A small molecule fluorescent photosensitizer containing a gefitinib backbone structure and an AIE group, its preparation and application

Abstract

This invention relates to a small-molecule fluorescent photosensitizer containing a gefitinib backbone structure and an AIE group, its preparation, and its application, belonging to the field of fluorescent photosensitizer technology. The fluorescent photosensitizer includes an AIE group, a linking group, and a recognition group. On one hand, the AIE group backbone structure consists of a pyridine cation (electron acceptor, A), an ethylene group (π-bridge), a thiophene fragment (π-bridge and electron donor, D), and a triphenylamine segment (D), which is a typical D-π-A molecular configuration, thus exhibiting near-infrared fluorescence emission. On the other hand, by introducing heavy atoms to enhance the spin-orbit coupling of the photosensitizer, the efficiency of singlet oxygen generation is improved. The fluorescent photosensitizer can precisely target EGFR-overexpressing non-small cell lung cancer cells without illuminating normal cells. The fluorescent photosensitizer exhibits aggregation-induced emission properties, avoiding the ACQ effect during aggregation, greatly improving the ROS generation efficiency and promoting its tumor-killing effect.

Description

Technical Field

[0001] This invention relates to a small molecule fluorescent photosensitizer containing a gefitinib backbone structure and an AIE group, its preparation and application, belonging to the field of fluorescent photosensitizer technology. Background Technology

[0002] Lung cancer is one of the leading causes of cancer-related deaths worldwide, with an estimated 2.2 million new cases annually (11.4% of all new cancer cases) and 1.79 million deaths annually (18.4% of all cancer deaths). Histologically, lung cancer is broadly classified into non-small cell lung cancer (NSCLC) (accounting for approximately 85% of confirmed cases) and small cell lung cancer (accounting for approximately 15% of confirmed cases). Early-stage NSCLC is often asymptomatic, leading most cases to be diagnosed as locally advanced or metastatic, missing the optimal time for surgical treatment. Patients with epidermal growth factor receptor (EGFR) mutations make up a significant proportion of clinical cases. The most common NSCLC-associated EGFR mutations are point mutations in exon 21 (L858R) and in-frame deletions in exon 19 (19del), accounting for approximately 85% of all EGFR mutations and typically associated with adenocarcinoma histology. These common mutations have been identified as oncogenic drivers of NSCLC and confer enhanced sensitivity to epidermal growth factor-tyrosine kinase inhibitors (EGFR-TKIs). Therefore, EGFR has received widespread attention from all sectors of society as an effective target for the treatment of non-small cell lung cancer.

[0003] EGFR, the earliest discovered prototypical member of the receptor tyrosine kinase (RTK) family, is a crucial component of cell signaling pathways, playing a vital role in regulating cancer cell proliferation, invasion, differentiation, and migration. EGFR binding to ligands (EGF and TGF-α) leads to conformational changes and the formation of homodimers and heterodimers with other members of the EGFR family. Subsequently, with the assistance of aptamers (such as SHC and GRB-2), the cytoplasmic tyrosine kinase (TK) domain undergoes autophosphorylation, triggering downstream signaling. EGFR overexpression plays a vital role in multiple intracellular and extracellular signaling pathways, particularly in the cell proliferation, differentiation, and death processes of cancers such as non-small cell lung cancer, head and neck cancer, renal cell carcinoma, ovarian cancer, and colon cancer. The complexity of EGFR structure and function presents challenges for EGFR protein visualization, especially fluorescence visualization.

[0004] With the rise of targeted therapy for diseases, selective small molecule inhibitors are constantly being developed and applied to the efficient diagnosis and treatment of diseases. Just as EGFR anticancer antibody drugs (i.e., cetuximab) can bind to fluorescent dyes as specific fluorescent probes, small molecule inhibitors (such as gefitinib and erlotinib) can serve as ideal recognition units for a new class of molecular fluorescent probes due to their small size and high specificity. Wang Fuyi et al. used fluorescein to label gefitinib and obtained a class of quinazoline fluorescent probes capable of imaging EGFR. This probe has a strong inhibitory effect on EGFR and is uniformly distributed on the cell membrane after interacting with various cells, which can be used for imaging cancer cells. Shengnan Liu et al. developed novel small molecule fluorescent probes Cy3-AFTN and Cy5-AFTN as effective dual-target inhibitors for the effective detection of HER1 / HER2 expression in cancer cells and in vivo tumor diagnostic imaging. Hui Deng et al. developed a multicolor fluorescence activated cell sorting (FACS) system and used an EGFR-TKI-based fluorescent probe (HX103) to analyze active EGFR in tumors. Currently, small molecule fluorescent probes based on EGFR-TKIs can only be used for the precise diagnosis of lung cancer and cannot overcome the bottleneck of integrated diagnosis and treatment.

[0005] Furthermore, patients with EGFR activating mutations benefit from EGFR-TKIs (such as gefitinib and erlotinib) for less than a year before developing resistance. For example, gefitinib blocks tyrosine kinases, inhibiting phosphorylation of mutant EGFR on the cell membrane and terminating downstream EGFR signaling. However, EGFR pathway activation does not follow a linear pattern and is a rather complex process. Therefore, drug resistance in non-small cell lung cancer patients remains a challenging problem to be solved. To address this issue, many researchers have attempted to combine two or more anticancer drugs to enhance tumor growth inhibition and overcome secondary resistance. This approach, which uses different drugs targeting different receptors or different sites of action on the same receptor to achieve a synergistic effect of multiple anticancer mechanisms, intuitively seems to more effectively inhibit cancer cell growth and even minimize the development of resistance mutations. However, in the clinical setting, several limiting factors often restrict the implementation of combination therapy, including multiple drugs, complex pharmacokinetic characteristics, unclear dosage regimens, and drug interactions. It is noteworthy that fluorescence imaging has been widely reported to enable faster, non-invasive, real-time, and precise monitoring of responses during cancer treatment interventions, making it a crucial technology for addressing the challenge of drug resistance. On one hand, while photodynamic therapy (PDT) holds great potential in overcoming drug resistance, excessive light exposure or photosensitizer (PS) loading inevitably leads to harmful side effects, thus creating a high demand for real-time monitoring of photodynamic or cellular responses. On the other hand, compared to traditional aggregation-quenched emission (ACQ) probes, aggregation-induced emission (AIE) probes induce high-intensity fluorescence by restricting intramolecular motion, offering advantages such as high signal-to-noise ratio, good biocompatibility, and low fluorescence background. In conclusion, if a small-molecule fluorescent photosensitizer containing the gefitinib backbone structure and AIE groups can be developed, it could not only address the intractable drug resistance problem through PDT but also report treatment responses via fluorescence monitoring. Summary of the Invention

[0006] In view of this, the present invention aims to provide a small molecule fluorescent photosensitizer containing the gefitinib backbone structure and the AIE group, its preparation, and its application. The fluorescent photosensitizer retains the selectivity of the tyrosine kinase inhibitor gefitinib for epidermal growth factor receptor (EGFR). Once the fluorescent ligand enters the ATP-binding pocket of EGFR, a fluorescence signal is activated, thereby enabling fluorescence visualization of EGFR-overexpressing cells. Furthermore, the fluorescent photosensitizer exhibits aggregation-induced emission properties, avoiding aggregation quenching (ACQ) during aggregation, significantly improving the generation efficiency of reactive oxygen species (ROS) and promoting their tumor-killing effect. In addition, the fluorescent photosensitizer TTVP-GETN can address the challenging problem of drug resistance through phototherapy (PDT) and can also report treatment response through fluorescence monitoring.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows.

[0008] A small molecule fluorescent photosensitizer containing a gefitinib analog and an AIE group, wherein the structural formula of the fluorescent photosensitizer is as follows:

[0009]

[0010] Where n is an integer from 1 to 5;

[0011] R1 is H or Br; R2 is H or Br;

[0012] X- represents a halide ion, perchlorate ion, or hexafluorophosphate ion.

[0013] Preferably, the fluorescent photosensitizer has the following structural formula:

[0014]

[0015] A method for preparing a small molecule fluorescent photosensitizer containing a gefitinib backbone structure and an AIE group, as described in this invention, comprising the following steps:

[0016] (1) Triphenylphosphine reacts with 4-chloromethylpyridine hydrochloride to produce triphenyl(4-pyridine-methyl)phosphine chloride hydrochloride;

[0017] (2) After 4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-ol and potassium carbonate are thoroughly mixed, the first reactant is added, and the first intermediate is obtained by Williamson ether synthesis reaction;

[0018] (3) After potassium tert-butoxide (t-BuOK) and triphenyl(4-pyridine-methyl)phosphine chloride hydrochloride are thoroughly mixed, the second reactant is added, and the reaction yields the second intermediate;

[0019] (4) The second intermediate reacts with the first intermediate to obtain a small molecule fluorescent photosensitizer containing a gefitinib analog and an AIE group;

[0020] The structural formula of the first reactant is: n is an integer from 1 to 5;

[0021] The structural formula of the first intermediate is:

[0022] The structural formula of the second reactant is:

[0023] The structural formula of the second intermediate is:

[0024] R1 is H or Br; R2 is H or Br.

[0025] Preferably, in step (1), triphenylphosphine and 4-chloromethylpyridine hydrochloride are heated under reflux for 12-24 h in a first organic solvent at 70-90 °C to obtain triphenyl(4-pyridine-methyl)phosphine chloride hydrochloride; the molar ratio of triphenylphosphine to 4-chloromethylpyridine hydrochloride is 1:1-1.2; and the first organic solvent is acetonitrile.

[0026] Preferably, in step (2), 4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-ol and a weak Lewis base are thoroughly mixed in a second organic solvent, and then the first reactant is added. The mixture is stirred for more than 24 hours, the organic phase is collected, concentrated, and purified to obtain the first intermediate. The molar ratio of 4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-ol, potassium carbonate, and the first reactant is 1:3 to 5:15 to 30. The second organic solvent is N,N-dimethylformamide.

[0027] Preferably, in step (3), t-BuOK and triphenyl(4-pyridine-methyl)phosphorus chloride hydrochloride are thoroughly mixed at 0-5°C in a third organic solvent. After adding the second reactant, the mixture is reacted at room temperature for 7-24 hours to obtain the second intermediate. The molar ratio of t-BuOK, triphenyl(4-pyridine-methyl)phosphorus chloride hydrochloride, and the second reactant is 2-3:1-1.2:1. The third organic solvent is tetrahydrofuran.

[0028] Preferably, in step (4), the second intermediate and the first intermediate are mixed in the fourth organic solvent, stirred and reacted at room temperature for more than 24 hours, and after the fourth organic solvent is removed under reduced pressure, the mixture is purified to obtain a small molecule fluorescent photosensitizer containing a gefitinib analog and an AIE group; the molar ratio of the second intermediate to the first intermediate is 1:1 to 1.2; the fourth organic solvent is acetonitrile.

[0029] The application of the small molecule fluorescent photosensitizer containing the gefitinib backbone structure and AIE group described in this invention in the preparation of fluorescence imaging products for EGFR overexpression in cells.

[0030] The application of a small molecule fluorescent photosensitizer containing the gefitinib backbone structure and AIE group as described in this invention in the preparation of non-therapeutic and diagnostic cell imaging products for non-small cell lung cancer.

[0031] The present invention discloses the application of a small molecule fluorescent photosensitizer containing a gefitinib backbone structure and an AIE group in the preparation of a non-small cell lung cancer tumor-targeting drug; more preferably, the non-small cell lung cancer tumor-targeting drug is a photodynamic therapy non-small cell lung cancer tumor-targeting drug.

[0032] Beneficial effects

[0033] This invention provides a small-molecule fluorescent photosensitizer containing a gefitinib backbone structure and an AIE group. The fluorescent photosensitizer includes an AIE group, a linking group, and a recognition group. On one hand, the AIE group backbone structure consists of a pyridine cation (electron acceptor, A), an ethylene group (π-bridge), a thiophene segment (π-bridge and electron donor, D), and a triphenylamine segment (D), exhibiting a typical D-π-A molecular configuration and thus near-infrared fluorescence emission. On the other hand, by introducing heavy atoms to enhance the spin-orbit coupling of the photosensitizer, the efficiency of singlet oxygen generation is improved. The mechanism of the fluorescent photosensitizer specifically targeting EGFR is shown in the diagram below. Figure 1 As shown: This fluorescent photosensitizer can retain the selectivity of the tyrosine kinase inhibitor gefitinib for the epidermal growth factor receptor (EGFR). Once the fluorescent ligand enters the ATP-binding pocket of EGFR, it will activate the fluorescence signal, thereby enabling fluorescence visualization of EGFR-overexpressing cells.

[0034] This invention provides a small-molecule fluorescent photosensitizer containing the gefitinib backbone structure and AIE group. This photosensitizer can precisely target EGFR-overexpressing non-small cell lung cancer (NSCLC) cells without illuminating normal cells. The photosensitizer exhibits aggregation-induced emission properties, avoiding the ACQ effect during aggregation, significantly improving ROS generation efficiency and promoting its tumor-killing effect. Research results show that the photosensitizer has better therapeutic efficacy than the clinical drug gefitinib. Due to the heavy atom effect, the photosensitizer 2BrTTVP-GETN has a stronger killing ability against NSCLC cells (normal strains A549 and H460); due to its small molecular structure, it more easily enters the cell and binds to the ATP pocket on the inner side of the cell membrane. The photosensitizer TTVP-GETN can better address the drug resistance problem of NSCLC cells (drug-resistant strain HCC829-MATC) through the PDT process. The aforementioned photosensitizer combines small molecule inhibitors with photodynamic therapy, which can solve the problem of resistance to the molecularly targeted drug gefitinib through the PDT process, while simultaneously enabling fluorescence monitoring to report treatment response; it is expected to achieve integrated diagnosis and treatment of non-small cell lung cancer. Attached Figure Description

[0035] Figure 1 This is a schematic diagram illustrating the mechanism of EGFR staining using the fluorescent photosensitizer described in this invention.

[0036] Figure 2 The AIE characteristics of TTVP-GETN and 2BrTTVP-GETN described in Example 2.

[0037] Figure 3The images shown are confocal fluorescence micrographs obtained under 488nm laser irradiation after staining normal HUVEC and L02 cells and non-small cell lung cancer cells H460, A549, and HCC827-MATC with TTVP-GETN and 2BrTTVP-GETN as described in Example 3. The acquisition wavelength is 660-730nm (red channel), and the scale bar is 100μm. The concentration of the fluorescent photosensitizer is 5μM.

[0038] Figure 4 The images show confocal fluorescence images of cancer cells A549 and H460 in Example 4 after pretreatment with gefitinib. The excitation wavelength was 488 nm, and the acquisition wavelength was 660-730 nm.

[0039] Figure 5 The cytotoxicity of AIEgens (or gefitinib) described in Example 5 was evaluated using the MTT assay with TTVP-GETN and 2BrTTVP-GETN. Absorption at 570 nm was recorded in each well using a microplate reader.

[0040] Figure 6 The confocal fluorescence images of A549 cells were observed using DCFH-DA as the fluorescent photosensitizer, as described in Example 6; confocal fluorescence images of HCC827-MATC cells were also observed using DCFH-DA as the fluorescent photosensitizer with TTVP-GETN. DCFH-DA was used to excite fluorescence at 485 nm, and fluorescence images were collected at 520-600 nm. The concentration of the fluorescent photosensitizer and DCFH-DA was both 5 μM.

[0041] Figure 7 The confocal fluorescence images of A549 cells were observed using the 2BrTTVP-GETN with Calcein AM / PI indicator as described in Example 7; confocal fluorescence images of HCC827-MATC cells were observed using the TTVP-GETN with Calcein AM / PI indicator. A white light laser was used as the light source for the confocal microscope. Calcein AM was excited at 485 nm, and green fluorescence images were collected at 510-520 nm. PI was excited at 561 nm, and red fluorescence images were collected at 610-620 nm. Detailed Implementation

[0042] The present invention will be further described in detail below with reference to specific embodiments.

[0043] Example 1: Preparation of a small molecule fluorescent photosensitizer containing the gefitinib backbone structure and AIE group

[0044] (1) Triphenylphosphine (1.68 g, 6.4 mmol) was added to a suspension of 4-chloromethylpyridine hydrochloride (1.00 g, 6.1 mmol) in acetonitrile (20 mL). The mixture was heated (78 °C) under reflux for 18 h, cooled, and filtered into 20 mL of toluene. The collected solid was washed with toluene and dried under vacuum to give triphenyl(4-pyridylmethyl)phosphine chloride hydrochloride (2.2 g, 84.6%), a pale yellow solid.

[0045] (2) 4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-ol (2.08 g, 6.5 mmol) and potassium carbonate (3.60 g, 26 mmol) were mixed in 30 mL of N,N-dimethylformamide (DMF). Then 1,4-diiodobutane (3 mL) was added, and the mixture was stirred at room temperature for 24 h. The mixture was poured into 50 mL of water and extracted with ethyl acetate (30 mL × 3). The organic layers were assembled and dried over anhydrous sodium sulfate. After concentration, the mixture was rapidly chromatographically analyzed on silica gel using ethyl acetate / petroleum ether (1:1) as the eluent to obtain N-(3-chloro-4-fluorophenyl)-6-(4-iodobutoxy)-7-methoxyquinazoline-4-amine (175 mg, yield 5.4%).

[0046] (3) Synthesis of (E)-n,n-diphenyl-4-(5-(2-(pyridin-4-yl)vinyl)thiophen-2-yl)aniline: t-BuOK (246.86 mg, 2.20 mmol) was added to a mixture of triphenyl(4-pyridin-methyl)phosphochloride (426.32 mg, 1.00 mmol) and tetrahydrofuran (THF) (25 mL). The mixture was stirred at 0 °C for 30 min, and 5-(4-(diphenylamine)phenyl)thiophen-2-acetaldehyde (355.46 mg, 1.00 mmol) was added. The mixture was stirred at room temperature for 24 h, and then poured into 50 mL of water. The mixture was extracted with dichloromethane (30 mL × 3), and the organic layers were combined and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the solid residue was purified by flash chromatography using petroleum ether / ethyl acetate (5:1, v / v) as the eluent to give (E)-n,n-diphenyl-4-(5-(2-(pyridin-4-yl)vinyl)thiophen-2-yl)aniline as an orange solid (215 mg, yield 50%).

[0047] (4) Synthesis of (E)-1-(4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinoline-6-yl)butyl-4-(2-(5-(4-(diphenyl)phenyl)thiophen-2-yl)vinyl)pyridine-1-iodide: (E)-N,N-diphenyl-4-(5-(2-(pyridine-4-yl)vinyl)thiophen-2-yl)aniline (86.03 mg, 0.2 mmol) and N-(3-chloro-4-fluorophenyl)-6-(4-iodobutoxy)-7-methoxyquinoline-4-amine (100.2 mg, 0.2 mmol) were mixed in 10 mL CH3CN. The resulting mixture was stirred at room temperature for 24 h, the solvent was removed under reduced pressure, and the solid residue was purified by flash chromatography using dichloromethane / methanol (10:1, v / v) as the eluent to give (E)-1-(4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxybutyl)-4-(2-(5-(4-(diphenylamine)phenyl)thiophene-2-yl)vinyl)pyridine-1-iodide as a red solid (60 mg, yield 37.5%), namely the fluorescent photosensitizer TTVP-GETN.

[0048] The second reactant in step (3), 5-(4-(diphenylamine)phenyl)thiophen-2-acetaldehyde, was replaced with 5-(4-(bis(4-bromophenyl)amino)phenyl)thiophen-2-acetaldehyde, and the remaining steps were the same as above. (E)-4-(2-(5-(4-(bis(4-bromophenyl)amino)phenyl)thiophen-2-yl)vinyl)-1-(4-(4-(3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)butyl)pyridine-1-iodide was obtained as an orange solid (60 mg, yield 37.5%), namely the fluorescent photosensitizer 2BrTTVP-GETN.

[0049] The reaction equation is as follows:

[0050]

[0051] The 1H NMR spectrum of the fluorescent photosensitizer is as follows:

[0052] TTVP-GETN: 1H NMR(400MHz,DMSO-d6),δ(ppm):9.57(s,1H),8.94(d,J=6.6Hz,2H),8.50(s,1H),8.24–8.15(m,3H),8.13(dd,J=6.8,2.6Hz,1H),7.85–7.76(m,2H),7.67–7.59(m,2H),7.52(d,J=3.9Hz,1H),7.51–7.47(m,1H),7.44(d,J=9.1Hz,1H),7.40–7.31(m,4H),7.24(s,1H),7.17–7.12(m,2H),7.12–7.06(m,5H),7.02–6.96(m,2H),4.60(t,J=7.1Hz,2H),4.21(t,J=6.0Hz,2H),3.96(s,3H),2.15(t,J=7.5Hz,2H),1.86(t,J=7.3Hz,2H).HRMS m / z:calcd forC 48 H 40 ClFN5O2S + 804.2570,found 804.1974.

[0053] 2BrTTVP-GETN: 1 H NMR(400MHz,DMSO-d6),δ(ppm):9.58(s,1H),8.96(d,J=6.7Hz,2H),8.50(s,1H),8.25–8.16(m,3H),8.13(dd,J=6.8,2.7Hz,1H),7.87–7.79(m,2H),7.71–7.64(m,2H),7.56(d,J=3.9Hz,1H),7.55–7.49(m,5H),7.45(t,J=9.1Hz,1H),7.24(s,1H),7.14(d,J=15.9Hz,1H),7.09–7.05(m,2H),7.04(d,J=2.1Hz,2H),7.02(d,J=2.2Hz,2H),4.61(t,J=7.0Hz,2H),4.22(t,J=6.0Hz,2H),3.96(s,3H),2.15(t,J=7.5Hz,2H),1.88(q,J=9.2,7.3Hz,2H).HRMS m / z:calcd for C 48 H 38 Br2ClFN5O2S + 960.0780,found 960.0803.

[0054] The results showed that the red solid was (E)-1-(4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)oxybutyl)-4-(2-(5-(4-(diphenylamine)phenyl)thiophen-2-yl)vinyl)pyridine-1-iodide, denoted as TTVP-GETN. The orange solid was (E)-4-(2-(5-(4-(bis(4-bromophenyl)amino)phenyl)thiophen-2-yl)vinyl)-1-(4-(4-(3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-yl)butyl)pyridine-1-iodide, denoted as 2BrTTVP-GETN.

[0055] AIE characteristics of Example 2TTVP-GETN and 2BrTTVP-GETN

[0056] Prepare EtOH / H2O mixtures with water volume fractions of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, and 98%. Dilute the TTVP-GETN and 2BrTTVP-GETN stock solutions with the above EtOH / H2O mixtures to prepare 10 μM working solutions. Place the solutions in a 1 cm wide quartz cuvette and measure the fluorescence emission spectra using an FS5-SS fluorescence spectrometer.

[0057] like Figure 2 As shown, for different H2O components (f W Studies of the AIE characteristics of the EtOH / H2O mixture showed that for TTVP-GETN, as the H2O fraction increased from 0 to 70%, the emission in the fluorescence spectrum decreased with a red shift due to the enhanced TICT effect. When the water fraction increased from 70% to 80%, the emission in the fluorescence spectrum increased significantly, exhibiting AIE characteristics. When the water fraction continued to increase from 80%, the PL intensity decreased slightly, which may be due to changes in the morphology and size of the aggregates.

[0058] Example 3: Culture of cancer cells (H460, A549, HCC827-MATC) and normal cells (HUVEC, L02)

[0059] Human lung adenocarcinoma cell lines A549 (CCL-185) and H460 (CCL-185) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). A549 and H460 were cultured in RPIM 1640 (Gibco) medium containing 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin). The gefitinib-resistant human lung adenocarcinoma cell line HCC827-MTAC was purchased from the Chinese Academy of Sciences Type Culture Collection / Stem Cell Bank. HCC827-MTAC was cultured in DMEM (Gibco) medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin). All cells were stored in a humidified incubator at 37°C and 5% CO2. All cell lines were identified and tested negative for mycoplasma.

[0060] Example 3: TTVP-GETN and 2BrTTVP-GETN staining experiments on cancer cells (H460, A549, HCC827-MATC) and normal cells (HUVEC, L02).

[0061] 1 mL of cell suspension (cell density 10 w / mL) was seeded into confocal microscopy dishes and cultured for 24–48 h. The culture medium was then replaced with 1 mL of complete culture medium (0.1% DMSO) containing 5 μM of the fluorescent photosensitizer TTVP-GETN or 2BrTTVP-GETN, and incubated for 1 h in a humidified incubator at 37 °C and 5% CO2, followed by washing three times with PBS. Differential interference contrast (DIC) and fluorescence images were obtained using a Nikon confocal microscope. A white light laser was used as the light source for the confocal microscope. Fluorescence was excited at 485 nm by the fluorescent photosensitizers TTVP-GETN and 2BrTTVP-GETN, and fluorescence images were collected at 630–710 nm. The fluorescence intensity of the confocal images was analyzed using ImageJ software.

[0062] Fluorescence confocal imaging studies showed that TTVP-GETN and 2BrTTVP-GETN bound abundantly in live cells A549 and HCC827-MATC, bound significantly less to H460, and bound negligibly to EGFR-negative cells HUVEC and L02.

[0063] Example 4: Competitive Experiments Mediated by Confocal Deposition of TTVP-GETN and 2BrTTVP-GETN

[0064] 1 mL of cell suspension (cell density 10 w / mL) was seeded into confocal microplates and cultured for 24–48 h. The gefitinib pretreatment group had its culture medium replaced with 1 mL of complete medium (0.1% DMSO) containing 50 μM gefitinib and incubated for 1 h in a humidified incubator at 37°C and 5% CO2. Afterward, the medium was replaced with 1 mL of complete medium (0.1% DMSO) containing 5 μM TTVP-GETN or 2BrTTVP-GETN and co-incubated for 1 h. The untreated group was directly incubated with 1 mL of complete medium (0.1% DMSO) containing 5 μM TTVP-GETN or 2BrTTVP-GETN for 1 h. Finally, the cells were washed three times with PBS. Differential interference contrast (DIC) and fluorescence images were obtained using a Nikon confocal microscope. A white light laser was used as the light source for the confocal microscope. Fluorescent photosensitizers TTVP-GETN and 2BrTTVP-GETN are excited to fluorescence at 485 nm, and emission spectra are collected in the range of 630-710 nm.

[0065] Imaging results showed that the uptake of TTVP-GETN and 2BrTTVP-GETN in the gefitinib pretreatment group was significantly reduced compared to the untreated group. This result demonstrates that gefitinib pretreatment significantly inhibited receptor binding, internalization, and cytoplasmic diffusion of the fluorescent photosensitizer in EGFR-expressing cells, indicating that the fluorescent photosensitizer shares the same binding target as gefitinib, namely EGFR.

[0066] Example 5: MTT assay of TTVP-GETN and 2BrTTVP-GETN

[0067] The cytotoxicity of the photosensitizer (or gefitinib) was evaluated using the MTT assay. A549 cells were seeded at 6000 cells / well (HCC827-MATC cells at 10000 cells / well) in 96-well plates. After 48 h of culture, the medium in each well was replaced with 100 μL of fresh medium containing different concentrations of AIEgens (or gefitinib). The DMSO concentration was less than 0.1%. After 1.5 h of incubation, the plates containing A549 (or HCC827-MATC) cells were exposed to white light (50 mW / cm²). 2 After 30 minutes of incubation, another group of plates containing cells was placed in the dark as a control. 24 hours later, 15 μL of MTT solution (5 mg / mL in PBS) was added to each well. After 3.5 hours of incubation, 100 μL of DMSO was added to each well, and the absorbance at 570 nm was recorded using a microplate reader. Each experiment was performed in six parallel wells, and each experiment was repeated at least three times.

[0068] Dose-dependent cytotoxicity studies have shown that fluorescent photosensitizers exhibit low cytotoxicity under dark conditions, indicating good biocompatibility, which is one of the fundamental characteristics of photosensitizers used in PDT applications. Figure 5 As shown, under low-concentration dark conditions, the cell viability of cells incubated with gefitinib was slightly higher than that of cells incubated with photosensitizers. This is because the molecular weight of the fluorescent photosensitizer is larger than that of gefitinib, resulting in a slight decrease in the rate and ability to enter the cells. Under white light irradiation, the viability of A549 cells gradually decreased, and 10 μM TTVP-GETN significantly reduced the viability of A549 cells to 40%, which is twice the killing effect of the clinical drug gefitinib. Furthermore, to further investigate whether TTVP-GETN also has a PDT effect on drug-resistant non-small cell lung cancer strains, a dose-dependent cytotoxicity experiment was conducted using the gefitinib-resistant strain HCC827-MACT as a model. Under white light irradiation, 5 μM TTVP-GETN still showed a good PDT effect on HCC827-MACT. This indicates that the killing ability of this fluorescent photosensitizer holds promise for addressing the problem of tyrosine kinase inhibitor resistance in the treatment of non-small cell lung cancer.

[0069] Example 6: In vivo ROS testing of TTVP-GETN and 2BrTTVP-GETN

[0070] Cells A549 and HCC827-MATC were observed using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) as a photosensitizer. A549 or HCC827-MATC cells were seeded in confocal dishes at a density of 14 w / mL and divided into five groups: Control group, Light group, photosensitizer group, photosensitizer + Light group, and NAC + photosensitizer + Light group. N-acetyl-L-cysteine ​​(NAC) is a ROS inhibitor. After 48 h of incubation, the validation group was pretreated with NAC (15 mM) for 1.5 h, washed three times with PBS, and then cultured with photosensitizer (5 μM) for 1.5 h. DCFH-DA staining was performed for 30 min, followed by irradiation with white light for 30 s. Differential interference contrast (DIC) and green fluorescence images were obtained using a Nikon confocal microscope. A white light laser was used as the light source for the confocal microscope. DCFH-DA is excited at 485 nm and fluorescence images are collected in the 520-600 nm range.

[0071] like Figure 6As shown, after irradiation with white light, DCFH-DA emitted bright green fluorescence, while the control group, the photosensitizer group, and the white light-only group showed almost no obvious fluorescence signal. This is because the photosensitizer can efficiently generate ROS in A549 and HCC827-MATC cells. Notably, the green fluorescence signal was significantly weakened in the NAC-pretreated photosensitizer group irradiated with white light. This further demonstrates that the photosensitizer can accumulate in cancer cells, thereby achieving the purpose of phototherapy.

[0072] Example 7: Live / dead staining experiment of TTVP-GETN and 2BrTTVP-GETN

[0073] The state of A549 and HCC827-MATC cells was observed using Calcein AM / PI indicators. The experiment consisted of five groups: Control group, Light group, fluorescent photosensitizer group, and fluorescent photosensitizer + Light group. A549 or HCC827-MATC cells were seeded in confocal microplates at a density of 14 w / mL and incubated for 48 h. The culture medium was then replaced with 5 μM fluorescent photosensitizer and incubated for 1.5 h, followed by 30 min of light irradiation and 2 h of culture in a humidified incubator at 37°C and 5% CO2. Cells were gently washed 2-3 times with PBS to remove any active esterases from the culture medium. 100 μL of staining working solution was added to each plate, and the plates were incubated at 37°C for 30 min. Differential interference contrast (DIC) and fluorescence images were obtained using a Nikon confocal microscope. A white light laser was used as the light source for the confocal microscope. Calcein AM was used to excite fluorescence at 485 nm, and green fluorescence images were collected at 510-520 nm. PI was excited to produce fluorescence at 561 nm, and red fluorescence images were collected in the 610-620 nm range.

[0074] like Figure 7 As shown, after Calcein AM / PI double staining, the control group cells were in good condition, while the light-illuminated group and the non-illuminated photosensitizer group had a small number of dead cells. The photosensitizer group with added light had a strong killing effect on cells. This is because TTVP-GETN and 2BrTTVP-GETN are photosensitizers themselves, which produce a large amount of ROS under white light irradiation, thus playing a phototherapy role.

[0075] This confirms that the photosensitizers TTVP-GETN and 2BrTTVP-GETN retain the selectivity of the tyrosine kinase inhibitor gefitinib for epidermal growth factor receptor (EGFR), exhibit aggregation-induced emission properties, and significantly improve the efficiency of ROS generation, thus promoting its tumor-killing effect. Among them, the photosensitizer TTVP-GETN, due to its small molecular structure, more easily enters the cell and binds to the ATP pocket on the inner side of the cell membrane, resulting in significantly enhanced imaging effects. It has also been shown to enable integrated diagnosis and treatment of the drug-resistant non-small cell lung cancer strain HCC827-MATC.

[0076] In summary, the invention includes, but is not limited to, the above embodiments. Any equivalent substitutions or partial improvements made under the spirit and principles of this invention shall be considered to be within the protection scope of this invention.

Claims

1. A small molecule fluorescent photosensitizer containing a gefitinib analog and an AIE group, characterized in that: The structural formula of the fluorescent photosensitizer is: or .

2. A method for preparing a small molecule fluorescent photosensitizer containing the gefitinib backbone structure and AIE group as described in claim 1, characterized in that: The method steps include: (1) Triphenylphosphine reacts with 4-chloromethylpyridine hydrochloride to produce triphenyl(4-pyridine-methyl)phosphine chloride hydrochloride; (2) After 4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-ol and potassium carbonate are thoroughly mixed, the first reactant is added, and the first intermediate is obtained by Williamson ether synthesis reaction; (3) After potassium tert-butoxide and triphenyl(4-pyridine-methyl)phosphorus chloride hydrochloride are thoroughly mixed, the second reactant is added, and the reaction yields the second intermediate; (4) The second intermediate reacts with the first intermediate to obtain a small molecule fluorescent photosensitizer containing a gefitinib analog and an AIE group; The structural formula of the first reactant is: n is 4; The structural formula of the first intermediate is: ; The structural formula of the second reactant is: or ; The structural formula of the second intermediate is: or .

3. The method for preparing a small molecule fluorescent photosensitizer containing a gefitinib backbone structure and an AIE group as described in claim 2, characterized in that: In step (1), triphenylphosphine and 4-chloromethylpyridine hydrochloride are heated and refluxed in a first organic solvent at 70-90°C for 12-24 hours to obtain triphenyl(4-pyridine-methyl)phosphine chloride hydrochloride; the molar ratio of triphenylphosphine to 4-chloromethylpyridine hydrochloride is 1:1-1.2; the first organic solvent is acetonitrile.

4. The method for preparing a small molecule fluorescent photosensitizer containing a gefitinib backbone structure and an AIE group as described in claim 2, characterized in that: In step (2), 4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-ol and a weak Lewis base are thoroughly mixed in a second organic solvent, and then the first reactant is added. The mixture is stirred for more than 24 hours, the organic phase is collected, concentrated, and purified to obtain the first intermediate. The molar ratio of 4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazoline-6-ol, potassium carbonate, and the first reactant is 1:3~5:15~30. The second organic solvent is N,N-dimethylformamide.

5. The method for preparing a small molecule fluorescent photosensitizer containing a gefitinib backbone structure and an AIE group as described in claim 2, characterized in that: In step (3), t-BuOK and triphenyl(4-pyridine-methyl)phosphorus chloride hydrochloride are thoroughly mixed at 0~5℃ in the third organic solvent. After adding the second reactant, the mixture is reacted at room temperature for 7~24h to obtain the second intermediate. The molar ratio of potassium tert-butoxide, triphenyl(4-pyridine-methyl)phosphorus chloride hydrochloride and the second reactant is 2~3:1~1.2:

1. The third organic solvent is tetrahydrofuran.

6. The method for preparing a small molecule fluorescent photosensitizer containing a gefitinib backbone structure and an AIE group as described in claim 2, characterized in that: In step (4), the second intermediate and the first intermediate are mixed in the fourth organic solvent and stirred at room temperature for more than 24 hours. After removing the fourth organic solvent under reduced pressure, the mixture is purified to obtain a small molecule fluorescent photosensitizer containing a gefitinib analog and an AIE group. The molar ratio of the second intermediate to the first intermediate is 1:1 to 1.

2. The fourth organic solvent is acetonitrile.

7. The application of a small molecule fluorescent photosensitizer containing the gefitinib backbone structure and AIE group as described in claim 1 in the preparation of a fluorescent imaging product for EGFR overexpression in cells.

8. The use of a small molecule fluorescent photosensitizer containing the gefitinib backbone structure and AIE group as described in claim 1 in the preparation of non-therapeutic and diagnostic cell imaging products for non-small cell lung cancer.

9. The use of a small molecule fluorescent photosensitizer containing the gefitinib backbone structure and AIE group as described in claim 1 in the preparation of a non-small cell lung cancer tumor-targeting drug.

10. The application as described in claim 9, characterized in that: The targeted drug for non-small cell lung cancer is a photodynamic therapy drug for non-small cell lung cancer.

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