WO2023087169A1 - Two prostate-specific membrane antigen-targeting fluorescent probes, method for preparing same and application thereof - Google Patents

Two prostate-specific membrane antigen-targeting fluorescent probes, method for preparing same and application thereof Download PDF

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WO2023087169A1
WO2023087169A1 PCT/CN2021/131122 CN2021131122W WO2023087169A1 WO 2023087169 A1 WO2023087169 A1 WO 2023087169A1 CN 2021131122 W CN2021131122 W CN 2021131122W WO 2023087169 A1 WO2023087169 A1 WO 2023087169A1
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prostate cancer
pharmaceutically acceptable
acceptable salt
targeting
fluorescent probe
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PCT/CN2021/131122
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French (fr)
Chinese (zh)
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邢念增
胡海宇
武岭岭
赵钦欣
王庆华
张青扬
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中国医学科学院肿瘤医院
中国医学科学院药物研究所
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom

Definitions

  • the invention relates to two prostate-specific membrane antigen-targeted fluorescent probes, a preparation method and an application thereof, and belongs to the fields of biotechnology and medicine preparation.
  • PCa prostate cancer
  • PSMA Prostate-specific membrane antigen
  • PSMA Prostate-specific membrane antigen
  • the relative molecular mass is about 100*10 3 . 750 amino acids, including 707 in the extracellular segment, 24 in the transmembrane segment, and 19 in the intracellular segment. Its intracellular and extracellular segments contain multiple antigenic epitopes, which are closely related to various proteins, and affect the molecular characteristics and protein localization of PSMA to a large extent.
  • oleic acid is a monounsaturated fatty acid, which can not only enhance the affinity between fluorescent molecules and cell membranes, but also inhibit or kill tumor cells (Eiber M, Fendler WP, Rowe SP, Calais J, Hofman MS , Maurer T, et al. Prostate-Specific Membrane Antigen Ligands for Imaging and Therapy. J Nucl Med. 2017, 58:67s-76s.). Therefore, fluorescent probes combined with anti-PSMA ligands are of great significance in fluorescence-guided targeted prostate surgery.
  • the object of the present invention is to provide the Cy fluorescent probe compound targeting prostate cancer or its acceptable salt and the Cy fluorescent probe compound targeting prostate cancer and cell membrane or its acceptable salt, its preparation method and its application .
  • the present invention provides following technical scheme:
  • F represents a marker molecule
  • the marker molecule refers to a marker that can directly or indirectly generate a detectable signal.
  • L 1 and L 2 represent linking groups, and L 1 connects the marker molecule F and the linking group L 2 .
  • L 2 connects linking group L 1 , targeting groups T 1 and T 2 .
  • T 1 and T 2 represent targeting groups respectively, and T 1 can recognize related proteins in tumor cells for detection of tumor cells.
  • L 2 and T 2 may or may not exist.
  • F represents a labeling molecule
  • the labeling molecule refers to a label that can directly or indirectly generate a detectable signal.
  • radioisotopes fluorophores, fluorescent proteins.
  • the radioisotope is selected from 111 In( ⁇ ), 99 mTc( ⁇ );
  • the fluorophore is selected from 7-dimethylaminocoumarin-4-acetic acid succinimidyl ester, 5/6-carboxyfluorescein and tetramethylrhodan Ming, BODIPY-493/503, BODIPY-FL, BODIPY-TMR, BODIPY-TMR-X, BODIPY-TR-X, BODIPY630/550-X, BODIPY-650/665-X, Alexa 350, Alexa 488, Alexa 532 , Alexa 546, Alexa 555, Alexa 635, Alexa 647, Cyanine 3 (Cy 3), Cyanine 3B (Cy 3B), Cyanine 5
  • L2 represents a linking group, which can be absent or present, and is selected from serine, threonine, cysteine, tyrosine, and lysine;
  • T1 stands for Targeted Prostate Cancer Specific Membrane Antigen Group, which can be selected from:
  • T2 represents targeting cell membrane groups, including trans-oleic acid, linoleic acid, C10-30 alkyl acid, C10-30 alkenyl acid, and C10-30 alkynyl acid.
  • the Cy fluorescent probe compound targeting prostate cancer shown in the above formula I can specifically be:
  • the targeted prostate cancer-specific membrane antigen group can be selected from:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 are independently selected from hydrogen atom, sulfonic acid group, sulfonamide, hydroxyl, amino, F, Cl, Br, I, nitro, C1 ⁇ 6 alkyl, C1 ⁇ 6 alkenyl, C1 ⁇ 6 Alkynyl, C1 ⁇ 6 alkoxy, C1 ⁇ 6 alkylamino, C1 ⁇ 6 carboxylic acid, C1 ⁇ 6 methyl carboxylate, C1 ⁇ 6 alkylamide (specifically hydrogen atom, sulfonic acid group, methyl, ethyl); or R 1 and R 2 , R 2 and R 3 , R 3 and R 4 , R 6 and R 7 , R 14 and R 15 , R 16 and R 17 , R
  • R 20 , R 21 , R 22 , R 23 are independently selected from hydrogen atom, sulfonic acid group, sulfonamide, hydroxyl, amino, F, Cl, Br, I, nitro or C1 ⁇ 6 alkyl, C1 ⁇ 6 alkenes Base, C1 ⁇ 6 alkynyl; said C1 ⁇ 6 is selected from C1, C2, C3, C4, C5, C6;
  • R 24 is selected from independently selected from hydrogen, sulfonic acid group, sulfonamide group, hydroxyl, amino, F, Cl, Br, I, or C1 ⁇ 6 alkyl, C1 ⁇ 6 alkenyl, C1 ⁇ 6 alkynyl;
  • n represents an integer of 0-4, specifically 0, 1, 2, 3, 4;
  • n an integer of 0-4, specifically 0, 1, 2, 3, 4;
  • p represents an integer from 0 to 9, specifically 0, 1, 2, 3, 4, 5, 6, 7, 8, 9;
  • X is selected from F, Cl, Br, I;
  • L1 is selected from C1 ⁇ 9 alkyl, C1 ⁇ 9 alkenyl, C1 ⁇ 9 alkynyl, and said C1 ⁇ 9 is selected from C1, C2, C3, C4, C5, C6, C7, C8, C9;
  • R 25 and R 26 are each independently selected from
  • the Cy-type fluorescent probe compound targeting prostate cancer shown in the above formula I is selected from Cy3, Cy3.3, Cy5, Cy5.5, Cy7, Cy7 except for the L2 -targeting prostate cancer-specific membrane antigen group. .5, the structure is as follows:
  • the targeted prostate cancer specific membrane antigen group is selected from
  • the targeting cell membrane group can be selected from oleic acid, trans oleic acid, linoleic acid, octadecanoic acid;
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 are independently selected from hydrogen atom, sulfonic acid group, sulfonamide, hydroxyl, amino, F, Cl, Br, I, nitro, C1 ⁇ 6 alkyl, C1 ⁇ 6 alkenyl, C1 ⁇ 6 Alkynyl, C1 ⁇ 6 alkoxy, C1 ⁇ 6 alkylamino, C1 ⁇ 6 carboxylic acid, C1 ⁇ 6 methyl carboxylate, C1 ⁇ 6 alkylamide; or R 1 and R 2 , R 2 and R 3 , R 3 and R 4 , R 6 and R 7 , R 14 and R 15 , R 16 and R 17 , R 17 and R 18 , R 18 and R 19 are adjacent to benzene ring, n
  • R 20 , R 21 , R 22 , R 23 are independently selected from hydrogen atom, sulfonic acid group, sulfonamide, hydroxyl, amino, F, Cl, Br, I, nitro or C1 ⁇ 6 alkyl, C1 ⁇ 6 alkenes Base, C1 ⁇ 6 alkynyl; said C1 ⁇ 6 is selected from C1, C2, C3, C4, C5, C6;
  • R 24 is selected from independently selected from hydrogen, sulfonic acid group, sulfonamide group, hydroxyl, amino, F, Cl, Br, I, or C1 ⁇ 6 alkyl, C1 ⁇ 6 alkenyl, C1 ⁇ 6 alkynyl;
  • n represents an integer representing 0-4, specifically 0, 1, 2, 3, 4;
  • n an integer of 0-4, specifically 0, 1, 2, 3, 4;
  • p represents an integer from 0 to 9, specifically 0, 1, 2, 3, 4, 5, 6, 7, 8, 9;
  • X represents F, Cl, Br, I
  • L1 is selected from C1 ⁇ 9 alkyl, C1 ⁇ 9 alkenyl, C1 ⁇ 9 alkynyl, and said C1 ⁇ 9 is selected from C1, C2, C3, C4, C5, C6, C7, C8, C9;
  • L2 is selected from C1-20 alkyl, C1-20 alkenyl, C1-20 alkynyl, and the C1-20 is selected from C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20;
  • L3 is selected from serine, threonine, cysteine, tyrosine, lysine;
  • R 25 , R 26 , R 27 are independently selected from
  • Cy-type fluorescent probe compound targeting prostate cancer shown in the above formula I can be:
  • the first aspect of the technical solution of the present invention is a Cy-type fluorescent probe compound targeting prostate cancer or a pharmaceutically acceptable salt thereof, and the pharmaceutically acceptable salt is an organic acid salt or an inorganic acid salt of the compound,
  • the pharmaceutically acceptable salt is an organic base salt or an inorganic base salt of the compound, and the organic base can be pyridine, triethylamine, diethylamine, N-methylmorpholine, tetramethylethylenediamine No said organic base can be phosphate, hydrogen phosphate, carbonate, bicarbonate, potassium carbonate, bicarbonate, hypochlorite, hypobromite, silicate.
  • the second aspect of the technical solution of the present invention is to provide the application of the Cy-type fluorescent probe compound targeting prostate cancer in the first aspect or a pharmaceutically acceptable salt thereof in the preparation of a reagent for detecting prostate cancer-specific membrane antigen.
  • the detection is the detection of prostate cancer specific membrane antigen at the molecular level.
  • the third aspect of the technical solution of the present invention is to provide the application of Cy-type fluorescent probe compounds targeting prostate cancer or pharmaceutically acceptable salts thereof in the preparation of reagents for targeted detection of prostate cancer. Biopsy-level targeted detection of prostate cancer.
  • the prostate cancer is a prostate cancer expressing PSMA at a high level, which can be distinguished from normal prostate cells and tissues.
  • the fourth aspect of the technical solution of the present invention also provides the application of the Cy-type fluorescent probe compound targeting prostate cancer or a pharmaceutically acceptable salt thereof in the preparation of a prostate cancer diagnostic reagent.
  • the Cy fluorescent probe compound targeting prostate cancer of the present invention has good affinity and specificity to prostate cancer cells expressing PSMA.
  • Cy-like fluorescent probe compounds targeting prostate cancer and cell membranes can increase fluorescent signals, reduce background signals, and have good stability within a certain period of time, as well as temporal and spatial resolutions in small animal live imaging .
  • the Cy-type fluorescent probe compound targeting prostate cancer has no inhibitory effect on cells at a concentration of 25 ⁇ M within 24 hours, that is, the toxicity is low.
  • the invention designs and synthesizes a novel fluorescent probe molecule specifically targeting prostate cancer, which greatly improves the specificity and sensitivity of prostate cancer detection, provides high-precision and high-sensitivity real-time imaging navigation for individualized precision surgery, and realizes prostate cancer accurate diagnosis and treatment.
  • Fig. 1 is a synthesis route diagram of probe molecules 1 and 2 in the embodiment of the present invention.
  • Figure 2 shows the absorption and emission spectra results of probes 1 and 2.
  • Fig. 3 shows the cell viability results of probes 1 and 2 on prostate cancer cells (C4-2) within 24 hours.
  • Figure 4 shows the co-localization fluorescence imaging results of probes 1 and 2 in prostate cancer cells (C4-2, PC3).
  • Figure 5 shows the flow cytometric results of probes 1 and 2 in prostate cancer cells (C4-2, PC3).
  • Fig. 6 shows the imaging results of probe 2 of the present invention in small animals.
  • Figure 7a shows fluorescence imaging of prostate cancer in mice under fluorescence laparoscopy
  • Figure 7b shows real-time tumor resection under fluorescence guidance
  • Figure 7c shows fluorescence imaging after tumor resection
  • Figure 7d shows surgical specimens confirmed by HE staining as prostate cancer.
  • Figure 8a shows prostate cancer tissue (upper part) and normal prostate tissue (lower part);
  • Figure 8b shows prostate cancer with high expression of PSMA (upper part) and prostate cancer with low expression, even normal prostate tissue with no expression (lower part);
  • the results in Fig. 8c and Fig. 8d suggest that the probe 2 has good imaging performance on clinical prostate cancer samples, but not on normal prostate tissue.
  • the invention designs and synthesizes a novel fluorescent probe molecule specifically targeting prostate cancer, which greatly improves the specificity and sensitivity of prostate cancer detection, provides high-precision and high-sensitivity real-time imaging navigation for individualized precision surgery, and realizes prostate cancer accurate diagnosis and treatment.
  • Substrate 1 (10g, 62.8mmol) and bromoethane (6.84g, 62.8mmol) were dissolved in toluene solvent (32mL). After the reaction was complete, the reaction system was distilled off the solvent under reduced pressure, washed, dried under vacuum, and directly for the next step. Finally, 4.8 g of reddish-brown solid product 2 was obtained with a yield of 28.5%.
  • Substrates 6 (2.0g, 11.6mmol) and 5 (3.1g, 11.6mmol) were dissolved in acetic anhydride (33mL). After the reaction was over, they were distilled under reduced pressure, and the residue was re-dissolved in absolute ethanol (33mL), followed by adding 4 (4.9 g, 1.2 mmol) and sodium acetate (2.85 g, 34.8 mmol). After the reaction, vacuum distillation, washing, extraction, and vacuum distillation, the residue was purified by column chromatography to obtain product 7 as a dark green solid, 2.4 g, with a yield of 30%.
  • Substrate 8 (930 mg, 2.73 mmol) was added to dichloromethane solvent (27 mL), EDCI (629 mg, 3.28 mmol), HOBt (443 mg, 3.28 mmol) and DIPEA (424 mg, 3.28 mmol) were added, and substrate 9 was added (1.33g, 2.73mmol), after the reaction was completed, extraction, vacuum distillation, and column chromatography of the residue gave compound 10 as a white solid, 1.6g, yield 70%.
  • Substrate 10 (1.6 g, 1.97 mmol) and 10% palladium on carbon (160 mg) were dispersed in methanol solvent (32 mL) for catalytic hydrogenation. After the reaction was completed, suction filtration, vacuum distillation, and chromatographic purification of the residue gave product 11 as a colorless oil, 1.3 g, with a yield of 97%.
  • Substrate 7 (80mg, 0.138mmol) was dissolved in dichloromethane (1.4mL), EDCI (32mg, 0.166mmol), HOBt (22mg, 0.166mmol) and DIPEA (21.5mg, 0.166mmol) were added sequentially, and the substrate After 11 (83mg, 0.138mmol), continue to stir at room temperature for 1.5h. After the reaction was completed, it was washed and distilled under reduced pressure. After the residue was purified by column chromatography, it was dissolved in dichloromethane (2.76 mL), and trifluoroacetic acid (2.76 mL) was added. After the reaction was completed, it was purified by direct chromatography.
  • Probe 1 was obtained as a green solid, 29 mg, with a two-step yield of 21.7%.
  • Substrate 12 (814mg, 1.62mmol) was uniformly dispersed in dichloromethane solvent (16mL), EDCI (372mg, 1.94mmol), HOBt (262mg, 1.94mmol) and DIPEA (96mg, 1.94mmol) were added in sequence, and the bottom Compound 11 (1.1g, 1.62mmol). After the reaction was completed, the solvent was extracted and distilled off under reduced pressure. The residue was purified by column chromatography to obtain compound 13 as a colorless oil, 1.49g, with a yield of 77%.
  • Substrate 13 (1.49g, 1.28mmol) and 10% palladium on carbon (149mg) were dispersed in methanol solvent (20mL) for catalytic hydrogenation to give product 14 as a white solid, 1.04g, with a yield of 78.9%. used directly in the next step.
  • Substrate 15 (342mg, 1.01mmol) was uniformly dispersed in dichloromethane solvent (10mL), and after adding EDCI (232mg, 1.21mmol), HOBt (164mg, 1.21mmol) and DIPEA (156mg, 1.21mmol), added Compound 14 (1.04g, 1.01mmol), after the completion of the reaction, was extracted, evaporated under reduced pressure, and purified by column chromatography to obtain Compound 16 as a white solid, 0.559g, with a yield of 42.8%.
  • Substrate 16 (559 mg, 0.443 mmol) was dissolved in acetonitrile solvent (13.5 mL), and diethylamine (3.17 g, 43.3 mmol) was added. The reaction system was stirred at room temperature for 40 min. The solvent was distilled off under reduced pressure, and the residue was purified by column chromatography to obtain product 17 as a white solid, 200 mg, with a yield of 43.2%, which was directly used in the next step.
  • Substrate 7 (40mg, 0.067mmol) was dissolved in dichloromethane (1.34mL), EDCI (19.4mg, 0.101mmol) and HOBt (13.6mg, 0.101mmol) were added successively, followed by substrate 17 (57mg, 0.054mmol ), after the completion of the reaction, washing, distillation under reduced pressure, after the residue was purified by column chromatography, after the product was dissolved in dichloromethane solution (2.68mL), trifluoroacetic acid (2.68mL) was added, and the reaction was completed under argon protection. Afterwards, the solvent was distilled off under reduced pressure, and the residue was directly purified by HPLC.
  • Probe 2 was obtained as a green solid, 15.2 mg, with a two-step yield of 16.3%.
  • 1 H NMR 600MHz,DMSO-d 6 ) ⁇ 8.27(s,2H),7.72(s,1H),7.64(s,2H),7.49-7.42(m,3H),7.30(s,2H), 6.36-6.30(m,2H),6.25(s,1H),5.34-5.28(m,2H),4.26(s,2H),4.20(s,2H),4.08(s,1H),3.99(s, 1H),3.85(s,2H),3.63(s,2H),3.59-3.545(m,4H),3.53-3.49(m,3H),3.39(s,2H),3.18(s,2H),3.07 (s,2H),2.98(s,2H),2.79-2.65(m,4H),2.26(s,2H),2.14(s,2H),2.00(s,2H
  • Embodiment 2 probe 1, the measurement of the absorption emission spectrum of 2
  • Embodiment 3 probe 1, 2 are to prostate cancer cell (C4-2) cytotoxic experiment
  • Cell viability was determined by MTS method.
  • One experimental group and nine control groups were set up.
  • the adherent cells (C4-2 cells) in the 96-well plate were sequentially treated with probe 2 at concentrations of 0.2 ⁇ M, 0.4 ⁇ M, 0.8 ⁇ M, 1.6 ⁇ M, 3.2 ⁇ M, 6.25 ⁇ M, 12.5 ⁇ M and 25 ⁇ M, at 37° C.
  • 20 ⁇ L MTS was added to each well, and then cultured at 37 °C and 5% CO2 for 4 hours.
  • the absorbance OD value of each group was measured on a microplate reader, and the wavelength was set at 490 nm.
  • cell survival rate (%) (OD of experimental group/OD of control group) ⁇ 100%.
  • Probes 1 and 2 did not exhibit cytotoxicity to C4-2 at a concentration of 25 ⁇ M, indicating that probes 1 and 2 had low cytotoxicity. See Figure 3.
  • Fig. 3 shows the cell viability results of probes 1 and 2 on prostate cancer cells (C4-2) within 24 hours.
  • Prostate cancer cells (C4-2, PC3) were inoculated into 8-well plates (1 ⁇ 106 cells), and formed a monolayer within 48 hours. Incubate with fresh medium (200uL) containing probes 1, 2 and the small molecule inhibitor 2-PMPA, respectively, and then stain with Hoechst 33342. After the incubation is complete, confocal cells are imaged.
  • probes 1 and 2 had a good imaging effect in PSMA-positive prostate cell C4-2, and probe 2 was stronger than probe 1, but did not image in PSMA-negative prostate cell PC3;
  • the PSMA inhibitor 2-PMPA was added, the cell imaging was significantly weakened, indicating that probes 1 and 2 had good affinity and specificity for PSMA-positive prostate cancer cells. See Figure 4.
  • Embodiment 5 the flow cytometric experiment of probe 1,2 in prostate cancer cell (C4-2, PC3)
  • Prostate cancer cells (C4-2, PC3) were seeded into 24-well plates. Incubate with fresh medium (400uL) containing probes 1 and 2, respectively. After incubation, flow cytometry was performed.
  • BALB/c nude mice with an average weight of 20 grams at 6-8 weeks were subcutaneously injected with 200 ⁇ L of prostate cell C4-2 cell suspension at the end of the left hind limb to successfully establish a subcutaneous xenograft tumor model. After about two weeks, small animal live imaging tests were performed. . Probe 2 was injected separately into the tail vein. Live imaging monitoring of small animals was performed at 2h, 4h, 6h, 8h, 12h, 24h, 36h, 48h, and 72h (excitation wavelength 745nm, emission wavelength 820nm).
  • probe 2 could be used to specifically target PSMA imaging, and the fluorescence intensity reached the maximum at 12h and lasted until 36h; in addition, the signal-to-noise ratio of probe 2 was the highest at 24h, reaching 3.64 ⁇ 0.16. It shows that probe 2 has good temporal and spatial resolution for PSMA-targeted imaging. See Figure 6.
  • Example 7 Evaluation of the application of probe 2 in real-time fluorescence laparoscopic surgical resection of mouse prostate cancer
  • Figure 8a shows that the above is the prostate Cancer, the bottom is the normal prostate tissue, further immunohistochemistry of PSMA, Figure 8b shows that the prostate cancer with high expression of PSMA on the top, the prostate cancer with low expression, or even the normal prostate tissue without expression, confirms our prediction. Then we co-incubated the postoperative frozen sections and clinical fresh samples with our probe at 37 degrees Celsius for 1h. The results in Figure 8c and Figure 8d suggest that our probe and clinical prostate cancer samples have good imaging properties, and and Normal prostate tissue is not imaged. It shows that probe 2 has a good targeting imaging property for prostate cancer. See Figure 8.
  • the invention designs and synthesizes a novel fluorescent probe molecule specifically targeting prostate cancer, which greatly improves the specificity and sensitivity of prostate cancer detection, provides high-precision and high-sensitivity real-time imaging navigation for individualized precision surgery, and realizes prostate cancer accurate diagnosis and treatment.

Abstract

Provided are two prostate-specific membrane antigen-targeting fluorescent probes, a method for preparing same and the application thereof. The probes have a good affinity and specificity to prostate cancer cells which express a PSMA. In small-animal in vivo imaging, fluorescence signals can be increased, background signals can be reduced, and a good stability, time resolution and spatial resolution are achieved within a certain time. New specific prostate cancer targeting fluorescent probe molecules are designed and synthesized, thereby greatly improving the specificity and sensitivity of prostate cancer detection; same can be used for testing a prostate cancer clinical sample; and high-precision and high-sensitivity real-time imaging navigation is also provided for an individualized accurate surgery, thereby realizing accurate prostate cancer diagnosis and treatment.

Description

两种前列腺特异性膜抗原靶向荧光探针及其制备方法与应用Two kinds of prostate-specific membrane antigen targeting fluorescent probes and their preparation methods and applications 技术领域technical field
本发明涉及两种前列腺特异性膜抗原靶向荧光探针及其制备方法及其应用，属于生物技术及医药制备领域。The invention relates to two prostate-specific membrane antigen-targeted fluorescent probes, a preparation method and an application thereof, and belongs to the fields of biotechnology and medicine preparation.
背景技术Background technique
前列腺癌(prostate cancer，PCa)发病率在欧美国家已居男性恶性肿瘤首位，是癌症病死率的第二位原因(Siegel RL,Miller KD,Jemal A.Cancer Statistics,2017.CA Cancer J Clin.2019,69(1):7-34)。在我国，随着人们生活水平的提高、人口结构的老龄化以及饮食结构的改变，前列腺癌发病率呈上升趋势，成为威胁男性健康的主要因素(Chen W,Zheng R,Baade PD,Zhang S,Zeng H,Bray F,et al.Cancer statistics in China,2015.CA Cancer J Clin.2016,66:115-132.)。The incidence of prostate cancer (PCa) has ranked first in male malignant tumors in European and American countries, and it is the second cause of cancer mortality (Siegel RL, Miller KD, Jemal A. Cancer Statistics, 2017. CA Cancer J Clin.2019 , 69(1):7-34). In my country, with the improvement of people's living standards, the aging of population structure and the change of diet structure, the incidence of prostate cancer is on the rise, becoming the main factor threatening men's health (Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, et al. Cancer statistics in China, 2015. CA Cancer J Clin. 2016, 66:115-132.).
目前，根治性前列腺切除术和/或盆腔淋巴结清扫术是可能治愈局限性前列腺癌最有效的方法之一。但手术治疗前列腺癌面临两个重要问题(Mottet N,Bellmunt J,Bolla M,Briers E,Cumberbatch MG,De Santis M,et al.EAU-ESTRO-SIOG Guidelines on Prostate Cancer.Part 1:Screening,Diagnosis,and Local Treatment with Curative Intent.Eur Urol.2017,71:618-29.；Sopko NA,Burnett AL.Erection rehabilitation following prostatectomy--current strategies and future directions.Nat Rev Urol.2016,13:216-25.)。其一就是手术切缘的问题，由于术中很难评估前列腺癌的侵犯程度，所以完全切除肿瘤使切缘阴性仍然很困难(Yossepowitch O,Briganti A,Eastham JA,Epstein J,Graefen M,Montironi R,et al.Positive surgical margins after radical prostatectomy:a systematic review and contemporary update.Eur Urol.2014,65:303-13)。第二大问题就是淋巴结转移的问题。在手术过程中，对于大多数体积小的转移淋巴结，外科医生的肉眼是看不见的，容易漏掉一些微转移的一些病灶(Rocha R,Fiorelli RK,Buogo G, Rubistein M,Mattos RM,Frota R,Coelho RF,Palmer K,Patel V.Robotic-assisted laparoscopic prostatectomy(RALP):a new way to training.J Robot Surg.2016,10:19-25)。为了增加完全切除所有前列腺肿瘤组织的机会，需要更敏感、更特异的技术，以便在术中实时检测所有癌组织，包括最微小的病变，减少术中没必要的损伤，减少术后并发症和局部复发率，从而提高患者术后生活质量和生存期。Currently, radical prostatectomy and/or pelvic lymph node dissection is one of the most effective treatments for potentially curative localized prostate cancer. However, surgical treatment of prostate cancer faces two important issues (Mottet N, Bellmunt J, Bolla M, Briers E, Cumberbatch MG, De Santis M, et al. EAU-ESTRO-SIOG Guidelines on Prostate Cancer. Part 1: Screening, Diagnosis, and Local Treatment with Curative Intent.Eur Urol.2017,71:618-29.;Sopko NA,Burnett AL.Erection rehabilitation following prostatetomy--current strategies and future directions.Nat Rev Urol.2016,13:216-25.) . One is the problem of surgical margins. Since it is difficult to assess the extent of prostate cancer invasion during surgery, it is still difficult to completely resect the tumor with negative margins (Yossepowitch O, Briganti A, Eastham JA, Epstein J, Graefen M, Montironi R , et al. Positive surgical margins after radical prostateectomy: a systematic review and contemporary update. Eur Urol.2014,65:303-13). The second major problem is the problem of lymph node metastasis. During the operation, most of the small metastatic lymph nodes are invisible to the naked eye of the surgeon, and it is easy to miss some lesions of micrometastases (Rocha R, Fiorelli RK, Buogo G, Rubistein M, Mattos RM, Frota R , Coelho RF, Palmer K, Patel V. Robotic-assisted laparoscopic prostateectomy (RALP): a new way to training. J Robot Surg. 2016, 10:19-25). To increase the chances of complete resection of all prostate tumor tissue, more sensitive and specific techniques are needed to detect all cancerous tissue, including the tiniest of lesions, in real time intraoperatively, to reduce unnecessary intraoperative trauma, and to reduce postoperative complications and Local recurrence rate, thereby improving the quality of life and survival of patients after surgery.
近年来，荧光引导手术(Fluorescence guided surgery,FGS)在术中检测恶性组织方面取得了重大进展，这是一种敏感的技术，可以利用积聚在肿瘤组织中的荧光染料改善手术中阳性切除边缘的可视化。有许多关于荧光探针用于前列腺癌的手术治疗的报道，但结果不是很理想，主要体现为荧光探针敏感性、特异性差，肿瘤与周围组织的比率显影差(Tumor-to-background ratio，TBR)，影响了前列腺的手术效果。In recent years, significant progress has been made in the intraoperative detection of malignant tissue in fluorescence guided surgery (FGS), a sensitive technique that utilizes fluorescent dyes that accumulate in tumor tissue to improve the detection of positive resection margins during surgery. visualization. There are many reports about the use of fluorescent probes in the surgical treatment of prostate cancer, but the results are not very satisfactory, mainly reflected in the poor sensitivity and specificity of fluorescent probes, and the poor contrast between tumor and surrounding tissue (Tumor-to-background ratio, TBR), affecting the surgical effect of the prostate.
前列腺特异性膜抗原(Prostate-specific membrane antigen,PSMA)是一种II型跨膜糖蛋白，PSMA由前列腺上皮细胞分泌，其氨基端位于细胞膜内，相对分子质量约为100*10 3，共含750个氨基酸，其中胞外段707个，跨膜段24个，胞内段19个。其胞内段和胞外段含有多个抗原表位，与多种蛋白有着密切联系，在很大程度上影响着PSMA的分子特性和蛋白定位。因为PSMA在前列腺癌细胞表面的表达量是其生理性表达部位的100-1000倍，并且其表达量与前列腺癌的分级、分期呈正相关，在晚期前列腺癌细胞表面表达量升高更显著(Eiber M,Fendler WP,Rowe SP,Calais J,Hofman MS,Maurer T,et al.Prostate-Specific Membrane Antigen Ligands for Imaging and Therapy.J Nucl Med.2017,58:67s-76s.)。此外，油酸(oleic acid，OA)是一种单不饱和脂肪酸，不仅可以增强荧光分子与细胞膜的亲和力，同时抑制或是杀伤肿瘤细胞(Eiber M,Fendler WP,Rowe SP,Calais J,Hofman MS,Maurer T,et al.Prostate-Specific Membrane Antigen Ligands for Imaging and Therapy.J Nucl Med.2017,58:67s-76s.)。所以，以抗PSMA配体结合的荧光探针在进行荧光引导的靶向前列腺手术治疗具有非常重要的意义。 Prostate-specific membrane antigen (PSMA) is a type II transmembrane glycoprotein. PSMA is secreted by prostate epithelial cells, and its amino terminal is located in the cell membrane. The relative molecular mass is about 100*10 3 . 750 amino acids, including 707 in the extracellular segment, 24 in the transmembrane segment, and 19 in the intracellular segment. Its intracellular and extracellular segments contain multiple antigenic epitopes, which are closely related to various proteins, and affect the molecular characteristics and protein localization of PSMA to a large extent. Because the expression level of PSMA on the surface of prostate cancer cells is 100-1000 times that of its physiological expression site, and its expression level is positively correlated with the grade and stage of prostate cancer, and the expression level on the surface of advanced prostate cancer cells increases more significantly (Eiber M, Fendler WP, Rowe SP, Calais J, Hofman MS, Maurer T, et al. Prostate-Specific Membrane Antigen Ligands for Imaging and Therapy. J Nucl Med. 2017, 58:67s-76s.). In addition, oleic acid (OA) is a monounsaturated fatty acid, which can not only enhance the affinity between fluorescent molecules and cell membranes, but also inhibit or kill tumor cells (Eiber M, Fendler WP, Rowe SP, Calais J, Hofman MS , Maurer T, et al. Prostate-Specific Membrane Antigen Ligands for Imaging and Therapy. J Nucl Med. 2017, 58:67s-76s.). Therefore, fluorescent probes combined with anti-PSMA ligands are of great significance in fluorescence-guided targeted prostate surgery.
发明公开invention disclosure
本发明的目的是提供靶向前列腺癌的Cy类荧光探针化合物或其可接受的盐和靶向前列腺癌和细胞膜的Cy类荧光探针化合物或其可接受的盐、其制备方法以及其应用。The object of the present invention is to provide the Cy fluorescent probe compound targeting prostate cancer or its acceptable salt and the Cy fluorescent probe compound targeting prostate cancer and cell membrane or its acceptable salt, its preparation method and its application .
本发明提供如下技术方案：The present invention provides following technical scheme:
本发明技术方案的第一方面是提供了如式I所示的靶向前列腺癌的Cy类荧光探针化合物或其药学上可接受的盐：The first aspect of the technical solution of the present invention is to provide a Cy fluorescent probe compound targeting prostate cancer as shown in formula I or a pharmaceutically acceptable salt thereof:
Figure PCTCN2021131122-appb-000001
Figure PCTCN2021131122-appb-000001
其中：F代表标记分子，标记分子是指能够直接或间接产生可检测信号的标记物。L 1和L 2代表连接基团，L 1连接标记分子F和连接基团L 2。L 2连接连接基团L 1，靶向基团T 1和T 2。T 1和T 2分别代表靶向基团，T 1可以识别肿瘤细胞中相关蛋白，用于肿瘤细胞的检测。其中，L 2和T 2可以存在，也可以不存在。 Wherein: F represents a marker molecule, and the marker molecule refers to a marker that can directly or indirectly generate a detectable signal. L 1 and L 2 represent linking groups, and L 1 connects the marker molecule F and the linking group L 2 . L 2 connects linking group L 1 , targeting groups T 1 and T 2 . T 1 and T 2 represent targeting groups respectively, and T 1 can recognize related proteins in tumor cells for detection of tumor cells. Wherein, L 2 and T 2 may or may not exist.
F代表标记分子，标记分子是指能够直接或间接产生可检测信号的标记物。例如，放射性同位素、荧光团、荧光蛋白。放射性同位素选自 111In(γ)， 99mTc(γ)；荧光团选自7-二甲氨基香豆素-4-乙酸琥珀酰亚胺酯、5/6-羧基荧光素和四甲基罗丹明、BODIPY-493/503、BODIPY-FL、BODIPY-TMR、BODIPY-TMR-X、BODIPY-TR-X、BODIPY630/550-X、BODIPY-650/665-X、Alexa 350、Alexa 488、Alexa 532、Alexa 546、Alexa 555、Alexa 635、Alexa 647、花菁3(Cy 3),花菁3B(Cy 3B),花菁5(Cy 5),花菁5.5(Cy 5.5),花菁7(Cy7),花菁7.5(Cy 7.5),Cy7-Cl，ATTO 488,ATTO 532,ATTO 650,ATTO 650，DY-505，DY-547，DY-632，DY-647，IRDye78，ZW800+3C，S0456，AF647，Dylight680，Sulfo‐Cy5；荧光蛋白，例如绿色荧光蛋和具有不同吸收/发射特性的绿色荧光蛋白修饰物； F represents a labeling molecule, and the labeling molecule refers to a label that can directly or indirectly generate a detectable signal. For example, radioisotopes, fluorophores, fluorescent proteins. The radioisotope is selected from 111 In(γ), 99 mTc(γ); the fluorophore is selected from 7-dimethylaminocoumarin-4-acetic acid succinimidyl ester, 5/6-carboxyfluorescein and tetramethylrhodan Ming, BODIPY-493/503, BODIPY-FL, BODIPY-TMR, BODIPY-TMR-X, BODIPY-TR-X, BODIPY630/550-X, BODIPY-650/665-X, Alexa 350, Alexa 488, Alexa 532 , Alexa 546, Alexa 555, Alexa 635, Alexa 647, Cyanine 3 (Cy 3), Cyanine 3B (Cy 3B), Cyanine 5 (Cy 5), Cyanine 5.5 (Cy 5.5), Cyanine 7 (Cy7 ), Cyanine 7.5 (Cy 7.5), Cy7-Cl, ATTO 488, ATTO 532, ATTO 650, ATTO 650, DY-505, DY-547, DY-632, DY-647, IRDye78, ZW800+3C, S0456, AF647, Dylight680, Sulfo‐Cy5; fluorescent proteins such as green fluorescent eggs and modified green fluorescent proteins with different absorption/emission properties;
L1代表连接基团，选自
Figure PCTCN2021131122-appb-000002
其中，X代表羰基，磺酰基，亚磺酰基；m＝3-12，具体可为3,4,5,6,7,8,9,10,11,12；Y代表O或是S，NH，NCH 3，NCH 2CH 3； L1 represents a linking group selected from
Figure PCTCN2021131122-appb-000002
Among them, X represents carbonyl, sulfonyl, sulfinyl; m=3-12, specifically 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; Y represents O or S, NH , NCH 3 , NCH 2 CH 3 ;
L2代表连接基团，可以不存在，或是存在1个，选自丝氨酸，苏氨酸，半胱氨酸，酪氨酸，赖氨酸；L2 represents a linking group, which can be absent or present, and is selected from serine, threonine, cysteine, tyrosine, and lysine;
T1代表靶向前列腺癌特异性膜抗原基团，可选自：T1 stands for Targeted Prostate Cancer Specific Membrane Antigen Group, which can be selected from:
Figure PCTCN2021131122-appb-000003
Figure PCTCN2021131122-appb-000003
T2代表靶向细胞膜基团，包括反式油酸，亚油酸，C10～30烷基酸，C10～30烯基酸，C10～30炔基酸。T2 represents targeting cell membrane groups, including trans-oleic acid, linoleic acid, C10-30 alkyl acid, C10-30 alkenyl acid, and C10-30 alkynyl acid.
上述式I所示的靶向前列腺癌的Cy类荧光探针化合物具体可为：The Cy fluorescent probe compound targeting prostate cancer shown in the above formula I can specifically be:
Figure PCTCN2021131122-appb-000004
Figure PCTCN2021131122-appb-000004
其中：所述靶向前列腺癌特异性膜抗原基团可选自：Wherein: the targeted prostate cancer-specific membrane antigen group can be selected from:
Figure PCTCN2021131122-appb-000005
Figure PCTCN2021131122-appb-000005
R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 10、R 11、R 12、R 13、R 14、R 15、R 16、R 17、R 18、R 19独立地选自氢原子，磺酸基，磺酰胺，羟基，氨基，F,Cl,Br,I，硝基，C1～6烷基，C1～6烯基，C1～6炔基，C1～6烷氧基，C1～6烷氨基，C1～6羧酸，C1～6羧酸甲酯，C1～6烷基酰胺(具体可为氢原子、磺酸基、甲基、乙基)；或R 1与R 2，R 2与R 3，R 3与R 4，R 6与R 7，R 14与R 15，R 16与R 17，R 17与R 18，R 18 与R 19相邻位置以苯环，萘环，蒽环，菲环形式相连，其中所述苯环，萘环，蒽环，菲环无取代基，或有单取代磺酸基，双取代磺酸基，三取代磺酸基，四取代磺酸基；所述的C1～6选自C1,C2,C3,C4,C5,C6； R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 are independently selected from hydrogen atom, sulfonic acid group, sulfonamide, hydroxyl, amino, F, Cl, Br, I, nitro, C1~6 alkyl, C1~6 alkenyl, C1~6 Alkynyl, C1~6 alkoxy, C1~6 alkylamino, C1~6 carboxylic acid, C1~6 methyl carboxylate, C1~6 alkylamide (specifically hydrogen atom, sulfonic acid group, methyl, ethyl); or R 1 and R 2 , R 2 and R 3 , R 3 and R 4 , R 6 and R 7 , R 14 and R 15 , R 16 and R 17 , R 17 and R 18 , R 18 and The adjacent positions of R 19 are connected in the form of benzene ring, naphthalene ring, anthracene ring, and phenanthrene ring, wherein the benzene ring, naphthalene ring, anthracene ring, and phenanthrene ring have no substituents, or have single-substituted sulfonic acid groups, double-substituted sulfonic acid groups group, three-substituted sulfonic acid group, four-substituted sulfonic acid group; the C1-6 are selected from C1, C2, C3, C4, C5, C6;
R 20、R 21、R 22、R 23独立地选自氢原子，磺酸基，磺酰胺，羟基，氨基，F,Cl,Br,I，硝基或C1～6烷基，C1～6烯基，C1～6炔基；所述的C1～6选自C1,C2,C3,C4,C5,C6； R 20 , R 21 , R 22 , R 23 are independently selected from hydrogen atom, sulfonic acid group, sulfonamide, hydroxyl, amino, F, Cl, Br, I, nitro or C1~6 alkyl, C1~6 alkenes Base, C1～6 alkynyl; said C1～6 is selected from C1, C2, C3, C4, C5, C6;
R 24选自独立地选自氢、磺酸基，磺酰胺基，羟基，氨基，F,Cl,Br,I，或C1～6烷基，C1～6烯基，C1～6炔基； R 24 is selected from independently selected from hydrogen, sulfonic acid group, sulfonamide group, hydroxyl, amino, F, Cl, Br, I, or C1～6 alkyl, C1～6 alkenyl, C1～6 alkynyl;
m表示0-4的整数，具体可为0,1,2,3,4；m represents an integer of 0-4, specifically 0, 1, 2, 3, 4;
n表示0-4的整数，具体可为0,1,2,3,4；n represents an integer of 0-4, specifically 0, 1, 2, 3, 4;
p表示0-9的整数，具体可为0,1,2,3,4,5,6,7,8,9；p represents an integer from 0 to 9, specifically 0, 1, 2, 3, 4, 5, 6, 7, 8, 9;
X选自F,Cl,Br,I；X is selected from F, Cl, Br, I;
L 1选自C1～9烷基，C1～9烯基，C1～9炔基，所述C1～9选自C1,C2,C3,C4,C5,C6,C7,C8,C9； L1 is selected from C1～9 alkyl, C1～9 alkenyl, C1～9 alkynyl, and said C1～9 is selected from C1, C2, C3, C4, C5, C6, C7, C8, C9;
L 2选自聚乙二醇，
Figure PCTCN2021131122-appb-000006
其中n＝1-15，具体可为1,2,3,4,5,6,7,8,9,10,11,12,13,14,15； L2 is selected from polyethylene glycol,
Figure PCTCN2021131122-appb-000006
Where n=1-15, specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15;
R 25、R 26各自独立地选自
Figure PCTCN2021131122-appb-000007
Figure PCTCN2021131122-appb-000008
R 25 and R 26 are each independently selected from
Figure PCTCN2021131122-appb-000007
Figure PCTCN2021131122-appb-000008
上述式I所示的靶向前列腺癌的Cy类荧光探针化合物除L 2-靶向前列腺癌特异性膜抗原基团部分外选自Cy3,Cy3.3,Cy5,Cy5.5,Cy7,Cy7.5，结构如下： The Cy-type fluorescent probe compound targeting prostate cancer shown in the above formula I is selected from Cy3, Cy3.3, Cy5, Cy5.5, Cy7, Cy7 except for the L2 -targeting prostate cancer-specific membrane antigen group. .5, the structure is as follows:
Figure PCTCN2021131122-appb-000009
Figure PCTCN2021131122-appb-000009
其中：所述靶向前列腺癌特异性膜抗原基团选自Wherein: the targeted prostate cancer specific membrane antigen group is selected from
Figure PCTCN2021131122-appb-000010
Figure PCTCN2021131122-appb-000010
所述靶向细胞膜基团可选自油酸，反式油酸，亚油酸，十八烷酸；The targeting cell membrane group can be selected from oleic acid, trans oleic acid, linoleic acid, octadecanoic acid;
R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 10、R 11、R 12、R 13、R 14、R 15、R 16、R 17、R 18、R 19独立地选自氢原子，磺酸基，磺酰胺，羟基，氨基，F,Cl,Br,I，硝基，C1～6烷基，C1～6烯基，C1～6炔基，C1～6烷氧基，C1～6烷氨基，C1～6羧酸，C1～6羧酸甲酯，C1～6烷基酰胺；或R 1与R 2，R 2与R 3，R 3与R 4，R 6与R 7，R 14与R 15，R 16与R 17，R 17与R 18，R 18与R 19相邻位置以苯环，萘环，蒽环，菲环形式相连，其中所述苯环，萘环，蒽环，菲环无取代基，或有单取代磺酸基，双取代磺酸基，三取代磺酸基，四取代磺酸基；所述的C1～6选自C1,C2,C3,C4,C5,C6； R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 are independently selected from hydrogen atom, sulfonic acid group, sulfonamide, hydroxyl, amino, F, Cl, Br, I, nitro, C1~6 alkyl, C1~6 alkenyl, C1~6 Alkynyl, C1~6 alkoxy, C1~6 alkylamino, C1~6 carboxylic acid, C1~6 methyl carboxylate, C1~6 alkylamide; or R 1 and R 2 , R 2 and R 3 , R 3 and R 4 , R 6 and R 7 , R 14 and R 15 , R 16 and R 17 , R 17 and R 18 , R 18 and R 19 are adjacent to benzene ring, naphthalene ring, anthracene ring, phenanthrene ring The benzene ring, the naphthalene ring, the anthracene ring, and the phenanthrene ring have no substituents, or have a single-substituted sulfonic acid group, a double-substituted sulfonic acid group, a tri-substituted sulfonic acid group, and a four-substituted sulfonic acid group; the described C1～6 are selected from C1, C2, C3, C4, C5, C6;
R 20、R 21、R 22、R 23独立地选自氢原子，磺酸基，磺酰胺，羟基，氨基，F,Cl,Br,I，硝基或C1～6烷基，C1～6烯基，C1～6炔基；所述的C1～6选自C1,C2,C3,C4,C5,C6； R 20 , R 21 , R 22 , R 23 are independently selected from hydrogen atom, sulfonic acid group, sulfonamide, hydroxyl, amino, F, Cl, Br, I, nitro or C1~6 alkyl, C1~6 alkenes Base, C1～6 alkynyl; said C1～6 is selected from C1, C2, C3, C4, C5, C6;
R 24选自独立地选自氢、磺酸基，磺酰胺基，羟基，氨基，F,Cl,Br,I，或C1～6烷基，C1～6烯基，C1～6炔基； R 24 is selected from independently selected from hydrogen, sulfonic acid group, sulfonamide group, hydroxyl, amino, F, Cl, Br, I, or C1～6 alkyl, C1～6 alkenyl, C1～6 alkynyl;
m表示表示0-4的整数，具体可为0,1,2,3,4；m represents an integer representing 0-4, specifically 0, 1, 2, 3, 4;
n表示0-4的整数，具体可为0,1,2,3,4；n represents an integer of 0-4, specifically 0, 1, 2, 3, 4;
p表示0-9的整数，具体可为0,1,2,3,4,5,6,7,8,9；p represents an integer from 0 to 9, specifically 0, 1, 2, 3, 4, 5, 6, 7, 8, 9;
X表示F,Cl,Br,I；X represents F, Cl, Br, I;
L 1选自C1～9烷基，C1～9烯基，C1～9炔基，所述C1～9选自C1,C2,C3,C4,C5,C6,C7,C8,C9； L1 is selected from C1～9 alkyl, C1～9 alkenyl, C1～9 alkynyl, and said C1～9 is selected from C1, C2, C3, C4, C5, C6, C7, C8, C9;
L 2选自C1～20烷基，C1～20烯基，C1～20炔基，所述的C1～20选自C1,C2,C3,C4,C5,C6,C7,C8,C9,C10,C11,C12,C13,C14,C15,C16,C17,C18,C19,C20； L2 is selected from C1-20 alkyl, C1-20 alkenyl, C1-20 alkynyl, and the C1-20 is selected from C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20;
L 3选自丝氨酸，苏氨酸，半胱氨酸，酪氨酸，赖氨酸； L3 is selected from serine, threonine, cysteine, tyrosine, lysine;
R 25，R 26，R 27独立选自
Figure PCTCN2021131122-appb-000011
Figure PCTCN2021131122-appb-000012
R 25 , R 26 , R 27 are independently selected from
Figure PCTCN2021131122-appb-000011
Figure PCTCN2021131122-appb-000012
上述式II所示的靶向前列腺癌和细胞膜的Cy类荧光探针化合物除Cy fluorescent probe compounds targeting prostate cancer and cell membranes shown in the above formula II except
Figure PCTCN2021131122-appb-000013
外选自Cy3,Cy3.3,Cy5,Cy5.5,Cy7,Cy7.5，结构如下：
Figure PCTCN2021131122-appb-000013
Externally selected from Cy3, Cy3.3, Cy5, Cy5.5, Cy7, Cy7.5, the structure is as follows:
Figure PCTCN2021131122-appb-000014
Figure PCTCN2021131122-appb-000014
更具体地，上述式I所示的靶向前列腺癌的Cy类荧光探针化合物可为：More specifically, the Cy-type fluorescent probe compound targeting prostate cancer shown in the above formula I can be:
Figure PCTCN2021131122-appb-000015
Figure PCTCN2021131122-appb-000015
Figure PCTCN2021131122-appb-000016
Figure PCTCN2021131122-appb-000016
本发明技术方案的第一方面是靶向前列腺癌的Cy类荧光探针化合物或其药学上可接受的盐，所述药学上可接受的盐为所述化合物的有机酸盐或无机酸盐，所述药学上可接受的盐为所述化合物的有机碱盐或无机碱盐，所述有机碱可为吡啶，三乙胺，二乙胺，N-甲基吗啉，四甲基乙二胺；无所述机碱可为磷酸盐，磷酸氢盐，碳酸盐，碳酸氢盐，碳酸钾，碳酸氢盐，次氯酸盐，次溴酸盐，硅酸盐。The first aspect of the technical solution of the present invention is a Cy-type fluorescent probe compound targeting prostate cancer or a pharmaceutically acceptable salt thereof, and the pharmaceutically acceptable salt is an organic acid salt or an inorganic acid salt of the compound, The pharmaceutically acceptable salt is an organic base salt or an inorganic base salt of the compound, and the organic base can be pyridine, triethylamine, diethylamine, N-methylmorpholine, tetramethylethylenediamine No said organic base can be phosphate, hydrogen phosphate, carbonate, bicarbonate, potassium carbonate, bicarbonate, hypochlorite, hypobromite, silicate.
本发明技术方案的第二方面是提供第一方面所述靶向前列腺癌的Cy类荧光探针化合物或其药学上可接受的盐在制备对前列腺癌特异性膜抗原检测的试剂中的应用。所述检测为在分子水平对前列腺癌特异性膜抗原检测。The second aspect of the technical solution of the present invention is to provide the application of the Cy-type fluorescent probe compound targeting prostate cancer in the first aspect or a pharmaceutically acceptable salt thereof in the preparation of a reagent for detecting prostate cancer-specific membrane antigen. The detection is the detection of prostate cancer specific membrane antigen at the molecular level.
本发明技术方案的第三方面是提供靶向前列腺癌的Cy类荧光探针化合物或其药学上可接受的盐在制备对前列腺癌靶向检测的试剂中的应用，所述检测为在细胞或活体组织样本水平对前列腺癌靶向检测。The third aspect of the technical solution of the present invention is to provide the application of Cy-type fluorescent probe compounds targeting prostate cancer or pharmaceutically acceptable salts thereof in the preparation of reagents for targeted detection of prostate cancer. Biopsy-level targeted detection of prostate cancer.
所述前列腺癌为高水平表达PSMA的前列腺癌，可以与正常的前列腺细胞和组织做区分。The prostate cancer is a prostate cancer expressing PSMA at a high level, which can be distinguished from normal prostate cells and tissues.
本发明技术方案的第四方面还提供所述靶向前列腺癌的Cy类荧光探针化合物或其药学上可接受的盐在制备前列腺癌诊断试剂中的应用。The fourth aspect of the technical solution of the present invention also provides the application of the Cy-type fluorescent probe compound targeting prostate cancer or a pharmaceutically acceptable salt thereof in the preparation of a prostate cancer diagnostic reagent.
本发明的靶向前列腺癌的Cy类荧光探针化合物，对表达PSMA的前列 腺癌细胞具有较好的亲和性和特异性。The Cy fluorescent probe compound targeting prostate cancer of the present invention has good affinity and specificity to prostate cancer cells expressing PSMA.
靶向前列腺癌和细胞膜的Cy类荧光探针化合物，在小动物活体成像中，能够增加荧光信号，降低背景信号，并在一定时间内具有很好的稳定性，以及时间分辨率和空间分辨率。Cy-like fluorescent probe compounds targeting prostate cancer and cell membranes can increase fluorescent signals, reduce background signals, and have good stability within a certain period of time, as well as temporal and spatial resolutions in small animal live imaging .
通过对细胞的生长抑制的检测，证明了靶向前列腺癌的Cy类荧光探针化合物，24小时内，在25μM的浓度下对细胞无抑制作用，即毒性低。Through the detection of cell growth inhibition, it is proved that the Cy-type fluorescent probe compound targeting prostate cancer has no inhibitory effect on cells at a concentration of 25 μM within 24 hours, that is, the toxicity is low.
本发明设计与合成新型的特异性靶向前列腺癌荧光探针分子，极大提高前列腺癌检测的特异性和灵敏度，为个体化精准手术提供高精度和高灵敏度的实时影像学导航，实现前列腺癌的精准诊断和治疗。The invention designs and synthesizes a novel fluorescent probe molecule specifically targeting prostate cancer, which greatly improves the specificity and sensitivity of prostate cancer detection, provides high-precision and high-sensitivity real-time imaging navigation for individualized precision surgery, and realizes prostate cancer accurate diagnosis and treatment.
附图说明Description of drawings
图1为本发明实施例中探针分子1，2的合成路线图。Fig. 1 is a synthesis route diagram of probe molecules 1 and 2 in the embodiment of the present invention.
图2表示探针1,2吸收和发射光谱结果。Figure 2 shows the absorption and emission spectra results of probes 1 and 2.
图3表示探针1，2在24小时内对前列腺癌细胞(C4-2)的细胞存活率结果。Fig. 3 shows the cell viability results of probes 1 and 2 on prostate cancer cells (C4-2) within 24 hours.
图4表示探针1，2在前列腺癌细胞(C4-2，PC3)共定位荧光成像结果。Figure 4 shows the co-localization fluorescence imaging results of probes 1 and 2 in prostate cancer cells (C4-2, PC3).
图5表示探针1，2在前列腺癌细胞(C4-2，PC3)流式细胞结果。Figure 5 shows the flow cytometric results of probes 1 and 2 in prostate cancer cells (C4-2, PC3).
图6表示本发明探针2小动物成像结果。Fig. 6 shows the imaging results of probe 2 of the present invention in small animals.
图7a显示荧光腹腔镜下小鼠前列腺癌荧光成像，图7b表示荧光指导下的实时肿瘤切除，图7c表示肿瘤前程切除后的荧光成像图，图7d表示手术标本HE染色证实为前列腺癌。Figure 7a shows fluorescence imaging of prostate cancer in mice under fluorescence laparoscopy, Figure 7b shows real-time tumor resection under fluorescence guidance, Figure 7c shows fluorescence imaging after tumor resection, and Figure 7d shows surgical specimens confirmed by HE staining as prostate cancer.
图8a表示前列腺癌组织(上部)与正常的前列腺组织(下部)；图8b图提示PSMA高表达的前列腺癌(上部)与低表达的前列腺癌，甚至是不表达的正常前列腺组织(下部)；图8c，图8d图结果提示了探针2和临床前列腺癌样本有很好的成像性，而和正常前列腺组织不成像。Figure 8a shows prostate cancer tissue (upper part) and normal prostate tissue (lower part); Figure 8b shows prostate cancer with high expression of PSMA (upper part) and prostate cancer with low expression, even normal prostate tissue with no expression (lower part); The results in Fig. 8c and Fig. 8d suggest that the probe 2 has good imaging performance on clinical prostate cancer samples, but not on normal prostate tissue.
实施发明的最佳方式The best way to practice the invention
下述实施例中的实验方法，如无特别说明，均为常规方法Experimental methods in the following examples, if no special instructions, are conventional methods
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径 得到。The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.
本发明设计与合成新型的特异性靶向前列腺癌荧光探针分子，极大提高前列腺癌检测的特异性和灵敏度，为个体化精准手术提供高精度和高灵敏度的实时影像学导航，实现前列腺癌的精准诊断和治疗。The invention designs and synthesizes a novel fluorescent probe molecule specifically targeting prostate cancer, which greatly improves the specificity and sensitivity of prostate cancer detection, provides high-precision and high-sensitivity real-time imaging navigation for individualized precision surgery, and realizes prostate cancer accurate diagnosis and treatment.
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。The experimental methods used in the following examples are conventional methods unless otherwise specified.
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.
实施例1Example 1
化合物2的制备Preparation of Compound 2
将底物1(10g，62.8mmol)和溴乙烷(6.84g，62.8mmol)溶解于甲苯溶剂(32mL)，反应完全后，反应体系减压蒸馏除去溶剂，洗涤，真空条件下干燥后，直接用于下一步。最终得到红棕色固体产物2，4.8g，产率为28.5％。 1H NMR(500MHz,DMSO-d 6)δ8.04(d,J＝6.8Hz,1H),7.90(d,J＝6.4Hz,1H),7.69–7.60(m,2H),4.56(q,J＝7.5Hz,2H),2.91(s,3H),1.57(s,6H),1.47(t,J＝8.1Hz,4H)； 13C NMR(125MHz,DMSO-d 6)δ196.60,142.41,141.18,129.77,129.38,124.04,115.81,54.57,43.56,22.33,14.46,13.17. Substrate 1 (10g, 62.8mmol) and bromoethane (6.84g, 62.8mmol) were dissolved in toluene solvent (32mL). After the reaction was complete, the reaction system was distilled off the solvent under reduced pressure, washed, dried under vacuum, and directly for the next step. Finally, 4.8 g of reddish-brown solid product 2 was obtained with a yield of 28.5%. 1 H NMR (500MHz, DMSO-d 6 )δ8.04(d, J=6.8Hz, 1H), 7.90(d, J=6.4Hz, 1H), 7.69–7.60(m, 2H), 4.56(q, J＝7.5Hz, 2H), 2.91(s, 3H), 1.57(s, 6H), 1.47(t, J＝8.1Hz, 4H); 13 C NMR (125MHz, DMSO-d 6 ) δ196.60, 142.41, 141.18 ,129.77,129.38,124.04,115.81,54.57,43.56,22.33,14.46,13.17.
化合物4的制备Preparation of Compound 4
将底物1(10g，62.8mmol)和6-溴己酸(12.2g，62.8mmol)溶解于甲苯溶剂(32mL)，反应完全后，旋蒸，洗涤，真空条件下干燥后，直接用于下一步。最终得到白色固体产物4，9.7g，产率为43.5％。 1H NMR(500MHz,DMSO-d 6)δ12.00(s,1H),δ8.07–7.95(m,1H),7.91–7.80(m,1H),7.67–7.56(m,2H),4.47(t,J＝7.7Hz,2H),2.87(s,3H),2.22(t,J＝7.3Hz,2H),1.84(p,J＝7.8Hz,2H),1.59–1.51(m,8H),1.42(p,J＝8.0,7.4Hz,2H)； 13C NMR(125MHz,DMSO-d 6)δ196.99,174.80,142.33,141.52,129.84,129.40,124.00,115.99,54.63,47.93,33.83,27.42,25.86,24.49,22.47,14.59.HRMS(ESI):理论分子量C17H24NO2+:274.1802[M]+；实测值:274.1802。 Substrate 1 (10g, 62.8mmol) and 6-bromohexanoic acid (12.2g, 62.8mmol) were dissolved in toluene solvent (32mL). After the reaction was complete, rotary evaporation, washing, and drying under vacuum were used directly in the following step. Finally, 9.7 g of white solid product 4 was obtained with a yield of 43.5%. 1 H NMR (500MHz,DMSO-d 6 )δ12.00(s,1H),δ8.07–7.95(m,1H),7.91–7.80(m,1H),7.67–7.56(m,2H),4.47 (t,J=7.7Hz,2H),2.87(s,3H),2.22(t,J=7.3Hz,2H),1.84(p,J=7.8Hz,2H),1.59–1.51(m,8H) , 1.42 (p, J=8.0, 7.4Hz, 2H); 13 C NMR (125MHz, DMSO-d 6 ) δ196.99, 174.80, 142.33, 141.52, 129.84, 129.40, 124.00, 115.99, 54.63, 47.93, 33.83, 27. 42, 25.86, 24.49, 22.47, 14.59. HRMS (ESI): theoretical molecular weight C17H24NO2+: 274.1802 [M]+; measured value: 274.1802.
化合物7(Cy7-Cl)的制备Preparation of Compound 7 (Cy7-Cl)
将底物6(2.0g，11.6mmol)和5(3.1g，11.6mmol)溶解于醋酸酐(33mL)，反应结束后，减压蒸馏，残余物重新溶于无水乙醇(33mL)，依次加入4(4.9g，1.2mmol)和醋酸钠(2.85g，34.8mmol)。反应结束后，减压蒸馏，洗涤，萃取，减压蒸馏，残余物用柱色谱纯化，得到产物7为深绿色固体，2.4g，产率为30％。 1H NMR(500MHz,CDCl 3)δ8.37(d,J＝14.1Hz,1H),8.32(d,J＝13.9Hz,1H),7.44–7.33(m,4H),7.25–7.17(m,2H),7.13(d,J＝8.0Hz,1H),6.26(d,J＝14.1Hz,1H),6.13(d,J＝14.0Hz,1H),4.20–4.12(m,4H),2.74(t,J＝6.1Hz,2H),2.70(t,J＝6.0Hz,2H),2.60(t,J＝7.2Hz,2H),2.00(p,J＝6.2Hz,2H),1.87(p,J＝7.8Hz,2H),1.79(p,J＝7.3Hz,2H),1.71(s,12H),1.57(q,J＝7.5,7.0Hz,2H),1.44(t,J＝7.2Hz,3H)； 13C NMR(125MHz,CDCl 3)δ176.01,172.97,171.33,150.88,145.20,144.10,142.09,141.95,141.21,141.12,129.10,128.92,128.06,127.62,125.69,125.15,122.39,122.33,111.27,110.47,101.93,100.42,49.59,49.28,44.75,39.62,34.73,28.25,28.22,27.01,26.71,26.65,26.31,24.64,20.79,12.43；HRMS(ESI):m/z理论值为C38H46ClN2O2+:597.3242[M]+；实测值为597.3242。 Substrates 6 (2.0g, 11.6mmol) and 5 (3.1g, 11.6mmol) were dissolved in acetic anhydride (33mL). After the reaction was over, they were distilled under reduced pressure, and the residue was re-dissolved in absolute ethanol (33mL), followed by adding 4 (4.9 g, 1.2 mmol) and sodium acetate (2.85 g, 34.8 mmol). After the reaction, vacuum distillation, washing, extraction, and vacuum distillation, the residue was purified by column chromatography to obtain product 7 as a dark green solid, 2.4 g, with a yield of 30%. 1 H NMR (500MHz, CDCl 3 ) δ8.37(d, J=14.1Hz, 1H), 8.32(d, J=13.9Hz, 1H), 7.44–7.33(m, 4H), 7.25–7.17(m, 2H), 7.13(d, J=8.0Hz, 1H), 6.26(d, J=14.1Hz, 1H), 6.13(d, J=14.0Hz, 1H), 4.20–4.12(m, 4H), 2.74( t,J=6.1Hz,2H),2.70(t,J=6.0Hz,2H),2.60(t,J=7.2Hz,2H),2.00(p,J=6.2Hz,2H),1.87(p, J＝7.8Hz, 2H), 1.79(p, J＝7.3Hz, 2H), 1.71(s, 12H), 1.57(q, J＝7.5, 7.0Hz, 2H), 1.44(t, J＝7.2Hz, 3H); 13 C NMR (125MHz, CDCl 3 ) δ176.01, 172.97, 171.33, 150.88, 145.20, 144.10, 142.09, 141.95, 141.21, 141.12, 129.10, 128.92, 128.06, 127.62, 125.69, 125.15, 122.39, 122.33, 111.27, 110.47, 101.93, 100.42, 49.59, 49.28, 44.75, 39.62, 34.73, 28.25, 28.22, 27.01, 26.71, 26.65, 26.31, 24.64, 20.79, 12.43; HRMS (ESI): m/z theoretical value is C38H 46ClN2O2+:597.3242[M ]+; the measured value is 597.3242.
化合物10的制备Preparation of compound 10
将底物8(930mg，2.73mmol)加到二氯甲烷溶剂(27mL)中，加入EDCI(629mg,3.28mmol)，HOBt(443mg,3.28mmol)和DIPEA(424mg,3.28mmol)，加入底物9(1.33g，2.73mmol)后，反应完毕后，萃取，减压蒸馏，残余物柱色谱纯化得化合物10为白色固体，1.6g，产率70％。 1H NMR(400MHz,CDCl 3)δ7.36–7.34(m,2H),7.34–7.32(m,2H),7.31(m,1H),7.05(t,J＝6.0Hz,1H),5.57(t,J＝5.9Hz,1H),5.43(d,J＝8.0Hz,1H),5.38(d,J＝8.2Hz,1H),5.09(s,2H),4.41–4.21(m,2H),3.96(q,J＝5.0Hz,2H),3.63(s,8H),3.56(t,J＝5.1Hz,2H),3.39(d,J＝5.4Hz,2H),3.33–3.16(m,2H),2.29(td,J＝10.0,6.1Hz,2H),2.12–1.97(m,2H),1.87–1.69(m,2H),1.55–1.47(m,2H),1.43(s,18H),1.41(s,9H),1.37–1.22(m,2H)； 13C NMR(100MHz,CDCl 3)δ172.62,172.52,172.32,170.25,157.16,156.74,136.63,128.60,128.26,128.21,81.88,81.61,80.54,70.95,70.47,70.26,70.20,66.85,53.41,52.90,40.91,38.36,32.34,31.73,29.24,28.70,28.19,28.12,22.32.HRMS(ESI):理论分子量C 40H 67N 4O 13 +:811.4699[M] +；实测值:811.4697。 Substrate 8 (930 mg, 2.73 mmol) was added to dichloromethane solvent (27 mL), EDCI (629 mg, 3.28 mmol), HOBt (443 mg, 3.28 mmol) and DIPEA (424 mg, 3.28 mmol) were added, and substrate 9 was added (1.33g, 2.73mmol), after the reaction was completed, extraction, vacuum distillation, and column chromatography of the residue gave compound 10 as a white solid, 1.6g, yield 70%. 1 H NMR (400MHz, CDCl 3 ) δ7.36–7.34(m,2H),7.34–7.32(m,2H),7.31(m,1H),7.05(t,J=6.0Hz,1H),5.57( t,J=5.9Hz,1H),5.43(d,J=8.0Hz,1H),5.38(d,J=8.2Hz,1H),5.09(s,2H),4.41–4.21(m,2H), 3.96(q, J=5.0Hz, 2H), 3.63(s, 8H), 3.56(t, J=5.1Hz, 2H), 3.39(d, J=5.4Hz, 2H), 3.33–3.16(m, 2H ),2.29(td,J＝10.0,6.1Hz,2H),2.12–1.97(m,2H),1.87–1.69(m,2H),1.55–1.47(m,2H),1.43(s,18H), 1.41(s,9H),1.37–1.22(m,2H); 13 C NMR(100MHz,CDCl 3 )δ172.62,172.52,172.32,170.25,157.16,156.74,136.63,128.60,128.26,128.21,81.88 ,81.61,80.54 ,70.95,70.47,70.26,70.20,66.85,53.41,52.90,40.91,38.36,32.34,31.73,29.24,28.70,28.19,28.12,22.32.HRMS(ESI):Theoretical molecular weight C 40 H 67 N 4 O 13 + : 811.4699[M] + ; Found: 811.4697.
探针1的制备Preparation of Probe 1
将底物10(1.6g，1.97mmol)和10％钯碳(160mg)分散于甲醇溶剂(32mL)，催化氢化。反应完毕后，抽滤，减压蒸馏，残余物色谱纯化，得到产物11为无色油状，1.3g，产率为97％。Substrate 10 (1.6 g, 1.97 mmol) and 10% palladium on carbon (160 mg) were dispersed in methanol solvent (32 mL) for catalytic hydrogenation. After the reaction was completed, suction filtration, vacuum distillation, and chromatographic purification of the residue gave product 11 as a colorless oil, 1.3 g, with a yield of 97%.
将底物7(80mg，0.138mmol)溶于二氯甲烷(1.4mL)，依次加入EDCI(32mg,0.166mmol)，HOBt(22mg,0.166mmol)和DIPEA(21.5mg,0.166mmol)，加入底物11(83mg，0.138mmol)后，继续室温搅拌1.5h。反应完毕后，洗涤，减压蒸馏，残余物柱色谱纯化后，用二氯甲烷(2.76mL)溶解，加入三氟乙酸(2.76mL)，反应完毕后，直接色谱纯化。得到的探针1为绿色固体，29mg，两步产率为21.7％。 1H NMR(400MHz,DMSO-d 6)δ8.28(s,1H),8.24(s,1H),7.82(s,1H),7.70–7.61(m,3H),7.48–7.40(m,4H),7.34–7.25(m,2H),6.37–6.27(m,3H),5.76(s,1H),4.22(d,J＝7.3Hz,4H),4.14–4.07(m,1H),4.07–3.98(m,1H),3.84(s,2H),3.57–3.53(m,6H),3.53–3.48(m,4H),3.19–3.13(m,2H),3.11–3.03(m,2H),2.75–2.67(m,4H),2.31(t,J＝7.2Hz,2H),2.28–2.20(m,2H),2.10–2.05(m,2H),1.91–1.84(m,2H),1.77–1.71(m,4H),1.67(s,9H),1.62–1.51(m,5H),1.43–1.35(m,5H),1.29–1.23(m,4H)； 13C NMR(150MHz,DMSO)δ174.44,174.06,173.63,173.14,172.31,172.12,171.92,168.94,157.21,147.98,143.07,142.86,142.04,142.00,141.06,141.01,129.60,128.60,126.16,126.09,125.20,125.10,122.49,111.55,111.46,101.67,101.47,70.16,69.93,69.68,69.54,69.52,69.11,54.86,52.24,51.61,51.13,49.00,48.94,43.58,38.37,37.90,34.95,33.05,31.76,29.85,28.86,27.53,27.45,26.75,26.61,25.79,25.72,25.52,24.81,24.09,22.52,20.37.HRMS(ESI):理论分子量C 58H 80ClN 6O 12 +:1087.5517[M] +；实测值:1087.5485。 Substrate 7 (80mg, 0.138mmol) was dissolved in dichloromethane (1.4mL), EDCI (32mg, 0.166mmol), HOBt (22mg, 0.166mmol) and DIPEA (21.5mg, 0.166mmol) were added sequentially, and the substrate After 11 (83mg, 0.138mmol), continue to stir at room temperature for 1.5h. After the reaction was completed, it was washed and distilled under reduced pressure. After the residue was purified by column chromatography, it was dissolved in dichloromethane (2.76 mL), and trifluoroacetic acid (2.76 mL) was added. After the reaction was completed, it was purified by direct chromatography. Probe 1 was obtained as a green solid, 29 mg, with a two-step yield of 21.7%. 1 H NMR (400MHz,DMSO-d 6 )δ8.28(s,1H),8.24(s,1H),7.82(s,1H),7.70–7.61(m,3H),7.48–7.40(m,4H ),7.34–7.25(m,2H),6.37–6.27(m,3H),5.76(s,1H),4.22(d,J＝7.3Hz,4H),4.14–4.07(m,1H),4.07– 3.98(m,1H),3.84(s,2H),3.57–3.53(m,6H),3.53–3.48(m,4H),3.19–3.13(m,2H),3.11–3.03(m,2H), 2.75–2.67(m,4H),2.31(t,J＝7.2Hz,2H),2.28–2.20(m,2H),2.10–2.05(m,2H),1.91–1.84(m,2H),1.77– 1.71(m,4H),1.67(s,9H),1.62–1.51(m,5H),1.43–1.35(m,5H),1.29–1.23(m,4H); 13 C NMR(150MHz,DMSO)δ174 .44, 174.06, 173.63, 173.14, 172.31, 172.12, 171.92, 168.94, 157.21, 147.98, 143.07, 142.86, 142.04, 142.00, 141.06, 141.01, 129.60, 128.6 0,126.16,126.09,125.20,125.10,122.49,111.55,111.46,101.67 ,101.47,70.16,69.93,69.68,69.54,69.52,69.11,54.86,52.24,51.61,51.13,49.00,48.94,43.58,38.37,37.90,34.95,33.05,31.76,29.85 ,28.86,27.53,27.45,26.75,26.61 , 25.79, 25.72, 25.52, 24.81, 24.09, 22.52, 20.37. HRMS (ESI): theoretical molecular weight C 58 H 80 ClN 6 O 12 + : 1087.5517[M] + ; measured value: 1087.5485.
化合物13的制备Preparation of Compound 13
将底物12(814mg，1.62mmol)均匀分散到二氯甲烷溶剂(16mL)中，依次加入EDCI(372mg,1.94mmol)，HOBt(262mg,1.94mmol)和DIPEA(96mg,1.94mmol)，加入底物11(1.1g，1.62mmol)，反应完毕后，萃取减压蒸馏除去溶剂，残余物柱色谱纯化得化合物13为无色油状，1.49g，产率77％。 1H  NMR(600MHz,CDCl 3)δ7.66(d,J＝7.6Hz,2H),7.51(d,J＝7.6Hz,2H),7.30(t,J＝7.5Hz,2H),7.26-7.23(m,3H),7.23-7.21(m,2H),7.19(s,1H),7.15(s,1H),7.02(s,1H),6.12(d,J＝8.4Hz,1H),5.78(d,J＝8.4Hz,1H),5.67(d,J＝7.8Hz,1H),5.19(s,1H),4.99(s,2H),4.34-4.29(m,2H),4.29-4.20(m,2H),4.18-4.07(m,2H),3.93(s,2H),3.57-3.53(m,2H),3.53-3.47(m,4H),3.46(t,J＝5.3Hz,2H),3.40-3.32(m,2H),3.22-3.14(m,1H),3.14-3.05(m,3H),2.29-2.16(m,2H),1.98(ddt,J＝14.9,10.3,5.4,1H),1.81-1.69(m,2H),1.68-1.58(m,2H),1.55-1.43(m,3H),1.43-1.38(m,2H),1.35(s,9H),1.34(s,9H),1.33(s,9H),1.25-1.16(m,3H),1.07(t,J＝5.4Hz,1H)； 13C NMR(150MHz,CDCl 3)δ172.85,172.63,172.46,172.43,170.29,157.32,156.65,156.52,143.92,143.84,141.29,136.71,128.49,128.08,128.04,127.73,127.15,127.10,125.21,125.17,119.98,119.96,81.92,81.44,80.47,70.68,70.43,70.15,69.69,67.13,66.56,54.85,53.35,52.81,47.16,40.64,39.42,38.61,32.62,32.44,31.70,29.43,29.23,28.74,28.10,28.05,28.04,22.63,22.50。HRMS(ESI):理论分子量C 61H 89N 6O 16 +:1161.6330[M] +；实测值:1161.6322。 Substrate 12 (814mg, 1.62mmol) was uniformly dispersed in dichloromethane solvent (16mL), EDCI (372mg, 1.94mmol), HOBt (262mg, 1.94mmol) and DIPEA (96mg, 1.94mmol) were added in sequence, and the bottom Compound 11 (1.1g, 1.62mmol). After the reaction was completed, the solvent was extracted and distilled off under reduced pressure. The residue was purified by column chromatography to obtain compound 13 as a colorless oil, 1.49g, with a yield of 77%. 1 H NMR (600MHz, CDCl 3 ) δ7.66(d, J=7.6Hz, 2H), 7.51(d, J=7.6Hz, 2H), 7.30(t, J=7.5Hz, 2H), 7.26-7.23 (m,3H),7.23-7.21(m,2H),7.19(s,1H),7.15(s,1H),7.02(s,1H),6.12(d,J＝8.4Hz,1H),5.78( d,J=8.4Hz,1H),5.67(d,J=7.8Hz,1H),5.19(s,1H),4.99(s,2H),4.34-4.29(m,2H),4.29-4.20(m ,2H),4.18-4.07(m,2H),3.93(s,2H),3.57-3.53(m,2H),3.53-3.47(m,4H),3.46(t,J＝5.3Hz,2H), 3.40-3.32(m,2H),3.22-3.14(m,1H),3.14-3.05(m,3H),2.29-2.16(m,2H),1.98(ddt,J＝14.9,10.3,5.4,1H) ,1.81-1.69(m,2H),1.68-1.58(m,2H),1.55-1.43(m,3H),1.43-1.38(m,2H),1.35(s,9H),1.34(s,9H) ,1.33(s,9H),1.25-1.16(m,3H),1.07(t,J=5.4Hz,1H); 13 C NMR(150MHz,CDCl 3 )δ172.85,172.63,172.46,172.43,170.29,157.32, 156.65,156.52,143.92,143.84,141.29,136.71,128.49,128.08,128.04,127.73,127.15,127.10,125.21,125.17,119.98,119.96,81.92,81. 44,80.47,70.68,70.43,70.15,69.69,67.13,66.56, 54.85, 53.35, 52.81, 47.16, 40.64, 39.42, 38.61, 32.62, 32.44, 31.70, 29.43, 29.23, 28.74, 28.10, 28.05, 28.04, 22.63, 22.50. HRMS (ESI): theoretical molecular weight C 61 H 89 N 6 O 16 + : 1161.6330 [M] + ; found value: 1161.6322.
化合物16的制备Preparation of compound 16
将底物13(1.49g，1.28mmol)和10％钯碳(149mg)分散于甲醇溶剂(20mL)，催化氢化，得到产物14为白色固体，1.04g，产率为78.9％。直接用于下一步。Substrate 13 (1.49g, 1.28mmol) and 10% palladium on carbon (149mg) were dispersed in methanol solvent (20mL) for catalytic hydrogenation to give product 14 as a white solid, 1.04g, with a yield of 78.9%. used directly in the next step.
将底物15(342mg，1.01mmol)均匀分散到二氯甲烷溶剂(10mL)中，加入EDCI(232mg,1.21mmol)，HOBt(164mg,1.21mmol)和DIPEA(156mg,1.21mmol)后，加入底物14(1.04g，1.01mmol)，反应完毕后，萃取，减压蒸馏，柱色谱纯化得化合物16为白色固体，0.559g，产率42.8％。 1H NMR(400MHz,CDCl 3)δ7.76(d,J＝7.5Hz,2H),7.60(d,J＝8.2Hz,2H),7.39(t,J＝7.4Hz,2H),7.33–7.28(m,2H),7.25-7.19(m,1H),7.15–7.06(m,1H),6.14(d,J＝8.4Hz,1H),5.96–5.90(m,1H),5.85(d,J＝8.3Hz,1H),5.74(d,J＝8.1Hz,1H),5.35–5.31(m,2H),4.38(q,J＝8.7,7.2Hz,3H),4.30(dd,J＝11.7,7.5Hz,1H),4.26–4.16(m,2H),4.03(s,2H),3.67–3.63(m,2H),3.63-3.61(m,2H),3.60–3.58(m,4H),3.57–3.53(m,2H),3.49-3.42(m,2H),3.28-3.16(m,4H),2.39–2.25(m,2H),2.19–2.08(m,2H),2.05–1.95(m,6H),1.87–1.76(m,2H),1.79–1.66(m,2H),1.66–1.55(m,4H), 1.54-1.46(m,4H),1.44–1.41(m,27H),1.29-1.23(m,22H),0.89–0.85(m,3H). 13C NMR(150MHz,CDCl 3)δ173.58,172.60,157.42,143.92,141.41,134.82,130.10,129.88,129.22,127.87,127.26,127.21,125.29,124.47,120.44,120.09,82.04,80.63,70.79,70.61,70.56,70.27,69.84,67.30,54.85,53.48,52.94,47.27,39.55,39.12,38.71,36.94,32.63,32.04,31.85,29.90,29.88,29.83,29.66,29.51,29.46,29.44,29.37,29.33,29.22,28.83,28.24,28.22,28.17,27.36,27.34,25.98,22.81,22.60,14.24。HRMS(ESI):理论分子量C 71H 115N 6O 15 +:1291.8415[M] +；实测值:1291.8418。 Substrate 15 (342mg, 1.01mmol) was uniformly dispersed in dichloromethane solvent (10mL), and after adding EDCI (232mg, 1.21mmol), HOBt (164mg, 1.21mmol) and DIPEA (156mg, 1.21mmol), added Compound 14 (1.04g, 1.01mmol), after the completion of the reaction, was extracted, evaporated under reduced pressure, and purified by column chromatography to obtain Compound 16 as a white solid, 0.559g, with a yield of 42.8%. 1 H NMR (400MHz, CDCl 3 ) δ7.76(d, J=7.5Hz, 2H), 7.60(d, J=8.2Hz, 2H), 7.39(t, J=7.4Hz, 2H), 7.33–7.28 (m,2H),7.25-7.19(m,1H),7.15–7.06(m,1H),6.14(d,J＝8.4Hz,1H),5.96–5.90(m,1H),5.85(d,J =8.3Hz,1H),5.74(d,J=8.1Hz,1H),5.35–5.31(m,2H),4.38(q,J=8.7,7.2Hz,3H),4.30(dd,J=11.7, 7.5Hz,1H),4.26–4.16(m,2H),4.03(s,2H),3.67–3.63(m,2H),3.63-3.61(m,2H),3.60–3.58(m,4H),3.57 –3.53(m,2H),3.49-3.42(m,2H),3.28-3.16(m,4H),2.39–2.25(m,2H),2.19–2.08(m,2H),2.05–1.95(m, 6H),1.87–1.76(m,2H),1.79–1.66(m,2H),1.66–1.55(m,4H), 1.54-1.46(m,4H),1.44–1.41(m,27H),1.29- 1.23(m,22H),0.89–0.85(m,3H). 13 C NMR(150MHz,CDCl 3 )δ173.58,172.60,157.42,143.92,141.41,134.82,130.10,129.88,129.22,127.87,127. 26,127.21,125.29 . 63, 32.04, 31.85, 29.90, 29.88, 29.83 ,29.66,29.51,29.46,29.44,29.37,29.33,29.22,28.83,28.24,28.22,28.17,27.36,27.34,25.98,22.81,22.60,14.24. HRMS (ESI): Theoretical molecular weight C 71 H 115 N 6 O 15 + : 1291.8415[M] + ; found value: 1291.8418.
探针2的制备Preparation of Probe 2
将底物16(559mg，0.443mmol)溶于乙腈溶剂(13.5mL)，加入二乙胺(3.17g，43.3mmol)。反应体系室温搅拌40min。减压蒸馏除去溶剂，残余物用柱色谱纯化，得到产物17为白色固体，200mg，产率为43.2％，直接用于下一步。Substrate 16 (559 mg, 0.443 mmol) was dissolved in acetonitrile solvent (13.5 mL), and diethylamine (3.17 g, 43.3 mmol) was added. The reaction system was stirred at room temperature for 40 min. The solvent was distilled off under reduced pressure, and the residue was purified by column chromatography to obtain product 17 as a white solid, 200 mg, with a yield of 43.2%, which was directly used in the next step.
将底物7(40mg，0.067mmol)溶于二氯甲烷(1.34mL)，依次加入EDCI(19.4mg,0.101mmol)和HOBt(13.6mg,0.101mmol)后，加入底物17(57mg，0.054mmol)，反应完毕后，洗涤，减压蒸馏，残余物柱色谱纯化后，产物用二氯甲烷溶液(2.68mL)溶解后，加入三氟乙酸(2.68mL)，反应在氩气保护下，反应完毕后，减压蒸馏除去溶剂，残余物直接HPLC纯化。得到的探针2为绿色固体,15.2mg，两步产率为16.3％。 1H NMR(600MHz,DMSO-d 6)δ8.27(s,2H),7.72(s,1H),7.64(s,2H),7.49-7.42(m,3H),7.30(s,2H),6.36-6.30(m,2H),6.25(s,1H),5.34-5.28(m,2H),4.26(s,2H),4.20(s,2H),4.08(s,1H),3.99(s,1H),3.85(s,2H),3.63(s,2H),3.59-3.545(m,4H),3.53-3.49(m,3H),3.39(s,2H),3.18(s,2H),3.07(s,2H),2.98(s,2H),2.79-2.65(m,4H),2.26(s,2H),2.14(s,2H),2.00(s,2H),1.98-1.92(m,4H),1.87(s,2H),1.75(s,2H),1.73-1.61(m,12H),1.60-1.52(m,4H),1.51-1.42(m,4H),1.42-1.36(m,4H),1.36-1.30(m,5H),1.28-1.18(m,24H),0.86(s,3H). 13C NMR(150MHz,DMSO)δ174.24,172.54,172.03,171.88,171.84,168.97,168.96,157.50,157.15,148.01,143.18,142.89,142.04,141.61,141.19,141.00,129.62,129.59,129.57,128.65,128.60,126.13,126.07,125.21,125.10,122.55,122.48,111.46,111.33,101.43,101.37,70.19,69.93,69.70,69.55,68.91,52.40,52.28,51.71,51.30,49.02,48.94,45.86,43.70,38.45,38.18,37.95,37.82,35.41,35.10,34.87,31.84,31.24,29.09, 29.06,29.00,28.95,28.84,28.79,28.72,28.67,28.64,28.55,28.51,28.19,28.12,27.47,27.36,26.70,26.56,26.53,25.85,25.77,25.29,25.09,24.89,22.76,22.54,22.05,20.38,13.91,12.20.HRMS(ESI):理论分子量C 82H 124ClN 8O 14 +:1479.8920[M] +；实测值:1479.8921。 Substrate 7 (40mg, 0.067mmol) was dissolved in dichloromethane (1.34mL), EDCI (19.4mg, 0.101mmol) and HOBt (13.6mg, 0.101mmol) were added successively, followed by substrate 17 (57mg, 0.054mmol ), after the completion of the reaction, washing, distillation under reduced pressure, after the residue was purified by column chromatography, after the product was dissolved in dichloromethane solution (2.68mL), trifluoroacetic acid (2.68mL) was added, and the reaction was completed under argon protection. Afterwards, the solvent was distilled off under reduced pressure, and the residue was directly purified by HPLC. Probe 2 was obtained as a green solid, 15.2 mg, with a two-step yield of 16.3%. 1 H NMR (600MHz,DMSO-d 6 )δ8.27(s,2H),7.72(s,1H),7.64(s,2H),7.49-7.42(m,3H),7.30(s,2H), 6.36-6.30(m,2H),6.25(s,1H),5.34-5.28(m,2H),4.26(s,2H),4.20(s,2H),4.08(s,1H),3.99(s, 1H),3.85(s,2H),3.63(s,2H),3.59-3.545(m,4H),3.53-3.49(m,3H),3.39(s,2H),3.18(s,2H),3.07 (s,2H),2.98(s,2H),2.79-2.65(m,4H),2.26(s,2H),2.14(s,2H),2.00(s,2H),1.98-1.92(m,4H ),1.87(s,2H),1.75(s,2H),1.73-1.61(m,12H),1.60-1.52(m,4H),1.51-1.42(m,4H),1.42-1.36(m,4H ),1.36-1.30(m,5H),1.28-1.18(m,24H),0.86(s,3H). 13 C NMR(150MHz,DMSO)δ174.24,172.54,172.03,171.88,171.84,168.97,168.96,157.50 ,157.15,148.01,143.18,142.89,142.04,141.61,141.19,141.00,129.62,129.59,129.57,128.65,128.60,126.13,126.07,125.21,125.10,1 22.55, 122.48, 111.46, 111.33, 101.43, 101.37, 70.19, 69.93 ,69.70,69.55,68.91,52.40,52.28,51.71,51.30,49.02,48.94,45.86,43.70,38.45,38.18,37.95,37.82,35.41,35.10,34.87,31.84,31.24, 29.09, 29.06, 29.00, 28.95, 28.84 ,28.79,28.72,28.67,28.64,28.55,28.51,28.19,28.12,27.47,27.36,26.70,26.56,26.53,25.85,25.77,25.29,25.09,24.89,22.76,22.54, 22.05, 20.38, 13.91, 12.20. HRMS (ESI): theoretical molecular weight C 82 H 124 ClN 8 O 14 + : 1479.8920[M] + ; measured value: 1479.8921.
实施例2、探针1，2的吸收发射光谱测定 Embodiment 2, probe 1, the measurement of the absorption emission spectrum of 2
取5mM的实施例1制备的probe1，probe2各1.0μL，加入499μL的Tris-HCl缓冲液，然后在酶标仪上检测荧光探针的吸收和发射光谱。见图2。Take 5 mM probe1 and 1.0 μL of probe2 prepared in Example 1, add 499 μL of Tris-HCl buffer solution, and then detect the absorption and emission spectra of the fluorescent probe on a microplate reader. See Figure 2.
实施例3、探针1，2对前列腺癌细胞(C4-2)细胞毒实验 Embodiment 3, probe 1, 2 are to prostate cancer cell (C4-2) cytotoxic experiment
采用MTS法测定细胞存活率。设一组实验组和九组对照组。将96孔板中贴壁的细胞(C4-2细胞)，依次用浓度为0.2μM，0.4μM，0.8μM，1.6μM，3.2μM，6.25μM，12.5μM和25μM的探针2处理，37℃、5％CO2条件下分别培养24h后，每孔加入20μL MTS，然后37℃、5％CO2培养4h，酶标仪上测每组的吸收OD值，波长设为490nm。根据各孔OD值，通过公式计算细胞存活率。公式如下：细胞存活率(％)＝(实验组OD/对照组OD)×100％。探针1，2在25μM浓度下均未表现出对C4-2细胞毒性，说明探针1，2具有低细胞毒性。见图3。图3表示探针1，2在24小时内对前列腺癌细胞(C4-2)的细胞存活率结果。Cell viability was determined by MTS method. One experimental group and nine control groups were set up. The adherent cells (C4-2 cells) in the 96-well plate were sequentially treated with probe 2 at concentrations of 0.2 μM, 0.4 μM, 0.8 μM, 1.6 μM, 3.2 μM, 6.25 μM, 12.5 μM and 25 μM, at 37° C. After culturing for 24 hours under 5% CO2 and 5% CO2 conditions, 20 μL MTS was added to each well, and then cultured at 37 °C and 5% CO2 for 4 hours. The absorbance OD value of each group was measured on a microplate reader, and the wavelength was set at 490 nm. According to the OD value of each well, the cell viability was calculated by the formula. The formula is as follows: cell survival rate (%)=(OD of experimental group/OD of control group)×100%. Probes 1 and 2 did not exhibit cytotoxicity to C4-2 at a concentration of 25 μM, indicating that probes 1 and 2 had low cytotoxicity. See Figure 3. Fig. 3 shows the cell viability results of probes 1 and 2 on prostate cancer cells (C4-2) within 24 hours.
实施例4、探针1,2在在前列腺癌细胞(C4-2，PC3)中的共聚集荧光成像Example 4. Co-aggregation fluorescence imaging of probes 1 and 2 in prostate cancer cells (C4-2, PC3)
将前列腺癌细胞(C4-2,PC3)接种到8孔板中(1×106个)，并在48h内形成单层。分别用含探针1,2和小分子抑制剂2-PMPA的新鲜培养基(200uL)孵育，然后用Hoechst 33342染色。孵育完成后，共聚焦细胞成像。Prostate cancer cells (C4-2, PC3) were inoculated into 8-well plates (1×106 cells), and formed a monolayer within 48 hours. Incubate with fresh medium (200uL) containing probes 1, 2 and the small molecule inhibitor 2-PMPA, respectively, and then stain with Hoechst 33342. After the incubation is complete, confocal cells are imaged.
结果表明，探针1，2应用在PSMA表达阳性的前列腺细胞C4-2中具有很好的成像效果，并且探针2强于探针1，而在PSMA表达阴性的前列腺细 胞PC3中不成像；此外，当加入PSMA抑制剂2-PMPA后细胞成像明显减弱，说明探针1，2对PSMA表达阳性的前列腺癌细胞具有良好的亲和性和特异性。见图4。The results showed that probes 1 and 2 had a good imaging effect in PSMA-positive prostate cell C4-2, and probe 2 was stronger than probe 1, but did not image in PSMA-negative prostate cell PC3; In addition, when the PSMA inhibitor 2-PMPA was added, the cell imaging was significantly weakened, indicating that probes 1 and 2 had good affinity and specificity for PSMA-positive prostate cancer cells. See Figure 4.
实施例5、探针1,2在在前列腺癌细胞(C4-2，PC3)中的流式细胞实验Embodiment 5, the flow cytometric experiment of probe 1,2 in prostate cancer cell (C4-2, PC3)
将前列腺癌细胞(C4-2,PC3)接种到24孔板中。分别用含探针1,2的新鲜培养基(400uL)孵育。孵育完成后，进行流式细胞检测。Prostate cancer cells (C4-2, PC3) were seeded into 24-well plates. Incubate with fresh medium (400uL) containing probes 1 and 2, respectively. After incubation, flow cytometry was performed.
结果表明，探针1，2应用在PSMA表达阳性的前列腺细胞C4-2中峰图相比对照组出现右移，而且探针2右移更加明显，而在PSMA表达阴性的前列腺细胞PC3中不出现右移；此外，当加入PSMA抑制剂2-PMPA后前列腺细胞C4-2中峰图出现左移，说明探针1，2对PSMA表达阳性的前列腺癌细胞具有良好的亲和性和特异性。见图5。图5表示探针1，2在前列腺癌细胞(C4-2，PC3)流式细胞结果The results showed that probes 1 and 2 were applied to the PSMA-positive prostate cell C4-2, and the peak pattern shifted to the right compared with the control group, and the right-shift of probe 2 was more obvious, but not in the PSMA-negative prostate cell PC3. There was a right shift; in addition, when the PSMA inhibitor 2-PMPA was added, the peak pattern in the prostate cell C4-2 shifted to the left, indicating that probes 1 and 2 have good affinity and specificity for PSMA-positive prostate cancer cells . See Figure 5. Figure 5 shows the flow cytometric results of probe 1 and 2 in prostate cancer cells (C4-2, PC3)
实施例6、探针2在小动物活体成像应用Example 6. Application of Probe 2 in Live Imaging of Small Animals
将6-8周，平均体重20克的BALB/c裸鼠，左后肢末端皮下注射前列腺细胞C4-2细胞混悬浮液200μL，成功建立皮下移植瘤模型，二周左右，进行小动物活体成像检测。尾静脉分别注射探针2。分别2h，4h，6h，8h,12h，24h，36h,48h，72h进行小动物活体成像监测(激发波长745nm，发射波长820nm)。BALB/c nude mice with an average weight of 20 grams at 6-8 weeks were subcutaneously injected with 200 μL of prostate cell C4-2 cell suspension at the end of the left hind limb to successfully establish a subcutaneous xenograft tumor model. After about two weeks, small animal live imaging tests were performed. . Probe 2 was injected separately into the tail vein. Live imaging monitoring of small animals was performed at 2h, 4h, 6h, 8h, 12h, 24h, 36h, 48h, and 72h (excitation wavelength 745nm, emission wavelength 820nm).
结果表明，探针2可以用于特异性靶向PSMA成像，且在12h荧光强度达到最大,且持续到36h；另外，24h探针2的信燥比最高，达到3.64±0.16。说明探针2对PSMA靶向成像具有很好的时间和空间分辨率。见图6。The results showed that probe 2 could be used to specifically target PSMA imaging, and the fluorescence intensity reached the maximum at 12h and lasted until 36h; in addition, the signal-to-noise ratio of probe 2 was the highest at 24h, reaching 3.64±0.16. It shows that probe 2 has good temporal and spatial resolution for PSMA-targeted imaging. See Figure 6.
实施例7、评估探针2在荧光腹腔镜下荧光实时对小鼠前列腺癌手术切除的应用Example 7. Evaluation of the application of probe 2 in real-time fluorescence laparoscopic surgical resection of mouse prostate cancer
同案例6方法一样，建立前列腺细胞C4-2小鼠皮下移植瘤，然后尾静脉分别注射探针2，24h后在荧光腹腔镜下进行探针实时成像，并进行手术 操作模拟，图7a显示的是荧光腹腔镜下小鼠前列腺癌荧光成像，图7b荧光指导下的实时肿瘤切除，图7c肿瘤前程切除后的荧光成像图，图7d手术标本HE染色证实为前列腺癌。见图7。In the same way as in Case 6, subcutaneous transplantation of prostate cell C4-2 mice was established, and probe 2 was injected into the tail vein respectively. After 24 hours, real-time imaging of the probe was performed under a fluorescent laparoscope, and surgical operation simulation was performed, as shown in Figure 7a Fluorescence imaging of prostate cancer in mice under fluorescence laparoscopy, Fig. 7b Real-time tumor resection under fluorescence guidance, Fig. 7c Fluorescence imaging after tumor resection, and Fig. 7d Surgical specimen confirmed to be prostate cancer by HE staining. See Figure 7.
实施例8、探针2在临床前列癌样本中的应用Example 8, Application of Probe 2 in Clinical Prostate Cancer Samples
我们从临床样本水平验证探针和前列腺癌的成像性能，我们取前列腺根治性术后预估的一部分正常组织，一部分前列腺癌组织，进行了术后快速冰冻HE染色，图8a图提示上面为前列腺癌，下面的是正常的前列腺组织，进一步进行了PSMA的免疫组化，图8b图提示上面提示PSMA高表达的前列腺癌，下面是低表达的前列腺癌，甚至是不表达的正常前列腺组织，证实了我们的预判。然后我们把术后冰冻切片和临床新鲜样本和我们的探针37摄氏度进行共孵育1h,图8c，图8d图结果提示了我们的探针和临床前列腺癌样本有很好的成像性，而和正常前列腺组织不成像。说明探针2对前列腺癌具有很好的靶向成像性。见图8。We verified the imaging performance of the probe and prostate cancer from the clinical sample level. We took a part of the normal tissue and a part of the prostate cancer tissue estimated after radical prostatectomy, and performed postoperative rapid frozen HE staining. Figure 8a shows that the above is the prostate Cancer, the bottom is the normal prostate tissue, further immunohistochemistry of PSMA, Figure 8b shows that the prostate cancer with high expression of PSMA on the top, the prostate cancer with low expression, or even the normal prostate tissue without expression, confirms our prediction. Then we co-incubated the postoperative frozen sections and clinical fresh samples with our probe at 37 degrees Celsius for 1h. The results in Figure 8c and Figure 8d suggest that our probe and clinical prostate cancer samples have good imaging properties, and and Normal prostate tissue is not imaged. It shows that probe 2 has a good targeting imaging property for prostate cancer. See Figure 8.
工业应用industrial application
本发明设计与合成新型的特异性靶向前列腺癌荧光探针分子，极大提高前列腺癌检测的特异性和灵敏度，为个体化精准手术提供高精度和高灵敏度的实时影像学导航，实现前列腺癌的精准诊断和治疗。The invention designs and synthesizes a novel fluorescent probe molecule specifically targeting prostate cancer, which greatly improves the specificity and sensitivity of prostate cancer detection, provides high-precision and high-sensitivity real-time imaging navigation for individualized precision surgery, and realizes prostate cancer accurate diagnosis and treatment.

Claims (9)

  1. 式I所示的靶向前列腺癌的Cy类荧光探针化合物或其药学上可接受的盐：Cy fluorescent probe compound targeting prostate cancer represented by formula I or a pharmaceutically acceptable salt thereof:
    Figure PCTCN2021131122-appb-100001
    Figure PCTCN2021131122-appb-100001
    其中：F代表标记分子，标记分子是指能够直接或间接产生可检测信号的标记物；Where: F represents a marker molecule, and a marker molecule refers to a marker that can directly or indirectly generate a detectable signal;
    L 1和L 2代表连接基团，连接标记分子F和靶向基团T 1和T 2；T 1和T 2分别代表靶向基团，可以识别肿瘤细胞中相关蛋白，用于肿瘤细胞的检测；其中，L 2和T 2可以存在，也可以不存在； L 1 and L 2 represent linking groups, linking marker molecule F and targeting groups T 1 and T 2 ; T 1 and T 2 represent targeting groups, which can identify related proteins in tumor cells, and are used for the detection of tumor cells. Detection; wherein, L 2 and T 2 may or may not exist;
    F代表标记分子，标记分子是指能够直接或间接产生可检测信号的标记物，选自：放射性同位素、荧光团、荧光蛋白；F represents a labeling molecule, and the labeling molecule refers to a label that can directly or indirectly generate a detectable signal, selected from: radioisotopes, fluorophores, and fluorescent proteins;
    放射性同位素选自111In(γ)，99mTc(γ)；The radioactive isotope is selected from 111In(γ), 99mTc(γ);
    荧光团选自7-二甲氨基香豆素-4-乙酸琥珀酰亚胺酯、5/6-羧基荧光素和四甲基罗丹明、BODIPY-493/503、BODIPY-FL、BODIPY-TMR、BODIPY-TMR-X、BODIPY-TR-X、BODIPY630/550-X、BODIPY-650/665-X、Alexa 350、Alexa488、Alexa 532、Alexa 546、Alexa 555、Alexa 635、Alexa 647、Cy 3),花菁3B(Cy 3B),花菁5(Cy 5),花菁5.5(Cy 5.5),花菁7(Cy 7),花菁7.5(Cy 7.5),Cy7-Cl，ATTO 488,ATTO 532,ATTO 650,ATTO 650，DY-505，DY-547，DY-632，DY-647，IRDye78，ZW800+3C，S0456，AF647，Dylight680，Sulfo‐Cy5；Fluorophores selected from 7-dimethylaminocoumarin-4-acetate succinimidyl ester, 5/6-carboxyfluorescein and tetramethylrhodamine, BODIPY-493/503, BODIPY-FL, BODIPY-TMR, BODIPY-TMR-X, BODIPY-TR-X, BODIPY630/550-X, BODIPY-650/665-X, Alexa 350, Alexa488, Alexa 532, Alexa 546, Alexa 555, Alexa 635, Alexa 647, Cy 3), Cyanine 3B (Cy 3B), Cyanine 5 (Cy 5), Cyanine 5.5 (Cy 5.5), Cyanine 7 (Cy 7), Cyanine 7.5 (Cy 7.5), Cy7-Cl, ATTO 488, ATTO 532, ATTO 650, ATTO 650, DY-505, DY-547, DY-632, DY-647, IRDye78, ZW800+3C, S0456, AF647, Dylight680, Sulfo-Cy5;
    荧光蛋白选自绿色荧光蛋白和绿色荧光蛋白修饰物；The fluorescent protein is selected from green fluorescent protein and modified green fluorescent protein;
    L1代表连接基团，选自
    Figure PCTCN2021131122-appb-100002
    其中，X代表羰基，磺酰基，亚磺酰基；m＝3-12；Y代表O或是S，NH，NCH3，NCH2CH3；     L1 represents a linking group selected from
    Figure PCTCN2021131122-appb-100002
    Among them, X represents carbonyl, sulfonyl, sulfinyl; m=3-12; Y represents O or S, NH, NCH3, NCH2CH3;
    L2代表连接基团，可以不存在，或是存在1个，选自丝氨酸，苏氨酸，半胱氨酸，酪氨酸，赖氨酸；L2 represents a linking group, which can be absent or present, and is selected from serine, threonine, cysteine, tyrosine, and lysine;
    T1代表靶向前列腺癌特异性膜抗原基团，选自：T1 stands for Targeting Prostate Cancer Specific Membrane Antigen Group, selected from:
    Figure PCTCN2021131122-appb-100003
    Figure PCTCN2021131122-appb-100003
    T2代表靶向细胞膜基团，包括反式油酸，亚油酸，C10～30烷基酸，C10～30烯基酸，C10～30炔基酸。T2 represents targeting cell membrane groups, including trans-oleic acid, linoleic acid, C10-30 alkyl acid, C10-30 alkenyl acid, and C10-30 alkynyl acid.
  2. 根据权利要求1所述的化合物，其特征在于：式I所示的靶向前列腺癌的Cy类荧光探针化合物为：The compound according to claim 1, characterized in that: the Cy fluorescent probe compound targeting prostate cancer shown in formula I is:
    Figure PCTCN2021131122-appb-100004
    Figure PCTCN2021131122-appb-100004
    Figure PCTCN2021131122-appb-100005
    Figure PCTCN2021131122-appb-100005
  3. 根据权利要求1-2中任一项所述的式I所示的靶向前列腺癌的Cy类荧光探针化合物或其药学上可接受的盐，其特征在于：According to the Cy-type fluorescent probe compound or pharmaceutically acceptable salt thereof targeting prostate cancer shown in formula I according to any one of claims 1-2, it is characterized in that:
    所述药学上可接受的盐为所述化合物的有机酸盐或无机酸盐，The pharmaceutically acceptable salt is an organic or inorganic acid salt of the compound,
    所述药学上可接受的盐为所述化合物的有机碱盐或无机碱盐，The pharmaceutically acceptable salt is an organic base salt or an inorganic base salt of the compound,
    所述有机碱为吡啶，三乙胺，二乙胺，N-甲基吗啉，四甲基乙二胺；Described organic base is pyridine, triethylamine, diethylamine, N-methylmorpholine, tetramethylethylenediamine;
    无所述机碱为磷酸盐，磷酸氢盐，碳酸盐，碳酸氢盐，碳酸钾，碳酸氢盐，次氯酸盐，次溴酸盐，硅酸盐。The organic bases not mentioned are phosphate, hydrogen phosphate, carbonate, bicarbonate, potassium carbonate, bicarbonate, hypochlorite, hypobromite, silicate.
  4. 权利要求1-3中任一项所述的式I所示的靶向前列腺癌的Cy类荧光探针化合物或其药学上可接受的盐在制备对前列腺癌特异性膜抗原检测的试剂中的应用。The Cy-type fluorescent probe compound targeting prostate cancer shown in formula I described in any one of claims 1-3 or a pharmaceutically acceptable salt thereof in the preparation of reagents for prostate cancer-specific membrane antigen detection application.
  5. 权利要求1-3中任一项所述的式I所示的靶向前列腺癌的Cy类荧光探针化合物或其药学上可接受的盐在制备对前列腺癌靶向检测的试剂中的应用。Use of the Cy-type fluorescent probe compound targeting prostate cancer represented by formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1-3 in the preparation of a reagent for targeted detection of prostate cancer.
  6. 权利要求1-3中任一项所述的式I所示的靶向前列腺癌的Cy类荧光探针化合物或其药学上可接受的盐在制备前列腺癌诊断试剂中的应用。Use of the Cy-type fluorescent probe compound targeting prostate cancer represented by formula I according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof in the preparation of a prostate cancer diagnostic reagent.
  7. 权利要求1-3中任一项所述的式I所示的靶向前列腺癌的Cy类荧光探针化合物或其药学上可接受的盐在前列腺癌临床样检测中的应用。Use of the Cy-type fluorescent probe compound targeting prostate cancer represented by formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1-3 in the clinical detection of prostate cancer.
  8. 一种诊断前列腺癌的试剂盒，含有权利要求1-3中任一项所述的式I所示的靶向前列腺癌的Cy类荧光探针化合物或其药学上可接受的盐。A kit for diagnosing prostate cancer, comprising the Cy-type fluorescent probe compound targeting prostate cancer represented by formula I according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof.
  9. 一种诊断前列腺癌的方法，为将权利要求1-3中任一项所述的式I所示的靶向前列腺癌的Cy类荧光探针化合物或其药学上可接受的盐与待检测样品共孵育，如有很好的成像性，则判定所述检测样品为前列腺癌组织。A method for diagnosing prostate cancer, comprising the Cy-type fluorescent probe compound targeting prostate cancer shown in formula I described in any one of claims 1-3 or a pharmaceutically acceptable salt thereof and a sample to be detected After co-incubation, if there is good imaging performance, it is determined that the detection sample is prostate cancer tissue.
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