NIRF / PA dual-modal probe based on NTR response and its preparation method and application

Abstract

The present invention discloses a NIRF / PA dual-modal probe based on NTR response, a preparation method thereof, and an application thereof, belonging to the field of biomedicine technology. The NIRF / PA dual-modal probe based on NTR response can rapidly respond to NTR, with response saturation in about 5 minutes, good selectivity for nitroreductase, and high sensitivity. The probe absorbs and emits in the near-infrared region, which can effectively overcome interference from biological background signals. The probe is a fluorescence / photoacoustic dual-modal ratiometric probe, which can achieve self-correction during the imaging process of biological systems and improve imaging accuracy. The probe realizes near-infrared fluorescence / photoacoustic dual-modal ratiometric imaging of pancreatic cancer tumors in mice, reflecting the clinical application prospects of the present invention in imaging-mediated pancreatic cancer tumor surgery.

Description

Technical Field

[0001] The present invention relates to an NTR response-based NIRF / PA dual-modal probe and a preparation method and application thereof, belonging to the technical field of biomedicine. Background Art

[0002] Nitroreductase (NTR) plays a crucial role in the detection and imaging of malignant tumors, serving as a key marker for tumor hypoxia. Hypoxia is a specific marker of the tumor microenvironment and is closely associated with the development and metastasis of cancer. A hypoxic microenvironment not only promotes tumor cell proliferation and invasion but also reduces tumor sensitivity to chemotherapy, radiotherapy, and photodynamic therapy, severely impacting the effectiveness of cancer treatment. As a key marker for tumor hypoxia, NTR expression is closely correlated with the degree of tumor hypoxia.

[0003] So far, many fluorescent probes have been reported for the detection of NTR, but these probes are generally single-channel enhanced probes. In order to solve the problems of insufficient penetration depth of near-infrared fluorescence (NIRF) and low sensitivity of photoacoustic (PA) imaging probes, it is of great significance to develop ratiometric NIRF / PA dual-modality probes. PA imaging can diagnose and locate tumors non-invasively before surgery, while NIRF can accurately image pancreatic cancer tissue and micrometastases during intraoperative surgical navigation. This multimodal imaging strategy meets the multifaceted needs of detection and treatment of different stages of cancer and minimizes external interference. In addition, most fluorescent probes respond slowly to NTR, usually taking more than 20 minutes. Considering the clinical demand for rapid pathological evaluation and fluorescence-guided tumor resection, fluorescent probes that can respond quickly to NTR are particularly important for bedside pathological diagnosis and intraoperative surgical navigation.

[0004] To accurately image pancreatic cancer by reflecting tumor hypoxia levels, NTR probes must meet several requirements: high penetration depth, high imaging resolution, 3D spatial imaging, rapid response, and excellent selectivity. Furthermore, given the complex microenvironment within the tumor, the probe must possess robust anti-interference and self-calibration capabilities to significantly reduce false-positive signals caused by interfering substances. Therefore, developing a NIRF / PA dual-modality probe that meets these requirements is crucial. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the purpose of the present invention is to provide a NIRF / PA dual-modal probe based on NTR response, as well as its preparation method and application, to solve the problems that the existing fluorescence / photoacoustic probes cannot be effectively used for high-sensitivity detection and high-contrast imaging of NTR in tumors due to high biological background fluorescence / photoacoustic signals and slow NTR response speed, and cannot be effectively used for imaging-mediated surgical treatment of pancreatic cancer.

[0006] The present invention also provides a NIRF / PA dual-modality probe (SiRho-SHD-NTR) based on NTR response, the structural formula of the probe is as follows:

[0007]

[0008] The mechanism of the probe (SiRho-SHD-NTR) detecting nitroreductase is as follows:

[0009]

[0010] The present invention also provides a method for preparing the NIRF / PA dual-modality probe based on NTR response, comprising the following steps:

[0011] (1) Compound 1 was dissolved in anhydrous acetonitrile and transferred to a round-bottom flask under an inert atmosphere. Phosphorus oxychloride was then slowly added to the reaction system. The mixture was stirred at a set temperature for reaction. After the reaction was completed, the solvent was removed by rotary evaporation to obtain Compound 2.

[0012] (2) 1-Boc-piperazine and compound 2 were dissolved in anhydrous acetonitrile, and triethylamine was slowly added. The reaction mixture was stirred at room temperature, and the reaction mixture was concentrated under reduced pressure. Compound 3 was obtained after extraction and purification;

[0013] (3) Compound 3 was dissolved in a mixture of trifluoroacetic acid and dichloromethane and stirred at room temperature. After the reaction was completed, the solvent was removed by rotary evaporation and the round-bottom flask was placed in a vacuum drying oven overnight to obtain compound 4;

[0014] (4) Compound 5 and 3-hydroxythiophenol were dissolved in anhydrous N,N-dimethylformamide under an inert atmosphere, and triethylamine was subsequently added. The mixture was stirred at a predetermined temperature. After the reaction was completed, the reaction mixture was diluted with dichloromethane and washed and extracted with a saturated sodium chloride solution for multiple times. The mixture was then dried, rotary evaporated to remove the solvent, and purified to obtain compound 6.

[0015] (5) Compound 6 was dissolved in methanol, and then concentrated hydrochloric acid was added and stirred at room temperature to react. After the reaction was completed, compound 7 was obtained;

[0016] (6) Compound 7 was dissolved in dichloromethane, and then acetic anhydride and triethylamine were added in sequence and reacted at room temperature. After the reaction was completed, the reaction mixture was washed and extracted with saturated sodium chloride solution several times, and then dried, rotary evaporated to remove the solvent, and purified to obtain compound 8;

[0017] (7) Compound 4 and Compound 8 were dissolved in dichloromethane, and HATU and DIPEA were subsequently added in sequence. The mixture was stirred at room temperature. After the reaction was completed, the reaction mixture was washed and extracted with saturated sodium chloride solution several times, and then dried, rotary evaporated to remove the solvent, and purified to obtain Compound 9;

[0018] (8) Compound 9 was dissolved in methanol, and then concentrated hydrochloric acid was added and stirred at room temperature to react. After the reaction was completed, the dye (SiRho-SHD-OH) was obtained;

[0019] (9) The dye obtained in step (8) is dissolved in anhydrous N,N-dimethylformamide, and then potassium carbonate and sodium iodide are added, and the reaction is stirred at a set temperature. After the reaction is completed, a tetrahydrofuran solution of p-nitrobenzyl bromide is added under an inert atmosphere, and the mixture is stirred at room temperature overnight. The reaction mixture is then washed and extracted with a saturated sodium chloride solution for multiple times, and then dried, rotary evaporated to remove the solvent, and purified to obtain a NIRF / PA dual-modality probe based on NTR response (SiRho-SHD-NTR);

[0020] The reaction formula is:

[0021]

[0022] In step (1), the molar ratio of compound 1 to phosphorus oxychloride is 1:(0.5-1).

[0023] In step (2), the molar ratio of compound 2, 1-Boc-piperazine and triethylamine is 1:(1.5-2):(1.5-2).

[0024] In step (3), the amount of compound 3 added is 0.1-0.4 mmol, the volume ratio of trifluoroacetic acid and dichloromethane is 1:(4.0-5.0), and the total volume of the solvent is 20-30 mL.

[0025] In step (4), the molar ratio of compound 5, 3-hydroxythiophenol and triethylamine is 1:(1.2-1.5):(1.2-1.5).

[0026] In step (5), the molar ratio of compound 6 to concentrated hydrochloric acid is 1:(3-5), and the mass fraction of concentrated hydrochloric acid is 36-38%.

[0027] In step (6), the molar ratio of compound 7, acetic anhydride and triethylamine is 1:(1.2-1.5):(1.2-1.5).

[0028] In step (7), the molar ratio of compound 4, compound 8, HATU and DIPEA is 1:(1.1-1.2):(1.2-1.5):(2-3).

[0029] In step (8), the molar ratio of compound 9 to concentrated hydrochloric acid is 1:(3-5), and the mass fraction of concentrated hydrochloric acid is 36-38%.

[0030] In step (9), the molar ratio of the dye, p-nitrobenzyl bromide, potassium carbonate and sodium iodide is 1:(1.1-1.3):(1.5-2):(2-3).

[0031] The present invention also provides applications of the NTR-responsive NIRF / PA dual-modality probe, which is used to detect NTR in samples, cells, tissues, and living mice.

[0032] The present invention also provides a detection solution of the NIRF / PA dual-modal probe based on NTR response, and the preparation process is as follows:

[0033] The probe is dissolved in dimethyl sulfoxide and then added into a DPBS buffer solution. The probe concentration is 5 to 10 μM and the volume ratio of dimethyl sulfoxide is 1 to 30%.

[0034] The best test results can be obtained when the probe concentration is 5-10 μM and the volume ratio of dimethyl sulfoxide is 10%.

[0035] The present invention proposes a strategy for detecting NTR based on a dual-mode ratiometric signal of FRET and designs a NIRF / PA dual-mode ratiometric probe SiRho-SHD-NTR. The probe itself has a strong fluorescence at 680nm (FL 680nm ) and photoacoustic (PA 680nm ) signal. When the probe reacts with NTR, the fluorescence emission and photoacoustic signal of the FRET system receptor are enhanced. The photoacoustic (PA) 740nm ) signal is enhanced, and the fluorescence (FL) at 760 nm 760nm ) signal is enhanced, while FL 680nm and PA 680nmRemaining unchanged, ratiometric fluorescence / photoacoustic signal detection of NTR can be achieved. The probe has a strong interaction with the NTR enzyme, and molecular docking simulations show that the binding energy between NTR and the probe is -9.279 kcal / mol (a value generally lower than -7 kcal / mol indicates a strong binding affinity). It is worth noting that the lower affinity value indicates that the binding energy between the probe and NTR is lower, making it easier for NTR to dissociate from the reduced probe and participate in the subsequent catalytic process, confirming the rapid response ability of the probe and NTR, which is beneficial to the imaging of living tumors and intraoperative surgical navigation applications. Therefore, the probe realizes rapid, highly sensitive detection of NTR in living tumors and near-infrared fluorescence / photoacoustic dual-modal ratio imaging. The present invention can well overcome the interference of biological background fluorescence / photoacoustic signals, and is expected to improve the accuracy and sensitivity of in vivo imaging analysis of NTR, and be applied to surgical navigation of pancreatic cancer and metastatic tumors.

[0036] The beneficial technical effects of the present invention are:

[0037] 1) The NTR-responsive NIRF / PA dual-modal probe can rapidly respond to NTR, reaching saturation in about 5 minutes, with good selectivity for nitroreductase and high sensitivity (detection limit 50 ng / mL);

[0038] 2) The probe absorbs and emits in the near-infrared, which can effectively overcome the interference of biological background signals;

[0039] 3) The probe is a fluorescence / photoacoustic dual-modality ratiometric probe that can achieve self-correction during the imaging process of biological systems, thereby improving imaging accuracy;

[0040] 4) The probe achieved near-infrared fluorescence / photoacoustic dual-modality ratiometric imaging of pancreatic cancer tumors in mice, reflecting the clinical application prospects of imaging-mediated pancreatic cancer tumor surgery of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The UV spectrum (a) and fluorescence spectrum (b) of the probe SiRho-SHD-NTR in response to 0-20 μg / mL NTR.

[0042] Figure 2 The full photoacoustic spectrum of the SiRho-SHD-NTR probe's response to NTR (a), the quantification of the photoacoustic intensity after the probe's response to 0-20 μg / mL NTR (b, c), and its photoacoustic imaging (d) are shown. The inset shows the linear relationship between the photoacoustic ratio signal of the probe's response to low concentrations of NTR.

[0043] Figure 3 The photoacoustic selectivity image (a), photoacoustic ratio signal selectivity (b), fluorescence selectivity (c) and chemical kinetic curve (d) of the probe SiRho-SHD-NTR.

[0044] Figure 4 Confocal imaging of intracellular NTR using the probe SiRho-SHD-NTR (a), schematic diagram of the mechanism of cobalt chloride-induced cell hypoxia (b), and quantification bar graph of cell fluorescence ratio signals (c).

[0045] Figure 5 The figures are the ratiometric photoacoustic imaging image of the probe SiRho-SHD-NTR on the living tumor (a), the photoacoustic intensity quantification images of the tumor area (b) and normal tissue (c), and the photoacoustic ratio signal quantification histogram of the tumor area and normal tissue (d).

[0046] Figure 6 The photoacoustic scanning imaging images of each section of a living tumor using the probe SiRho-SHD-NTR (a), the photoacoustic ratio signal quantification images of each section of normal tissue (b) and tumor area (c).

[0047] Figure 7 Schematic diagram of the experimental model for ratiometric near-infrared fluorescence imaging of living tumors by the probe SiRho-SHD-NTR (a), ratiometric near-infrared fluorescence imaging image (b), quantification of near-infrared fluorescence intensity of normal tissue (c) and tumor area (d), and quantification of near-infrared fluorescence ratio signals of tumor area and normal tissue (e).

[0048] Figure 8 This is the near-infrared fluorescence imaging of living in situ tumors by spraying the probe SiRho-SHD-NTR.

[0049] Figure 9 This is the detection principle of the probe SiRho-SHD-NTR. DETAILED DESCRIPTION

[0050] The present invention is further described below with reference to specific examples, but the present invention is not limited to the following examples. The methods described are conventional methods unless otherwise specified, and the raw materials described can be obtained from public commercial channels unless otherwise specified.

[0051] Example 1

[0052] Synthesis of probe SiRho-SHD-NTR

[0053] (1) Compound 1 was dissolved in 50 mL of anhydrous acetonitrile, placed in a 250 mL round-bottom flask, and operated under nitrogen protection. Then, 2 mL of phosphorus oxychloride was slowly added to the reaction system. The mixture was stirred at 88°C for 5 hours. After the reaction was completed, the solvent was removed by rotary evaporation without further purification to obtain compound 2;

[0054] (2) 1-Boc-piperazine and compound 2 were dissolved in 50 mL of anhydrous acetonitrile. 1 mL of triethylamine was then slowly added to the reaction system and stirred at room temperature for 30 minutes. The mixture was concentrated under reduced pressure and extracted with a saturated sodium chloride solution and dichloromethane. The crude product was then separated and purified by column chromatography using dichloromethane-ethanol as the mobile phase to obtain compound 3.

[0055] (3) Compound 3 was dissolved in a mixture of trifluoroacetic acid and dichloromethane at a volume ratio of 1:(4.0-5.0) and stirred at room temperature for 2 hours. After the reaction was complete, the solvent was removed by rotary evaporation and the round-bottom flask was placed in a vacuum drying oven overnight. Compound 4 was obtained without further purification.

[0056] (4) Compound 5 and 3-hydroxyphenol were dissolved in 50 mL of anhydrous N,N-dimethylformamide under a nitrogen atmosphere. After the reactants were dissolved, 1 mL of triethylamine was slowly added and the reaction was stirred at 60°C for 8 hours. After the reaction was completed, the reaction mixture was diluted with 50 mL of dichloromethane and washed and extracted several times with a saturated sodium chloride solution. The aqueous phase was discarded and the organic phase was retained. Anhydrous sodium sulfate was then added to the organic phase and evaporated under reduced pressure to obtain a crude product. The crude product was separated and purified by column chromatography using dichloromethane-methanol as the mobile phase to obtain compound 6;

[0057] (5) Compound 6 was dissolved in 30 mL of methanol and placed in a round-bottom flask. 5 mL of concentrated hydrochloric acid was then added and stirred for 2 hours. After the reaction was completed, methanol was removed by evaporation under reduced pressure. The residue was washed and extracted three times with 50 mL of dichloromethane and saturated brine. The organic phase was collected, dried over anhydrous sodium sulfate, and rotary evaporated to obtain a crude product. Finally, compound 7 was separated and purified by column chromatography using dichloromethane-methanol as the mobile phase.

[0058] (6) Compound 7 was dissolved in 30 mL of dichloromethane, and then 1 mL of acetic anhydride and 1 mL of triethylamine were added. The mixture was reacted at room temperature for 2 hours. After the reaction was completed, the reaction mixture was washed with saturated brine and extracted three times. The organic phase was collected and dried over anhydrous sodium sulfate. The solvent was removed by evaporation under reduced pressure to obtain a crude product. The crude product was separated and purified by column chromatography using dichloromethane-methanol as the mobile phase to obtain compound 8;

[0059] (7) Compound 4 and Compound 8 were dissolved in 30 mL of dichloromethane. 2 mL of HATU and 100 mg of DIPEA were then added in sequence and stirred at room temperature for two hours. After the reaction, the mixture was washed with saturated brine and extracted three times. The mixture was then dried over anhydrous sodium sulfate and the solvent was removed by rotary evaporation to obtain Compound 9 without further purification.

[0060] (8) Compound 9 was dissolved in 30 mL of methanol, and 5 mL of concentrated hydrochloric acid was added, and the mixture was stirred for 2 hours. After the reaction was completed, the methanol was removed by evaporation under reduced pressure. The residue was washed and extracted three times with 50 mL of dichloromethane and saturated brine. The organic phase was collected, dried over anhydrous sodium sulfate, and then rotary evaporated to obtain a crude product. Finally, the dye SiRho-SHD-OH was separated and purified by column chromatography using dichloromethane-methanol as the mobile phase.

[0061] Structure of the dye SiRho-SHD-OH:

[0062]

[0063] Mass spectrometry and NMR characterization of dye SiRho-SHD-OH:

[0064] 1 H NMR (500MHz, DMSO-d6) δ9.92(s,1H),8.20(d,J=14.2Hz,2H),7.77(d,J=7.4Hz,2H),7.68(d,J=8.0Hz,2H),7.57-7.52(t,J= 7.55Hz,2H),7.45(t,J=7.5Hz,2H),7.39(s,2H),7.19(d,J=1.9Hz,2H),6.97(m,1H),6.95(m,1H),6.90(d,J=7.7Hz,1H),6. 69(d,J=8.1Hz,1H),6.62(d,J=14.3Hz,2H),4.40(t,J=7.0Hz,4H),3.55(s,12H),2.76-2.69(m,4H),2.66(t,J=5.6Hz,4H), 2.31(t,J=7.3Hz,4H),1.75(s,6H),1.63-1.54(m,4H),1.46-1.36(m,3H),1.13(t,J=7.1Hz,2H),1.05(s,3H),0.85(s,3H);

[0065] ESI-MS: calculated for C 61 H 69 N5O3SSi 2+ [M] 2+ ,489.7440;Found,490.12.

[0066] (9) The dye SHD-SiRho-OH was dissolved in anhydrous N,N-dimethylformamide (1.5 mL), and then potassium carbonate and sodium iodide were added and stirred at 40°C for 10 minutes. Subsequently, p-nitrobenzyl bromide dissolved in anhydrous tetrahydrofuran was added and stirred overnight under a nitrogen atmosphere. After the reaction, the product was washed and extracted three times with 50 mL of dichloromethane and saturated sodium chloride. The organic phase was collected, dried over anhydrous sodium sulfate, and then evaporated under reduced pressure to remove the solvent to obtain a crude product. Finally, the probe SiRho-SHD-NTR was separated and purified by column chromatography using dichloromethane-methanol as the mobile phase to obtain a blue-purple solid.

[0067] The structure of the probe SiRho-SHD-NTR:

[0068]

[0069] Mass spectrometry and NMR characterization of the probe SiRho-SHD-NTR:

[0070] 1 H NMR (500MHz, CDCl3) δ8.43(s,1H),8.28(d,J=6.1Hz,2H),7.71(s,2H),7.57(d,J=8.6Hz,2H),7.47(d,J=8.6Hz,2H),7 .39(s,2H),7.17(dd,J=8.5,2.4Hz,2H),7.08(d,J=8.6Hz,1H),6.87(t,J=13.1Hz,1H),6.37-6.26(m,2H),6.16(d,J= 16.2Hz,2H),4.32(s,2H),3.67(d,J=18.4Hz,8H),3.54(d,J=12.2Hz,2H),2.85(s,3H),2.80(s,2H),2.47(s,2H),2.4 2-2.35(m,2H),2.30-2.22(m,2H),2.05(d,J=5.8Hz,6H),1.71-1.59(m,12H),1.52(s,3H),1.07(s,9H),0.92(s,2H).

[0071] 13C NMR(126MHz,DMSO-d6)δ178.19,171.01,165.59,159.45,153,94,151.65,147,38,144.60,141.75,135.35,1 28.83,124.14,121.67,110.40,69.14,55.40,51.15,45.72,31.60,29.48,27.99,25.12,22.54,14.40,8.84.

[0072] ESI-MS: calculated for C 68 H 74 N6O5SSi 2+ [M] 2+ ,557.2600;Found,557.68.

[0073] Example 2

[0074] Detection of NTR in a buffer system using the probe SiRho-SHD-NTR

[0075] 11.16 mg of the probe was dissolved in 10 mL of DMSO to prepare a 1 mM stock solution, which was stored at -20°C. The detection system was a DPBS buffer solution (10 mM, pH 7.4, containing 10% DMSO). The reaction system of the probe SiRho-SHD-NTR and NTR was shaken at room temperature for 30 minutes, and then its fluorescence emission and UV-visible absorption spectra were measured. The excitation wavelength of the fluorescence instrument was set to 650 nm and 730 nm, and the emission wavelength receiving range was set to 660-740 nm and 740-840 nm. The measurement range of the absorption spectrum was set to 500-800 nm. The results are as follows. Figure 1 As shown, from Figure 1 It can be seen that the probe SiRho-SHD-NTR has a good response to NTR, the near-infrared fluorescence spectrum shows nearly 18-fold fluorescence enhancement, and both the absorption spectrum and the fluorescence spectrum show ratiometric changes.

[0076] The experimental steps of the photoacoustic response of the probe SiRho-SHD-NTR to NTR in the buffer system are the same as those of the absorption spectrum. The sample after the reaction is placed in a photoacoustic imager to measure the photoacoustic spectrum of the sample. The results are as follows: Figure 2 As shown, from Figure 2 It can be seen that the probe SiRho-SHD-NTR has a good photoacoustic response to NTR, and the photoacoustic response result is similar to the absorption spectrum.

[0077] Example 3

[0078] Investigation of the detection selectivity and chemical kinetics of NTR by the probe SiRho-SHD-NTR in a buffer system

[0079] 11.16 mg of the probe was dissolved in 10 mL of DMSO to prepare a 1 mM stock solution, which was stored at -20°C. The detection system was a DPBS buffer solution (10 mM, pH 7.4, containing 10% DMSO). The reaction system of the probe SiRho-SHD-NTR, NTR, and various test substances was shaken at room temperature for 15 minutes, and then its fluorescence emission spectrum and photoacoustic imaging were measured. The excitation wavelength of the fluorescence instrument was set to 650 nm and 730 nm, and the emission wavelength receiving range was 660-740 nm and 740-840 nm. The results are as follows Figure 3 As shown in ac, from Figure 3 As can be seen from ac, the probe SiRho-SHD-NTR has good selectivity for NTR, and has little interference from GSH, Hcy, etc. Figure 3 As shown in d, the probe SiRho-SHD-NTR has a very fast reaction rate to NTR, and basically reaches the reaction platform in about 5 minutes.

[0080] Example 4

[0081] Imaging detection of intracellular NTR using the probe SiRho-SHD-NTR

[0082] 11.16 mg of the probe was dissolved in 10 mL of DMSO to prepare a 1 mM stock solution and stored at -20°C. 5 μL of the probe was incubated in the cells and the confocal microscope was set to an excitation wavelength of 640 nm and an emission wavelength range of 650-750 nm and 720-780 nm. Figure 4 As shown, from Figure 4 It can be seen that the probe SiRho-SHD-NTR has a good response to the nitroreductase content induced by cobalt chloride in cells, and the imaging contrast is very high.

[0083] Example 5

[0084] Photoacoustic imaging detection of NTR in living tumors and tumor cross-sections using the SiRho-SHD-NTR probe

[0085] 11.16 mg of probe was dissolved in 10 mL of DMSO to prepare a 1 mM stock solution and stored at -20°C. The probe was prepared to 200 μM and dissolved in DPBS / DMSO, v / v = 7:3, pH 7.4. 20 μL of probe solution was injected intratumorally, and then small animal photoacoustic imaging was performed. The excitation wavelength of the small animal photoacoustic imager was set to 680 and 740 nm. The results are shown in Figure 2. Figure 5 As shown, from Figure 5It can be seen that the probe SiRho-SHD-NTR has a good response to NTR in the tumor, and the photoacoustic ratio imaging contrast is very high. Figure 6 It can be seen that the probe SiRho-SHD-NTR has a good response to NTR in various sections of the tumor, and the photoacoustic ratio imaging contrast is very high.

[0086] Example 6

[0087] Near-infrared fluorescence imaging detection of NTR in living tumors using the probe SiRho-SHD-NTR

[0088] Dissolve 11.16 mg of the probe in 10 mL of DMSO to prepare a 1 mM stock solution and store it at -20°C. Prepare the probe to 200 μM and dissolve it in DPBS / DMSO, v / v=7:3, pH 7.4. Take 20 μL of the probe solution and inject it into the tumor, and then perform in vivo fluorescence imaging of small animals. Set the excitation wavelength of the fluorescence instrument to 640 nm and 740 nm, and the emission wavelength receiving range to 650-700 nm and 750-800 nm. The results are as follows Figure 7 As shown, from Figure 7 It can be seen that the probe SiRho-SHD-NTR has a good response to NTR in the tumor, and the near-infrared fluorescence ratio imaging contrast is very high.

[0089] Example 7

[0090] Near-infrared fluorescence imaging of in situ tumors using the probe SiRho-SHD-NTR by spraying

[0091] Dissolve 11.16 mg of the probe in 10 mL of DMSO to prepare a 1 mM stock solution, and store it at -20°C. Prepare the probe to 200 μM and dissolve it in DPBS / DMSO, v / v=7:3, pH 7.4. Take 2 mL of the probe solution and place it in a 3 mL small spray bottle, spray it on the dissected in situ tumor mice, and then perform in vivo fluorescence imaging of the small animals. Set the excitation wavelength of the fluorescence instrument to 640 nm and 740 nm, and the emission wavelength receiving range to 650-700 nm and 750-800 nm. The results are as follows Figure 8 As shown, from Figure 8 It can be seen that the probe SiRho-SHD-NTR has a good response to NTR in situ tumors, and the near-infrared fluorescence ratio imaging contrast is very high. Its detection principle is as follows Figure 9 shown.

[0092] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A NIRF / PA dual-modality probe based on NTR response, characterized in that: The probe comprises a cation of the following formula: 。 2. The method for preparing the NIRF / PA dual-modality probe based on NTR response according to claim 1, characterized in that: The following steps are involved: (1) Compound 1 was dissolved in anhydrous acetonitrile and transferred to a round-bottom flask under an inert atmosphere. Phosphorus oxychloride was then slowly added to the reaction system. The mixture was stirred at a set temperature. After the reaction was completed, the solvent was removed by rotary evaporation to obtain compound 2. (2) 1-Boc-piperazine and compound 2 were dissolved in anhydrous acetonitrile, and triethylamine was slowly added. The reaction mixture was stirred at room temperature, and the reaction mixture was concentrated under reduced pressure. Compound 3 was obtained after extraction and purification. (3) Compound 3 was dissolved in a mixture of trifluoroacetic acid and dichloromethane and stirred at room temperature. After the reaction was completed, the solvent was removed by rotary evaporation and the round-bottom flask was placed in a vacuum drying oven overnight to obtain compound 4; (4) Compound 5 and 3-hydroxythiophenol were dissolved in anhydrous N,N-dimethylformamide under an inert atmosphere, and triethylamine was subsequently added. The mixture was stirred at a predetermined temperature. After the reaction was completed, the reaction mixture was diluted with dichloromethane and washed and extracted with a saturated sodium chloride solution several times. The mixture was then dried, the solvent was removed by rotary evaporation, and the mixture was purified to obtain Compound 6. (5) Compound 6 was dissolved in methanol, and then concentrated hydrochloric acid was added and stirred at room temperature to react. After the reaction was completed, compound 7 was obtained; (6) Compound 7 was dissolved in dichloromethane, and then acetic anhydride and triethylamine were added in sequence and reacted at room temperature. After the reaction was completed, the reaction mixture was washed and extracted with saturated sodium chloride solution several times, and then dried, rotary evaporated to remove the solvent, and purified to obtain compound 8; (7) Compound 4 and Compound 8 were dissolved in dichloromethane, and HATU and DIPEA were subsequently added in sequence. The mixture was stirred at room temperature. After the reaction was completed, the reaction mixture was washed and extracted with saturated sodium chloride solution several times, and then dried, rotary evaporated to remove the solvent, and purified to obtain Compound 9. (8) Compound 9 was dissolved in methanol, and then concentrated hydrochloric acid was added and stirred at room temperature to obtain a dye. (9) The dye obtained in step (8) is dissolved in anhydrous N, N-dimethylformamide, and then potassium carbonate and sodium iodide are added, and the reaction is stirred at a set temperature. After the reaction is completed, a tetrahydrofuran solution of p-nitrobenzyl bromide is added under an inert atmosphere, and the mixture is stirred at room temperature overnight. The reaction mixture is then washed and extracted multiple times with a saturated sodium chloride solution, and then dried, rotary evaporated to remove the solvent, and purified to obtain a NIRF / PA dual-modal probe based on NTR response; The reaction formula is: 。 3. The method for preparing the NIRF / PA dual-modality probe based on NTR response according to claim 2, characterized in that: In step (1), the molar ratio of compound 1 to phosphorus oxychloride is 1:(0.5-1).

4. The method for preparing the NIRF / PA dual-modality probe based on NTR response according to claim 2, characterized in that: In step (2), the molar ratio of compound 2, 1-Boc-piperazine and triethylamine is 1:(1.5-2):(1.5-2).

5. The method for preparing the NIRF / PA dual-modality probe based on NTR response according to claim 2, characterized in that: In step (3), the amount of compound 3 added is 0.1~0.4 mmol, the volume ratio of trifluoroacetic acid and dichloromethane is 1:(4.0~5.0), and the total volume of the solvent is 20~30 mL.

6. The method for preparing the NIRF / PA dual-modality probe based on NTR response according to claim 2, characterized in that: In step (4), the molar ratio of compound 5, 3-hydroxythiophenol and triethylamine is 1:(1.2-1.5):(1.2-1.5).

7. The method for preparing the NIRF / PA dual-modality probe based on NTR response according to claim 2, characterized in that: In step (5), the molar ratio of compound 6 to concentrated hydrochloric acid is 1:(3-5), and the mass fraction of concentrated hydrochloric acid is 36-38%.

8. The method for preparing the NIRF / PA dual-modality probe based on NTR response according to claim 2, characterized in that: In step (6), the molar ratio of compound 7, acetic anhydride and triethylamine is 1:(1.2-1.5):(1.2-1.5); In step (7), the molar ratio of compound 4, compound 8, HATU and DIPEA is 1:(1.1-1.2):(1.5-2):(2-3).

9. The method for preparing the NIRF / PA dual-modality probe based on NTR response according to claim 2, characterized in that: In step (8), the molar ratio of compound 9 to concentrated hydrochloric acid is 1:(3-5), and the mass fraction of concentrated hydrochloric acid is 36-38%; In step (9), the molar ratio of the dye, p-nitrobenzyl bromide, potassium carbonate and sodium iodide is 1:(1.1-1.3):(1.5-2):(2-3).

10. The use of the NIRF / PA dual-modality probe based on NTR response according to claim 1, characterized in that: It is used to prepare drugs for detecting NTR in samples, cells, tissues and living mice.

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