz,1H),2.69(dddd,J=15.9,9.0,4.9,2.0Hz,1 H),2.04(ddd,J=16.0,6.3,4.7Hz,1H),1.77-1.69(m,2H),1.63(ddd,J=16.0,8.4,4.7Hz,1H),1.18(t,J=7.0Hz,6H). 13 C NMR (151MHz, CDCl3) δ170.22,152.58,152.43,149.54,147.18,140.71,134.58,129.38,129.10,128.71,128.18,127.83 ,127.33,126.60,125.12,123.66,118.39,108.96,108.25,105.04,97.48,44.57,27.64,23.03,22.11,12.73.ESI-MS(M) + [m / z] 470.17844, calculated as 470.17844, M=C 29 H 28 NO3S + .

[0051] (2) Synthesis of compound 2b+2t:

[0052] Compound 2b (463 mg, 0.97 mmol) was added to a 100 mL round-bottom flask and dissolved in 20 mL of acetic acid. 2,2-Bithiophene-5-acetaldehyde (376 mg, 1.94 mmol) was then added to the flask and refluxed overnight at 110°C. After the reaction, the mixture was cooled and concentrated under reduced pressure. The resulting product was purified by column chromatography (dichloromethane:methanol = 200:1 to 100:7 v / v) to obtain the blue target product (yield: 318 mg, 50.27%). The NMR and mass spectra of the product are as follows:

[0053] 1 H NMR(600MHz,Chloroform-d)δ8.01(d,J=7.7Hz,1H),7.65-7.60(m,2H),7.58-7.53(m,1H), 7.25-7.21(m,3H),7.20-7.16(m,2H),7.03(dd,J=5.1,3.6Hz,1H),6.57(d,J=8.9Hz,1H),6 .54(d,J=2.4Hz,1H),6.43(dd,J=9.1,2.5Hz,1H),3.41(q,J=7.1Hz,4H),2.91(d,J=16.3Hz ,1H),2.73(t,J=12.4Hz,1H),2.15-2.09(m,1H),1.82-1.72(m,3H),1.20(t,J=7.1Hz,6H). 13 C NMR (151MHz, CDCl3) δ169.84,139.47,137.38,134.17,131.47,129.49,128.98,128.16,127.44 ,126.24,125.03,124.40,124.10,124.04,97.28,44.85,27.69,23.50,21.91,12.75.ESI-MS(M) + [m / z] 552.16632, calculated as 552.16616, M=C 33 H 30 NO3S2 + .

[0054] (3) Synthesis of compound 2b+3t

[0055] Compound 2b (457 mg, 0.96 mmol) was added to a 100 mL round-bottom flask and dissolved in 20 mL of acetic acid. 2,2':5',2"-terthiophene-5-carboxaldehyde (582 mg, 2.11 mmol) was then added to the round-bottom flask and refluxed at 110°C overnight. After the reaction, the mixture was cooled and concentrated under reduced pressure. The resulting product was purified by column chromatography (dichloromethane:methanol = 200:1-100; 11 v / v) to obtain the green target product (yield 387 mg, 54.90%). The NMR and mass spectrometry information of the product are as follows:

[0056] 1H NMR(600MHz,Chloroform-d)δ7.99(d,J=7.7Hz,1H),7.63(t,J=7.4Hz,1H),7.57-7.52(m,2H),7.24-7.22( m,1H),7.18(td,J=8.6,4.3Hz,4H),7.12(d,J=3.8Hz,1H),7.09(d,J=3.8Hz,1H),7.02(dd,J=5.1,3.6Hz,1H ),6.53(d,J=8.9Hz,1H),6.49(d,J=2.5Hz,1H),6.39(dd,J=8.9,2.6Hz,1H),3.39(q,J=7.1Hz,4H),2.91(d ,J=16.3Hz,1H),2.75-2.67(m,1H),2.08(dd,J=14.6,6.5Hz,1H),1.74-1.67(m,3H),1.19(t,J=7.1Hz,6H). 13 C NMR (151MHz, CDCl3) δ129.44,128.85,128.09,124.78,124.64,124.51,123.95,44.71,27.75,23.29,22.00,12.75.ESI-MS(M) + [m / z] 634.15388, calculated as 634.15388, M=C 37 H 32 NO3S3 + .

[0057] 2. Characterization of photosensitizers

[0058] (1) Test method

[0059] 1) Nuclear Magnetic Resonance (NMR) Test: An AVANCE III NMR spectrometer manufactured by Bruker, Germany, was used to determine the structure of the compound by dissolving the compound in deuterated chloroform (CDCl 3 ) at room temperature with the frequency set at 400 / 600 MHz.

[0060] 2) Mass spectrometry: A Q Exactive HF-X liquid chromatography-mass spectrometer produced by Thermo Fisher Scientific was used to determine the molecular weight of the target product.

[0061] 3) Fluorescence Emission Spectroscopy: Using an FLS980 steady-state fluorescence spectrometer manufactured in Edinburgh, UK, we measured the fluorescence emission intensity of the target product under different experimental conditions. Fluorescence emission spectra were plotted under different conditions and analyzed to determine the fluorescence characteristics of each compound. Sensitivity was achieved with a water Raman peak signal-to-noise ratio >12,000:1, and a transient wavelength range of 300-1,700 nm.

[0062] 4) UV Absorption Spectroscopy: Using a UV-2700i UV-visible spectrophotometer manufactured by Shimadzu Corporation of Japan, we experimentally measured the UV absorption intensity of the target product under various experimental conditions. UV absorption spectra were plotted under these conditions and analyzed to determine the singlet oxygen generation profile of each compound. All spectrophotometric and fluorescence measurements were performed using quartz cuvettes (width = 1 cm).

[0063] (2) Test results

[0064] 1) 1 H NMR, 13 C NMR and mass spectrometry characterization: The prepared compounds 2b+t, 2b+2t and 2b+3t were dissolved in deuterated chloroform and characterized by 1 H NMR ( Figure 1-3 ), 13 C NMR ( Figure 4-6 ) and mass spectrometry ( Figure 7-9 ) proved the structures of 2b+t, 2b+2t and 2b+3t.

[0065] 2) Spectroscopic determination: By measuring the photophysical properties of the three photosensitizers in solution, we can know ( Figure 10 ), with the increase of the amount of thiophene in the compound, the absorption wavelength of the photosensitizer red-shifted. Among them, the maximum absorption wavelength of 2b+t is 575nm, the maximum absorption wavelength of 2b+2t is 610nm, and the maximum absorption wavelength of 2b+3t is 632nm.

[0066] 3) Detection of reactive oxygen species: The compound 2'-7'-dichlorodihydrofluorescein (DCFH) was used as an indicator for detecting reactive oxygen species in the solution. When reactive oxygen species are generated in the system, DCFH, which is not fluorescent, will be oxidized and emit obvious fluorescence at 525nm. 10μM 2b+t, 2b+2t, and 2b+3t were dissolved in 5mL DMSO / PBS = 1:9 (v / v) containing 5μM DCFH. The mixture was then placed in a test tube and the concentration was measured with 3mW·cm -2 The samples were irradiated with LED light (400-800 nm) and the fluorescence intensity changes of the samples at 525 nm were recorded using a fluorescence spectrometer.

[0067] Through Figure 11 From the analysis of (a), (b), and (c), we can see that with the continuous increase of illumination time, the fluorescence emission intensity of the three photosensitizers at 525nm continues to increase, and then gradually stabilizes. This phenomenon indicates that active oxygen is generated in these three photosensitizers. Figure 11From the analysis in (d), it can be seen that the amount of active oxygen generated by these three photosensitizers as the illumination time increases is as follows: 2b+2t>2b+3t>2b+t.

[0068] 4) Singlet oxygen test: The compound 1,3-diphenylisobenzofuran (DPBF) is used as an indicator to detect the amount of singlet oxygen produced in the solution. When singlet oxygen is produced in the solution, DPBF will be oxidized by the singlet oxygen, resulting in a decrease in the absorbance of the solution at 410nm. 10μM 2b+t, 2b+2t and 2b+3t were dissolved in 5mL DMSO / PBS = 1:9 (v / v) containing 25μM DPBF. The mixture was then placed in a test tube and the ionization temperature was set at 3mW·cm -2 The samples were irradiated with an LED light (400-800 nm) and the absorption changes of the samples at 410 nm were recorded using a UV-visible spectrophotometer.

[0069] Through Figure 12 The analysis of (a), (b), and (c) shows that the UV absorption intensity of the three photosensitizers at 410 nm decreases as the illumination time increases, and eventually stabilizes. This phenomenon indicates that the three photosensitizers generate a large amount of singlet oxygen after illumination. Figure 12 From the analysis in (d), we can see that with the increase of illumination time, the amount of singlet oxygen produced by these three photosensitizers is as follows: 2b+3t>2b+t>2b+2t.

[0070] 5) Theoretical Calculations: To explore the relationship between the photophysical properties and structures of the photosensitizers, density functional theory (DFT) was used with the B3LYP method and the 6-31G basis set to calculate the HOMO-LUMO energy levels and orbital compositions of the frontier molecular orbitals (FMOs). Tables 1 and 2 show the energy levels and orbital compositions of the FMOs.

[0071] According to the HOMO and LOMO data in Tables 1 and 2, the HOMO-LUMO energy gap of the three compounds is 2.447 eV for 2b+t, 2.175 eV for 2b+2t, and 1.981 eV for 2b+3t. The energy gap of 2b+t is the largest, and the energy gap of 2b+3t is the smallest. This indicates that the energy required for electronic transitions in the molecules of the 2b+t compound is higher, while the energy required for electronic transitions in the 2b+3t compound is lower, making it easier to be excited and resulting in a longer excitation wavelength.

[0072] Table 1 Energy levels of HOMO-LUMO orbitals of three photosensitizers

[0073]

[0074] Table 2 HOMO-LUMO orbitals of three photosensitizers

[0075]

[0076]

[0077] Example 2: Synthesis and characterization of pH-activated photosensitizers

[0078] 1. Synthesis of pH-activated photosensitizer

[0079] The synthetic reaction formula of pH-activated photosensitizer is as follows:

[0080]

[0081] The specific synthesis method is as follows: Compound 2b + 3t (55 mg, 0.075 mmol) was added to a 100 mL round-bottom flask and dissolved in 20 mL of dichloromethane. NHS (11.85 mg, 0.103 mmol) and EDC (14.4 mg, 0.075 mmol) were then added to the round-bottom flask in the dark. The mixture was then refluxed and stirred at 40°C for 5 h. After stirring, adamantane (36 mg, 0.238 mmol) and three drops of triethylamine were added to the round-bottom flask. Reflux and stirring were continued at 40°C overnight. After the reaction, the resulting reactants were concentrated under reduced pressure using a rotary evaporator and the product was purified by column chromatography (dichloromethane:ethanol = 100:1-100:5) to obtain the target product, a pH-activated photosensitizer, as a yellow solid (yield 14.7 mg, yield 67.88%). The NMR and mass spectrometry information of the product are as follows:

[0082] 1H NMR(400MHz,Chloroform-d)δ7.69(dd,J=6.7,1.6Hz,1H),7.36(d,J=2.1Hz,1H),7.35-7.25(m,2H),7.15(dd,J=5.1,1.1Hz,1H),7.10(dd,J=5.8,3 .9Hz,2H),7.06(d,J=3.9Hz,1H),7.03(q,J=3.8Hz,2H),6.97-6.95(m,1H ),6.93(dd,J=7.5,1.9Hz,1H),6.44(d,J=8.8Hz,1H),6.31(d,J=2.5Hz,1H ),6.21(dd,J=8.9,2.6Hz,1H),3.29(q,J=7.1Hz,4H),2.86(dt,J=16.0,5 .6Hz,1H),2.70-2.60(m,1H),2.32(d,J=11.8Hz,3H),2.14(d,J=11.8Hz,3 H),2.05(ddd,J=16.1,6.5,4.4Hz,1H),1.96-1.89(m,3H),1.67(ddt,J=18 .1,10.0,5.0Hz,3H),1.53(q,J=13.9,13.1Hz,6H),1.12(t,J=7.0Hz,6H). 13 C NMR (101MHz, CDCl3) δ169.67,152.52,151.25,148.72,144.44,140.16,137.74,137.21,136.4 4,136.32,132.15,131.51,129.76,128.66,128.51,128.02,127.94,124.66,124.57,124.39,1 24.20,124.10,123.83,123.81,123.74,122.56,122.36,116.84,113.03,108.26,107.89,98. 17,67.47,59.35,53.53,44.38,39.30,36.59,30.08,28.24,23.34,22.38,12.75.ESI-MS(M+H) + [m / z] 767.27972, calculated as 767.27997, M=C 47 H 46 N2O2S3.

[0083] 2. Characterization of pH-activated photosensitizers:

[0084] 1) 1 HNMR,13 C NMR and mass spectrometry characterization: A new compound pH-activated photosensitizer was obtained by modifying the 2b+3T structure and then purified. This activated photosensitizer was dissolved in deuterated chloroform and 1 H NMR ( Figure 13 ), 13 C NMR ( Figure 14 ) and mass spectrometry ( Figure 15 ) detection, which confirmed the structure of the pH-activated photosensitizer.

[0085] 2) Reactive oxygen species test before and after the addition of pH-activated photosensitizer to the marker: Measure the reactive oxygen species at different illumination times (min) before and after the addition of pH-activated photosensitizer to the marker, such as Figure 16 As shown in the figure, the fluorescence of the activated photosensitizer is significantly enhanced after the addition of the marker, indicating that the activated photosensitizer can be successfully activated to generate a large amount of reactive oxygen species.

[0086] 3) NIR-II spectrum of pH-activated photosensitizers: The fluorescence emission spectrum of the NIR-II region of the activated photosensitizers was investigated, such as Figure 17 As shown. Figure 17 Analysis shows that this activated photosensitizer + As the content of α-hydroxy-1,1-dione increases, the fluorescence emission intensity in the NIR-II region continues to increase. This shows that this activated photosensitizer has good fluorescence imaging potential.

[0087] In summary, the present invention synthesized three photosensitizers by modifying rhodamine derivatives and controlling the number of attached thiophenes. Based on the heavy atom effect, the presence of sulfur atoms in these three photosensitizers enhances spin-orbit coupling, thereby promoting intersystem crossing and generating more reactive oxygen species. Experiments revealed that the photosensitizer 2b+3t, which contains the most thiophenes, produces the most singlet oxygen. This work provides new insights into the construction of heavy metal-free photosensitizers.

[0088] Furthermore, the present invention modified photosensitizers 2b+3t to create a pH-activated photosensitizer. This activated photosensitizer retains the properties of rhodamine dyes. Upon specific activation by tumor markers, it generates a large amount of singlet oxygen through ring opening. It exhibits advantages such as strong stability, ease of synthesis, and high biocompatibility. Experiments have shown that this activated photosensitizer produces strong fluorescence emission in the NIR-II region, enabling in vivo NIR-II imaging of cancerous sites. This work provides a new strategy for integrated diagnosis and treatment in cancer treatment, with broad prospects for development.

[0089] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. It is apparent to those skilled in the art that various changes, modifications, substitutions, and variations of these embodiments may be made without departing from the principles and spirit of the present invention, and the changes still fall within the scope of protection of the present invention.

Claims

1. A pH-activated photosensitizer, characterized in that The structural formula of the pH-activated photosensitizer is shown below:

2. The method for preparing the pH-activated photosensitizer according to claim 1, wherein: According to the following reaction formula, the preparation method comprises the following steps: S1. Dissolve compound 2b in acetic acid, add 2,2':5',2"-terthiophene-5-carboxaldehyde, and reflux at 100-120°C for 8-15 hours. After the reaction, cool under reduced pressure, concentrate, and purify by column chromatography to obtain compound 2b+3t. S2. Dissolve compound 2b+3t in dichloromethane, add NHS and EDC, and reflux with stirring at 35°C-50°C for 4-7 hours. Then add adamantane and triethylamine, and continue to reflux with stirring at 35°C-50°C for 8-15 hours. After the reaction, concentrate under reduced pressure and purify by column chromatography to obtain a pH-activated photosensitizer.

3. The method for preparing a pH-activated photosensitizer according to claim 2, wherein: In S1, the molar ratio of the compound 2b to 2,2':5',2"-terthiophene-5-carboxaldehyde is 0.8-1.0:2.0-3.

0.

4. The method for preparing a pH-activated photosensitizer according to claim 2, wherein: The column chromatography purification in S1 uses dichloromethane:methanol=200:1-100; v / v as the elution solvent.

5. The method for preparing a pH-activated photosensitizer according to claim 2, wherein: In S2, the molar ratio of the compound 2b+3t, NHS, EDC and adamantine is 0.06-0.09: 0.1-0.2: 0.06-0.09: 0.2-0.

3.

6. The method for preparing a pH-activated photosensitizer according to claim 2, wherein: In S2, the concentration of the compound 2b+3t in dichloromethane is 40-60 mg / 20 mL.

7. The method for preparing a pH-activated photosensitizer according to claim 2, wherein: In S2, NHS and EDC were added under light-protected conditions.

8. The method for preparing a pH-activated photosensitizer according to claim 2, wherein: The column chromatography purification in S2 uses dichloromethane:ethanol = 100:1-100:v / v as the elution solvent.

9. Use of the pH-activated photosensitizer according to claim 1 in the preparation of an integrated tumor diagnosis and treatment preparation.

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