Nitroreductase response fluorescence and magnetic resonance bimodal probe NO2-Cy-Gxtb-Gd-Biotin designed based on gemcitabine prodrug as well as synthesis method and application of nitroreductase response fluorescence and magnetic resonance bimodal probe NO2-Cy-Gxtb-Gd-Biotin

Abstract

The invention discloses a nitroreductase response fluorescence and magnetic resonance bimodal probe NO2-Cy-Gxtb-Gd-Biotin designed on the basis of a gemcitabine prodrug as well as a synthesis method and application of the nitroreductase response fluorescence and magnetic resonance bimodal probe NO2-Cy-Gxtb-Gd-Biotin. The probe has relatively low fluorescence signals and magnetic resonance signals, and has relatively low biotoxicity. After the nitroreductase responds, on one hand, the gemcitabine anti-cancer drug is released, and tumor cell apoptosis is promoted; on the other hand, a molecular skeleton can generate a series of electron transfer to form an intermediate structure of methylquinone, and the intermediate structure of methylquinone is very high in reaction activity and is easily attacked by an external residue nucleophilic group of protein, so that the probe is anchored on nitroreductase, and a fluorescence signal and a magnetic resonance signal are remarkably enhanced; therefore, tumor enrichment is increased, an imaging time window is expanded, and early monitoring of tumors is facilitated. The medicine and the imaging probe are integrated, so that the treatment of monitoring tumors is facilitated.

Description

Technical Field

[0001] The present invention belongs to the technical field of magnetic resonance imaging, and in particular relates to a nitroreductase-responsive fluorescence and magnetic resonance dual-modal probe NO2-Cy-Gxtb-Gd-Biotin designed based on gemcitabine prodrug, and a synthesis method and application thereof. Background Art

[0002] Lung cancer remains the leading cause of cancer death worldwide. Lung cancer is histologically divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), of which NSCLC accounts for about 90% of diagnosed patients, and most patients are diagnosed with locally advanced or metastatic disease.

[0003] The rapid growth and reproduction of tumor cells will lead to increased tissue oxygen consumption, resulting in varying degrees of tissue hypoxia. In addition, overexpression of hypoxia-inducible factor (HIF) will lead to the accumulation of related enzymes and proteins, and participate in tumor immune escape and promote tumorigenesis. Nitroreductase (NTR) is the most representative enzyme in solid tumors, and hypoxia directly leads to the upregulation of NTR. Therefore, probes for NTR detection and imaging are of great significance for the detection of lung cancer.

[0004] Cancer diagnosis and imaging examinations play an important role in prognosis, staging, treatment planning, and evaluation of treatment response. Non-invasive imaging techniques are often used in clinical diagnosis, such as magnetic resonance imaging (MRI), X-ray imaging, computed tomography (CT) imaging, single photon emission computed tomography (SPECT) imaging, positron emission tomography (PET) imaging, ultrasound imaging, and optical imaging. Each of these imaging techniques has its own characteristics and applicable usage scenarios. As shown in Table 1, the advantages and disadvantages of each imaging technique are summarized.

[0005] Table 1 Advantages and disadvantages of different imaging techniques

[0006]

[0007]

[0008] Many fluorescent probes and magnetic resonance contrast agents for detecting and real-time imaging of NTR activity in lung cancer have been reported. However, single-modality imaging cannot take into account sensitivity, resolution, penetration depth, etc. at the same time, and it is difficult to meet the requirements for accurate measurement of molecules in organisms. Multimodal molecular imaging integrates the advantages of different modalities in sensitivity, resolution, and penetration depth to image the same individual, which helps to accurately measure biological molecules in vivo and promote disease diagnosis. For example, near-infrared (NIR) fluorescence imaging can generate highly sensitive images for detecting low concentrations of tumor-related biomarkers (such as overexpressed enzymes) and image-guided tumor surgery. Magnetic resonance imaging (MRI) can produce anatomical images with unlimited tissue penetration depth and high spatial resolution to facilitate preoperative detection of deep tumors. Therefore, combining the two imaging methods can achieve high-sensitivity imaging of deep tumors.

[0009] At present, most activatable probes can achieve variable imaging of enzymes, but they inevitably tend to diffuse from the original active site of the enzyme or even shift out of the cell, seriously impairing the in situ high-resolution observation of the enzyme. While in situ imaging of enzymes can be achieved by signal-labeled probes or antibodies that are always on, these signals cannot achieve activatable imaging of enzymes. Therefore, imaging probes with "activatable" and "in situ" properties will be able to conduct high-resolution studies of enzymes in living systems, thereby achieving early diagnosis of diseases. At present, in situ imaging of enzymes is mainly achieved in covalent and non-covalent forms, in which covalently linked probes, also called irreversible probes, directly cross-link signal units to target enzymes. The active group (electrophilic site) is an indispensable component of the probe, which can react specifically with the nucleophilic group exposed outside the enzyme to form a stable covalent bond. For example, quinone methides (QMs) are transient molecules with extremely high electrophilicity. Based on the introduction of good leaving groups (fluoride, carbamate, etc.) into QMs imaging agents, the quinone methide formed after reaction with the enzyme acts as an electrophilic intermediate with high reactivity and can be reacted with nucleophilic groups (hydroxyl, amino or thiol) outside the enzyme or nearby proteins to form covalent bonds, causing probe retention, thereby visualizing enzyme activity in a dynamic environment. In order to avoid the problems of short circulation time and rapid metabolism of small molecule probes, tumor targeting groups such as biotin and RGD peptides are usually introduced to extend the blood circulation time of small molecules, increase the aggregation of tumor sites, and improve imaging effects.

[0010] At present, the clinical treatment of tumors mainly relies on surgical resection, radiotherapy and chemotherapy, but these treatment methods all have some disadvantages. Surgical resection is prone to late infection and tumor cell metastasis, while radiotherapy is prone to cause radiation damage to local body tissues. Chemotherapy is a commonly used treatment method, but it is prone to multidrug resistance and toxic side effects to normal tissues. Therefore, the development of tumor-specific activated original drugs has attracted widespread attention. Inactive original drugs are activated when they are subjected to specific stimuli (such as pH, redox state, temperature, light, enzymes, etc.) in the tumor, thereby reducing toxic side effects on normal tissues and achieving drug accumulation in tumor tissues. Summary of the invention

[0011] Based on the above-mentioned prior art, the present invention provides a nitroreductase-responsive fluorescence and magnetic resonance dual-modal probe NO2-Cy-Gxtb-Gd-Biotin designed based on gemcitabine prodrug and its synthesis method and application. The compound itself has relatively low fluorescence signal and magnetic resonance signal, but after the nitroreductase response, on the one hand, it will release gemcitabine anticancer drugs to promote tumor cell apoptosis; on the other hand, a series of electron transfers will occur in the molecular skeleton to form a methylquinone intermediate structure. This methylquinone intermediate structure has high reactivity and is easily attacked by the nucleophilic group of the protein external residue, so that the probe is anchored on the nitroreductase, resulting in a significant enhancement of the fluorescence signal and the magnetic resonance signal, thereby increasing the enrichment of the tumor, expanding the imaging time window, and facilitating the early monitoring of the tumor. The drug and the imaging probe are integrated to facilitate the monitoring of tumor treatment.

[0012] In order to achieve the above object, the present invention adopts the following technical solutions:

[0013] A nitroreductase-responsive fluorescence and magnetic resonance dual-modal probe NO2-Cy-Gxtb-Gd-Biotin designed based on gemcitabine prodrug, the structural formula of which is as follows:

[0014]

[0015] A method for synthesizing a nitroreductase-responsive fluorescence and magnetic resonance dual-modal probe NO2-Cy-Gxtb-Gd-Biotin designed based on gemcitabine prodrug comprises the following steps:

[0016] 1. Mix N2-Fmoc-N6-biotin-lysine and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate in N,N-dimethylformamide, add N,N-diisopropylethylamine and 2,2',2"-(10-(2-((2-aminoethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate in N,N-dimethylformamide, stir at 25-40°C for 4-8 hours to obtain compound 4, the reaction formula is as follows:

[0017]

[0018] 2. Mix azido-PEG4-acetic acid and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate in N,N-dimethylformamide, add N,N-diisopropylethylamine and the N,N-dimethylformamide solution after de-Fmoc of compound 4, and stir at 25-40°C for 4-8 hours to obtain compound 5. The reaction formula is as follows:

[0019]

[0020] 3. Dissolve compound 5 in a hydrochloric acid ethyl acetate solution, stir at 25-40°C for 4-10 hours, then add gadolinium chloride hexahydrate, stir at 25-40°C for 6-12 hours to obtain compound 6. The reaction formula is as follows:

[0021]

[0022] 4. Dissolve compound 8, p-nitrobenzyl bromide, potassium iodide and potassium carbonate in acetone and stir at 25-60°C for 2-3 days to obtain compound 9. The reaction formula is as follows:

[0023]

[0024] 5. Dissolve compound 9, p-nitrophenyl chloroformate and pyridine in dichloromethane and stir at 25-60° C. for 6-12 hours to obtain compound 10. The reaction formula is as follows:

[0025]

[0026] 6. Dissolve compound 10 and gemcitabine in N,N-dimethylformamide, add N,N-diisopropylethylamine and 4-dimethylaminopyridine, and stir at room temperature for 8-24 hours to obtain compound 11. The reaction formula is as follows:

[0027]

[0028] 7. Dissolve compound 11 and compound 6 in N,N-dimethylformamide, add aqueous solution of copper sulfate and sodium ascorbate, and stir at room temperature for 2-8 hours to obtain probe NO2-Cy-Gxtb-Gd-Biotin. The reaction formula is as follows:

[0029]

[0030] The above compound 8 can be purchased from commercial sources, or can be synthesized according to the following method:

[0031] 1. Add a dichloromethane solution of tert-butyldimethylsilyl chloride dropwise to a dichloromethane solution containing 2,4-dihydroxybenzaldehyde and imidazole, and stir at 25-40°C for 6-12 hours to obtain compound d; add sodium borohydride in batches to a methanol solution containing compound d under an ice bath, and stir at 25-40°C for 2-8 hours to obtain compound e. The reaction formula is as follows:

[0032]

[0033] 2. Add the N,N-dimethylformamide solution of compound 7 to the N,N-dimethylformamide solution of compound e and sodium hydride, and stir the mixture at 25-60° C. for 8-16 hours to obtain compound 8. The reaction formula is as follows:

[0034]

[0035] Application of nitroreductase-responsive fluorescence and magnetic resonance dual-modal probe NO2-Cy-Gxtb-Gd-Biotin designed based on gemcitabine prodrug in the preparation of developers for magnetic resonance imaging and fluorescence imaging: In a specific embodiment of the present invention, the probe NO2-Cy-Gxtb-Gd-Biotin is administered to mice with orthotopic lung cancer model by pulmonary instillation, and the tumor sites of the mice with orthotopic lung cancer model are photographed at different time points (0h, 2h, 4h, 8h, 12h, 24h, 36h) by a visual camera, and the changes in the fluorescence intensity of the tumor sites are measured. The results show that with the extension of time, the probe NO2-Cy-Gxtb-Gd-Bi The fluorescence signal of NO2-Cy-Gxtb-Gd-Biotin at the tumor site gradually increased and reached the highest value at 12 hours; the probe NO2-Cy-Gxtb-Gd-Biotin was administered to mice with orthotopic lung cancer model by pulmonary instillation, and the tumor site of the mice with orthotopic lung cancer model was imaged at different time points (0h, 2h, 4h, 6h, 8h, 12h, 24h, and 36h) using a 9.4T magnetic resonance spectrometer, and the changes in the magnetic resonance intensity of the tumor site were measured. The results showed that with the extension of time, the magnetic resonance signal of NO2-Cy-Gxtb-Gd-Biotin at the tumor site gradually increased and reached the highest value at 8 hours, indicating that the probe has good tumor imaging ability.

[0036] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0037] 1. After the NO2-Cy-Gxtb-Gd-Biotin compound responds to nitroreductase, the UV absorption shift changes, and the fluorescence emission wavelength is in the near-infrared region, which effectively reduces the interference of background fluorescence signals and greatly improves the sensitivity and accuracy of fluorescence imaging.

[0038] 2. After the NO2-Cy-Gxtb-Gd-Biotin compound responds, a series of electron transfers will occur to form a methylquinone intermediate structure. This methylquinone intermediate structure has high reactivity and is easily attacked by nucleophilic groups of protein external residues, thereby anchoring the compound to the protein, resulting in enhanced magnetic resonance signals, which is beneficial to improving the magnetic resonance imaging effect of the tumor site.

[0039] 3. NO2-Cy-Gxtb-Gd-Biotin compound can release gemcitabine anticancer drug after response, and has a good therapeutic effect on lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 (a) The UV-visible absorption spectrum and (b) the fluorescence emission spectrum of the NO2-Cy-Gxtb-Gd-Biotin compound prepared in Example 1.

[0041] Figure 2 The fluorescence response (a) and magnetic resonance response test results (b) of the NO2-Cy-Gxtb-Gd-Biotin compound in Example 2 under different analytes.

[0042] Figure 3 The drug release test results of the NO2-Cy-Gxtb-Gd-Biotin compound under nitroreductase response in Example 2. a) is the HPLC analysis result, and b) is the peak area quantitative curve.

[0043] Figure 4 These are the fluorescence and magnetic resonance imaging results of the NO2-Cy-Gxtb-Gd-Biotin compound in Example 3 in A549 lung cancer cells.

[0044] Figure 5 These are the fluorescence imaging and magnetic resonance imaging images of the NO2-Cy-Gxtb-Gd-Biotin compound in Example 4 in an in situ lung cancer mouse model. DETAILED DESCRIPTION

[0045] The present invention is described in detail below with reference to specific embodiments.

[0046] Example 1 Synthesis of NO2-Cy-Gxtb-Gd-Biotin Compound

[0047] 1. Synthesis of compound d

[0048] Dissolve 2,4-dihydroxybenzaldehyde (5.0g, 36.2mmol) and imidazole (14.8g, 217.2mmol) in CH2Cl2 (400mL), drop tert-butyldimethylsilyl chloride (16.4g, 108.6mmol) in CH2Cl2 (150mL), stir at room temperature for 6h, add saturated NH4Cl (350mL), separate the two phases, and extract the aqueous layer with CH2Cl2 (3×100mL). The collected organic layer was dried over anhydrous Na2SO4 and concentrated. The residue was purified by chromatography on a silica gel column (volume ratio of petroleum ether: ethyl acetate = 50:1) to obtain compound d (10.5g, yield 78.9%) as a light yellow oil. The reaction formula is as follows:

[0049]

[0050] 2. Synthesis of compound e

[0051] Compound d (16.0 g, 43.6 mmol) was dissolved in MeOH (300 mL), and sodium borohydride (1.65 g, 43.6 mmol) was added at 0°C. After stirring at room temperature for 2 h, methanol was evaporated in vacuo. Saturated NH4Cl (300 mL) solution and CH2Cl2 (150 mL) were added respectively, the two phases were separated, and the aqueous layer was extracted with CH2Cl2 (3×100 mL). The collected organic layer was dried over anhydrous Na2SO4 and concentrated. The residue was purified by chromatography on a silica gel column (petroleum ether: ethyl acetate volume ratio = 20:1) to obtain compound e (10.5 g, yield 78.9%) as a light yellow oil. The reaction formula is as follows:

[0052]

[0053] 3. Synthesis of compound 4

[0054] N2-Fmoc-N6-biotin-lysine (CAS: 146987-10-2, purchased from Bidex Pharmaceuticals) (2.8 g, 4.7 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 1.5 g, 3.9 mmol) were mixed in N,N-dimethylformamide (DMF, 50 mL), and N,N-diisopropylethylamine (DIPEA, 756.1 mg, 5.9 mmol) and compound 3 were added. A solution of tri-tert-butyl 2,2',2"-(10-(2-((2-aminoethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (2.4 g, 3.9 mmol) in DMF (50 mL) was stirred at room temperature for 4 h, and then extracted with CH2Cl2 (200 mL) and H2O (200 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was purified by chromatography on a silica gel column (CH2Cl2:MeOH volume ratio = 100:3) to obtain compound 4 (4.1 g, yield 88.5%) as a white solid. The reaction formula is as follows:

[0055]

[0056] 4. Synthesis of Compound 5

[0057] Azide-PEG4-acetic acid (1.4 g, 4.7 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 1.5 g, 3.9 mmol) were mixed in N,N-dimethylformamide (DMF, 50 mL), and a DMF (50 mL) solution of N,N-diisopropylethylamine (DIPEA, 756.1 mg, 5.9 mmol) and compound 4 (4.6 g, 3.9 mmol) was added, stirred at room temperature for 4 h, and then extracted with CH2Cl2 (200 mL) and H2O (200 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was purified by chromatography on a silica gel column (CH2Cl2: MeOH volume ratio = 100:3) to obtain compound 5 (3.3 g, yield 68.8%) as a white solid. The reaction formula is as follows:

[0058]

[0059] 5. Synthesis of Compound 6

[0060] Compound 5 was dissolved in a hydrochloric acid ethyl acetate solution, stirred at 25°C for 6 hours, filtered to obtain a residue, and then water was added to dissolve the residue, and the pH value was adjusted to about 6.0 using a 5% NaOH solution, and gadolinium chloride hexahydrate was added, stirred for 12 hours, and separated by HPLC preparation (1%-99% acetonitrile, 40 minutes), and freeze-dried to obtain a white powder. The reaction formula is as follows:

[0061]

[0062] 6. Synthesis of Compound 8

[0063] Compound e (2.1 g, 5.6 mmol) and NaH (224.0 mg, 5.6 mmol) in DMF (10 mL) were added to the DMF solution (10 mL), stirred at room temperature for 8 h, then extracted with CH2Cl2 (150 mL) and saturated NH4Cl (100 mL), and the aqueous layer was extracted with CH2Cl2 (3×50 mL). The collected organic layer was dried over anhydrous Na2SO4 and concentrated. Purified by chromatography on a silica gel column (CH2Cl2: MeOH volume ratio = 20:1) to obtain compound 8 (860.0 mg, yield 25.3%) as a blue solid. The reaction formula is as follows:

[0064]

[0065] 7. Synthesis of Compound 9

[0066] Compound 8 (400 mg, 0.6 mmol), 4-nitrobenzyl bromide (172.8 mg, 0.8 mmol), KI (219.1 mg, 1.3 mmol) and K2CO3 (109.2 mg, 0.8 mmol) were dissolved in acetone (10 mL), stirred at room temperature for 3 d, extracted with CH2Cl2 (150 mL) and saturated NH4Cl (100 mL), and the aqueous layer was extracted with CH2Cl2 (3×50 mL). The collected organic layer was dried over anhydrous Na2SO4 and concentrated. Purified by chromatography on a silica gel column (CH2Cl2: MeOH volume ratio = 100:3) to obtain compound 9 (237.8 mg, yield 54.4%) as a blue solid. The reaction formula is as follows:

[0067]

[0068] 8. Synthesis of Compound 10

[0069] Compound 9 (920 mg, 0.9 mmol), p-nitrophenyl chloroformate (362.8 mg, 1.8 mmol) and pyridine (356.0 mg, 4.5 mmol) were dissolved in CH2Cl2 (15 mL) and stirred at room temperature for 6 h. The liquid was extracted with CH2Cl2 (150 mL) and saturated NH4Cl (100 mL). The aqueous layer was extracted with CH2Cl2 (3×50 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated. The compound 10 (550.9 mg, yield 68.5%) was obtained as a blue solid by chromatography on a silica gel column (CH2Cl2: MeOH volume ratio = 25:1). The reaction formula is as follows:

[0070]

[0071] 9. Synthesis of Compound 11

[0072] Compound 10 (268.1 mg, 0.3 mmol), gemcitabine (473.7 mg, 1.8 mmol), N,N-diisopropylethylamine (77.5 mg, 0.6 mmol) and 4-dimethylaminopyridine (36.7 mg, 0.3 mmol) were dissolved in CH2Cl2 (10 mL) and stirred at room temperature for 12 h. After vacuum concentration, the mixture was purified by preparative RP-HPLC on a C18 column (1%-99% acetonitrile, 60 min) to obtain compound 11 (64.4 mg, yield 21.1%) as a blue solid. The reaction formula is as follows:

[0073]

[0074] 10. Synthesis of compound NO2-Cy-Gxtb-Gd-Biotin

[0075] Compound 11 (20.0 mg, 19.6 μmol), compound 6 (22.0 mg, 32.1 μmol), CuSO4 (1.6 mg, 6.4 μmol), ascorbic acid (5.1 mg, 25.7 μmol), DMF (0.5 mL), and H2O (0.2 mL) were stirred at room temperature for 4 h, concentrated in vacuo, and purified by preparative RP-HPLC on a C18 column to obtain the product NO2-Cy-Gxtb-Gd-Biotin (10.0 mg, yield 22.7%) as a blue solid. The reaction formula is as follows:

[0076]

[0077] The prepared NO2-Cy-Gxtb-Gd-Biotin compound was subjected to UV-visible absorption spectral analysis and fluorescence emission spectral analysis. 0.8 mL of the solution to be tested was transferred into a micro cuvette for UV-visible light spectral test, and another 2 mL of the solution to be tested was transferred into a cuvette for fluorescence spectral test. The prepared NO2-Cy-Gxtb-Gd-Biotin compound was incubated with NADH and / or NTR at 37°C for 70 min. 0.8 mL of the mixed solution was transferred into a micro cuvette for UV-visible light spectral test, and another 2 mL of the solution to be tested was transferred into a cuvette for fluorescence spectral test. The obtained UV-visible absorption spectrum and fluorescence emission spectrum are shown in Fig. Figure 1 As shown, the UV absorption peak of the NO2-Cy-Gxtb-Gd-Biotin compound itself showed shoulder peaks at 600nm and 660nm, and the fluorescence intensity was weak. After incubation with NADH and NTR, the UV maximum absorption wavelength red-shifted, a single peak appeared at 690nm, and the fluorescence intensity at 720nm was significantly enhanced.

[0078] Example 2 Fluorescence response and magnetic resonance response test results of NO2-Cy-Gxtb-Gd-Biotin compound under different analytes

[0079] Experimental method: The NO2-Cy-Gxtb-Gd-Biotin compound prepared in Example 1 was prepared into 1 mL of a 5 μM solution using PBS solution, and different analytes (NADH, NADH+ nitroreductase, glutathione, cysteine, tyrosinase, trypsin, sodium ascorbate and inhibitor dihydroxycoumarin) were added at a final concentration of 500 μM, and the fluorescence intensity was tested using a fluorescence spectrometer.

[0080] The NO2-Cy-Gxtb-Gd-Biotin compound prepared in Example 1 was prepared into 1 mL of a 100 μM solution using PBS solution, and different analytes (NADH, NADH+ nitroreductase, glutathione, cysteine, tyrosinase, trypsin, sodium ascorbate and inhibitor dihydroxycoumarin) were added at a final concentration of 5 mM, and the T1 time was measured using a 20 MHz nuclear magnetic resonance spectrometer.

[0081] Test results: The fluorescence intensity of the NO2-Cy-Gxtb-Gd-Biotin compound prepared in Example 1 changes under the action of different analytes as shown in Figure 2 As shown, the fluorescence intensity will be enhanced and the T1 time will be shortened only when NADH and nitroreductase exist at the same time.

[0082] Example 3 Drug release test results of NO2-Cy-Gxtb-Gd-Biotin compound under nitroreductase response

[0083] Experimental method: Take 2.4 mg of the NO2-Cy-Gxtb-Gd-Biotin compound prepared in Example 1 and dissolve it in 1 mL PBS to prepare 1 mL 1 mM NO2-Cy-Gxtb-Gd-Biotin solution, take 100 μL NO2-Cy-Gxtb-Gd-Biotin solution, add 5 μL 1000 μg / mL nitroreductase solution to prepare 1 mL solution, mix well and place on a 37°C shaker for shaking, take samples at different time points (0 min, 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min), and measure the change in the peak area of ​​the released gemcitabine by HPLC (1%-99% acetonitrile, 30 min).

[0084] Test results: The peak area of ​​gemcitabine released by the NO2-Cy-Gxtb-Gd-Biotin compound prepared in Example 1 under the action of nitroreductase changes as follows: Figure 3 As shown in the figure, with the extension of incubation time, the amount of released gemcitabine gradually increased and was completely released within 60 min.

[0085] Example 4 Fluorescence and magnetic resonance imaging results of NO2-Cy-Gxtb-Gd-Biotin compound in A549 lung cancer cells

[0086] Experimental method: Lung cancer cells A549 were cultured in T75 culture flasks and incubated in normoxic and hypoxic incubators for 12 hours. 2.4 mg of the NO2-Cy-Gxtb-Gd-Biotin compound prepared in Example 1 was dissolved in 1 mL of PBS solution to prepare 1 mL of 1 mM NO2-Cy-Gxtb-Gd-Biotin solution. 100 μL of NO2-Cy-Gxtb-Gd-Biotin solution was dissolved in F12K culture medium, configured to 5 mL, added to a T75 culture flask, incubated with A549 cells for 8 hours, digested with trypsin, and the cells were transferred to a 1.5 mL centrifuge tube to test the fluorescence imaging and magnetic resonance imaging results. The cells were incubated with the inhibitor (500 μM dihydroxycoumarin) for 30 min, and then 5 mL of NO2-Cy-Gxtb-Gd-Biotin (20 μM) was added. After incubation for 8 h, the cells were digested with trypsin and transferred to a 1.5 mL centrifuge tube to test their fluorescence imaging and magnetic resonance imaging results.

[0087] Experimental results: After incubation with cells, the fluorescence intensity and magnetic resonance intensity of the NO2-Cy-Gxtb-Gd-Biotin compound prepared in Example 1 were significantly enhanced compared with the inhibitor group, such as Figure 4 As shown, it shows that NO2-Cy-Gxtb-Gd-Biotin has a good imaging effect.

[0088] Example 5 Fluorescence imaging and magnetic resonance imaging of NO2-Cy-Gxtb-Gd-Biotin compound in an orthotopic lung cancer mouse model

[0089] Test method: Take 4.8 mg of the NO2-Cy-Gxtb-Gd-Biotin compound prepared in Example 1 and dissolve it in 1 mL of PBS solution to prepare 1 mL of 2 mM NO2-Cy-Gxtb-Gd-Biotin solution. Take 50 μL of each sample and dilute it to 100 μM 1mL solution was administered to the orthotopic lung cancer model mice by pulmonary instillation of 100μL (100μM) NO2-Cy-Gxtb-Gd-Biotin solution, and the lungs of the orthotopic lung cancer model mice were photographed at different time points (0h, 2h, 4h, 8h, 12h, 24h, 36h) using a visual camera, and the changes in the fluorescence intensity of the tumor site were measured; 100μL (2mM) NO2-Cy-Gxtb-Gd-Biotin solution was administered to the orthotopic lung cancer model mice by pulmonary instillation, and the lungs of the orthotopic lung cancer model mice were imaged at different time points (0h, 2h, 4h, 8h, 12h, 24h, 36h) using a 9.4T nuclear magnetic resonance spectrometer, and the changes in the magnetic resonance intensity of the tumor site were measured.

[0090] Test results: The fluorescence intensity change of the tumor site of the mouse in situ lung cancer model with the NO2-Cy-Gxtb-Gd-Biotin compound prepared in Example 1 under the action of nitroreductase in the tumor area is shown in the figure below: Figure 5 As shown, with the extension of time, the fluorescence intensity of the tumor area gradually increased, reaching the highest fluorescence intensity at 12 hours; the magnetic resonance signal of the tumor area also gradually increased, reaching the highest value at 8 hours, indicating that the compound NO2-Cy-Gxtb-Gd-Biotin has a good tumor imaging effect.

Claims

1. A nitroreductase-responsive fluorescence and magnetic resonance dual-modal probe NO2-Cy-Gxtb-Gd-Biotin designed based on gemcitabine prodrug, characterized in that: Its structural formula is as follows:

2. The method for synthesizing the probe NO2-Cy-Gxtb-Gd-Biotin according to claim 1, characterized in that: The steps include: (1) N2-Fmoc-N 6 -Biotin-lysine and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate were mixed in N,N-dimethylformamide, and N,N-diisopropylethylamine and 2,2',2"-(10-(2-((2-aminoethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate in N,N-dimethylformamide were added, and stirred at 25-40°C for 4-8 hours to obtain compound 4, and the reaction formula is as follows: (2) Azide-PEG4-acetic acid and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate were mixed in N,N-dimethylformamide, and N,N-diisopropylethylamine and the N,N-dimethylformamide solution of compound 4 after de-Fmoc were added, and stirred at 25-40° C. for 4-8 hours to obtain compound 5. The reaction formula is as follows: (3) Compound 5 is dissolved in a hydrochloric acid ethyl acetate solution, stirred at 25-40° C. for 4-10 hours, and then gadolinium chloride hexahydrate is added, and stirred at 25-40° C. for 6-12 hours to obtain compound 6. The reaction formula is as follows: (4) Compound 8, p-nitrobenzyl bromide, potassium iodide and potassium carbonate are dissolved in acetone and stirred at 25-60° C. for 2-3 days to obtain compound 9. The reaction formula is as follows: (5) Compound 9, p-nitrophenyl chloroformate and pyridine are dissolved in dichloromethane and stirred at 25-60° C. for 6-12 hours to obtain compound 10. The reaction formula is as follows: (6) Compound 10 and gemcitabine were dissolved in N,N-dimethylformamide, N,N-diisopropylethylamine and 4-dimethylaminopyridine were added, and stirred at room temperature for 8-24 hours to obtain compound 11. The reaction formula is as follows: (7) Compound 11 and compound 6 were dissolved in N,N-dimethylformamide, and aqueous solutions of copper sulfate and sodium ascorbate were added, and stirred at room temperature for 2-8 hours to obtain the probe NO2-Cy-Gxtb-Gd-Biotin. The reaction formula is as follows:

3. Use of the probe NO2-Cy-Gxtb-Gd-Biotin according to claim 1 in the preparation of a developer for magnetic resonance imaging and fluorescence imaging.

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