A non-heavy atom type near-infrared two-photon photosensitive dye TMBDP, a preparation method and application thereof

Abstract

This invention discloses a non-heavy atom near-infrared two-photon photosensitive dye, TMBDP, its preparation method, and its applications. This TMBDP photosensitive dye contains no heavy atoms, possesses a fluoroboron dipyrrole dimer structure and two-photon absorption properties, and can simultaneously generate singlet oxygen and superoxide anion radicals through a spin vibrational coupling mechanism. The non-heavy atom two-photon photosensitive dye TMBDP is prepared from triphenylamine fluoroboron dipyrrole dimer and 1-(2-morpholinoethyl)-1H-indole-3-carboxaldehyde via a multi-step tandem reaction. Under near-infrared laser excitation, the non-heavy atom two-photon photosensitive dye TMBDP effectively releases reactive oxygen species through two-photon absorption, promoting tumor cell apoptosis through oxidative damage. As a high-performance near-infrared non-heavy atom two-photon photosensitive dye, it has wide applications in two-photon photodynamic therapy, two-photon fluorescence imaging, and two-photon fluorescence labeling.

Description

Technical Field

[0001] This invention relates to a photosensitive dye, specifically to a non-heavy atom type two-photon photosensitive dye TMBDP, its preparation method, and its applications. Background Technology

[0002] Under near-infrared laser excitation, two-photon photosensitive dyes achieve photocontrolled energy transfer or electron transfer through a two-photon absorption mechanism, generating singlet oxygen or reactive oxygen free radicals at the tumor site. This promotes apoptosis of tumor cells, achieving two-photon photodynamic therapy (PDT) of tumor cells. PDT has many advantages, such as near-infrared laser excitation wavelength, large penetration depth, minimal tissue trauma, short treatment time, and wide applicability. However, there are few reports on near-infrared two-photon photosensitive dyes in current technology. Most photosensitive dyes used for PDT are single-photon excited, with excitation wavelengths in the visible light region. These dyes have poor biocompatibility and shallow penetration depth, failing to meet the requirements of PDT in biological systems and greatly limiting their application.

[0003] Non-heavy atom near-infrared two-photon photosensitive dyes can effectively reduce photodamage, significantly enhance penetration depth in biological tissues, improve reactive oxygen species generation efficiency, and are safe and reliable. The design and preparation of high-performance non-heavy atom near-infrared two-photon photosensitive dyes is a recent research hotspot. Summary of the Invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a high-performance, non-heavy atom type near-infrared two-photon photosensitive dye TMBDP that has a fluoroboron dipyrrole dimer structure and two-photon absorption characteristics, can simultaneously generate singlet oxygen and superoxide anion radicals through a spin vibration coupling mechanism, has good biocompatibility, and is non-heavy atom type near-infrared two-photon photosensitive dye.

[0005] Another technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned non-heavy atom type near-infrared two-photon photosensitive dye TMBDP.

[0006] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned non-heavy atom type near-infrared two-photon photosensitizer TMBDP as a two-photon photodynamic therapy photosensitizer.

[0007] The final technical problem to be solved by the present invention is to provide the application of the above-mentioned non-heavy atom type near-infrared two-photon photosensitive dye TMBDP in near-infrared two-photon fluorescence imaging and fluorescence labeling.

[0008] Technical Solution: To solve the above-mentioned technical problems, the present invention provides a non-heavy atom type near-infrared two-photon photosensitive dye TMBDP, comprising the following structural formula:

[0009]

[0010] The material TMBDP is prepared by a multi-step, one-bottle reaction of triphenylamine fluoroboron dipyrrole dimer and 1-(2-morpholinoethyl)-1H-indole-3-carboxaldehyde. The structural formula of the triphenylamine fluoroboron dipyrrole dimer is as follows:

[0011]

[0012] The present invention discloses a method for preparing a non-heavy atom type two-photon photosensitive dye TMBDP, comprising the following steps: under inert gas protection and at 110°C, triphenylamine fluoroboron dipyrrole dimer and 1-(2-morpholinoethyl)-1H-indole-3-carboxaldehyde are prepared by a "one-bottle multi-step" reaction to obtain the two-photon photosensitive dye TMBDP.

[0013] The molar ratio of the triphenylamine fluoroboron dipyrrole dimer to 1-(2-morpholinoethyl)-1H-indole-3-carboxaldehyde is 1:6 to 1:10.

[0014] The “one bottle, multiple steps” reaction includes: dissolving triphenylamine fluoroboron dipyrrole dimer and 1-(2-morpholinoethyl)-1H-indole-3-carboxaldehyde in dry toluene, continuing to add acetic acid and piperidine dropwise to the system, gradually raising the temperature to 110°C, and stirring the reaction for 7.5 h.

[0015] The present invention also includes the application of the non-heavy atom two-photon photosensitizer TMBDP as a photosensitizer for near-infrared two-photon photodynamic therapy.

[0016] The application mentioned here is photodynamic therapy for tumor cells.

[0017] The present invention also includes the application of the non-heavy atom type two-photon photosensitive dye TMBDP in near-infrared fluorescence imaging, fluorescence sensing or fluorescence labeling.

[0018] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: The near-infrared two-photon photosensitive dye TMBDP described in this invention does not contain heavy atoms, possesses a fluoroboron dipyrrole dimer structure and two-photon absorption characteristics, and can simultaneously generate singlet oxygen and superoxide anion radicals through a spin vibrational coupling mechanism. Under 1000nm laser excitation, singlet oxygen and superoxide anion radicals are simultaneously generated through two-photon absorption, promoting tumor cell apoptosis through oxidative damage. As a high-performance non-heavy atom near-infrared two-photon photosensitive dye, it has wide applications in two-photon photodynamic therapy, two-photon fluorescence imaging, and two-photon fluorescence labeling. Attached Figure Description

[0019] Figure 1The electron paramagnetic resonance spectrum of superoxide anion radicals generated by the non-heavy atom type two-photon photosensitive dye TMBDP is shown.

[0020] Figure 2 This is a two-photon fluorescence image of the non-heavy atom two-photon photosensitive dye TMBDP in zebrafish. The concentration of TMBDP is 20 μM, and the scale bar is 300 μm.

[0021] Figure 3 The phototoxicity and dark toxicity experiments of the non-heavy atom two-photon photosensitive dye TMBDP on CNE-2 cells were conducted, with the concentration range of TMBDP being 0-24 μM. Detailed Implementation

[0022] Example 1

[0023] (1) Preparation of non-heavy atom type near-infrared two-photon photosensitive dye TMBDP:

[0024] Triphenylamine fluoroboron dipyrrole dimer 50 mg and 1-(2-morpholinoethyl)-1H-indole-3-carboxaldehyde were mixed under nitrogen protection and anhydrous and oxygen-free conditions. 140mg Dissolve in 15 mL of dry toluene, then add 100 μL of acetic acid and 100 μL of piperidine dropwise to the system, gradually raise the temperature to 110 °C, and stir for 7.5 h. After the reaction is complete, purify by vacuum distillation and column chromatography to obtain the non-heavy atom two-photon fluoroboron dipyrrole dimer photosensitive dye TMBDP, with a yield of 28%.

[0025] The synthetic route for the non-heavy atom two-photon fluoroboron dipyrrole dimer photosensitive dye TMBDP is as follows:

[0026]

[0027] Among them, the triphenylamine-boron dipyrrole dimer was prepared according to the literature method (Linfeng Wang and Ying Qian, Modification of a SOCT-ISC type triphenylamine-BODIPY photosensitizer by a multipolar dendrimer design for photodynamic therapy and two-photonfluorescence imaging, Biomater. Sci., 2023, 11, 1459-1469).

[0028] (2) Structural characterization of the non-heavy atom two-photon photosensitive dye TMBDP:

[0029] 1H NMR spectrum: 1H NMR(600MHz, CDCl3)δ8.16–8.11(m,4H),7.79(d,J=16.2Hz,4H),7.52–7.4 6(m,8H),7.38–7.35(m,6H),7.33–7.29(m,8H),7.25–7.20(m,8H),7.19–7 .17(m,2H),7.13(t,J=7.3Hz,1H),6.68(s,4H),4.26–4.20(m,8H),3.70(t ,J=4.6Hz,16H),2.79–2.74(m,8H),2.50(t,J=4.4Hz,16H),1.68(s,12H).

[0030] High-resolution mass spectrometry (HRMS): Exact Mass calcd for C 104 H 106 B2F4N 13 O4[M+H + ]:1698.8613found:1698.8462

[0031] Therefore, the structural formula of the obtained non-heavy atom type two-photon fluoroboron dipyrrole dimer photosensitive dye TMBDP is confirmed as follows:

[0032]

[0033] Example 2: Preparation of the non-heavy atom type near-infrared two-photon photosensitive dye TMBDP

[0034] The preparation of the triphenylamine fluoroboron dipyrrole dimer was the same as in Example 1, wherein 50 mg of the triphenylamine fluoroboron dipyrrole dimer and 1-(2-morpholinoethyl)-1H-indole-3-carboxaldehyde were mixed under nitrogen protection and anhydrous and oxygen-free conditions. 105mg Dissolve in 15 mL of dry toluene, then add 100 μL of acetic acid and 100 μL of piperidine dropwise to the system, gradually raise the temperature to 110 °C, and stir for 7.5 h. After the reaction is complete, purify by vacuum distillation and column chromatography to obtain the non-heavy atom two-photon fluoroboron dipyrrole dimer photosensitive dye TMBDP, with a yield of 16%.

[0035] Example 3

[0036] The preparation of triphenylamine fluoroboron dipyrrole dimer was the same as in Example 1. Under nitrogen protection and anhydrous and oxygen-free conditions, 50 mg of triphenylamine fluoroboron dipyrrole dimer and 175 mg of 1-(2-morpholinoethyl)-1H-indole-3-carboxaldehyde were dissolved in 15 mL of dry toluene. Then, 100 μL of acetic acid and 100 μL of piperidine were added dropwise to the system, and the temperature was gradually increased to 110 °C. The reaction was stirred for 7.5 h. After the reaction was completed, the product was purified by vacuum distillation and column chromatography to obtain the non-heavy atom two-photon fluoroboron dipyrrole dimer photosensitive dye TMBDP, with a yield of 22%.

[0037] Example 4

[0038] like Figure 1 As shown, using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a superoxide anion radical scavenger, the superoxide anion radicals were detected by electron paramagnetic resonance spectroscopy. The photosensitive dye TMBDP prepared in Example 1 could generate superoxide anion radicals under near-infrared light excitation. Simultaneously, using 1,3-diphenylisobenzofuran (DPBF) as a singlet oxygen scavenger, the release of singlet oxygen by TMBDP under near-infrared light excitation was detected. The non-heavy atom two-photon photosensitive dye TMBDP can generate both superoxide anion radicals and singlet oxygen under near-infrared light excitation. The photosensitive dyes TMBDP prepared in Examples 2 and 3 also achieved the same effect.

[0039] Example 5

[0040] like Figure 2 As shown, 15 μL of the photosensitive dye TMBDP (concentration of 2 × 10⁻⁶) prepared in Example 1 was used. -3 M) was added to a culture dish containing zebrafish (containing 2 mL of culture medium) and incubated for 6 h, followed by two-photon fluorescence imaging. Two-photon imaging results showed that TMBDP could be absorbed by zebrafish, exhibiting significant red fluorescence in the head, abdomen, and tail. The non-heavy-atom two-photon photosensitive dye TMBDP possesses excellent biocompatibility and two-photon imaging capabilities. Using 2',7'-dichlorodihydrofluorescein (DCFH) as a reactive oxygen species indicator, the photosensitive dye TMBDP (concentration 2 × 10⁻⁶) was irradiated with a 1000 nm femtosecond laser. -3 M) and DCFH (concentration of 10) -2 After 30 minutes of mixing with M), the fluorescence of DCFH at 525 nm was enhanced, indicating that the two-photon photosensitive dye TMBDP generates reactive oxygen species under 1000 nm femtosecond laser excitation.

[0041] like Figure 3As shown, after co-incubating the TMBDP photosensitive dye prepared in Example 1 with human nasopharyngeal carcinoma CNE-2 tumor cells for 24 hours, dark toxicity and phototoxicity experiments were performed. The final concentration of TMBDP was 0-24 μM (including 0, 4, 8, 12, 16, 10, and 24 μM). In the absence of light, the cell viability exceeded 90%. When irradiated with near-infrared laser for 30 minutes, the cell viability decreased significantly with increasing concentration. Under near-infrared laser excitation, the photosensitive dye TMBDP rapidly generated reactive oxygen species within CNE-2 tumor cells, promoting tumor cell apoptosis through oxidative damage.

[0042] In summary, the non-heavy atom near-infrared two-photon photosensitive dye TMBDP possesses a fluoroboron dipyrrole dimer structure and two-photon absorption properties, and can simultaneously generate singlet oxygen and superoxide anion radicals through a spin vibrational coupling mechanism. Under near-infrared laser excitation, reactive oxygen species are effectively released through two-photon absorption, promoting tumor cell apoptosis through oxidative damage. TMBDP exhibits strong two-photon absorption and two-photon fluorescence emission, good biocompatibility, and can serve as a high-performance non-heavy atom near-infrared two-photon photosensitive dye with wide applications in two-photon photodynamic therapy, two-photon fluorescence imaging, and two-photon fluorescence labeling.

Claims

1. A non-heavy atom type near-infrared two-photon photosensitive dye TMBDP, characterized in that, Its structural formula is:

2. The method for preparing the non-heavy atom type two-photon photosensitive dye TMBDP according to claim 1, characterized in that, Includes the following steps: Under inert gas protection and at 110°C, triphenylamine fluoroboron dipyrrole dimer was reacted with 1-(2-morpholinoethyl)-1H-indole-3-carboxaldehyde to obtain the non-heavy atom two-photon photosensitive dye. The structural formula of the triphenylamine fluoroboron dipyrrole dimer is as follows:

3. The method for preparing the non-heavy atom type two-photon photosensitive dye TMBDP according to claim 2, characterized in that, The molar ratio of the triphenylamine fluoroboron dipyrrole dimer to 1-(2-morpholinoethyl)-1H-indole-3-carboxaldehyde is 1:6 to 1:

10.

4. The method for preparing the non-heavy atom type two-photon photosensitive dye TMBDP according to claim 2, characterized in that, The reaction comprises: dissolving triphenylamine fluoroboron dipyrrole dimer and 1-(2-morpholinoethyl)-1H-indole-3-carboxaldehyde in dry toluene, continuing to add acetic acid and piperidine dropwise to the system, gradually raising the temperature to 110°C, and stirring the reaction for 7.5 h.

5. The application of the non-heavy atom type two-photon photosensitizer TMBDP as a photosensitizer in near-infrared two-photon photodynamic therapy, wherein the application does not involve methods for diagnosing or treating diseases.

6. The application according to claim 5, characterized in that, The application described is photodynamic therapy for tumor cells, and does not involve the diagnosis or treatment of diseases.

7. The application of the non-heavy atom type two-photon photosensitive dye TMBDP according to claim 1 in near-infrared two-photon fluorescence imaging, wherein the application does not involve methods for the diagnosis and treatment of diseases.

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