Micromolecular bioluminescent probe capable of being used for monitoring in vivo / in vitro dipeptidyl peptidase IV in real time, and preparation method and application of micromolecular bioluminescent probe

Abstract

The invention discloses a micromolecular bioluminescent probe capable of being used for monitoring in vivo / in vitro dipeptidyl peptidase IV in real time and a preparation method and application thereof.The probe is marked as DPP4-PD, during preparation, N-[t-butyloxycarbonyl] glycyl-L-proline and 6-amino-2-benzothiazole formonitrile serve as starting materials and then react with L-cysteine hydrochloric acid, and finally Boc is removed. The synthesis raw materials are easy to obtain, operation is simple, and popularization is easy. The micromolecular bioluminescence probe provided by the invention can specifically respond to DPP4 enzyme in vivo / in vitro in real time; and the probe has the remarkable advantages of high selectivity, high sensitivity, high stability, high biological safety, high signal-to-noise ratio (S / N) and the like during imaging, and has wide application prospects in the fields of pathological research, efficacy evaluation and new drug research and development.

Description

Technical Field

[0001] The present invention belongs to the technical field of the detection of dipeptidyl peptidase IV, and particularly relates to a small molecule bioluminescent probe capable of real-time monitoring of dipeptidyl peptidase IV in vivo / in vitro, a preparation method and an application thereof. Background Art

[0002] Dipeptidyl peptidase-4 (DPP4), also known as CD26, is a cell transmembrane glycoprotein that participates in a variety of physiological and pathological processes. In particular, its role in the tumor microenvironment has gradually attracted attention. DPP4 not only degrades bioactive peptides as an enzyme, but also participates in functions such as cell signal transduction, immune regulation and cell migration. DPP4 plays an important role in the pathogenesis and progression of tumors such as liver cancer, melanoma, rectal cancer, pancreatic cancer, etc. The function of DPP4 is mainly reflected in its regulation of chemokines. The N-terminal cleavage mediated by DPP4 can limit the functions of chemokines (such as CXCL10 and CXCL12), thereby affecting the migration of T cells and natural killer cells. This mechanism has been confirmed in models such as melanoma and colorectal cancer. Animal models treated with DPP4 inhibitors (such as sitagliptin) show that DPP4 inhibition can enhance the anti-tumor immune response. This indicates that DPP4 may promote tumor growth by regulating the functions of immune cells in the tumor microenvironment. However, the function and role of DPP4 in breast cancer have not been fully elucidated.

[0003] Therefore, the development of an optical probe that specifically responds to DPP4 is of great significance for studying the pathogenesis of breast cancer and drug screening by responding to DPP4 in breast cancer. So far, however, fluorescent probes for DPP4 in diabetes, thyroid tumor disease models, and human blood and urine samples have been reported. However, there is no literature report on the application of DPP4 in breast cancer tumor models and anti-tumor drug screening. In addition, the self-limitations of fluorescent probes themselves, such as photobleaching and interference from the autofluorescence of the detected samples, cannot meet the purpose of highly sensitive detection of DPP4. Summary of the Invention

[0004] Object of the Invention: The technical problem to be solved by the present invention is to provide a small molecule bioluminescent probe capable of real-time monitoring of dipeptidyl peptidase IV in vivo / in vitro, a preparation method and an application thereof. This bioluminescent probe can detect DPP4 enzyme in real time, with high sensitivity, high selectivity and high contrast, effectively filling the defects of existing fluorescent probes in detecting DPP4 enzyme.

[0005] Technical Solution: The small molecule bioluminescent probe of the present invention capable of real-time monitoring of dipeptidyl peptidase IV in vivo / in vitro is denoted as DPP4-PD, and its chemical structure is shown as follows:

[0006]

[0007] Bioluminescence imaging is a non-invasive imaging method with high sensitivity and high signal-to-noise ratio, and is widely used in cell imaging, in vivo animal imaging, and drug screening. In the presence of specific substrates, such as luciferin or other luminescent molecules, after binding to luciferase, it catalyzes the substrate to be converted into an excited intermediate. When the excited intermediate returns to the ground state, a bioluminescence signal with a specific wavelength that can be detected is generated and captured and detected by a specific instrument. Usually, the light wavelength produced by the substrate is green light with a wavelength of 520-530 nm. After optimizing the structure of the substrate luciferin, the emission wavelength can reach 600 nm or even longer, which not only increases the biological penetration depth but also improves the stability.

[0008] As an optical imaging method different from fluorescence imaging, bioluminescence (BL) can generate strong optical signals without an external excitation light source. Therefore, BL can effectively avoid the current disadvantages of fluorescence probes in in vivo imaging and has significant advantages in imaging accuracy and biological safety.

[0009] Based on the existence of a long and narrow cavity structure in the active center of DPP4 enzyme, in the early stage of the present invention, a series of potentially active compounds were designed according to the protein crystal form characteristics of DPP4 enzyme, and the structures are shown as follows:

[0010]

[0011] And through computer-aided molecular docking technology (Discovery studio software), the optimal DPP4 enzyme bioluminescence probe structure DPP4-PD was screened out from the series of compounds. As Figure 1 shown: Among the series of probes, DPP4-PD can show strong hydrogen bond interactions with the His156, AGR125, and SER209 amino acid residues in the DPP4 enzyme protein crystal form (PDB code 2ONC), and shows the highest enzyme affinity (Cdocker interaction energy: -60.7327).

[0012] In vitro and in vivo imaging experiments showed that the DPP4-responsive bioluminescent probe DPP4-PD exhibited excellent sensitivity, high selectivity, high contrast, and a relatively large emission wavelength. The mechanism is as follows: The small-molecule bioluminescent probe DPP4-PD consists of a luciferin bioluminescence core and an L-proline fragment. Due to quenching, DPP4-PD itself has no optical signal. When it is specifically catalyzed by DPP4, the L-proline in the molecular structure can be selectively removed to generate compound (IV), which can rapidly generate compound (VI) under the action of firefly luciferase (fLuc) along with a strong optical signal.

[0013]

[0014] The method for preparing the above-mentioned small-molecule bioluminescent probe in the present invention has the following preparation route:

[0015]

[0016] The preparation method includes the following steps:

[0017] (1) Preparation of compound (II): Compound (I) and 6-amino-2-benzothiazolecarbonitrile are dissolved in an organic solvent at a molar ratio of 1:(1-4), and a condensing agent is added at -20 to 0 °C, and the mixture is stirred and reacted at 0 to 100 °C under the protection of an inert gas for 1 to 24 h to obtain the intermediate compound (II);

[0018] (2) Preparation of compound (III): Compound (II) and D-cysteine hydrochloride are stirred and reacted in a solvent at a molar ratio of 1:(1-2) at 0 to 100 °C to obtain compound (III);

[0019] (3) Preparation of DPP4-PD: Compound (III) is dissolved in an organic solvent, and ZnBr2 is added and stirred to react to obtain DPP4-PD.

[0020] Furthermore, in steps (1) and (3) of the preparation method of the present invention, the organic solvent is tetrahydrofuran, methanol, isopropanol, ethanol, ethyl acetate, acetone, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, chloroform, ether, or methyl tert-butyl ether.

[0021] Furthermore, in step (1) of the preparation method of the present invention, the addition amount of the condensing agent is 1-4 times the molar amount of compound (I), and it includes 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, thionyl chloride, Carter's condensing agent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N,N'-dicyclohexylcarbodiimide, 1-[bis(1H-benzotriazolyl)-methyl]-1,1,3,3-tetramethylammonium hydroxide trifluoroacetate, O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylurea tetrafluorophosphate, O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylaminotetrafluorophosphate or phenyl dihydrobenzotriazolyl tetrafluorophosphate.

[0022] Furthermore, in step (1) of the preparation method of the present invention, the inert protective gas is nitrogen or argon.

[0023] Furthermore, in step (2) of the preparation method of the present invention, the solvent is a mixed solvent of methanol and water with a volume ratio of (2-4):1.

[0024] Furthermore, in step (3) of the preparation method of the present invention, the molar ratio of compound (III) to ZnBr2 is 1:(1-10).

[0025] The application of the above-mentioned small molecule bioluminescent probe of the present invention in the preparation of in vivo / in vitro detection reagents or detection elements for dipeptidyl peptidase IV, and the detection element is a kit or a test strip.

[0026] Beneficial effects: Compared with the prior art, the remarkable advantages of the present invention are as follows: The bioluminescent probe DPP4-PD can be used for accurate detection of DPP4 enzyme activity and content fluctuations in real time, with high sensitivity, high selectivity, and high contrast; and this bioluminescent probe DPP4-PD can generate strong optical signals without an external light source, thus avoiding the interference of autofluorescence of biological tissues caused by the excitation light source, as well as photobleaching and light scattering phenomena. It has outstanding advantages in imaging sensitivity and detection accuracy, and without an external excitation light source, it can effectively avoid tissue burns caused by long-term irradiation, significantly improving biological safety during imaging; at the same time, its synthesis steps are simple, easy to promote and use, and have broad application prospects in the fields of pathological research, drug efficacy evaluation, and new drug research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a graph showing the results of the enzyme affinity of a series of DPP4 enzyme bioluminescent probe structures;

[0028] Figure 2 For the compound (II) prepared in Example 1 of the present invention 1 1H-NMR spectrum;

[0029] Figure 3 13C-NMR spectrum of the compound (II) prepared in Example 1 of the present invention 13 ;

[0030] Figure 4 ESI-HRMS spectrum of the compound (II) prepared in Example 1 of the present invention;

[0031] Figure 5 1H-NMR spectrum of the compound (III) prepared in Example 1 of the present invention 1 ;

[0032] Figure 6 13C-NMR spectrum of the compound (III) prepared in Example 1 of the present invention 13 ;

[0033] Figure 7 ESI-HRMS spectrum of the compound (III) prepared in Example 1 of the present invention;

[0034] Figure 8 1H-NMR spectrum of the bioluminescent probe DPP4-PD prepared in Example 1 of the present invention 1 ;

[0035] Figure 9 13C-NMR spectrum of the bioluminescent probe DPP4-PD prepared in Example 1 of the present invention 13 ;

[0036] Figure 10 ESI-HRMS spectrum of the bioluminescent probe DPP4-PD prepared in Example 1 of the present invention;

[0037] Figure 11 UV and fluorescence spectra of the bioluminescent probe DPP4-PD before and after incubation with DPP4 in the present invention;

[0038] Figure 12 HPLC spectra of DPP4-PD and NH2-Luc after incubation of DPP4-PD with DPP4 at 37 °C for 1 and 4 h;

[0039] Figure 13 ESI-HRMS spectrum; wherein, (a) is the ESI-HRMS of the product after the reaction of fluorescein with DPP4; (b) is the ESI-HRMS of the product after the reaction of DPP4-PD with DPP4;

[0040] Figure 14 Bioluminescence kinetic curve of the reaction of DPP4-PD with DPP4;

[0041] Figure 15Experimental graphs of DPP4-PD reacting with 0, 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, 100 ng / mL DPP4; among them, (a) are bioluminescence imaging pictures; (b) are linear fitting results;

[0042] Figure 16 Experimental result graphs of DPP4-PD reacting with different analytes; among them, (a) are bioluminescence results of reacting with some enzymes in organisms, some ions, and some biological small molecules; (b) are bioluminescence statistical graphs of reacting with some enzymes in organisms; (c) are bioluminescence statistical graphs of reacting with some ions and some biological small molecules;

[0043] Figure 17 Inhibitory effects of different concentrations of vildagliptin on DPP4; among them, (a) are imaging graphs and (b) are statistical graphs;

[0044] Figure 18 Cell survival rates corresponding to different concentrations of DPP4-PD; among them, (a) is the cell survival rate after co-incubating different concentrations of DPP4-PD with 4T1 for 24 h; (b) is the cell survival rate after co-incubating different concentrations of DPP4-PD with MCF10A cells for 24 h;

[0045] Figure 19 Experimental graphs of DPP4 endocytosis inhibition; among them, (a) are imaging graphs and (b) are statistical graphs;

[0046] Figure 20 Experimental graphs of different numbers of 4T1-Luc cells (0, 1000, 2000, 4000, 8000, 10000, 12000, 16000, and 20000) incubated with the probe DPP4-PD at 37 °C for 2 minutes; among them, (a) are imaging graphs and (b) are linear fitting curve graphs;

[0047] Figure 21 Experimental graphs of DPP4-PD with or without the inhibitor VDLP and 4T1-Luc at different time points; among them, (a) are bioluminescence graphs, (b) are line graphs of bioluminescence intensity, and (c) is a statistical graph of with or without VDLP at 2 min;

[0048] Figure 22 Time-dependent experimental result graphs of 4T1-Luc tumor-bearing mice with tumors for 7, 14, 21 days and intraperitoneally injected with VDLP; among them, (a) are bioluminescence images and (c) are bioluminescence intensity statistical graphs, and (b) is the tumor volume;

[0049] Figure 23Experimental result graphs of different organs (heart, liver, spleen, lung, kidney, tumor) of 4T1-Luc tumor-bearing mice at 7, 14, and 21 days of tumor formation; among them, (a) bioluminescence image and (b) statistical graph of bioluminescence intensity;

[0050] Figure 24 Experimental result graphs of time dependence of normal mice and 4T1-Luc breast cancer lung metastasis mice; among them, (a) bioluminescence image and (b) quantitative analysis; (c) bright-field and bioluminescence images of different organs (heart, liver, spleen, lung, kidney, tumor) of normal mice and 4T1-Luc tumor-bearing mice and (d) statistical graph of bioluminescence intensity. Detailed implementation manners

[0051] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0052] It should be noted that the compound (I) used in the following embodiments of the present invention is N-[tert-butoxycarbonyl]glycyl-L-proline. The organic solvents used in the following embodiments of the present invention only need to be in excess.

[0053] The method for preparing a small molecule bioluminescence probe for real-time monitoring of dipeptidyl peptidase IV in vivo / in vitro of the present invention includes the following steps:

[0054] (1) Prepare compound (II), and its route is as follows: Dissolve compound (I) and 6-amino-2-benzothiazolecarbonitrile in an organic solvent at a molar ratio of 1:(1-4), add a condensing agent at -20 to 0 °C, and the addition amount is 1 to 4 times the molar amount of compound (I). Stir at 0 to 100 °C for 1 to 24 h under the protection of an inert gas. After the reaction is completed, the solution is concentrated in vacuo, and then purified by silica gel chromatography using dichloromethane / methanol to obtain a yellow solid, namely the intermediate compound (II); the inert protective gas can be nitrogen or argon.

[0055]

[0056] (2) Prepare compound (III), and its route is as follows: Mix compound (II) and D-cysteine hydrochloride at a molar ratio of 1:(1-2) in a solvent (methanol and water with a volume ratio of (2-4):1), add anhydrous K2CO3 to adjust the pH value of the reaction solution to 6-8 (preferably 7.4), and stir and react at 0 to 100 °C under the protection of an inert gas for 1 to 3 h; after the reaction is completed, rotate and evaporate to remove methanol, dropwise add an acid to adjust the pH value of the reaction solution to 2-4 (preferably 3), and filter and dry to obtain a yellow powder compound (III).

[0057]

[0058] (3) Preparation of DPP4-PD: Dissolve compound (III) in an organic solvent, and then add ZnBr2 according to the molar ratio of compound (III) to ZnBr2 being 1:(1 - 10). Stir the reaction mixture for 1 - 4 h. After the reaction is completed, pour it into water, and then extract with ethyl acetate. Concentrate the combined organic layers under vacuum to obtain the crude probe DPP4-PD. Finally, the obtained crude probe is separated and purified by preparative liquid chromatography to obtain a highly pure bioluminescent probe DPP4-PD, which is a pale yellow powdery solid.

[0059] Example 1

[0060] (1) Preparation of compound (II)

[0061] Add (EEDQ, 1.08 g, 3.6 mmol) to a solution of compound (I) (500 mg, 1.8 mmol) and 6-amino-2-benzothiazolecarbonitrile (643 mg, 3.6 mmol) in 20 mL of DCM at 0 °C. Then stir the mixture at room temperature for 12 h. After the reaction is completed, monitor by TLC, concentrate the obtained solution under vacuum to obtain the crude compound (II), and purify it by silica gel chromatography to obtain a yellow solid. As Figures 2 to 4 shown, 1 1H NMR (600 MHz, DMSO-d6) δ 10.47 (s, 1H, Ar NH CO), 8.68 (s, 1H, Ar- H ), 8.16 (d, 1H, J = 8.7 Hz, Ar- H ), 7.38 (d, 1H, J = 8.7 Hz, Ar- H ), 6.79 (s, 1H, NH CO), 4.48 - 4.45 (m, 1H, N CH ), 3.84 - 3.69 (m, 2H, NCH 2), 3.54 (d, 2H, J = 11.2 Hz, N CH 2), 2.16 - 2.09 (m, 2H, CH2 CH 2CH), 2.01 - 1.90 (m, 2H, CH 2CH2CH), 1.34 (s, 9H, C (CH 3)3); 13CNMR(151MHz,DMSO-d6) 13C NMR(151MHz, dmso) δ = 171.64, 168.09, 156.21, 148.02, 139.97, 137.08, 135.35, 125.16, 121.21, 113.99, 111.70, 78.34, 60.85, 46.32, 42.84, 29.66, 28.62, 24.94.; MS(ESI): calcd. for C 20 H 23 N5O4S [M+Na] + , 452.15; found 452.139。

[0062] (2) Preparation of compound (Ⅲ)

[0063] Compound (Ⅱ) (100 mg, 2.3 mmol), K2CO3 (32 mg, 2.3 mmol), and cysteine (40 mg, 2.3 mmol) were stirred at room temperature under nitrogen protection in a 10 mL solution of methanol:water with a volume ratio of 1:2. After the reaction was completed, it was monitored by TLC, the methanol was removed by rotary evaporation, and 1M HCl was added dropwise to adjust the pH of the reaction solution to 3. The yellow solid was collected by filtration and dried. As Figures 5 to 7 shown 1 1H NMR(600MHz,DMSO-d6) δ 13.16(bs, 1H, COOH), 10.36(s, 1H, Ar NH CO), 8.56(s, 1H, Ar- H ), 8.06(d, 1H, J = 8.7Hz, Ar- H ), 7.62(d, 1H, J = 8.7Hz, Ar- H ), 6.79(s, 1H, NH CO), 5.41(t, 1H, J = 8.9Hz, N CH ), 4.49 - 4.43(m, 1H, CH COOH), 3.86 - 3.77(m, 2H, NCH 2), 3.76 - 3.65(m, 2H, S CH 2), 3.54(d, 2H, J = 11.2Hz, N CH 2), 2.17 - 1.96(m, 2H, CH2 CH 2CH), 1.92 - 1.78(m, 2H, CH 2CH2CH), 1.34(s, 9H, C (CH 3)3); 1313C NMR (151 MHz, DMSO-d6) δ = 171.52, 171.46, 168.09, 164.84, 159.43, 156.23, 149.00, 138.80, 136.68, 124.63, 120.13, 111.95, 78.54, 78.34, 60.83, 46.33, 42.85, 40.10, 35.19, 29.66, 28.62, 24.92.; MS (ESI): calcd. for C 23 H 27 N5O6S2 [M+H] + : 534.14; found 534.149.

[0064] (3) Preparation of compound DPP4-PD

[0065] To a solution of compound (III) (60 mg, 0.09 mmol) in dichloromethane was added ZnBr2 (40 mg, 0.18 mmol). The reaction mixture was stirred for 30 min, then poured into water and subsequently extracted with ethyl acetate. The organic layer was concentrated in vacuo to give a yellow solid, which was further purified by preparative liquid chromatography to give the bioluminescence probe DPP4-PD. As Figures 8 to 10 shown, 1 1H NMR (600 MHz, DMSO-d6) δ 10.78 (s, 1H, Ar NH CO), 8.60 (s, 1H, Ar- H ), 8.26 (s, 2H, NH2), 8.06 (d, 1H, J = 8.7 Hz, Ar- H ), 7.76 (d, 1H, J = 8.7 Hz, Ar- H ), 5.41 (t, 1H, J = 8.9 Hz, N CH ), 4.60 - 4.54 (m, 1H, CH COOH), 3.88 - 3.82 (m, 2H, NCH 2), 3.80 - 3.74 (m, 2H, S CH 2), 3.71 - 3.58 (m, 2H, N CH 2), 2.24 - 2.08 (m, 2H, CH2 CH 2CH), 2.03 - 1.87 (m, 2H, CH 2CH2CH; 1313C NMR(151MHz,DMSO-d6) δ=171.48, 171.01, 165.08, 164.83, 159.40, 149.01, 138.91, 136.58, 124.50, 120.13, 111.88, 78.53, 66.79, 60.95, 46.55, 35.22, 30.06, 24.73.; MS(ESI): calcd. for C 18 H 19 N5O6S2 [M+H] + , 434.09; found 434.0953.

[0066] Performance Detection 1

[0067] Dilute with Tris-HCl (pH 7.4) to a final concentration of 2 mL of reaction solution containing DPP4-PD (10 μM), ATP (2 mM), Mg 2+ (10 mM), and luciferase (0.1 mg / mL). Immediately add the freshly prepared detection reaction solution sample to a quartz cuvette, and use a UV-visible spectrophotometer and a fluorescence spectrometer to detect the UV absorption spectrum and fluorescence spectrum of DPP4-PD before and after adding DPP4, respectively.

[0068] As Figure 11 shown, the UV (black) and fluorescence (red) spectra of the bioluminescent probe DPP4-PD in the present invention before (dashed line) and after (solid line) incubation with DPP4. The results show that both the UV and fluorescence change significantly after the reaction of DPP4-PD with DPP4, indicating that DPP4 can effectively act on the probe DPP4-PD.

[0069] As Figure 12 shown, the HPLC spectra of the reaction solution of the bioluminescent probe DPP4-PD in the present invention before incubation with DPP4 (black) and after incubation at 37 °C for 1 h (light blue) or 4 h (dark green), as well as the solution of the control compound luciferin (red). As Figure 13 shown, the mass spectrometry results show that the main product formed after the reaction of DPP4-PD with DPP is luciferin, which confirms the luminescence mechanism of DPP4-PD shown in the above flow chart of the DPP4-PD luminescence mechanism.

[0070] Performance Detection 2

[0071] As Figure 14 shown, use a microplate reader to investigate the enzymatic cleavage kinetics of DPP4-PD. The reaction solution containing luciferase (0.1 mg / mL), ATP (1 mM), Mg 2+After adding DPP4 to the Tris-HCl (50 mM, 7.4) solution of DPP4-PD (10 μM) (10 mM), the bioluminescence signal increased rapidly and reached its peak at 1 min, then decreased within 10 min. Compared with before adding DPP4, the bioluminescence signal intensity increased by approximately 241 times after reacting with DPP4.

[0072] Performance Detection 3

[0073] As Figure 15 shown, the working solution of DPP4-PD (10 μM) in the present invention contains 0.1 mg / mL luciferase, 1 mM ATP, 10 mM Mg 2+ of Tris-HCl (50 mM, 7.4) was co-incubated with DPP4 at different concentrations (0, 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, 100 ng / mL), and the bioluminescence signal was immediately recorded using a small animal imager. As the concentration of DPP4 enzyme in the reaction system increased, the bioluminescence signal intensity also increased. The mean values of the DPP4 enzyme concentration and the bioluminescence intensity in their corresponding wells were linearly fitted. In the range of 0 - 100 ng / mL, there was a good linear relationship between the bioluminescence signal intensity and the enzyme concentration. Subsequently, the LOD was calculated to be 0.095 ng / mL. The results indicate that in vitro experiments, DPP4-PD has high detection sensitivity for DPP4.

[0074] Performance Detection 4

[0075] As Figure 16 shown, some enzymes in the body were selected (carboxylesterase 1 (CES1, 100 ng / mL), carboxylesterase 2 (CES2, 100 ng / mL), DPPVIII (100 ng / mL), aminopeptidase N (APN, 100 ng / mL), butyrylcholinesterase (BuchE, 100 ng / mL), alkaline phosphatase (ALP, 100 ng / mL), cytochrome P450 enzyme 2J2 (CYP2J2, 100 ng / mL), DPP4 (100 ng / mL), ions (Ca 2+ (1 mM), K + (1 mM), Na + (1 mM), Mg 2+ (1 mM), HPO4 - (1 mM), H2PO4 - (1 mM)), reactive oxygen species (H2O2 (100 μM), ClO -(100 μM)) and some small biological molecules (glucose (500 μM), GSH (500 μM), glutamate (Glu, 500 μM), ascorbic acid (100 μM), uric acid (100 μM)). After adding these analytes into the DPP4-PD working solution, it was immediately detected with a small animal imager. Only adding DPP4 to the DPP4-PD working solution produced bright bioluminescence, while the bioluminescence intensity produced by adding the remaining analytes was so weak that it could be ignored.

[0076] Performance detection 5

[0077] As Figure 17 shown, the specificity of DPP4-PD for DPP4 detection was further investigated by the DPP4 inhibitor vildagliptin (VDLP). First, DPP4 (100 ng / mL) was mixed with different concentrations of VDLP (0, 1.25, 2.5, 5, 10, 20 μM), then added to the DPP4-PD working solution, and detected with a small animal imager and statistically analyzed. As the concentration of VDLP increased, the bioluminescence signal intensity generated by DPP4-PD and DPP4 also decreased significantly. Therefore, DPP4-PD has good selectivity and specificity for DPP4 detection in vitro.

[0078] Performance detection 6

[0079] As Figure 18 shown, different concentrations (0, 5, 10, 20, 40, 80 μM) of DPP4-PD were co-incubated with mouse breast cancer cells 4T1 and human normal mammary epithelial cells MCF10A for 24 h, and then the cell viability corresponding to different concentrations of DPP4-PD was calculated by the ratio of absorbance values. When the probe concentration was as high as 80 μM, the survival rates of the two cell lines were still greater than 85%, and no obvious growth inhibition was observed. The results showed that DPP4-PD had low cytotoxicity and high safety.

[0080] Performance detection 7

[0081] As Figure 19 shown, cells were pretreated with inhibitors of 4 endocytic mechanisms for 1 h: chlorpromazine (CZP) inhibits clathrin-mediated invagination, nystatin inhibits the caveolin pathway, amiloride hydrochloride inhibits Na 2+ / H + exchange (macropinocytosis), and energy-dependent endocytosis is inhibited when cultured at 4 °C. The bioluminescence intensity produced by 4T1-Luc cells pretreated with CZP was significantly lower than that of the control group without any treatment, and the bioluminescence intensity of the remaining inhibition groups only decreased slightly. Therefore, DPP4-PD mainly enters cells through clathrin-mediated invagination.

[0082] Performance detection 8

[0083] As Figure 20 shown, DPP4-PD in the present invention was added to 4T-1-Luc cells at different densities (0, 1000, 2000, 4000, 8000, 10000, 12000, 16000, 20000 cells / well), and the bioluminescence signals in each group of wells were immediately collected using a small animal imager. As the cell density increased, the bioluminescence signal intensity of DPP4-PD also increased. Therefore, the bioluminescence signal intensity of the probe was positively correlated with the cell density. The results of fitting the bioluminescence signal intensity and cell density showed that within the range of 1000 - 20000 cells per well, the bioluminescence signal intensity of the probe had a good linear relationship, and the linear regression equation was (Y = 52.47X + 19414.84, R 2 = 0.998), and the limit of detection LOD was 300 (3δ / k). The results showed that DPP4-PD had good sensitivity in cell imaging.

[0084] Performance detection 9

[0085] As Figure 21 shown, DPP4-PD was added to 4T1-Luc cells without inhibitor and 4T1-Luc cells pretreated with 20 μM DPP4 inhibitor VDLP for 5 min respectively. The bioluminescence signal intensity generated by DPP4-PD in 4T1-Luc cells without inhibitor reached the peak rapidly at 2 min and gradually decreased within the subsequent 8 min. In addition, the bioluminescence intensity generated by 4T1-Luc cells pretreated with vildagliptin for 5 min at 2 min was significantly lower than that of the cells in the group without inhibitor added.

[0086] Performance detection 10

[0087] As Figure 22As shown in the figure, according to the number of days of tumor formation and administration of DPP4 inhibitors, the experimental mice were divided into: 7-day tumor formation group, 14-day tumor formation group, 21-day tumor formation group, and inhibitor VDLP group. Before imaging, each mouse was given DPP4-PD (100 μM, 50 μL) via the tail vein, and then immediately photographed with a small animal imager to record the bioluminescence signal at the tumor site of each mouse at different time points. A strong bioluminescence signal was rapidly generated at the tumor site of the mice injected with DPP4-PD, reaching a peak at 2 min. Subsequently, it gradually decayed within 10 min. As the number of days of tumor formation increased and the tumor volume increased accordingly, the tumor volumes of the 21-day tumor formation group were 8.7 times and 3.3 times those of the 7-day group and 14-day group, respectively. The bioluminescence signals generated by the 21-day tumor formation group were 12 times and 4.5 times those of the 7-day group and 14-day group, respectively. After intraperitoneal injection of VDLP to the mice in the 21-day tumor formation group before imaging, the bioluminescence signal intensity was significantly reduced, and only a weak bioluminescence signal was generated. Compared with the control group, the signal intensity was reduced by 67.6 times. This indicates that under the hydrolysis of DPP4, a large amount of DPP4-PD can be rapidly converted into NH2-luciferin, which produces a bright bioluminescence signal under the catalysis of firefly luciferase in 4T1-Luc cells. When the DPP4 inhibitor VDLP was used, only a small amount of DPP4-PD was converted into NH2-luciferin. Therefore, the bioluminescence signal at the tumor site of the mice was significantly reduced. Based on the experimental results, it was found that after tail vein injection of DPP4-PD, it can be rapidly hydrolyzed by DPP4 enzyme to generate NH2-luciferin and produce a bioluminescence signal, and it can be used to track and monitor the growth volume of tumors. In addition, DPP4-PD can also become a screening tool for the development of DPP4 inhibitors, meeting the needs of screening experiments at the in vivo animal level.

[0088] Performance detection 10

[0089] As Figure 23 shown, after the imaging experiment, the hearts, livers, spleens, lungs, and tumor tissues of the mice were removed, and DPP4-PD was sprayed on their surfaces. Subsequently, a small animal imager was used to record the bioimaging of the four groups of different tissues. It can be found from the figure that DPP-PD only produced a bioluminescence signal at the tumor site, and its signal intensity increased with the increase of the tumor volume, and the bioluminescence signal of the VDLP inhibitor group was significantly weaker than that of the 21-day tumor formation control group.

[0090] Performance detection 11

[0091] As Figure 24As shown, 7 days after tail vein injection of PBS and 4T1-Luc cells, the mice were given DPP4-PD (100 μM, 50 μL) by tail vein injection, and then immediately subjected to bioluminescence imaging experiments using a small animal imager. Bioluminescence images were recorded in real time at each time point, and the bioluminescence signals in the area within the dotted line in the lungs were quantitatively analyzed. No bioluminescence signal was generated in the lung area of the normal control group of mice injected with PBS, while bright bioluminescence signals were generated in the lung area of the mice injected with 4T1-Luc cells by tail vein, and no bioluminescence signal was observed in the remaining areas. The bioluminescence signal rapidly reached its peak at 2 min, and then the intensity of the bioluminescence signal in the lung area gradually decayed over time. The bioluminescence signal at the 2-min time point was 98.4 times that of the normal control group. To verify that 4T1-Luc only metastasized to the lungs and thus produced bioluminescence signals, the mice after imaging were sacrificed and the heart, liver, spleen, lungs, and kidneys were removed. After spraying DPP4-PD on their surfaces, bright-field pictures of the organs were recorded using a camera and bioluminescence pictures taken by the small animal imager. The lungs of the mice with breast cancer lung metastasis were swollen and significantly larger than those of the normal control group of mice. Only bright bioluminescence signals were observed in the lungs of the breast cancer lung metastasis model mice in the bioluminescence imaging images, while no bioluminescence signals were observed in the other organs. Through quantitative statistics of the bioluminescence signals in each organ of the two groups of mice, it was found that the bioluminescence signals generated in the lungs of the mice with breast cancer lung metastasis were significantly higher than those of the normal control group. The bioluminescence signals generated in the other organs were very weak in both the normal control group of mice and the breast cancer lung metastasis group of mice. DPP4-PD can be used for bioluminescence imaging of tumor tissues in a breast cancer lung metastasis model. In addition, DPP4-PD has good tissue penetration and high imaging resolution.

[0092] In summary, the small molecule bioluminescence probe of the present invention can specifically respond to DPP4 enzyme in vivo / in vitro in real time; and this probe has significant advantages such as high selectivity, high sensitivity, high stability, high biosafety, and high signal-to-noise ratio (S / N) during imaging, and it has broad application prospects in the fields of pathological research, efficacy evaluation, and new drug development. It can be used to prepare in vivo / in vitro detection reagents or detection elements for dipeptidyl peptidase IV, and this detection element is a kit or a test strip.

[0093] Except for the above Example 1, the bioluminescence probe can be successfully prepared according to the preparation method of the present invention, and the same technical effects can be achieved, so no further examples will be given here.

Claims

1. A small molecule bioluminescent probe that can be used for real-time monitoring of dipeptidyl peptidase IV in vivo / in vitro, characterized in that: The bioluminescent probe is denoted as DPP4-PD, and its chemical structure is shown below:

2. A method for preparing the small molecule bioluminescent probe according to claim 1, characterized in that: The preparation route is as follows: The preparation method comprises the following steps: (1) Preparation of compound (II): dissolve compound (I) and 6-amino-2-benzothiazolecarbonitrile in an organic solvent at a molar ratio of 1:(1-4), add a condensing agent at -20-0°C, and react under inert gas protection at 0-100°C for 1-24 hours to obtain intermediate compound (II); (2) Preparation of compound (III): Compound (II) and D-cysteine ​​hydrochloride are reacted in a solvent at a molar ratio of 1:(1-2) under stirring at 0-100° C. to obtain compound (III); (3) Preparation of DPP4-PD: Compound (III) was dissolved in an organic solvent, and ZnBr2 was added and stirred to obtain DPP4-PD.

3. The method for preparing a small molecule bioluminescent probe according to claim 2, characterized in that: In step (1) and step (3), the organic solvent is tetrahydrofuran, methanol, isopropanol, ethanol, ethyl acetate, acetone, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, chloroform, diethyl ether or methyl tert-butyl ether.

4. The method for preparing a small molecule bioluminescent probe according to claim 2, characterized in that: In step (1), the amount of the condensing agent added is 1-4 times the molar amount of compound (I), which includes 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, thionyl chloride, Carter's condensing agent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N,N'-dicyclohexylcarbodiimide, 1-[bis(1H-benzotriazolyl)-methyl]-1,1,3,3-tetramethylammonium hydroxide trifluoroacetic acid, O-(benzotriazole-1-yl)-N,N,N',N'-tetramethyluronium tetrafluorophosphate, O-(benzotriazole-1-yl)-N,N,N',N'-tetramethylamino tetrafluorophosphate or phenyldihydrobenzotriazolyl tetrafluorophosphate.

5. The method for preparing a small molecule bioluminescent probe according to claim 2, characterized in that: In step (1), the inert protective gas is nitrogen or argon.

6. The method for preparing a small molecule bioluminescent probe according to claim 2, characterized in that: In step (2), the solvent is a mixed solvent of methanol and water in a volume ratio of (2-4):

1.

7. The method for preparing a small molecule bioluminescent probe according to claim 2, characterized in that: In step (3), the molar ratio of the compound (III) to ZnBr2 is 1:(1-10).

8. Use of the small molecule bioluminescent probe according to claim 1 in the preparation of an in vivo / in vitro detection reagent or detection element for dipeptidyl peptidase IV, wherein the detection element is a kit or a test paper.

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