Small-molecular fluorescent photosensitizer containing gefitinib skeleton structure and AIE group, preparation and application of small-molecular fluorescent photosensitizer

Abstract

The invention relates to a small molecular fluorescence photosensitizer containing a gefitinib skeleton structure and an AIE group, and preparation and application thereof, and belongs to the technical field of fluorescence photosensitizers. The fluorescent photosensitizer comprises an AIE group, a linking group and a recognition group, on one hand, a skeleton structure of the AIE group is composed of a pyridine cation (an electron acceptor, A), ethylene (a pi bridge), a thiophene fragment (the pi bridge and an electron donor, D) and a triphenylamine fragment (D), and the AIE group is of a typical D-pi-A molecular configuration, so that the fluorescent photosensitizer has near-infrared fluorescence emission; on the other hand, by introducing heavy atoms, the spin-orbit coupling effect of the photosensitizer is enhanced, and the generation efficiency of singlet oxygen is improved. The fluorescent photosensitizer can accurately target non-small cell lung cancer cells with over-expressed EGFR without lightening normal cells. The fluorescent photosensitizer has an aggregation-induced emission characteristic, can avoid an ACQ effect during aggregation, greatly improves the ROS generation efficiency, and promotes the killing effect of the fluorescent photosensitizer on tumors.

Description

Technical Field

[0001] The invention relates to a small molecule fluorescent photosensitizer containing a gefitinib skeleton structure and an AIE group, preparation and application thereof, and belongs to the technical field of fluorescent photosensitizers. Background Art

[0002] Lung cancer is one of the leading causes of cancer-related deaths worldwide, with an estimated 2.2 million new cases (11.4% of all new cancer cases) and 1.79 million deaths (18.4% of all cancer deaths) each year. Histologically, lung cancer is broadly divided into non-small cell lung cancer (about 85% of confirmed cases) and small cell lung cancer (about 15% of confirmed cases). Early non-small cell lung cancer (NSCLC) is often asymptomatic, resulting in most NSCLC being considered locally advanced or metastatic at diagnosis, missing the ideal time for surgical treatment. Patients with epidermal growth factor receptor (EGFR) mutations account for 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 about 85% of all EGFR mutations and usually 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 attracted widespread attention from all walks of life as an effective target for the treatment of non-small cell lung cancer.

[0003] EGFR is the earliest discovered prototype member of the receptor tyrosine kinase (RTKs) family. It is an important component of cell signaling pathways and plays an important role in regulating cancer cell proliferation, invasion, differentiation and migration. EGFR binding to ligands (EGF and TGF-α) leads to conformational changes in EGFR and forms homodimers and heterodimers with other members of the EGFR family. Subsequently, with the help of adaptor proteins (such as SHC and GRB-2), the cytoplasmic tyrosine kinase (TK) domain undergoes autophosphorylation, thereby triggering downstream signals. EGFR overexpression plays an important role in multiple signaling pathways inside and outside the cell, especially in the cell proliferation, differentiation and death processes of cancers such as non-small cell lung cancer, head and neck cancer, kidney cancer, ovarian cancer, and colon cancer. The complexity of EGFR structure and function makes the study of EGFR protein visualization challenging, especially fluorescence visualization.

[0004] With the rise of targeted disease therapy, 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 be combined with fluorescent dyes as specific fluorescent probes, small molecule inhibitors (such as gefitinib, erlotinib, etc.) can be used as ideal recognition units for a new class of molecular fluorescent probes due to their small size and high specificity. Wang Fuyi et al. labeled gefitinib with fluorescein to obtain a class of quinazoline fluorescent probes capable of imaging EGFR. The probe has a strong inhibitory effect on EGFR and is evenly distributed on the cell membrane after interacting with a variety of cells, which can be used for imaging cancer cells. Shengnan Liu et al. developed new small molecule fluorescent probes Cy3-AFTN and Cy5-AFTN as effective dual-targeted inhibitors for 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) using an EGFR-TKI-based fluorescent probe (HX103) to analyze active EGFR in tumors. At present, small molecule fluorescent probes based on EGFR-TKI can only be used for accurate diagnosis of lung cancer and cannot break through the bottleneck of integrated diagnosis and treatment.

[0005] In addition, patients with EGFR activating mutations benefit from EGFR-TKIs (such as gefitinib and erlotinib) for less than 1 year before developing resistance. For example, gefitinib blocks tyrosine kinases, blocks the phosphorylation of mutant EGFR on the cell membrane, and terminates downstream cell signaling of EGFR. However, the activation of the EGFR pathway does not follow a linear pattern and is a rather complex process. Therefore, drug resistance in patients with non-small cell lung cancer remains a challenging problem that needs to be solved. In order to solve this problem, many people have tried to combine two or more anticancer drugs to enhance the inhibition of tumor growth and overcome the problem of secondary drug resistance. This superposition of multiple tumor suppressor mechanisms by targeting different receptors with different drugs or different action sites of the same receptor can intuitively inhibit cancer cell growth more effectively and even avoid the generation of drug-resistant mutations as much as possible. However, in the clinical setting, some limiting factors often limit the implementation of drug combination therapy, including multiple drugs, complex pharmacokinetic characteristics, unclear dosage regimens, and drug interactions. It is worth noting that fluorescence imaging has been widely reported to enable faster, non-invasive, real-time and precise response monitoring during cancer treatment interventions, and therefore, this is exactly the technology that is urgently needed to address the challenge of drug resistance. On the one hand, although photodynamic therapy (PDT) has great potential in addressing drug resistance, excessive light exposure or photosensitizer (PS) loading will inevitably produce harmful side effects, so there is also a high demand for real-time monitoring of photodynamic response or cellular response. On the other hand, compared with traditional aggregation-quenched luminescence (ACQ) probes, aggregation-induced emission (AIE) probes induce high-intensity fluorescence by restricting intramolecular motion, which has the advantages of high signal-to-noise ratio, good biocompatibility, and low fluorescence background. In summary, if a small molecule fluorescent photosensitizer containing a gefitinib skeleton structure and an AIE group can be developed, it can not only solve the thorny problem of drug resistance through PDT, but also report the treatment response through fluorescence monitoring. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a small molecule fluorescent photosensitizer containing a gefitinib skeleton structure and an AIE group, preparation and application thereof. The fluorescent photosensitizer can retain the selectivity of the tyrosine kinase small molecule inhibitor gefitinib to the epidermal growth factor receptor (EGFR). Once the fluorescent ligand enters the ATP binding pocket of the EGFR, the fluorescent signal will be turned on, thereby realizing the fluorescent visualization of EGFR overexpressing cells. In addition, the fluorescent photosensitizer has aggregation-induced luminescence characteristics, which can avoid the aggregation quenching luminescence (ACQ) effect when aggregated, greatly improve the generation efficiency of reactive oxygen species (ROS), and promote its killing effect on tumors. In addition, the fluorescent photosensitizer TTVP-GETN can also solve the thorny drug resistance problem through PDT, and can also report the 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] Wherein, n is an integer from 1 to 5;

[0011] R 1 is H or Br; R 2 is H or Br;

[0012] X- is a halogen ion, a perchlorate ion or a hexafluorophosphate ion.

[0013] Preferably, the structural formula of the fluorescent photosensitizer is:

[0014]

[0015] A method for preparing a small molecule fluorescent photosensitizer containing a gefitinib skeleton structure and an AIE group according to the present invention, the method steps comprising:

[0016] (1) triphenylphosphine reacts with 4-chloromethylpyridine hydrochloride to generate triphenyl (4-pyridyl-methyl) phosphine chloride hydrochloride;

[0017] (2) After 4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-ol and potassium carbonate are fully mixed, the first reactant is added to obtain a first intermediate through Williamson ether synthesis reaction;

[0018] (3) After potassium tert-butoxide (t-BuOK) and triphenyl (4-pyridyl-methyl) phosphonium chloride hydrochloride are fully mixed, a second reactant is added to react to obtain a 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] Wherein, 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; R 2 is H or Br.

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

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

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

[0028] Preferably, in step (4), the second intermediate and the first intermediate are mixed in a fourth organic solvent, stirred and reacted at room temperature for more than 24 hours, and the fourth organic solvent is removed under reduced pressure and then 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; and the fourth organic solvent is acetonitrile.

[0029] A use of the small molecule fluorescent photosensitizer containing a gefitinib skeleton structure and an AIE group in the preparation of a fluorescent imaging product for overexpressing EGFR in cells.

[0030] A use of the small molecule fluorescent photosensitizer containing a gefitinib skeleton structure and an AIE group in the preparation of a non-small cell lung cancer cell imaging product for non-therapeutic and diagnostic purposes.

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

[0032] Beneficial Effects

[0033] The present invention provides a small molecule fluorescent photosensitizer containing a gefitinib skeleton structure and an AIE group. The fluorescent photosensitizer includes an AIE group, a linking group and a recognition group. On the one hand, the AIE group skeleton structure is composed of a pyridinium cation (electron acceptor, A), an ethylene (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, and thus has near-infrared fluorescence emission. On the other hand, heavy atoms are introduced to enhance the spin-orbit coupling effect of the photosensitizer, thereby improving the efficiency of singlet oxygen generation. The mechanism diagram of the fluorescent photosensitizer specifically targeting EGFR is shown in the figure. Figure 1 As shown: The 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, the fluorescent signal will be turned on, thereby achieving fluorescent visualization of EGFR overexpressing cells.

[0034] The present invention provides an application of a small molecule fluorescent photosensitizer containing a gefitinib skeleton structure and an AIE group, wherein the fluorescent photosensitizer can accurately target non-small cell lung cancer cells with overexpression of EGFR without lighting up normal cells. The fluorescent photosensitizer has aggregation-induced luminescence characteristics, can avoid the ACQ effect when aggregated, greatly improves the generation efficiency of ROS, and promotes its tumor-killing effect. The results of the study show that the fluorescent photosensitizer has a better therapeutic effect than the clinical drug gefitinib. Due to the heavy atom effect, the fluorescent photosensitizer 2BrTTVP-GETN has a stronger killing ability against non-small cell lung cancer cells (normal strains A549 and H460); due to the small molecular structure, it is easier to enter the cell and bind to the ATP pocket on the inner side of the membrane. The fluorescent photosensitizer TTVP-GETN can better solve the problem of drug resistance of non-small cell lung cancer cells (resistant strain HCC829-MATC) through the PDT process. The fluorescent photosensitizer combines a small molecule inhibitor with photodynamic therapy, which can solve the problem of resistance to the molecular targeted drug gefitinib through the PDT process, while realizing fluorescent monitoring and reporting of treatment response; it is expected to achieve integrated diagnosis and treatment of non-small cell lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a diagram showing the mechanism of EGFR staining using the fluorescent photosensitizer of the present invention.

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

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

[0038] Figure 4 This is a confocal fluorescence image of cancer cells A549 and H460 after pretreatment with gefitinib in Example 4, with an excitation wavelength of 488 nm and a collection band of 660-730 nm.

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

[0040] Figure 6 The confocal fluorescence images of A549 cells were observed by 2BrTTVP-GETN using DCFH-DA as a fluorescent photosensitizer in Example 6; the confocal fluorescence images of HCC827-MATC cells were observed by TTVP-GETN using DCFH-DA as a fluorescent photosensitizer. DCFH-DA excited fluorescence at 485nm, and the fluorescent images were collected at 520-600nm. The concentration of the fluorescent photosensitizer was 5μM, and the concentration of DCFH-DA was 5μM.

[0041] Figure 7 The confocal fluorescence images of A549 cells observed by 2BrTTVP-GETN using Calcein AM / PI indicator as described in Example 7; the confocal fluorescence images of HCC827-MATC cells observed by TTVP-GETN using Calcein AM / PI indicator. The confocal microscope uses a white light laser as a light source. Calcein AM excites fluorescence at 485nm, and green fluorescence images are collected at 510-520nm. PI excites fluorescence at 561nm, and red fluorescence images are collected at 610-620nm. DETAILED DESCRIPTION

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

[0043] Example 1 Preparation of a small molecule fluorescent photosensitizer containing a gefitinib skeleton structure and an AIE group

[0044] (1) Add triphenylphosphine (1.68 g, 6.4 mmol) to a suspension of 4-chloromethylpyridine hydrochloride (1.00 g, 6.1 mmol) in acetonitrile (20 mL). Heat the mixture (78°C) and reflux for 18 h, cool it, add it to 20 ml of toluene and filter it. The collected solid is washed with toluene and dried in vacuo to obtain triphenyl (4-pyridylmethyl) phosphonium chloride hydrochloride (2.2 g, 84.6%) as a light yellow solid.

[0045] (2) 4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-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 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 combined and dried over anhydrous sodium sulfate. After concentration, the product was flash chromatographed on silica gel with ethyl acetate / petroleum ether (1:1) as the eluent to obtain N-(3-chloro-4-fluorophenyl)-6-(4-iodobutoxy)-7-methoxyquinazolin-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)phosphonium chloride hydrochloride (426.32 mg, 1.00 mmol) in tetrahydrofuran (THF) (25 mL). The mixture was stirred at 0°C for 30 min, and 5-(4-(diphenylamino)phenyl)thiophene-2-acetaldehyde (355.46 mg, 1.00 mmol) was added. The mixture was stirred at room temperature for 24 h, poured into 50 mL of water, 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 with petroleum ether / ethyl acetate (5:1, v / v) as 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-methoxyquinolin-6-yl)butyl-4-(2-(5-(4-(diphenyl)phenyl)thiophen-2-yl)vinyl)pyridine-1-iodonium salt: (E)-N,N-diphenyl-4-(5-(2-(pyridin-4-yl)vinyl)thiophen-2-yl)aniline (86.03 mg, 0.2 mmol) and N-(3-chloro-4-fluorophenyl)-6-(4-iodobutyloxy)-7-methoxyquinolin-4-amine (100.2 mg, 0.2 mmol) were mixed in 10 mL of CH 3 CN. 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 obtain (E)-1-(4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxybutyl)-4-(2-(5-(4-(diphenylamine)phenyl)thiophen-2-yl)vinyl)pyridine-1-iodine salt as a red solid (60 mg, yield 37.5%), i.e., fluorescent photosensitizer TTVP-GETN.

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

[0049] The reaction equation is as follows:

[0050]

[0051] The H-NMR spectrum results of the fluorescent photosensitizer are 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 ClFN 5 O 2 S + 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 Br 2 ClFN 5 O 2 S+ 960.0780, found 960.0803.

[0054] The results showed that the red solid was (E)-1-(4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxybutyl)-4-(2-(5-(4-(diphenylamine)phenyl)thiophen-2-yl)vinyl)pyridine-1-iodine salt, recorded 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-methoxyquinazolin-6-yl)butyl)pyridine-1-iodine salt, recorded as 2BrTTVP-GETN.

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

[0056] Prepare EtOH / H2O with a volume fraction of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%. 2 O mixture. 2 The TTVP-GETN and 2BrTTVP-GETN stock solutions were diluted to 10 μM working solutions by mixing with 1% TTVP-GETN and 2BrTTVP-GETN. The working solutions were placed in a 1 cm wide quartz cuvette and the fluorescence emission spectrum was measured using a FS5-SS fluorescence spectrometer.

[0057] like Figure 2 As shown, for different H 2 O component (f W ) of EtOH / H 2 The study of the AIE characteristics in the O mixture shows that for TTVP-GETN, as the H 2 As the O fraction increases from 0 to 70%, the emission in the fluorescence spectrum decreases with a red shift due to the enhancement of the TICT effect. When the water fraction increases from 70% to 80%, the emission of the fluorescence spectrum increases significantly, showing an AIE feature. When the water fraction continues to increase from 80%, the PL intensity decreases slightly, which may be due to the change in the morphology and size of the aggregates.

[0058] Example 3 Cancer cell (H460, A549, HCC827-MATC) and normal cell (HUVEC, L02) culture

[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 (Gibco) and 1% antibiotics (penicillin and streptomycin) by volume. The gefitinib-resistant human lung adenocarcinoma cell line HCC827-MTAC was purchased from the Cell Bank / Stem Cell Bank of the Committee for the Preservation of Typical Cultures of the Chinese Academy of Sciences. HCC827-MTAC was cultured in DMEM (Gibco) medium containing 10% fetal bovine serum (Gibco) and 1% antibiotics (penicillin and streptomycin) by volume. All cells were stored at 37°C and 5% CO 2 All cell lines were authenticated and tested negative for mycoplasma.

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

[0061] 1 mL of cell suspension (cell density of 10w / mL) was inoculated into a confocal dish and cultured for 24-48 h. The culture medium was replaced with 1 mL of complete culture medium (0.1% DMSO) containing 5 μM fluorescent photosensitizer TTVP-GETN or 2BrTTVP-GETN and incubated at 37°C and 5% CO 2 The cells were incubated in a humidified incubator for 1 h and then 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. Fluorescence photosensitizers TTVP-GETN and 2BrTTVP-GETN were excited at 485 nm, 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 had abundant binding in living cells A549 and HCC827-MATC, significantly less binding to H460, and negligible binding to EGFR-negative cells HUVEC and L02.

[0063] Example 4 Confocal-mediated competition experiment of TTVP-GETN and 2BrTTVP-GETN

[0064] 1 mL of cell suspension (cell density of 10w / mL) was inoculated into a confocal dish and cultured for 24-48h. In the gefitinib pretreatment group, the culture medium was replaced with 1 mL of complete culture medium (0.1% DMSO) containing 50 μM gefitinib and incubated at 37°C and 5% CO 2 The cells were incubated in a humidified incubator for 1 hour, after which the culture medium was replaced with 1 mL of complete culture medium (0.1% DMSO) containing 5 μM fluorescent photosensitizer TTVP-GETN or 2BrTTVP-GETN and incubated for 1 hour. The untreated group was directly incubated for 1 hour with 1 mL of complete culture medium (0.1% DMSO) containing 5 μM fluorescent photosensitizer TTVP-GETN or 2BrTTVP-GETN. 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. The fluorescent photosensitizers TTVP-GETN and 2BrTTVP-GETN excited fluorescence at 485 nm, and the emission spectra were collected at 630-710 nm.

[0065] The imaging results showed that the uptake of TTVP-GETN and 2BrTTVP-GETN in the gefitinib pretreatment group was significantly lower than that in the untreated group. This result proves that the gefitinib pretreatment group significantly hindered the receptor binding, internalization and cytoplasmic diffusion of the fluorescent photosensitizer in EGFR-expressing cells, indicating that the fluorescent photosensitizer has the same binding target as gefitinib, namely EGFR.

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

[0067] The MTT method was used to evaluate the cytotoxicity of the photosensitizer (or gefitinib). A549 cells were seeded at 6000 cells / well (HCC827-MATC cells at 10000 cells / well) in a 96-well plate. After 48 hours of culture, the culture medium in each well was replaced with 100 μL of fresh culture medium containing different concentrations of AIEgens (or gefitinib). The volume fraction of DMSO was less than 0.1%. After incubation for 1.5 hours, the plate containing A549 cells (or HCC827-MATC cells) was exposed to white light (50 mW / cm 2 ) for 30 minutes, and another set of plates containing cells were placed in the dark as a control. After 24 hours, 15 μL of MTT solution (5 mg / mL in PBS) was added to each well. After incubation for 3.5 hours, 100 μL of DMSO was added to each well, and the absorption of each well at 570 nm was recorded by a microplate reader. Each test was performed in 6 parallel wells, and each test was repeated at least three times.

[0068] Dose-dependent cytotoxicity studies have shown that fluorescent photosensitizers exhibit lower cytotoxicity under dark conditions, indicating that they have good biocompatibility, which is also one of the basic characteristics of photosensitizers for PDT applications. Figure 5 As shown in the figure, under low-concentration dark conditions, the 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 cell. Under white light irradiation, the viability of A549 cells gradually decreased, and 10μM TTVP-GETN caused the viability of A549 cells to be significantly reduced to 40%, which is twice the killing effect of the clinical drug gefitinib. In addition, in order to further study whether TTVP-GETN also has a PDT effect on non-small cell lung cancer resistant strains, a dose-dependent cytotoxicity experiment was carried out using the gefitinib-resistant strain HCC827-MATC as a model. Under white light irradiation, 5μM TTVP-GETN still has a good PDT effect on HCC827-MACT. This shows that the killing ability of the fluorescent photosensitizer is expected to solve 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] A549 cells and HCC827-MATC cells were observed using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) as a fluorescent photosensitizer. A549 cells or HCC827-MATC cells were inoculated in a confocal dish with a cell density of 14w / mL and divided into 5 groups: Control group, Light group, fluorescent photosensitizer, fluorescent photosensitizer + Light group, NAC + fluorescent photosensitizer + Light group. Among them, N-acetyl-L-cysteine ​​(NAC) is a ROS inhibitor. After incubation for 48 hours, the validation group was pretreated with NAC (15mM) for 1.5 hours, washed three times with PBS, and then the fluorescent photosensitizer (5μM) was added to culture the cells for 1.5 hours. DCFH-DA staining was performed for 30 minutes, and then the cells were irradiated with white light for 30 seconds, and differential interference contrast (DIC) and green fluorescence images were obtained using a Nikon confocal microscope. The confocal microscope uses a white light laser as the light source. DCFH-DA was excited for fluorescence at 485 nm, and fluorescence images were collected at 520–600 nm.

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

[0072] Example 7 Dead and alive staining experiment of TTVP-GETN and 2BrTTVP-GETN

[0073] Calcein AM / PI indicator was used to observe the status of A549 and HCC827-MATC cells. The experiment was divided into 5 groups: Control group, Light group, fluorescent photosensitizer, and fluorescent photosensitizer + Light group. A549 or HCC827-MATC cells were inoculated in a confocal dish at a cell density of 14w / mL and incubated for 48h. The culture medium was replaced with 5μM fluorescent photosensitizer for 1.5h. After 30min of illumination, the cells were incubated in 5% CO 2 , cultured in a humidified incubator at 37°C for 2h. Gently wash the cells 2-3 times with PBS to ensure the removal of active esterases contained in the culture medium, add 100μL of staining working solution to each dish, and incubate at 37°C for 30min. Differential interference contrast (DIC) and fluorescence images were obtained using a Nikon confocal microscope. The confocal microscope used a white light laser as the light source. Calcein AM excited fluorescence at 485nm, and green fluorescence images were collected at 510-520nm. PI excited fluorescence at 561nm, and red fluorescence images were collected at 610-620nm.

[0074] like Figure 7 As shown, after Calcein AM / PI double staining, the cells in the control group were in good condition, there were a small number of dead cells in the illumination group and the fluorescent photosensitizer group without illumination, and the fluorescent photosensitizer group with illumination had a strong killing effect on the cells. This is because TTVP-GETN and 2BrTTVP-GETN themselves are photosensitizers, which will produce a large amount of ROS under white light irradiation, playing a phototherapy role.

[0075] This confirms that the fluorescent photosensitizers TTVP-GETN and 2BrTTVP-GETN can retain the selectivity of the tyrosine kinase inhibitor gefitinib for epidermal growth factor receptor (EGFR), have aggregation-induced luminescence characteristics, can greatly improve the efficiency of ROS generation, and promote its tumor-killing effect. Among them, the fluorescent photosensitizer TTVP-GETN, due to its small molecular structure, is easier to enter the cell and bind to the ATP pocket on the inner side of the membrane, and the imaging effect is significantly enhanced. It has also been proven that it can achieve 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 substitution or partial improvement made under the spirit and principle of the invention shall be deemed to be within the protection scope of the 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 as follows: Wherein, n is an integer from 1 to 5; R1 is H or Br; R2 is H or Br; X- is a halogen ion, a perchlorate ion or a hexafluorophosphate ion.

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

3. A method for preparing a small molecule fluorescent photosensitizer containing a gefitinib skeleton structure and an AIE group as claimed in claim 1 or 2, characterized in that: The method steps include: (1) triphenylphosphine reacts with 4-chloromethylpyridine hydrochloride to generate triphenyl (4-pyridyl-methyl) phosphine chloride hydrochloride; (2) After 4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-ol and potassium carbonate are fully mixed, the first reactant is added to obtain a first intermediate through Williamson ether synthesis reaction; (3) After potassium tert-butoxide and triphenyl(4-pyridyl-methyl)phosphonium chloride hydrochloride are fully mixed, a second reactant is added to react to obtain a 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; Wherein, the structural formula of the first reactant is: n is an integer from 1 to 5; The structural formula of the first intermediate is: The structural formula of the second reactant is: The structural formula of the second intermediate is: R1 is H or Br; R2 is H or Br.

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

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

6. The method for preparing a small molecule fluorescent photosensitizer containing a gefitinib skeleton structure and an AIE group according to claim 3, characterized in that: In step (3), t-BuOK and triphenyl (4-pyridine-methyl) phosphonium chloride hydrochloride are fully mixed at 0 to 5° C. in a third organic solvent, and after adding the second reactant, the mixture is reacted at room temperature for 7 to 24 hours to obtain a second intermediate, wherein the molar ratio of potassium tert-butoxide, triphenyl (4-pyridine-methyl) phosphonium chloride hydrochloride and the second reactant is 2 to 3:1 to 1.2:1; and the third organic solvent is tetrahydrofuran.

7. The method for preparing a small molecule fluorescent photosensitizer containing a gefitinib skeleton structure and an AIE group according to claim 3, characterized in that: In step (4), the second intermediate and the first intermediate are mixed in a fourth organic solvent, stirred and reacted at room temperature for more than 24 hours, and the fourth organic solvent is removed under reduced pressure, followed by purification 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; and the fourth organic solvent is acetonitrile.

8. Use of the small molecule fluorescent photosensitizer containing the gefitinib skeleton structure and the AIE group as claimed in claim 1 or 2 in preparing a fluorescent imaging product for overexpressing EGFR in cells.

9. Use of the small molecule fluorescent photosensitizer containing a gefitinib skeleton structure and an AIE group as claimed in claim 1 or 2 in the preparation of a non-small cell lung cancer cell imaging product for non-therapeutic and diagnostic purposes.

10. Use of the small molecule fluorescent photosensitizer containing a gefitinib skeleton structure and an AIE group as claimed in claim 1 or 2 in the preparation of a non-small cell lung cancer tumor targeted drug; more preferably, the non-small cell lung cancer tumor targeted drug is a photodynamic therapy non-small cell lung cancer tumor targeted drug.

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