Responsive self-luminous probe based on AIE natural product as well as preparation method and application of responsive self-luminous probe

Abstract

The invention discloses a response type self-luminous probe based on an AIE natural product. The structure of the response type self-luminous probe is shown as a formula I. The response type self-luminous probe disclosed by the invention not only can realize specific response and detection on ROS on cell and living body levels, but also can inhibit proliferation and metastasis of cancers and kill residual and metastatic focuses, so that the probability of in-situ recurrence and metastasis of tumors is effectively reduced. The invention further discloses application of the response type self-luminous probe based on the AIE natural product in preparation of reagents or drugs for screening or diagnosing tumors. The invention further discloses application of the response type self-luminous probe based on the AIE natural product in preparation of tumor treatment drugs. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the fields of molecular recognition, optical analysis, and small molecule self-luminescent probe detection, and particularly relates to the application of a class of AIE natural product-based responsive self-luminescent probes in the preparation of reagents or drugs for diagnosing tumor microlesions by detecting reactive oxygen species and in the preparation of drugs for inhibiting cancer proliferation and / or metastasis. Background Art

[0002] Reactive oxygen species (ROS) are molecules or compounds containing oxygen, hydrogen, or nitrogen atoms, including hydrogen peroxide (H2O2) and singlet oxygen ( 1 O2), etc. [1] In normal cells, ROS act as signaling molecules to maintain homeostasis, while the increased metabolic activity of cancer cells produces high concentrations of ROS, leading to a series of pro-tumor signals, such as proliferation, tumor angiogenesis, invasion, and metastasis. [2] Therefore, the sensitive and specific detection and consumption of ROS are of great significance for the accurate diagnosis and metastasis inhibition of cancer.

[0003] As a new imaging method with low cost, high safety, and strong sensitivity, optical imaging is widely used in biomedicine. [3] Among various optical imaging technologies, chemiluminescence (CL) is widely used in the fields of bioimaging and disease diagnosis because it does not require external excitation light and has the characteristics of high signal-to-noise ratio, low phototoxicity, and deep tissue penetration. [4] In 1982, Schaap et al. [5] first discovered a special luminescence pathway of adamantane-1,2-dioxetane derivatives. Since then, adamantane-1,2-dioxetane derivatives have been widely used for the detection of fluoride, esterase, etc.

[0004] Research has found that the properties of adamantane-1,2-dioxetane depend on the structure of its lumophore. By adjusting the structure of the lumophore in the probe, the chemiluminescence wavelength and quantum yield of the probe can be adjusted. [5] That is, the strategy of coupling adamantane-1,2-dioxetane with the structure of a fluorescent dye can significantly improve the chemiluminescence performance of the CL probe, enhancing the luminescence intensity and luminescence efficiency. Under physiological conditions, the spatial distance between the chemiluminescent probe and the fluorescent molecule conjugate is close, and energy transfer can be effectively carried out through chemiluminescence resonance energy transfer (CRET), thereby increasing the application of chemiluminescent probes.

[0005] However, traditional fluorescent molecules emit strongly in their individual molecular forms. When they aggregate, they exhibit varying degrees of aggregation-caused quenching (ACQ) effects, resulting in reduced luminescence or even quenching. [6] Natural products with aggregation-induced emission (AIE) properties, as the most abundant compounds in nature, contain multiple conjugated structures that can increase the emission wavelength. In addition, they have good biocompatibility and excellent pharmacological activities, such as anti-cancer, antioxidant, and anti-inflammatory effects.

[0006] Self-luminescent probe structures with near-infrared (NIR>600nm) emission based on adamantane-1,2-dioxetane have been successively developed. However, current research mainly focuses on the strategy of spatial CRET transfer based on nanoassemblies, where the energy transfer efficiency between chemical excitation intermediates and loaded dyes is low, significantly weakening the self-luminescent signal.

[0007] References:

[0008] [1] Reczek C R and Chandel N S. The Two Faces of Reactive Oxygen Species in Cancer. 2017, 1(2017): 79-98.

[0009] [2] Zhang J, Duan D, Song Z-L, et al. Small molecules regulating reactive oxygen species homeostasis for cancer therapy. 2021, 41(1): 342-394.

[0010] [3] He J, Li C, Ding L, et al. Tumor Targeting Strategies of Smart Fluorescent Nanoparticles and Their Applications in Cancer Diagnosis and Treatment. 2019, 31(40): 1902409.

[0011] [4]Wang Z,Huang J,Huang J,et al.Chemiluminescence:From mechanism to applications in biological imaging and therapy.2021,2(6):e140.

[0012] [5]Schaap AP and Gagnon SD.Chemiluminescence from a phenoxide-substituted 1,2-dioxetane:a model for firefly bioluminescence.Journal of the American Chemical Society,1982,104(12):3504-3506.

[0013] [6]Islam M M,Hu Z,Wang Q,et al.Pyrene-based aggregation-induced emission luminogens and their applications.Materials Chemistry Frontiers,2019,3(5):762-781. Summary of the Invention

[0014] The object of the present invention is to design a novel responsive self-luminescent probe with "aggregation-induced emission" property of long wavelength, and to provide a probe that can obtain strong chemiluminescence in the aggregated state. By coupling the CL probe with AIE natural products, minimizing the distance between the donor and the acceptor, improving the CRET efficiency, effectively realizing the intramolecular charge transfer and significantly increasing the emission wavelength, a high-performance "enhanced" self-luminescent platform is constructed.

[0015] The object of the present invention is achieved by the following technical solutions:

[0016] A responsive self-luminescent probe based on AIE natural products with the structure shown in Formula I (denoted as H-C):

[0017]

[0018] The described AIE natural product-based responsive self-luminescent probe includes a recognition element, a chemiluminescent donor element, and an AIE natural product receptor element. Among them, an adamantane-enol ether structure is used as the ROS-responsive recognition element; an adamantane-1,2-dioxetane structure (the adamantane-enol ether is oxidized to form an adamantane-1,2-dioxetane derivative) is used as the chemiluminescent donor element, and 10-hydroxycamptothecin (HCPT) with fluorescence emission at 556 nm and AIE properties is used as the receptor element, and the two are coupled together through benzyl alcohol to obtain it.

[0019] The maximum emission wavelength of the described H-C probe is around 629 nm.

[0020] Another object of the present invention is to provide a preparation method of the described AIE natural product-based responsive self-luminescent probe, and the synthetic route is as follows:

[0021]

[0022] It includes: using a mixed solvent of water and 4-methyl-tetrahydropyran as the reaction solvent, under the protection of N2, using Pd(PPh3)4 as the catalyst and K2CO3 as the acid-binding agent, reacting compound 6 and compound Int A to generate the probe shown in formula I;

[0023] The molar ratio of the described compound 6 to compound Int A is 1:1.0 - 1:1.5, preferably 1:1.1.

[0024] The molar ratio of the described compound 6 to Pd(PPh3)4 is 1:0.1 - 1:0.3, preferably 1:0.1.

[0025] The molar ratio of the described compound 6 to K2CO3 is 1:1 - 1:3, preferably 1:3.

[0026] The reaction temperature is 70 - 120 °C, preferably 100 °C.

[0027] The volume ratio of water to 4-methyl-tetrahydropyran is 1:2 - 1:9, preferably 1:4.

[0028] As a further preferred technical solution of the preparation method of the described AIE natural product-based responsive self-luminescent probe, it includes: after the reaction is completed, the reaction solution is concentrated under reduced pressure, and the residue is subjected to silica gel column chromatography, and the eluent is petroleum ether:ethyl acetate = 10:1 - 1:1 V / V, and the probe shown in formula I is purified.

[0029] The chemiluminescence principle of the responsive self-luminescent probe H-C based on AIE natural products is as follows: When the probe H-C recognizes ROS, the adamantane-enol ether structure in the probe is oxidized to adamantane-1,2-dioxetane; in the physiological environment, the phenol oxygen exposes the oxygen anion, and then triggers the Chemically Initiated Electron Exchange Luminescence (CIEEL) process through a chemical reaction for decomposition, generating an excited benzoate structure. Subsequently, the high-energy substrate rapidly transfers its energy to the AIE natural product molecule coupled thereto through CRET, causing the excited dye molecule to release energy in the form of luminescence and return to the ground state. During the CRET process, the excited benzoate and 10-hydroxycamptothecin serve as the energy donor and acceptor, respectively. Compared with traditional chemiluminescent probes that achieve CRET by preparing nanomaterials, the self-luminescent signal of adamantane-1,2-dioxetane coupled with AIE natural products is significantly amplified.

[0030] The responsive self-luminescent probe H-C based on AIE natural products can achieve specific responses to ROS and generate chemiluminescence in the presence of reactive oxygen species (including but not limited to singlet oxygen ( 1 O2), hydrogen peroxide (H2O2), etc.). The probe can specifically respond to the ROS reaction overexpressed in various types of cancers and produce a "turn on" type change in the chemical signal, realizing chemiluminescent imaging detection of overexpressed ROS in various cancer cells, differentiating cancerous from normal cells / tissues, identifying cancerous cells / tissues, and even being able to identify early solid tumors as small as 1 mm 3 in size. The probe of the present invention provides an effective method for the early and accurate diagnosis of pan-cancer, and realizes "navigation" surgical resection of tumors through visualizing tumor lesions, solving the problems or bottlenecks encountered in the current clinical diagnosis of early pan-cancer.

[0031] Another object of the present invention is to provide the application of the responsive self-luminescent probe based on AIE natural products in the preparation of reagents or drugs for screening or diagnosing tumors.

[0032] Preferably, the application is the application of the responsive self-luminescent probe based on AIE natural products in the preparation of reagents or drugs for early diagnosing tumors.

[0033] Preferably, the application is the application of the responsive self-luminescent probe based on AIE natural products in the preparation of reagents or drugs for diagnosing tumors with overexpressed ROS.

[0034] Preferably, the application is the application of the responsive self-luminescent probe based on AIE natural products in the preparation of reagents or drugs for specific imaging diagnosis of tumors based on ROS.

[0035] The responsive self-luminescent probe based on AIE natural products of the present invention can significantly inhibit the proliferation and metastasis of cancer, kill residual and metastatic lesions, and effectively reduce the probability of tumor recurrence and metastasis. It can provide an effective treatment method for cancer recurrence and metastasis.

[0036] Another object of the present invention is to provide the application of the responsive self-luminescent probe based on AIE natural products in the preparation of anti-tumor drugs.

[0037] Preferably, the application is the application of the responsive self-luminescent probe based on AIE natural products in the preparation of drugs for inhibiting the proliferation and / or metastasis of tumors.

[0038] Another object of the present invention is to provide a pharmaceutical composition for diagnosing and / or treating tumors, and the pharmaceutical composition uses the responsive self-luminescent probe based on AIE natural products as the main active ingredient or an active ingredient.

[0039] The tumors include but are not limited to breast cancer, liver cancer, lung cancer, colon cancer, melanoma, etc.

[0040] Compared with existing chemiluminescent probes, the advantages of the present invention are as follows:

[0041] (1) The present invention first couples AIE natural products with a self-luminescent probe to obtain a novel long-wavelength aggregation-induced chemiluminescent probe, and this probe can obtain a novel structure with strong self-luminescent emission in the aggregated state. Since the external excitation light source is avoided, the probe can effectively avoid background autofluorescence, has low phototoxicity, ultra-high signal-to-noise ratio and biological detection sensitivity, is suitable for deep tissue imaging and light "navigation" surgical resection, and perfectly solves the challenges in surgical imaging caused by instruments.

[0042] (2) Although currently, CL structures with near-infrared emission based on adamantane-1,2-dioxetane have been successively developed. However, the current research is mainly based on the strategy of spatial CRET transfer of nanoassemblies, in which the energy transfer efficiency between chemical excitation intermediates and loaded dyes is low, greatly weakening the self-luminescent signal. The chemiluminescent probe based on AIE natural products of the present invention can effectively overcome these problems.

[0043] On the one hand, AIE natural products have multiple conjugated structures, and there is a large degree of overlap between their excitation wavelengths and the emission wavelengths of CL probes. Moreover, energy transfer through bond energy has been proven to be more effective than energy transfer through space, so it can significantly increase the wavelength of the luminescent probe. On the other hand, the present invention combines the excellent anti-cancer activity of 10-hydroxycamptothecin and the characteristics of the adamantane-enol ether structure to respond to and consume ROS, enabling the responsive self-luminescent system based on AIE natural products to have an effect of 1 + 1 > 2, effectively inhibiting the proliferation and metastasis of malignant tumors.

[0044] (3) The self-luminescent probe based on AIE natural products of the present invention has stable properties, good selectivity for ROS, and a low detection limit. It can realize chemiluminescent imaging detection of overexpressed ROS in various cancer cells. Moreover, in vivo, this probe has achieved chemiluminescent imaging detection of ROS at the in vivo level and can identify solid tumors as small as 1 mm 3 . At the same time, this AIE-CL probe can significantly inhibit the metastasis and proliferation of cancer, kill residual and metastatic lesions, and effectively reduce the probability of in-situ proliferation and metastasis of tumors, providing a new idea and method for the early diagnosis and treatment of pan-cancer. Description of the Drawings

[0045] Figure 1 HNMR spectrum of the chemiluminescent molecular probe H-C 1 .

[0046] Figure 2 CNMR spectrum of the chemiluminescent molecular probe H-C 13 .

[0047] Figure 3 MS spectrum of the chemiluminescent molecular probe H-C

[0048] Figure 4 MS diagram of the probe H-C after recognizing ROS; (A) Mechanism diagram of the probe H-C responding to ROS; (B) MS spectrum of H-C-1,2-dioxetane; (C) MS spectrum of H-C-methyl benzoate.

[0049] Figure 5 Selectivity of the probe H-C for ROS; among them, a-x are Asp, Ser, Arg, Ala, TBHP, NaClO, H2O2, 1 O2, ·OtBu, ·OH, O2 - , Leu, Val, Lys, Phe, Trp, Thr, KCl, NaCl, FeCl3, DTT, MgCl2, ZnCl2, GSH.

[0050] Figure 6 Chemiluminescent spectrum of the probe H-C after adding ROS.

[0051] Figure 7 It is the chemiluminescence intensity diagram of probe H-C after recognizing ROS in systems with different water contents.

[0052] Figure 8 It is the chemiluminescence kinetic curve of the reaction between probe H-C and ROS.

[0053] Figure 9 It is for the chemiluminescence intensity of probe H-C in 1 the linear fitting curve of the concentration of O2 (0 - 50 mM).

[0054] Figure 10 It is the chemiluminescence intensity of probe H-C after being placed at 4°C for different times.

[0055] Figure 11 It is the chemiluminescence imaging images of probe H-C for different cells (HCCLM3, MDA-MB-231, HepG2, MCF-7, AGS, RAW 264.7-LPS, MCF-10A, L02, RAW 264.7).

[0056] Figure 12 It is the relative intensity of chemiluminescence imaging of probe H-C for different cells.

[0057] Figure 13 It is the in vivo imaging of probe H-C for nude mice bearing human triple-negative breast cancer MDA-MB-231; among them, (A) chemiluminescence images of tumor-bearing mice with different tumor sizes detected by H-C; (B) relative intensity of chemiluminescence imaging of tumors with different sizes by H-C

[0058] Figure 14 It is the result of the in vitro migration experiment of probe H-C inhibiting human triple-negative breast cancer MDA-MB-231; among them, (A) Transwell migration images of the control group (DMEM), the HCPT treatment group, and the probe H-C treatment group; (B) relative migration rate.

[0059] Figure 15 It is the result of the in vivo pharmacodynamic experiment of probe H-C inhibiting mice bearing human triple-negative breast cancer MDA-MB-231; among them, (A) relative body weight changes of different treatment groups; (B) relative tumor volume of MDA-MB-231 tumor-bearing nude mice during treatment; (C) tumor images of different treatment groups; (D) tumor weights after the end of treatment in different groups; (E) migration of triple-negative breast cancer after treatment in different groups. Specific implementation manners

[0060] The technical solutions of the present invention will be further described below through specific embodiments. However, the following is only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions in the following embodiments, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0061] The responsive self-luminescent probe proposed by the present invention can replace AIE natural products, so as to obtain a series of self-luminescent probes based on AIE natural products, not limited to 10-hydroxycamptothecin. The present invention uses 10-hydroxycamptothecin as an example to prove the feasibility of this scheme.

[0062] Example 1

[0063] Synthesis and Characterization of Probe H-C

[0064] (1) Synthesis of Compound 6: Using a multi-step chemical synthesis method, starting from 2-chloro-3-hydroxybenzaldehyde (Compound 1), Compound 6 was finally obtained after 5-step chemical reactions. The synthesis route of Compound 6 is as follows:

[0065]

[0066] Including:

[0067] Step (1a), Synthesis of Compound 2: Add trimethoxymethane (108 g, 1.02 mol, 112 mL, 1.60 eq) and tetrabutylammonium (30.8 g, 63.9 mmol, 0.10 eq) to a methanol (1000 mL) solution of Compound 1 (100 g, 639 mmol, 1.00 eq). The mixture was stirred at 20 °C for 12 hours. At this time, TLC showed that reactant 1 was completely consumed; add 150 mL of 0.01 M Na2HCO3 solution and 300 mL of ethyl acetate to the reaction solution, collect the organic layer, and the organic layer was dried over sodium sulfate, filtered, and concentrated; the residue was purified by silica gel column chromatography (eluent: petroleum ether:ethyl acetate = 100:1 - 20:1 V / V) to obtain Compound 2 (112 g, 553 mmol, yield 86.5%), a colorless oil. 1 H NMR(400MHz,CDCl3):δ7.22-7.20(m,2H),7.04-7.01(m,1H),5.96(s,1H),5.61(s,1H),3.40(s,6H).

[0068] Step (1b), Synthesis of Compound 3: Dissolve Compound 2 (112 g, 553 mmol, 1.00 eq) in 1200 mL of dichloromethane, add imidazole (75.3 g, 1.11 mol, 2.00 eq), tert-butyldimethylchlorosilane (TBSCl, 99.9 g, 663 mmol, 81.6 mL, 1.20 eq), stir at 20 °C for 12 h. At this time, TLC shows that Compound 2 is completely consumed; filter the reaction solution, concentrate the filtrate under reduced pressure, and purify the residue by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 100:1 - 20:1 V / V) to obtain Compound 3 (136 g, 429 mmol, yield 77.6%), a colorless oil. 1 H NMR (400 MHz, CDCl3): δ 7.26 (dd, J = 8.0, 2.0 Hz, 1H), 7.17 (t, J = 8.0 Hz, 1H), 6.90 (dd, J = 8.0, 1.6 Hz, 1H), 5.66 (s, 1H), 3.40 (s, 6H), 1.06 (s, 9H), 0.25 (s, 6H).

[0069] Step (1c), Synthesis of Compound 4: Dissolve Compound 3 (100 g, 316 mmol, 1.00 eq) and trimethyl phosphite (P(OMe)3, 50.9 g, 410 mmol, 48.5 mL, 1.30 eq) in dichloromethane (900 mL). Cool the reaction mixture to 0 °C, dropwise add titanium tetrachloride (TiCl4, 71.8 g, 379 mmol, 71.8 mL, 1.20 eq), and stir for 4 h. TLC shows that the reactant 3 is completely consumed; filter the reaction solution, concentrate the filtrate under reduced pressure, and purify the residue by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 100:1 - 20:1 V / V) to obtain Compound 4 (96.6 g, 213 mmol, yield 67.4%, purity 87.0%), a colorless oil. 1 HNMR (400 MHz, CDCl3) δ 7.31 (dt, J = 6.8, 1.2 Hz, 1H), 7.23 (t, J = 8.0 Hz, 1H), 6.91 (dt, J = 8.0, 1.6 Hz, 1H), 5.22 (d, J = 15.6 Hz, 1H), 3.81 (d, J = 10.4 Hz, 3H), 3.67 (d, J = 10.4 Hz, 3H), 3.37 (s, 3H), 1.05 (s, 9H), 0.25 (s, 6H).

[0070] Step (1d), Synthesis of Compound 5: Dissolve Compound 4 (20.0 g, 50.6 mmol, 1.00 eq) in tetrahydrofuran (200 mL), add lithium diisopropylamide (LDA, 2 M, 30.4 mL, 1.20 eq) dropwise at -65 °C, and stir at -65 °C for 0.6 h; then dissolve 2-adamantanone (11.4 g, 75.9 mmol, 1.50 eq) in THF (30 mL), stir at 20 °C for 7.4 h. At this time, TLC shows that reactant 4 is completely consumed and new spots are detected; add 200 mL of saturated aqueous NaHCO3 solution to slowly quench the reaction, and extract with ethyl acetate (80 mL × 3). Combine the organic phases. The organic phase is dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and the residue is purified by silica gel column chromatography (eluent: petroleum ether:ethyl acetate = 100:1 - 20:1 V / V) to obtain Compound 5 (9.60 g, 20.6 mmol, yield 40.7%, purity 90%), a white solid. 1 H NMR (400 MHz, CDCl3) δ 7.02 (t, J = 8.0 Hz, 1H), 6.82 - 6.77 (m, 2H), 3.24 (s, 3H), 3.20 (s, 1H), 1.98 (s, 1H), 1.88 - 1.61 (m, 12H), 0.97 (s, 9H), 0.16 (s, 6H).

[0071] Step (1e), Synthesis of Compound 6: In a 100 mL sealed tube, add bis(pinacolato)diboron (969 mg, 3.82 mmol, 1.60 eq), 4-tert-butyl-2-(4-tert-butyl-2-pyridyl)pyridine (BBBPY, 128 mg, 477 μmol, 0.20 eq), [Ir(OMe)(1,5-cod)]2 (158 mg, 239 μmol, 0.10 eq) to a THF solution (40 mL) of Compound 5 (1.00 g, 2.39 mmol, 1.00 eq). Stir at 100 °C for 15 h at 20 °C under N2 protection. Detect by TLC. At this time, it can be seen that most of reactant 5 is consumed and new spots are generated. Concentrate the reaction solution under reduced pressure, and separate and purify the residue by silica gel column chromatography (eluent: petroleum ether:ethyl acetate = 100:1 - 20:1 V / V) to obtain Compound 6 (9.63 g, 17.67 mmol, yield 49.3%), a colorless oil. 11H NMR: (400 MHz, CDCl3) δ 9.48 - 8.37 (m, 1H), 7.31 (s, 1H), 7.28 (s, 1H), 3.32 - 3.29 (m, 4H), 2.06 (s, 1H), 1.96 - 1.62 (m, 13H), 1.36 (s, 12H), 1.07 (s, 9H), 0.26 (s, 6H).

[0072] (2) Synthesis of compound Int A: 3 - bromobenzyl bromide (11.3 g, 45.3 mmol, 1.10 eq), 10 - hydroxycamptothecin (compound Int A1, 15.0 g, 41.2 mmol, 1.00 eq) and K2CO3 (17.1 g, 124 mmol, 3.00 eq) were dissolved in acetonitrile (MeCN, 180 mL), and stirred at 30 °C for 12 h. At this time, TLC showed that compound Int A1 was completely consumed; the reaction solution was quenched by adding 750 mL of water, extracted with ethyl acetate (150 mL × 4), the organic layers were combined, washed with saturated aqueous NaHCO3 solution (60 mL × 3), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue; the residue was ground with a mixed solvent of dichloromethane and methyl tert - butyl ether (DCM:MBTE = 1:5 V / V, 120 mL) at 20 °C for 30 min, repeated 2 times, filtered after each grinding, the filtrates were combined, and the combined filtrate was purified by silica gel column chromatography (eluent: petroleum ether:ethyl acetate = 50:1 - 20:1 V / V) to obtain compound Int A (11.2 g, 14.1 mmol, yield 34.2%, purity 67%), a brown solid. 1 1H NMR (400 MHz, DMSO - d6) δ 8.25 (s, 1H), 8.10 (d, J = 9.2 Hz, 1H), 7.64 (d, J = 8.3 Hz, 1H), 7.59 (d, J = 9.1 Hz, 1H), 7.62 (d, J = 1.9 Hz, 2H), 7.60 (d, J = 2.8 Hz, 1H), 7.58 (d, J = 2.7 Hz, 1H), 7.52 (d, J = 8.4 Hz, 2H), 6.51 (s, 1H), 5.42 (s, 1H), 5.29 (s, 2H), 5.27 (s, 2H), 1.86 (q, 2H), 0.90 - 0.87 (m, 3H).

[0073]

[0074] (3) Synthesis of chemiluminescent probe H-C: Dissolve compound Int A (5.60 g, 10.5 mmol, 1.10 eq) in a mixed solvent of 40 mL of H2O and 160 mL of MTHP (4-methyl-tetrahydropyran) to obtain a solution of compound Int A. Then add compound 6 (5.20 g, 9.54 mmol, 1.00 eq). Under the conditions of 20 °C and N2 protection, add Pd(PPh3)4 (10 g, 954 μmol, 0.10 eq) and K2CO3 (3.96 g, 28.6 mmol, 3.00 eq), and stir the reaction at 100 °C for 12 h. At this time, TLC shows that compound Int A is completely consumed, and a new spot with a larger polarity is detected. The reaction solution is concentrated under reduced pressure, and the residue is purified by silica gel column chromatography (the eluent is petroleum ether:ethyl acetate = 10:1 - 1:1 V / V) to obtain 5.20 g of the target compound (denoted as H-C) with the structure shown in formula I, 5.49 mmol, a yield of 57.6%, a purity of 80%, and a yellow solid.

[0075]

[0076] For compound H-C 1 1H NMR, 13 13C NMR and MS are shown respectively as Figure 1 、 Figure 2 and Figure 3 shown.

[0077] 1 1H NMR: (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 8.53 (s, 1H), 8.10 (d, J = 12.0 Hz, 1H), 7.64 (s, 4H), 7.58 (dd, J = 4.0, 8.0 Hz, 1H), 7.29 (s, 1H), 7.24 - 7.22 (m, 1H), 7.00 (d, J = 2.0 Hz, 1H), 6.51 (s, 1H), 5.42 (s, 2H), 5.34 (s, 2H), 5.26 (s, 2H), 3.26 (s, 3H), 3.20 (br s, 1H), 2.05 (br s, 1H), 1.96 - 1.63 (m, 14H), 0.89 (br t, J = 7.2 Hz, 3H). 1313C-NMR: (400 MHz, Chloroform-d) δ 173.0, 157.6, 157.3, 154.2, 150.8 150.5, 146.2, 144.5, 140.6, 139.2, 136.6, 135.9, 131.1, 130.8, 130.5, 129.8, 129.3, 129.2, 127.2, 123.7, 120.7, 120.5, 118.9, 114.5, 108.0, 96.6, 72.9, 69.9, 65.7, 56.7, 50.7, 40.6, 40.4, 40.2, 40.0, 39.8, 39.6, 39.4, 37.0, 32.9, 30.7, 29.4, 28.2, 28.1, 8.2. MS: m / z calculated for C 45 H 41 ClN2O7: 756.3; found, 757.3 [M+H] + .

[0078] Example 2

[0079] Study on the Response Mechanism of Probe H-C

[0080] To verify the reaction mechanism between probe H-C and ROS, the product after the reaction of probe H-C with ROS was verified by mass spectrometry (MS). 3.78 mg of probe H-C was accurately weighed and completely dissolved in 5 mL of DMSO to obtain a mother liquor of probe H-C with a concentration of 1 mM. 100 μL of the mother liquor of probe H-C was taken and diluted to 100 mL with ultrapure water to obtain a working solution of probe H-C with a concentration of 10 μM.

[0081] 0.55 mL of 30% hydrogen peroxide (calibrated value: 9.01 mol / L) was accurately pipetted and diluted to 50 mL with PBS (pH 7.4) to obtain a hydrogen peroxide stock solution with a concentration of 0.1 M; the hydrogen peroxide stock solution was diluted to a hydrogen peroxide solution with a concentration of 10 mM with PBS (pH 7.4) of 0.1 mol / L.

[0082] 6.67 mL of 10% sodium hypochlorite (calibrated value: 0.75 mol / L) was accurately pipetted and diluted to 50 mL with PBS (pH 7.4) to obtain a sodium hypochlorite stock solution with a concentration of 0.1 M. The 0.1 M sodium hypochlorite stock solution was diluted to a 10 mM sodium hypochlorite solution with PBS (pH 7.4) of 0.1 mol / L.

[0083] 0.08 mL of the working solution of probe H-C, 0.10 mL of the 10 mM hydrogen peroxide solution, and 0.10 mL of the 10 mM sodium hypochlorite solution were mixed, sampled, and freeze-dried. MS was used to analyze the response mechanism of the H-C probe.

[0084] The results are as Figure 4 shown, the probe H-C (Formula I) reacts with ROS to generate H-C-1,2-dioxetane (Formula II, m / z = 789.1 [C 45 H 41 ClN2O9+H] + ), and then is decomposed into methyl H-C-benzoate (Formula III, m / z = 683.4 [C 35 H 26 ClN2O8+2Na] + ).

[0085] Example 3

[0086] Study on the Detection Performance of Probe H-C for Reactive Oxygen Species

[0087] (1) Selectivity of the Probe for Reactive Oxygen Species

[0088] Accurately weigh 3.78 mg of probe H-C, and completely dissolve it with 5 mL of DMSO to obtain a stock solution of probe H-C with a concentration of 1 mM. Take 100 μL of the stock solution of probe H-C and dilute it to 100 mL with PBS to obtain a working solution of probe H-C with a concentration of 10 μM.

[0089] The preparation methods of various reactive oxygen species are as follows (all solutions are prepared and used immediately):

[0090] ① 10 mM H2O2 solution: Dilute the 0.1 mol / L H2O2 stock solution to 10 mM with PBS buffer solution.

[0091] ② 10 mM NaClO solution: Dilute the 0.1 mol / L NaClO stock solution to 10 mM with PBS buffer solution.

[0092] ③ 10 mM of 1 O2 (generated by the rapid reaction of H2O2 and ClO - ): Mix equal volumes of 10 mM H2O2 solution and 10 mM NaClO solution.

[0093] ④ 10 mM TBHP working solution: Provided by tert-butyl hydroperoxide (TBHP). Accurately transfer 700 μL of 70% TBHP (7.20 mol / L) and dilute it to 50 mL with PBS to prepare a stock solution of TBHP with a concentration of 1 M. Take 300 μL of the stock solution of TBHP and dilute it to 3 mL with PBS to obtain a working solution of TBHP with a concentration of 10 mM.

[0094] ⑤10 mM tert-butyl radical (·OtBu): Generated by the rapid reaction of TBHP and ferrous sulfate (FeSO4). Accurately weigh 1.52 mg of FeSO4 and prepare a 1 mM FeSO4 working solution (prepared immediately before use) using 10 mL of PBS buffer. Take 1 mL of a 10 mM TBHP working solution and 1 mL of a 1 mM FeSO4 solution, and mix them rapidly to generate ·OtBu.

[0095] ⑥Hydroxyl radical (·OH): Generated by the rapid Fenton reaction after mixing 1 mL of a 10 mM H2O2 solution and 1 mL of a 1 mM FeSO4 working solution.

[0096] ⑦Superoxide anion (O2 - ): Generated by the reaction of xanthine and xanthine oxidase. Accurately weigh 4.56 mg of xanthine and prepare a 3.0 mM xanthine solution using 10 mL of PBS buffer. Accurately weigh 1 mg of xanthine oxidase and prepare a 50 U / mL xanthine oxidase stock solution using 1 mL of PBS buffer. Take 50 μL of the xanthine oxidase stock solution, dilute it to 5 mL with PBS buffer to obtain a 0.5 U / mL xanthine oxidase working solution. Mix 1 mL of a 3.0 mM xanthine solution and 1 mL of a 0.5 U / mL xanthine oxidase working solution and react for 1 min to generate O2 - 。

[0097] ⑧Preparation of ion solutions with a concentration of 100 μM each: Accurately weigh a certain amount of potassium chloride, sodium chloride, ferric chloride, 2-mercaptoethanol, magnesium chloride, zinc chloride, and glutathione, and prepare potassium ions (KCl), sodium ions (NaCl), iron ions (FeCl3), magnesium ions (MgCl2), and zinc ions (ZnCl2) with a concentration of 100 μM using PBS buffer.

[0098] ⑨Preparation of amino acid solutions with a concentration of 100 μM each: Accurately weigh a certain amount of aspartic acid (Asp), serine (Ser), arginine (Arg), alanine (Ala), leucine (Leu), valine (Val), lysine (Lys), phenylalanine (Phe), tryptophan (Trp), and threonine (Thr), and prepare them using PBS buffer.

[0099] ⑩Preparation of a reducing solution with a concentration of 100 μM: Accurately weigh a certain amount of 2-mercaptoethanol (DTT) and glutathione (GSH), and prepare them using PBS buffer.

[0100] Add 80 μL of the probe H-C working solution with a concentration of 10 μM and 100 μL of different reactive oxygen species or interference solutions into an EP tube in sequence, and mix evenly. Take 200 μL of the mixed solution and add it into a full-white 96-well plate, and use a multifunctional microplate reader to detect the chemiluminescence signal after the reaction of the probe with different working solutions in the CL mode.

[0101] The experimental results are shown in Figure 5 , indicating that: under physiological conditions of pH 7.4, the probe H-C has a strong response intensity to several common reactive oxygen species, and there is no obvious reaction to a variety of amino acids and ions, indicating that the probe H-C has excellent selectivity for ROS. Among them, H2O2 and 1 O2 have a strong oxidation ability to H-C, which can greatly increase the chemiluminescence intensity. In particular, after adding 1 O2, the luminescence intensity of H-C is more than 2 times stronger than that of other types of ROS. Therefore, in order to further study the performance of the probe H-C in analytical chemistry and bioimaging, the inventors selected 1 O2 as a typical ROS.

[0102] (2) Chemiluminescence performance of the probe H-C

[0103] Accurately pipette 0.55 mL of 30% hydrogen peroxide (9.01 mol / L), and dilute it to 50 mL with PBS (pH 7.4) to obtain a hydrogen peroxide stock solution with a concentration of 0.1 M.

[0104] Accurately pipette 6.67 mL of 10% sodium hypochlorite solution (0.75 mol / L), and dilute it to 50 mL with PBS (pH 7.4) to obtain a sodium hypochlorite stock solution with a concentration of 0.1 M.

[0105] Accurately weigh 3.78 mg of the probe H-C, and completely dissolve it in 5 mL of DMSO to obtain a mother liquor of the probe H-C with a concentration of 1 mM. Take 100 μL of the mother liquor of the probe H-C and dilute it to 100 mL with PBS to obtain a working solution of the probe H-C with a concentration of 10 μM.

[0106] Under dark conditions, dilute the hydrogen peroxide stock solution with a concentration of 0.1 M to a hydrogen peroxide solution with a concentration of 10 mM with 0.1 mol / L PBS (pH 7.4), and dilute the sodium hypochlorite stock solution with a concentration of 0.1 M to a sodium hypochlorite solution with a concentration of 10 mM with 0.1 mol / L PBS (pH 7.4). Mix 80 μL of the probe H-C working solution, 100 μL of the hydrogen peroxide solution with a concentration of 10 mM, and 100 μL of the sodium hypochlorite solution with a concentration of 10 mM, and use a SpectraMax iD5 multifunctional microplate reader to detect the chemiluminescence signal intensity before and after the reaction of the probe H-C with 1 O2.

[0107] The chemiluminescence spectrum results of probe H-C are as follows Figure 6 shown. In the absence of 1 O2, the chemiluminescence signal of H-C is negligible; however, after adding 1 O2, the chemiluminescence intensity of the H-C probe increases significantly, and the maximum emission wavelength is around 629 nm.

[0108] (3) AIE properties of probe H-C

[0109] Accurately weigh 3.78 mg of probe H-C and completely dissolve it in 5 mL of tetrahydrofuran (THF) to obtain a stock solution of probe H-C with a concentration of 1 mM. Take 100 μL of the probe H-C stock solution and dilute it to 1 mL of probe H-C working solution with a concentration of 10 μM with ultrapure water and / or THF according to Table 1, with different water contents (volume fractions are 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%) respectively.

[0110] Table 1. Preparation method of probe H-C working solution with different water contents

[0111]

[0112] Note: Vs represents the volume of the probe H-C stock solution, V w represents the volume of ultrapure water, V THF represents the volume of tetrahydrofuran, f w represents the water content.

[0113] The experimental results are as follows Figure 7 shown. As the water content in the probe H-C working solution increases (from left to right, the water content increases in turn), the luminescence intensity of probe H-C gradually increases. When the water content in the probe H-C working solution reaches 90%, the luminescence intensity of probe H-C is nearly twice as strong as that when the water content is 0%, showing an obvious AIE effect.

[0114] (4) Chemiluminescence kinetic curve

[0115] Prepare a probe H-C working solution with a concentration of 10 μM, a hydrogen peroxide solution with a concentration of 10 mM, and a sodium hypochlorite solution with a concentration of 10 mM according to Example 2. Add 80 μL of the 10 μM H-C probe working solution, 100 μL of the 10 mM hydrogen peroxide solution, and 100 μL of the 10 mM sodium hypochlorite solution to an EP tube in sequence and mix.

[0116] Take 200 μL of the above mixed solution, add it to a full-white 96-well plate, and use a multifunctional microplate reader to detect the reaction of probe H-C with within 0 - 300 min in the CL mode1 The chemiluminescence kinetic curve of O2. The results are as follows Figure 8 , in 1 the presence of O2, the probe H-C can be effectively activated and can react rapidly with 1 O2 to generate a strong chemiluminescence signal, and can continuously emit light for the O2 in the system. By collecting the chemiluminescence intensity data from 0 to 300 min, it was found that the probe H-C could reach the maximum luminescence intensity at about 30 min, and the chemiluminescence signal could still be detected at 60 min, indicating that this probe can be used for long-term detection and tracking of 1 O2. 1

[0117] (5) Detection limit of the probe for reactive oxygen species

[0118] Dilute the 0.1 mol / L hydrogen peroxide stock solution with 0.1 mol / L PBS at pH 7.4 to obtain H2O2 solutions with different concentrations (concentrations are 0.1, 0.5, 1, 5, 10, 50, 100, 200, 500, 1000, 1500, 2000, 5000, 10000, 15000, 20000, 50000 μM); dilute the 0.1 mol / L sodium hypochlorite stock solution with 0.1 mol / L PBS at pH 7.4 to obtain NaClO solutions with different concentrations (concentrations are 0.1, 0.5, 1, 5, 10, 50, 100, 200, 500, 1000, 1500, 2000, 5000, 10000, 15000, 20000, 50000 μM). Prepare working solutions of different concentrations of 1 O2 (0 - 50 mM): Add 80 μL of the 10 μM probe H-C working solution, 100 μL of H2O2 solutions with different concentrations, and 100 μL of NaClO solutions with different concentrations to an EP tube in sequence and mix. Replace the probe H-C working solution with an equal volume of 0.1 mol / L PBS at pH 7.4 to obtain a blank sample. Take 200 μL of the mixed solution and add it to a full-white 96-well plate, and use a multifunctional microplate reader to detect the chemiluminescence signal after the probe H-C reacts with different concentrations of 1 O2.

[0119] The detection limit refers to the lowest concentration at which the target substance can be clearly distinguished during the analysis process. Generally, the detection limit (LOD) can be calculated by the following formula:

[0120] LOD = 3σ / k

[0121] where k represents the slope of the linear equation for the reaction of the probe H-C with 1 O2, and σ represents the standard deviation of the blank sample.

[0122] HC probe with different concentrations 1 The chemiluminescence intensity after O2 reaction is Figure 9 As shown, the chemiluminescence intensity of the probe HC is related to 0-50mM 1 O2 concentration shows a good linear relationship, linear regression equation: Y = 0.8873x + 101.12, R 2 =0.9996. Probe HC pair 1 The detection limit of O2 is 2.07 μM, indicating that the probe has good detection sensitivity and can quantitatively detect the content of ROS in organisms.

[0123] Example 4

[0124] Study on the stability of probe HC

[0125] Accurately weigh 3.78 mg of probe HC, dissolve it completely with 5 mL of DMSO to obtain a probe HC mother solution with a concentration of 1 mM. Take 100 μL of the probe HC mother solution and dilute it to 10 mL with PBS buffer (pH = 7.4) to obtain a probe HC working solution with a concentration of 10 μM. The probe HC working solution was placed in a 4°C refrigerator for 1, 3, 5, 7, 14, 21, and 30 days. Take an EP tube, add 80 μL of the probe HC working solution placed at 4°C for different days, 100 μL of a 10 mM hydrogen peroxide solution, and 100 μL of a 10 mM hydrogen peroxide solution in sequence, mix the reaction, and use a multifunctional microplate reader to detect the chemiluminescence intensity.

[0126] The experimental results are shown in Figure 10 , when the placement time was extended to the 14th day, the CL signal intensity of the probe HC was 82% of the value measured in the freshly prepared solution, indicating that the probe HC had good stability.

[0127] Example 5

[0128] Optical activity detection of probe HC cellular levels

[0129] Accurately weigh 2 mg of lipopolysaccharide (LPS) and dissolve it in DMSO to obtain a 2 mg / mL LPS stock solution. When used, dilute it with serum-free DMEM medium to a concentration of 1 μg / mL.

[0130] Construction of lipopolysaccharide-induced RAW264.7 inflammatory cells: Mouse mononuclear macrophage leukemia cells RAW264.7 were cultured in a 37°C, 5% CO2 incubator overnight. When the cells grew to 70%, 1 μg / mL of LPS was used to stimulate RAW264.7 cells for 24 hours to obtain LPS-induced RAW264.7 inflammatory cells.

[0131] Accurately weigh 3.78 mg of probe H-C, and completely dissolve it with 2.5 mL of DMSO to obtain a mother liquor of probe H-C with a concentration of 2 mM. When in use, dilute it to a concentration of 10 μM with serum-free DMEM medium.

[0132] Human hepatocellular carcinoma cells HCCLM3, HepG2, human normal hepatocytes L02, human breast cancer cells MDA-MB-231, MCF-7, human normal mammary epithelial cells MCF-10A, human gastric adenocarcinoma cells AGS, mouse mononuclear macrophage leukemia cells RAW264.7, lipopolysaccharide (LPS)-induced RAW264.7 inflammatory cells, with 3 replicate wells for each type of cell, were cultured adherently in a 37 °C, 5% CO2 incubator for 24 h. Discard the old medium, add DMEM medium containing probe H-C (10 μM), incubate for 10 min, and perform real-time imaging analysis using a small animal in vivo imaging system (bioluminescence mode).

[0133] The results are shown in Figure 11 and Figure 12 , indicating that: after normal cells MCF-10A, L02, and RAW264.7 were incubated with probe H-C respectively, almost no chemiluminescence signal was observed; while after different types of cancer cells or LPS-induced RAW264.7 inflammatory cells were incubated with probe H-C, obvious chemiluminescence signals could be observed. It shows that probe H-C can achieve chemiluminescence imaging of cancer cells or other cells with overexpressed ROS.

[0134] Example 6

[0135] In vivo chemiluminescence imaging of probe H-C

[0136] Accurately weigh 5 mg of probe H-C, and dissolve it with a mixed solvent of DMSO, PEG300, Tween-80, and normal saline with a volume ratio of 2:40:5:53 to obtain a probe H-C solution with a concentration of 2 mg / mL.

[0137] All animal experiments were conducted in accordance with the requirements of the Animal Ethics Committee of China Pharmaceutical University. Female / male specific pathogen-free BALB / c nude mice, weighing 18 ± 1.0 g, were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. (License number: SYXK (Su) 2023-0019). During the experiment, the nude mice were placed in a sterile isolation cage (temperature: 25 °C, air humidity: 60%, light-dark cycle: 12 hours). The nude mice were allowed to eat and drink freely and fasted for 24 h before the experiment.

[0138] Take 100 μL of different densities (densities are 5×10 5 、1×10 6 、1.5×10 6 、2×10 6 、2.5×106 、 3×10 6 、 4×10 6 cells / 100μL), MDA-MB-231 cells in the logarithmic growth phase, were respectively subcutaneously injected into the second mammary gland of BALB / c nude mice. After 7 days, nude mice models with tumor-bearing of different tumor volumes were obtained. Each nude mouse was injected with 0.1 mL of probe H-C solution into the tumor at a dose of 10 mg / kg. After 10 min, the mice were anesthetized and real-time imaging analysis was performed on a small animal in vivo imager (bioluminescence mode).

[0139] The imaging results are as Figure 13 shown, showing that probe H-C can detect tiny lesions as small as 1.03 mm 3 and obtain imaging signals that are clearly distinguishable from normal tissues. It indicates that probe H-C has great potential in the early diagnosis of tumors.

[0140] Example 7

[0141] Anticancer activity of probe H-C

[0142] (1) Cytotoxicity test of probe H-C on different cancer cells

[0143] The in vitro cytotoxicity of probe H-C on different cancer cells was determined by the MTT method.

[0144] Accurately weigh 4.12 mg of HCPT and completely dissolve it with 1.13 mL of DMSO to obtain an HCPT stock solution with a concentration of 10 mM. When in use, dilute it with serum-free DMEM medium.

[0145] Accurately weigh 3.78 mg of probe H-C and completely dissolve it with 2.5 mL of DMSO to obtain a mother liquor of probe H-C with a concentration of 2 mM. When in use, dilute it with serum-free DMEM medium.

[0146] Normal human hepatocytes L02 in the logarithmic growth phase, human hepatoma cells HCCLM3, MHCC-97H, human breast cancer cells MDA-MB-231, normal human mammary epithelial cells MCF-10A, mouse mononuclear macrophage leukemia cells RAW 264.7, human non-small cell lung cancer cells A549, colon cancer cells MC 38, human gastric adenocarcinoma cells AGS, and mouse skin melanoma cells B16F10 were all seeded in 96-well plates at a density of 5000 cells / well and cultured in an incubator for 12 h until about 80% confluent. The supernatant was carefully aspirated and replaced with 100 μL of DMEM incomplete medium (blank group) or 100 μL of DMEM incomplete medium containing different concentrations of probe H-C or HCPT (sample group). After culturing for 24 h, 100 μL of MTT solution at a concentration of 0.5 mg / mL was added under light-protected conditions and continued to be cultured for 2 h. Then the supernatant was discarded and 100 μL of DMSO was added. The OD value of each well was measured at a wavelength of 492 nm using a microplate reader, and the cell survival rate was calculated.

[0147] Cell survival rate (%) = OD value of sample group / OD value of blank group × 100%

[0148] Table 2. IC of HCPT and H-C against different cells 50 (n = 5)

[0149]

[0150] As shown in Table 2, normal and different cancer cells were treated with HCPT and probe H-C respectively. Compared with HCPT, probe H-C had more excellent in vitro cytotoxicity against different cancer cells; among them, the inhibitory effect on the proliferation activities of AGS, A549, HCCLM3, and MDA-MB-231 cells was the best. Meanwhile, at a high concentration of 50 μM (5 times the IC 50 ), probe H-C maintained a cell survival rate of more than 80% for normal human hepatocytes L02, indicating that probe H-C had low cytotoxicity to normal cells and good selectivity between cancer cells and normal cells.

[0151] (2) Probe H-C inhibits cancer metastasis

[0152] The Transwell migration assay was used to detect the ability of HCPT and probe H-C to inhibit the migration of MDA-MB-231 cells in vitro.

[0153] Accurately weigh 4.12 mg of HCPT and completely dissolve it in 1.13 mL of DMSO to obtain a HCPT stock solution with a concentration of 10 mM. When used, it was diluted to 10 μM with serum-free DMEM medium.

[0154] Accurately weigh 3.78 mg of probe H-C, and completely dissolve it in 2.5 mL of DMSO to obtain a stock solution of probe H-C with a concentration of 2 mM. When in use, dilute it to 10 μM with serum-free DMEM medium.

[0155] MDA-MB-231 cells in the logarithmic growth phase were digested with trypsin and counted. Probe H-C or HCPT treatment group: Add 200 μL of DMEM cell suspension containing probe H-C or HCPT to the upper chamber of Transwell, and add 600 μL of DMEM medium containing 10% fetal bovine serum (Gibco) to the lower chamber. Add 200 μL of DMEM cell suspension to the upper chamber as the control group. The cells and drugs were co-cultured for 24 h, the medium was aspirated, and the cells were washed with PBS; 1 mL of 4% paraformaldehyde was added to the upper and lower chambers respectively and fixed for 20 - 30 min; washed with PBS, and stained with crystal violet for more than 10 min; washed with a large amount of pure water to remove the excess crystal violet, and gently wiped off the upper-layer cells with a cotton swab. Observe under a microscope (take a global picture at 4× magnification and take 3 - 5 pictures for counting at 10× magnification)

[0156] The results are shown in Figure 14 , compared with the control group (i.e., Figure 14 DMEM in ), probe H-C showed a significant inhibitory effect on the migration of MDA-MB-231 cells (P < 0.01); compared with the HCPT treatment group, the inhibitory activity of H-C probe on MDA-MB-231 cells was better, which may be attributed to the fact that the H-C probe can consume the overexpressed ROS in cancer cells, thereby more effectively inhibiting the metastasis and invasion of tumor cells.

[0157] (3) In vivo efficacy evaluation of the probe

[0158] Human triple-negative breast cancer cells MDA-MB-231 were cultured in DMEM medium containing 10% fetal bovine serum. Cells in the logarithmic growth phase were collected, digested into cell suspension with 0.25% trypsin, centrifuged to collect cells, and resuspended with normal saline. The cells were inoculated at a density of 1×10 6 / 100 μL / rat at the second mammary gland of Balb / c nude mice to construct MDA-MB-231 breast tumor-bearing nude mice. When the tumor reached 100 mm 3 , group treatment was carried out.

[0159] Accurately weigh 5 mg of probe H-C, and dissolve it with a mixed solvent of DMSO, PEG300, Tween-80 and normal saline with a volume ratio of 2:40:5:53 to obtain a probe H-C solution with a concentration of 2 mg / mL.

[0160] Accurately weigh 5 mg of HCPT, dissolve it with a mixed solvent of DMSO, PEG300, Tween-80 and normal saline in a volume ratio of 2:40:5:53 to obtain an HCPT solution with a concentration of 2 mg / mL.

[0161] Wait until the tumor volume grows to 100 mm 3 , and the tumor-bearing mice are randomly divided into 3 groups (n = 6): probe H-C treatment group (single-dose 10 mg / kg, 2 times / week, intraperitoneal injection); normal saline group (saline); HCPT treatment group (single-dose 10 mg / kg, 2 times / week, intraperitoneal injection). Each group of nude mice is administered on the 1st, 4th, 8th, 11th, and 15th days respectively. During the experiment, the body weight and tumor volume of the mice are measured. They are sacrificed on the 18th day, and the tumor tissues are collected.

[0162] Record the body weight of the tumor-bearing nude mice throughout the dosing cycle, weigh and calculate the relative change rate of the nude mice's body weight compared to the first day, and plot the body weight-time curve. As Figure 15 shown in A, the body weight of the tumor-bearing nude mice in the PBS group decreased during the dosing period, while the body weights of the normal saline group and the probe H-C treatment group did not show an obvious downward trend, indicating that the probe H-C has high biosafety.

[0163] From Figure 15 B, C, and D, it can be seen that the tumors in the probe H-C treatment group are smaller, showing the strongest anti-tumor effect. Figure 15 E shows that it can be seen that obvious cancer metastases occurred in both the normal saline group and the HCPT treatment group, while the probe H-C treatment group significantly inhibited the distal metastasis of triple-negative breast cancer.

Claims

1. A responsive self-luminescent probe based on AIE natural products with a structure as shown in Formula I:

2. A preparation method of the responsive self-luminescent probe based on AIE natural products according to claim 1, characterized in that: The synthetic route is as follows: It includes: using a mixed solvent of water and 4-methyl-tetrahydropyran as the reaction solvent, under the protection of N2, using Pd(PPh3)4 as the catalyst and K2CO3 as the acid-binding agent, reacting compound 6 and compound Int A to generate the probe shown in Formula I.

3. The preparation method of the responsive self-luminescent probe based on AIE natural products according to claim 2, wherein: The molar ratio of the said compound 6 to compound Int A is 1:1.0 - 1:1.5, preferably 1:1.1; The molar ratio of the said compound 6 to Pd(PPh3)4 is 1:0.1 - 1:0.3, preferably 1:0.1; The molar ratio of the said compound 6 to K2CO3 is 1:1 - 1:4, preferably 1:3; The reaction temperature is 70 - 120 °C; The volume ratio of water to 4-methyl-tetrahydropyran is 1:2 - 1:9, preferably 1:

4.

4. Use of the responsive self-luminescent probe based on AIE natural products according to claim 1 in the preparation of reagents or drugs for screening or diagnosing tumors.

5. The application according to claim 4, wherein: The said use is the use of the responsive self-luminescent probe based on AIE natural products in the preparation of reagents or drugs for specifically imaging and diagnosing tumors based on ROS.

6. The application according to claim 4, characterized in that: The said use is the use of the responsive self-luminescent probe based on AIE natural products in the preparation of reagents or drugs for diagnosing tumors with overexpression of ROS.

7. Use of the responsive self-luminescent probe based on AIE natural products according to claim 1 in the preparation of anti-tumor drugs.

8. The application according to claim 7, wherein: The said use is the use of the responsive self-luminescent probe based on AIE natural products in the preparation of drugs for inhibiting the proliferation and / or metastasis of tumors.

9. The application according to any one of claims 4-7, characterized in that: The said tumors include breast cancer, liver cancer, lung cancer, colon cancer, melanoma.

10. A pharmaceutical composition for diagnosing and / or treating tumors, characterized in that: The said pharmaceutical composition uses the responsive self-luminescent probe based on AIE natural products according to claim 1 as the main active ingredient or active ingredient.

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