WO2022016690A1 - Luciferase substrates, preparation method therefor and application thereof - Google Patents

Luciferase substrates, preparation method therefor and application thereof Download PDF

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WO2022016690A1
WO2022016690A1 PCT/CN2020/114873 CN2020114873W WO2022016690A1 WO 2022016690 A1 WO2022016690 A1 WO 2022016690A1 CN 2020114873 W CN2020114873 W CN 2020114873W WO 2022016690 A1 WO2022016690 A1 WO 2022016690A1
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cycluc
solvent
acetonitrile
methanol
dichloromethane
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PCT/CN2020/114873
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French (fr)
Chinese (zh)
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李敏勇
杜吕佩
陈新新
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山东大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
    • C07D513/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains two hetero rings
    • C07D513/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/002—Heterocyclic compounds
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    • 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"
    • 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
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • 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
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • 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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G01N21/763—Bioluminescence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/05—Isotopically modified compounds, e.g. labelled
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    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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    • 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
    • C09K2211/1037—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with sulfur
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    • 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
    • C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
    • C09K2211/1051—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms with sulfur
    • 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"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Definitions

  • the invention belongs to the technical field of luciferase substrate preparation, and in particular relates to a luciferase substrate and a preparation method and application thereof.
  • Deuteration refers to the process of selectively replacing the position of protium hydrogen isotope in small molecule drugs, thereby realizing the process of deuterium hydrogen isotope drugs.
  • deuteration of drugs is most likely to affect pharmacokinetic properties, such as metabolism, rather than pharmacodynamic effects. Therefore, the metabolism of some drugs may be favorably affected when deuterated.
  • improper placement can lead to significant major isotopic effects.
  • Deuterium exhibits unique physicochemical properties and has the strongest kinetic isotope effect among all other elements.
  • a wide variety of morphological and physiological changes have been observed in deuterium-treated cells and organisms, including changes in fundamental processes such as cell division or energy metabolism.
  • Deuteration improved the metabolic profile without affecting the pharmacological effects of the drugs and reduced drug-drug interactions, which showed markedly enhanced stability in vitro.
  • deuterium is most commonly used to increase the stability of a drug, thereby increasing its half-life, and reducing its propensity to form reactive metabolites, while increasing its safety or improving how the drug is distributed.
  • Bioluminescence is a luminescence phenomenon that exists widely in nature. Some organisms can convert chemical energy into light energy through a series of oxidation reactions. In this process, the luminescence signal can be efficiently generated without external light excitation.
  • bioluminescence methods have been widely used as an alternative to fluorescence, greatly advancing biologically relevant research.
  • bioluminescence imaging technology has been widely used, mainly in food, tumor, heavy metal, various ions, enzymes and harmful gas detection and other fields. Research is still scarce.
  • the present invention provides a synthetic cyclic alkylamino luciferin, which allows the use of modified luciferase Ultra-Glo to emit stable red-shifted light, and the luminescence intensity is very strong, so It is a very valuable bioluminescent probe.
  • the present invention carries out deuterium modification, and replaces some of the carbon-hydrogen bonds in the cyclic alkylamino luciferin with carbon-deuterium bonds. Strong bioluminescence intensity and longer bioluminescence time, thus showing better bioluminescence advantages, so it has good practical application value.
  • the first aspect of the present invention provides a luciferase substrate, and the structural formula of the luciferase substrate is:
  • R takes hydrogen or deuterium, preferably deuterium
  • the luciferase substrate is cycluc, and its structural formula is:
  • the luciferase substrate is d 2 -cycluc, and its structural formula is:
  • a second aspect of the present invention provides a method for preparing the above-mentioned luciferase substrate, the preparation method comprising:
  • intermediate 10 reacts with ammonium chloride and zinc powder to obtain intermediate 11;
  • each of the above reaction steps is carried out under solvent conditions.
  • the solvent can be dichloromethane, acetonitrile, methanol or ethanol; more preferably dichloromethane;
  • the reaction temperature is 10 ⁇ 30°C, and the reaction time is 1 ⁇ 3h;
  • the molar ratio of the 5-nitroindoline, triethylamine and trifluoroacetic anhydride is 1:(1-1.5):(1-1.5).
  • the solvent is dichloromethane, acetonitrile, methanol or ethanol, more preferably ethanol;
  • the reaction temperature is 50 ⁇ 70°C, and the reaction time is 3 ⁇ 5h;
  • the molar ratio of the intermediate 1 and SnCl 2 ⁇ 2H 2 O is 1:(1-1.5).
  • Described solvent is glacial acetic acid or hydrochloric acid, more preferably glacial acetic acid;
  • the reaction temperature is 10 ⁇ 30°C, and the reaction time is 15 ⁇ 25h;
  • the molar ratio of the intermediate 2, liquid bromine and potassium thiocyanate is 1:1:(2-3).
  • the solvent is dichloromethane, acetonitrile, methanol or ethanol; more preferably acetonitrile;
  • the reaction temperature is 40 ⁇ 70°C, and the reaction time is 1 ⁇ 3h;
  • the molar ratio of the intermediate 3, t-butyl nitrite and cuprous chloride is 1:2:(1-3).
  • the solvent is methanol, dichloromethane or acetonitrile; more preferably methanol;
  • the reaction temperature is 10 ⁇ 30 °C, and the reaction time is 5 ⁇ 30min;
  • the molar ratio of compound 4 and NaBH 4 is 1:(3-5).
  • the solvent is dichloromethane, acetonitrile, methanol or ethanol; more preferably acetonitrile;
  • the reaction temperature is 50 ⁇ 100°C, and the reaction time is 1 ⁇ 3h;
  • the molar ratio of the compound 5, cyanotrimethylsilane and tetrabutylammonium fluoride is 1:(3-5):(3-5).
  • the solvent is an organic solvent that is mutually soluble in methanol, dichloromethane and water in any proportion;
  • Described inorganic base is potassium carbonate, cesium carbonate, sodium carbonate, sodium bicarbonate; further preferably potassium carbonate;
  • the reaction temperature is 20 ⁇ 50°C, and the reaction time is 1 ⁇ 5h;
  • the molar ratio of the intermediate 6, potassium carbonate and D-cysteine hydrochloride is 1:(1-3):(1-3).
  • the solvent is deuterated methanol, deuterated acetonitrile; more preferably deuterated methanol;
  • the reaction temperature is 30 ⁇ 70°C, and the reaction time is 50 ⁇ 80h;
  • the molar ratio of the 1H-indole and the palladium carbon is 10:1.
  • Described solvent is glacial acetic acid or hydrochloric acid; It is further preferably glacial acetic acid;
  • the reaction temperature is 50 ⁇ 100°C, and the reaction time is 1 ⁇ 3h;
  • the molar ratio of the intermediate 8 and acetyl chloride is 1:(4-6).
  • the solvent is dichloromethane, acetonitrile, methanol or ethanol; more preferably acetonitrile;
  • the reaction temperature is 40 ⁇ 70°C, and the reaction time is 1 ⁇ 3h;
  • the molar ratio of the intermediate 9 and Fe(NO 3 ) 3 ⁇ 9H 2 O is 2:1.
  • the solvent is dichloromethane, acetonitrile or ethanol; more preferably ethanol;
  • the reaction temperature is 10 ⁇ 30°C, and the reaction time is 1 ⁇ 3h;
  • the molar ratio of the intermediate 10, ammonium chloride and zinc powder is 1:10:20.
  • the solvent is dichloromethane, acetonitrile, methanol or ethanol; more preferably acetonitrile;
  • the reaction temperature is 10 ⁇ 30°C, and the reaction time is 10 ⁇ 30h;
  • the molar ratio of the intermediate 11, potassium thiocyanate and liquid bromine is 1:3-4:1.
  • the solvent is dichloromethane, anhydrous acetonitrile, methanol or ethanol; more preferably anhydrous acetonitrile;
  • the reaction temperature is 40 ⁇ 70°C, and the reaction time is 1 ⁇ 3h;
  • the molar ratio of the intermediate 12, nitroso-tert-butyl ester and cuprous chloride is 1:1-2:1.5.
  • the solvent is tetrahydrofuran, dichloromethane, DMF, methanol or ethanol; more preferably tetrahydrofuran;
  • the reaction temperature is -40 ⁇ -100°C, and the reaction time is 1 ⁇ 2h;
  • the molar ratio of the intermediate 13 and LiAlH 4 is 1-2:1.
  • the solvent is anhydrous acetonitrile, dichloromethane, DMF, methanol or ethanol; more preferably anhydrous acetonitrile;
  • the reaction temperature is 40 ⁇ 100°C, and the reaction time is 1 ⁇ 2h;
  • the molar ratio of the intermediate 14, cyanotrimethylsilane and tetrabutylammonium fluoride is 1-2:1:2.5.
  • the solvent is preferably an organic solvent that is mutually soluble in methanol, dichloromethane and water in any proportion,
  • the inorganic base is potassium carbonate, cesium carbonate, sodium carbonate or sodium bicarbonate; further preferably potassium carbonate;
  • the reaction temperature is 20 ⁇ 50°C, and the reaction time is 1 ⁇ 5h;
  • the molar ratio of the intermediate 15, potassium carbonate and D-cysteine hydrochloride is 1:1-2:2.5.
  • the third aspect of the present invention provides the application of the above-mentioned luciferase substrate in fluorescent products and/or the preparation of fluorescent products.
  • the fluorescent products include, but are not limited to, fluorescent probes and fluorescent detection kits.
  • a fourth aspect of the present invention provides a fluorescent probe comprising the above-mentioned luciferase substrate.
  • a fluorescence detection kit in a fifth aspect of the present invention, includes the above-mentioned luciferase substrate or fluorescent probe.
  • the sixth aspect of the present invention provides the application of the above-mentioned luciferase substrate, fluorescent probe and/or fluorescent detection kit in the following:
  • the biological analysis and detection includes, but is not limited to, the analysis and detection of the biological individual level, the organ level, the tissue level and the cellular level; preferably the cellular level.
  • Such organisms include, but are not limited to, fish, mice, rats, guinea pigs, chickens, rabbits, dogs, cats, monkeys, orangutans, humans, and the like.
  • the biological analysis and detection include using the above-mentioned luciferase substrates, fluorescent probes and/or fluorescent detection kits to fluorescently label cells, and use flow cytometry or fluorescence microscopy to sort fluorescently labeled cells. And use the in vivo fluorescence imaging device to observe the fluorescently labeled cells in vivo.
  • the above technical solutions provided by the luciferase substrate has good selectivity, high sensitivity, low detection line and good biocompatibility, etc; Further studies have shown that, in vitro, cellular, in vivo environment, d 2 -cycluc ratio The bioluminescence intensity of cycluc is strong, the bioluminescence time is long, and both luciferase substrates have good concentration dependence. At the same time, the preparation method provided by the above technical scheme is simple, operability and low cost, so it has the advantages of The value of good practical application.
  • Fig. 1 is the hydrogen nuclear magnetic resonance spectrum of the luciferase substrate cycluc prepared in Example 1;
  • Fig. 2 is the carbon nuclear magnetic resonance spectrum of the luciferase substrate cycluc prepared in Example 1;
  • Fig. 3 is the high-resolution mass spectrum of the luciferase substrate cycluc prepared in Example 1;
  • Fig. 4 is the HPLC of the luciferase substrate cycluc prepared in Example 1;
  • Fig. 5 is the hydrogen nuclear magnetic resonance spectrum of the luciferase substrate d 2 -cycluc prepared in Example 2;
  • Fig. 6 is the carbon nuclear magnetic resonance spectrum of the luciferase substrate d 2 -cycluc prepared in Example 2;
  • Figure 7 is the high-resolution mass spectrum of the luciferase substrate d 2 -cycluc prepared in Example 2;
  • Figure 8 is the HPLC of the luciferase substrate d 2 -cycluc prepared in Example 2;
  • FIG. 9 d is a substrate for luciferase Embodiment 2 -cycluc, in vitro metabolism studies cycluc; wherein, A is the 0 ⁇ 120min luciferase substrate d 2 -cycluc, cycluc change in intensity of bioluminescence in vitro FIG, B is 0 ⁇ 30min the luciferase substrate d 2 -cycluc, cycluc in FIG bioluminescence intensity variation in vitro, C is within 30 ⁇ 60min luciferase substrate d 2 -cycluc, cycluc in vitro Bioluminescence intensity change graph, D is the bioluminescence intensity change graph of luciferase substrates d 2 -cycluc and cycluc in vitro within 60-120 min, E is the bioluminescence intensity of compounds cycluc and d 2 -cycluc at 30 min, Difference, F is the bioluminescence intensity difference between compound cycluc and d 2 -cycl
  • FIG. 10 d is a substrate for luciferase Embodiment 2 -cycluc, concentration-dependent in vitro studies cycluc; wherein, A d 2 -cycluc different concentrations, cycluc FIG vitro imaging, B d 2 -cycluc different concentrations The relationship with bioluminescence intensity, C is the relationship between different concentrations of cycluc and bioluminescence intensity;
  • Figure 11 shows the cytotoxicity study of luciferase substrates d 2 -cycluc and cycluc in Example 6; wherein, A is the survival rate of ES-2-Fluc cells with different concentrations of d 2 -cycluc, and B is the effect of different concentrations of cycluc on ES-2-Fluc cells. The survival rate of ES-2-Fluc cells;
  • FIG. 12 is a luciferase substrate d 2 -cycluc, concentration-dependent study of cell cycluc; wherein, A d 2 -cycluc different concentrations, cycluc imaging cells, B to different concentrations and d 2 -cycluc
  • the relationship between bioluminescence intensity, C is the relationship between different concentrations of cycluc and bioluminescence intensity
  • D is the bioluminescence intensity at 1-60min when the concentration of the two compounds is 5 ⁇ M
  • E is when the concentration of the two compounds is 0-100 ⁇ M
  • d 2 - the relative ratio of cycluc and cycluc bioluminescence intensity
  • Figure 13 is the in vivo concentration-dependent study of the luciferase substrate d 2 -cycluc of Example 8 in FVB-luc + mice; wherein, A is the relationship between different concentrations of d 2 -cycluc and bioluminescence intensity, and B is different concentrations Mouse imaging results of d 2-cycluc;
  • Figure 14 is the in vivo comparative study of FVB-luc + mice of luciferase substrates d 2 -cycluc and cycluc in Example 9, wherein A is the intraperitoneal injection of cybluc and d 2 -cycluc (100 ⁇ M, A is FVB-luc + mice) 100 ⁇ L) at 60min, B is FVB-luc + mouse intraperitoneal injection of cybluc and d 2 -cycluc (100 ⁇ M, 100 ⁇ L) at 120 min, C is FVB-luc + mouse intraperitoneal injection of cybluc and d 2 -cycluc (100 ⁇ M, 100 ⁇ L) In vivo photon number at 240min, D is the in vivo photon number at 480min in FVB-luc + mouse intraperitoneal injection of cybluc and d 2 -cycluc (100 ⁇ M, 100 ⁇ L), E is FVB-luc + mouse intraperitoneal injection of cycl
  • Example 3 Experimental study on in vitro metabolism of luciferase substrates cycluc and d 2 -cycluc
  • Example 5 firefly luciferase substrate cycluc concentration dependent in vitro studies, and d is 2 -cycluc
  • Example 6 Toxicity assay of firefly luciferase substrates to cells
  • ES-2-Fluc cells in log phase were digested and centrifuged, they were dispersed with RPMI 1640 medium containing 10% fetal bovine serum, and the cells were counted to adjust the cell density to 4 ⁇ 10 4 cells/mL.
  • Cells were seeded in a 60-well area (100 ⁇ L/well) in the middle of a transparent 96-well plate with a row gun, and a circle around the 96-well plate was filled with common medium and placed in a cell incubator for incubation.
  • the concentrated stock solution of luciferase substrate was diluted with serum-free RPMI 1640 medium into different concentration gradients (0, 8 ⁇ M, 16 ⁇ M, 31 ⁇ M, 62 ⁇ M, 125 ⁇ M, 250 ⁇ M, 500 ⁇ M, 1000 ⁇ M) , 2000 ⁇ M) into a 96-well plate, three replicate wells were set for each concentration, and the luciferase substrate and cells were incubated in a cell incubator.
  • MTT 5mg/mL 20 ⁇ L/well
  • PBS phosphate buffered saline
  • the liquid in the 96-well plate was sucked off with a syringe, 150 ⁇ L of DMSO was added to each well, and placed on a shaker for 5 min to fully dissolve the formed crystals, and the absorbance at 490 nm was scanned under a microplate reader.
  • Fig. 11 in order to detect that the luciferase substrate has less biological toxicity to cells, subsequent cell experiments can be carried out, and we carried out cell MTT experiments.
  • the experimental results show that the two luciferase substrates have little toxicity to cells.
  • the IC 50 of cycluc 855.4 ⁇ M, and the IC 50 of d 2 -cycluc is greater than 5*10 5 ⁇ M, which is much larger than that used in cell experiments.
  • the luciferase substrate concentration can therefore be used for biological level detection.
  • Example 8 Intraperitoneal injection experiment of firefly luciferase substrate d 2 -cycluc with different concentrations of FVB-luc + mice
  • Compound d 2 -cycluc was formulated into solutions with final concentrations of 10 mM, 5 mM, 1 mM, 100 ⁇ M, 10 ⁇ M with normal saline and DMSO, and 200 ⁇ L was injected intraperitoneally per FVB-luc + mouse (transcriptional luciferase) (about 20 g). Chloral hydrate solution (40 mg/mL) was anesthetized. After anesthesia, 100 ⁇ L of compound d 2 -cycluc was intraperitoneally injected. Immediately, imaging was performed with a small animal in vivo imager, and the time was recorded as 1 min.
  • the bioluminescence intensity was photographed with a small animal in vivo imager.
  • the ROI value was calculated from other parts of FVB-luc + mice except the tail, and statistical calculation was performed with Graphpad.
  • Example 9 Intraperitoneal injection of firefly luciferase substrates cycluc and d 2 -cycluc to FVB-luc + mice
  • Cycluc and d 2 -cycluc were prepared into a solution with a final concentration of 100 ⁇ M in a ratio of 9:1 with normal saline and DMSO, and 6 adult female FVB-luc + mice (transcriptional luciferase) were taken and divided into 2 groups, Each FVB-luc + mouse (transcription luciferase) (about 20 g) was anesthetized by intraperitoneal injection of 200 ⁇ L of chloral hydrate solution (40 mg/mL), and the two groups of mice were intraperitoneally injected with 100 ⁇ L of cycluc and d 2 -cycluc, respectively, and immediately used The small animal in vivo imager was used for imaging, and the time was recorded as 1 min.
  • the bioluminescence intensity was photographed with a small animal in vivo imager.
  • the ROI values were calculated from other parts of FVB-luc + mice except the tail, and statistical calculations were performed with Graphpad.

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Abstract

Provided are luciferase substrates, a preparation method therefor and an application thereof. The luciferase substrates have the advantages of good selectivity, high sensitivity, a low detection line and good biocompatibility, and so on; and further research shows that d2-cycluc has stronger bioluminescence intensity and longer bioluminescence duration than cycluc in in vitro, cell, and in vivo environments. In addition, both luciferase substrates have a good concentration dependence, and the preparation method for the luciferase substrates is simple, has strong operability, low costs, and has good practical application values.

Description

一种萤光素酶底物及其制备方法和应用A kind of luciferase substrate and its preparation method and application 技术领域technical field
本发明属于荧光素酶底物制备技术领域，具体涉及一种萤光素酶底物及其制备方法和应用。The invention belongs to the technical field of luciferase substrate preparation, and in particular relates to a luciferase substrate and a preparation method and application thereof.
背景技术Background technique
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。The disclosure of information in this Background section is only for enhancement of understanding of the general background of the invention and should not necessarily be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.
许多药物都是基于碳的，碳氢键与理解药物分子的重要特性特别相关。氘化是指选择性取代氕氢同位素在小分子药物中的位置，从而实现氘氢同位素药物的过程。一方面，药物的氘化最有可能影响药代动力学特性，例如代谢，而不是药效学作用。因此，氘代时某些药物的代谢可能受到有利影响。可以从氘化作为改变药物药代动力学的策略中获益，在一系列微生物学和生化分析中，氘化似乎并未损害药物的疗效。另一方面，使用氘化时，放置不当会导致明显的主要同位素效应。当与重同位素的键是反应(或代谢转化)中的限速步骤时，就会发生主要的同位素效应，分子与重同位素的反应将进行得更慢，由于轻和重同位素之间的质量差异而产生了标记。Many drugs are carbon-based, and carbon-hydrogen bonds are particularly relevant for understanding important properties of drug molecules. Deuteration refers to the process of selectively replacing the position of protium hydrogen isotope in small molecule drugs, thereby realizing the process of deuterium hydrogen isotope drugs. On the one hand, deuteration of drugs is most likely to affect pharmacokinetic properties, such as metabolism, rather than pharmacodynamic effects. Therefore, the metabolism of some drugs may be favorably affected when deuterated. One could benefit from deuteration as a strategy to alter the pharmacokinetics of drugs, which did not appear to impair drug efficacy in a range of microbiological and biochemical assays. On the other hand, when using deuteration, improper placement can lead to significant major isotopic effects. Primary isotopic effects occur when the bond with the heavy isotope is the rate-limiting step in the reaction (or metabolic transformation), and the molecule will react more slowly with the heavy isotope due to the mass difference between the light and heavy isotopes mark is generated.
氘表现出独特的理化性质，并且在所有其他元素中具有最强的动力学同位素效应。此外，在经氘处理过的细胞和生物体中已观察到各种各样的形态和生理变化，包括诸如细胞分裂或能量代谢等基本过程的变化。氘化作用改善了代谢谱，同时不影响药物的药理作用，并且降低了药物之间的相互作用，它们在体外显示出明显增强的稳定性。通常，氘最常用于增加药物的稳定性，从而增加其半衰期，并降低其形成反应性代谢产物的倾向，同时增加其安全性或改善药物的分布方式。氘化药物项目的巨大优势是可获得来自其非氘化变体的人类临床试验数据，这使得该过程的财务需求降低了。尽管我们仍未完全了解氘的所有后果，但很显然，在科学应用、生物技术、药理学等领域具有利用这些效应的巨大潜力。此外，动力学同位素效应的表现是其他元素中无与伦比的，此类应用的首选同位素。Deuterium exhibits unique physicochemical properties and has the strongest kinetic isotope effect among all other elements. In addition, a wide variety of morphological and physiological changes have been observed in deuterium-treated cells and organisms, including changes in fundamental processes such as cell division or energy metabolism. Deuteration improved the metabolic profile without affecting the pharmacological effects of the drugs and reduced drug-drug interactions, which showed markedly enhanced stability in vitro. In general, deuterium is most commonly used to increase the stability of a drug, thereby increasing its half-life, and reducing its propensity to form reactive metabolites, while increasing its safety or improving how the drug is distributed. A huge advantage of the deuterated drug program is the availability of human clinical trial data from its non-deuterated variants, which makes the process less financially demanding. Although we still do not fully understand all the consequences of deuterium, it is clear that there is great potential to exploit these effects in scientific applications, biotechnology, pharmacology, and more. In addition, the performance of kinetic isotope effects is unmatched among other elements, the isotope of choice for such applications.
生物发光现象是自然界广泛存在的一种发光现象，一些生物可以通过一系列氧化反应将化学能转化为光能。在该过程中，可以在没有外部光激发的情况下有效地产生发光信号。在过去的十年中，生物发光方法作为荧光的替代方法得到了广泛应用，大大推动了生物学相关研究。目前生物发光成像技术已得到广泛的应用，主要应用于食品，肿瘤，重金属，各种离子，酶和有害气体检测等多个领域，然而，发明人发现，基于生物发光现象对动力学同位素效应的研究仍然较少。Bioluminescence is a luminescence phenomenon that exists widely in nature. Some organisms can convert chemical energy into light energy through a series of oxidation reactions. In this process, the luminescence signal can be efficiently generated without external light excitation. In the past decade, bioluminescence methods have been widely used as an alternative to fluorescence, greatly advancing biologically relevant research. At present, bioluminescence imaging technology has been widely used, mainly in food, tumor, heavy metal, various ions, enzymes and harmful gas detection and other fields. Research is still scarce.
发明内容SUMMARY OF THE INVENTION
针对目前现有技术的不足，本发明提供一种合成的环状烷基氨基萤光素，其允许使用修饰的萤光素酶Ultra-Glo发出稳定的红移光，并且发光强度很强，因此它是一种非常有应用价值的生物发光探针。本发明根据环状烷基氨基萤光素发光的优越性，对其进行氘代修饰，将环状烷基氨基萤光素中的部分碳氢键替换为碳氘键，经试验证明其具有更强的生物发光强度和更长的生物发光时间，从而展现出更加良好的生物发光优势，因此具有良好的实际应用之价值。In view of the shortcomings of the current prior art, the present invention provides a synthetic cyclic alkylamino luciferin, which allows the use of modified luciferase Ultra-Glo to emit stable red-shifted light, and the luminescence intensity is very strong, so It is a very valuable bioluminescent probe. According to the superiority of luminescence of cyclic alkylamino luciferin, the present invention carries out deuterium modification, and replaces some of the carbon-hydrogen bonds in the cyclic alkylamino luciferin with carbon-deuterium bonds. Strong bioluminescence intensity and longer bioluminescence time, thus showing better bioluminescence advantages, so it has good practical application value.
为了实现上述技术目的，本发明的技术方案如下：In order to realize the above-mentioned technical purpose, the technical scheme of the present invention is as follows:
本发明的第一个方面，提供一种萤光素酶底物，所述荧光素酶底物结构式为：The first aspect of the present invention provides a luciferase substrate, and the structural formula of the luciferase substrate is:
Figure PCTCN2020114873-appb-000001
Figure PCTCN2020114873-appb-000001
其中，R取氢或氘，优选为氘；Wherein, R takes hydrogen or deuterium, preferably deuterium;
当R取自氢时，所述萤光素酶底物为cycluc，其结构式为：When R is taken from hydrogen, the luciferase substrate is cycluc, and its structural formula is:
Figure PCTCN2020114873-appb-000002
Figure PCTCN2020114873-appb-000002
当R取自氘时，所述萤光素酶底物为d 2-cycluc，其结构式为： When R is taken from deuterium, the luciferase substrate is d 2 -cycluc, and its structural formula is:
Figure PCTCN2020114873-appb-000003
Figure PCTCN2020114873-appb-000003
本发明的第二个方面，提供上述荧光素酶底物的制备方法，所述制备方法包括：A second aspect of the present invention provides a method for preparing the above-mentioned luciferase substrate, the preparation method comprising:
Figure PCTCN2020114873-appb-000004
或，
Figure PCTCN2020114873-appb-000004
or,
Figure PCTCN2020114873-appb-000005
Figure PCTCN2020114873-appb-000005
具体的，所述cycluc的合成步骤为：Concretely, the synthetic step of described cycluc is:
(1)以5-硝基吲哚啉为原料和三乙胺反应，同时滴加三氟乙酸酐，将该混合物搅拌反应得到中间体1；(1) take 5-nitroindoline as raw material and triethylamine reaction, drip trifluoroacetic anhydride simultaneously, and this mixture is stirred and reacted to obtain intermediate 1;
(2)中间体1和SnCl 2·2H 2O进行反应，得到中间体2； (2) Intermediate 1 reacts with SnCl 2 ·2H 2 O to obtain Intermediate 2;
(3)中间体2、硫氰酸钾的冰醋酸溶液和液溴进行反应，得到中间体3；(3) the glacial acetic acid solution of intermediate 2, potassium thiocyanate reacts with liquid bromine to obtain intermediate 3;
(4)中间体3、亚硝酸叔丁酯和氯化亚铜进行反应，得到中间体4；(4) intermediate 3, tert-butyl nitrite and cuprous chloride react to obtain intermediate 4;
(5)中间体4和NaBH 4进行反应，得到中间体5； (5) Intermediate 4 and reaction of NaBH 4, to give intermediate 5;
(6)中间体5、氰基三甲基硅烷和四丁基氟化铵进行反应，得到中间体6；(6) intermediate 5, cyanotrimethylsilane and tetrabutylammonium fluoride react to obtain intermediate 6;
(7)中间体6、无机碱和D-半胱氨酸盐酸盐进行反应，得到cycluc；(7) react intermediate 6, inorganic base and D-cysteine hydrochloride to obtain cycluc;
所述d 2-cycluc的具体合成步骤为： The specific synthesis steps of the d 2 -cycluc are:
(8)1H-吲哚和钯碳在氘气条件下进行反应，得到中间体8；(8) 1H-indole and palladium carbon react under deuterium gas conditions to obtain intermediate 8;
(9)中间体8和乙酰氯进行反应，得到中间体9；(9) intermediate 8 reacts with acetyl chloride to obtain intermediate 9;
(10)中间体9和Fe(NO 3) 3·9H 2O进行反应，得到中间体10； (10) Intermediate 9 reacts with Fe(NO 3 ) 3 ·9H 2 O to obtain Intermediate 10;
(11)中间体10和氯化铵、锌粉进行反应，得到中间体11；(11) intermediate 10 reacts with ammonium chloride and zinc powder to obtain intermediate 11;
(12)中间体11和硫氰酸钾进行反应，得到中间体12；(12) intermediate 11 reacts with potassium thiocyanate to obtain intermediate 12;
(13)中间体12、亚硝基叔丁酯和氯化亚铜进行反应，得到中间体13；(13) intermediate 12, nitroso-tert-butyl ester and cuprous chloride react to obtain intermediate 13;
(14)中间体13和LiAlH 4进行反应，得到中间体14； (14) and the intermediate 13 is reacted LiAlH 4 to give intermediate 14;
(15)中间体14、氰基三甲基硅烷和四丁基氟化铵进行反应，得到中间体15；(15) Intermediate 14, cyanotrimethylsilane and tetrabutylammonium fluoride react to obtain Intermediate 15;
(16)中间体15、碳酸钾和D-半胱氨酸盐酸盐进行反应，得到中间体15；(16) intermediate 15, potassium carbonate and D-cysteine hydrochloride react to obtain intermediate 15;
优选的，上述各反应步骤均在溶剂条件下进行。Preferably, each of the above reaction steps is carried out under solvent conditions.
优选的，所述步骤(1)中，Preferably, in the step (1),
所述溶剂可以为二氯甲烷、乙腈、甲醇或乙醇；进一步优选为二氯甲烷；The solvent can be dichloromethane, acetonitrile, methanol or ethanol; more preferably dichloromethane;
反应温度为10～30℃，反应时间为1～3h；The reaction temperature is 10～30℃, and the reaction time is 1～3h;
所述5-硝基吲哚啉、三乙胺、三氟乙酸酐的摩尔比为1：(1-1.5)：(1-1.5)。The molar ratio of the 5-nitroindoline, triethylamine and trifluoroacetic anhydride is 1:(1-1.5):(1-1.5).
优选的，所述步骤(2)中，Preferably, in the step (2),
所述溶剂为二氯甲烷、乙腈、甲醇或乙醇，进一步优选为乙醇；The solvent is dichloromethane, acetonitrile, methanol or ethanol, more preferably ethanol;
反应温度为50～70℃，反应时间为3～5h；The reaction temperature is 50～70℃, and the reaction time is 3～5h;
所述中间体1、SnCl 2·2H 2O的摩尔比为1：(1-1.5)。 The molar ratio of the intermediate 1 and SnCl 2 ·2H 2 O is 1:(1-1.5).
优选的，所述步骤(3)中，Preferably, in the step (3),
所述溶剂为冰醋酸或盐酸，进一步优选为冰醋酸；Described solvent is glacial acetic acid or hydrochloric acid, more preferably glacial acetic acid;
反应温度为10～30℃，反应时间为15～25h；The reaction temperature is 10～30℃, and the reaction time is 15～25h;
所述中间体2、液溴和硫氰酸钾的摩尔比为1：1:(2-3)。The molar ratio of the intermediate 2, liquid bromine and potassium thiocyanate is 1:1:(2-3).
优选的，所述步骤(4)中，Preferably, in the step (4),
所述溶剂为二氯甲烷、乙腈、甲醇或乙醇；进一步优选为乙腈；The solvent is dichloromethane, acetonitrile, methanol or ethanol; more preferably acetonitrile;
反应温度为40～70℃，反应时间为1～3h；The reaction temperature is 40～70℃, and the reaction time is 1～3h;
所述中间体3、亚硝酸叔丁酯和氯化亚铜的摩尔比为1：2:(1-3)。The molar ratio of the intermediate 3, t-butyl nitrite and cuprous chloride is 1:2:(1-3).
优选的，所述步骤(5)中，Preferably, in the step (5),
所述溶剂为甲醇、二氯甲烷或乙腈；进一步优选为甲醇；The solvent is methanol, dichloromethane or acetonitrile; more preferably methanol;
反应温度为10～30℃，反应时间为5～30min；The reaction temperature is 10～30 ℃, and the reaction time is 5～30min;
所述化合物4、NaBH 4的摩尔比为1：(3-5)。 The molar ratio of compound 4 and NaBH 4 is 1:(3-5).
优选的，所述步骤(6)中，Preferably, in the step (6),
所述溶剂为二氯甲烷、乙腈、甲醇或乙醇；进一步优选为乙腈；The solvent is dichloromethane, acetonitrile, methanol or ethanol; more preferably acetonitrile;
反应温度为50～100℃，反应时间为1～3h；The reaction temperature is 50～100℃, and the reaction time is 1～3h;
所述化合物5、氰基三甲基硅烷、四丁基氟化铵的摩尔比为1：(3-5)：(3-5)。The molar ratio of the compound 5, cyanotrimethylsilane and tetrabutylammonium fluoride is 1:(3-5):(3-5).
优选的，所述步骤(7)中，Preferably, in the step (7),
所述溶剂为甲醇、二氯甲烷与水以任意比例互溶的有机溶剂；The solvent is an organic solvent that is mutually soluble in methanol, dichloromethane and water in any proportion;
所述无机碱为碳酸钾、碳酸铯、碳酸钠、碳酸氢钠；进一步优选碳酸钾；Described inorganic base is potassium carbonate, cesium carbonate, sodium carbonate, sodium bicarbonate; further preferably potassium carbonate;
反应温度为20～50℃，反应时间为1～5h；The reaction temperature is 20～50℃, and the reaction time is 1～5h;
所述中间体6、碳酸钾、D-半胱氨酸盐酸盐的摩尔比为1：(1-3)：(1-3)。The molar ratio of the intermediate 6, potassium carbonate and D-cysteine hydrochloride is 1:(1-3):(1-3).
优选的，所述步骤(8)中，Preferably, in the step (8),
所述溶剂为氘代甲醇、氘代乙腈；进一步优选为氘代甲醇；The solvent is deuterated methanol, deuterated acetonitrile; more preferably deuterated methanol;
反应温度为30～70℃，反应时间为50～80h；The reaction temperature is 30～70℃, and the reaction time is 50～80h;
所述1H-吲哚、钯碳的摩尔比为10：1。The molar ratio of the 1H-indole and the palladium carbon is 10:1.
优选的，所述步骤(9)中，Preferably, in the step (9),
所述溶剂为冰醋酸或盐酸；进一步优选为冰醋酸；Described solvent is glacial acetic acid or hydrochloric acid; It is further preferably glacial acetic acid;
反应温度为50～100℃，反应时间为1～3h；The reaction temperature is 50～100℃, and the reaction time is 1～3h;
所述中间体8、乙酰氯的摩尔比为1：(4-6)。The molar ratio of the intermediate 8 and acetyl chloride is 1:(4-6).
优选的，所述步骤(10)中，Preferably, in the step (10),
所述溶剂为二氯甲烷、乙腈、甲醇或乙醇；进一步优选为乙腈；The solvent is dichloromethane, acetonitrile, methanol or ethanol; more preferably acetonitrile;
反应温度为40～70℃，反应时间为1～3h；The reaction temperature is 40～70℃, and the reaction time is 1～3h;
所述中间体9、Fe(NO 3) 3·9H 2O的摩尔比为2：1。 The molar ratio of the intermediate 9 and Fe(NO 3 ) 3 ·9H 2 O is 2:1.
优选的，所述步骤(11)中，Preferably, in the step (11),
所述溶剂为二氯甲烷、乙腈或乙醇；进一步优选为乙醇；The solvent is dichloromethane, acetonitrile or ethanol; more preferably ethanol;
反应温度为10～30℃，反应时间为1～3h；The reaction temperature is 10～30℃, and the reaction time is 1～3h;
所述中间体10、氯化铵、锌粉的摩尔比为1：10：20。The molar ratio of the intermediate 10, ammonium chloride and zinc powder is 1:10:20.
优选的，所述步骤(12)中，Preferably, in the step (12),
所述溶剂为二氯甲烷、乙腈、甲醇或乙醇；进一步优选为乙腈；The solvent is dichloromethane, acetonitrile, methanol or ethanol; more preferably acetonitrile;
反应温度为10～30℃，反应时间为10～30h；The reaction temperature is 10～30℃, and the reaction time is 10～30h;
所述中间体11、硫氰酸钾、液溴的摩尔比为1：3～4：1。The molar ratio of the intermediate 11, potassium thiocyanate and liquid bromine is 1:3-4:1.
优选的，所述步骤(13)中，Preferably, in the step (13),
所述溶剂为二氯甲烷、无水乙腈、甲醇或乙醇；进一步优选为无水乙腈；The solvent is dichloromethane, anhydrous acetonitrile, methanol or ethanol; more preferably anhydrous acetonitrile;
反应温度为40～70℃，反应时间为1～3h；The reaction temperature is 40～70℃, and the reaction time is 1～3h;
所述中间体12、亚硝基叔丁酯、氯化亚铜的摩尔比为1：1～2：1.5。The molar ratio of the intermediate 12, nitroso-tert-butyl ester and cuprous chloride is 1:1-2:1.5.
优选的，所述步骤(14)中，Preferably, in the step (14),
所述溶剂为四氢呋喃、二氯甲烷、DMF、甲醇或乙醇；进一步优选为四氢呋喃；The solvent is tetrahydrofuran, dichloromethane, DMF, methanol or ethanol; more preferably tetrahydrofuran;
反应温度为-40～-100℃，反应时间为1～2h；The reaction temperature is -40～-100℃, and the reaction time is 1～2h;
所述中间体13、LiAlH 4的摩尔比为1～2：1。 The molar ratio of the intermediate 13 and LiAlH 4 is 1-2:1.
优选的，所述步骤(15)中，Preferably, in the step (15),
所述溶剂为无水乙腈、二氯甲烷、DMF、甲醇或乙醇；进一步优选为无水乙腈；The solvent is anhydrous acetonitrile, dichloromethane, DMF, methanol or ethanol; more preferably anhydrous acetonitrile;
反应温度为40～100℃，反应时间为1～2h；The reaction temperature is 40～100℃, and the reaction time is 1～2h;
所述中间体14、氰基三甲基硅烷、四丁基氟化铵的摩尔比为1～2：1：2.5。The molar ratio of the intermediate 14, cyanotrimethylsilane and tetrabutylammonium fluoride is 1-2:1:2.5.
优选的，步骤(16)中，Preferably, in step (16),
所述溶剂优选为甲醇、二氯甲烷与水以任意比例互溶的有机溶剂，The solvent is preferably an organic solvent that is mutually soluble in methanol, dichloromethane and water in any proportion,
所述无机碱为碳酸钾、碳酸铯、碳酸钠或碳酸氢钠；进一步优选为碳酸钾；The inorganic base is potassium carbonate, cesium carbonate, sodium carbonate or sodium bicarbonate; further preferably potassium carbonate;
反应温度为20～50℃，反应时间为1～5h；The reaction temperature is 20～50℃, and the reaction time is 1～5h;
所述中间体15、碳酸钾、D-半胱氨酸盐酸盐的摩尔比为1：1～2：2.5。The molar ratio of the intermediate 15, potassium carbonate and D-cysteine hydrochloride is 1:1-2:2.5.
本发明的第三个方面，提供上述萤光素酶底物在荧光产品和/或制备荧光产品中的应用。The third aspect of the present invention provides the application of the above-mentioned luciferase substrate in fluorescent products and/or the preparation of fluorescent products.
所述荧光产品包括但不限于荧光探针和荧光检测试剂盒。The fluorescent products include, but are not limited to, fluorescent probes and fluorescent detection kits.
本发明的第四个方面，提供一种荧光探针，所述荧光探针包含上述萤光素酶底物。A fourth aspect of the present invention provides a fluorescent probe comprising the above-mentioned luciferase substrate.
本发明的第五个方面，提供一种荧光检测试剂盒，所述荧光检测试剂盒包括上述萤光素酶底物或荧光探针。In a fifth aspect of the present invention, a fluorescence detection kit is provided, and the fluorescence detection kit includes the above-mentioned luciferase substrate or fluorescent probe.
本发明的第六个方面，提供上述萤光素酶底物、荧光探针和/或荧光检测试剂盒在如下中的应用：The sixth aspect of the present invention provides the application of the above-mentioned luciferase substrate, fluorescent probe and/or fluorescent detection kit in the following:
1)环境检测；1) Environmental testing;
2)分析化学；2) Analytical chemistry;
3)生物分析和检测。3) Biological analysis and detection.
其中，所述生物分析和检测包括但不限于对生物个体水平、器官水平、组织水平和细胞水平的分析与检测；优选为细胞水平。Wherein, the biological analysis and detection includes, but is not limited to, the analysis and detection of the biological individual level, the organ level, the tissue level and the cellular level; preferably the cellular level.
所述生物包括但不限于鱼、小鼠、大鼠、豚鼠、鸡、兔、狗、猫、猴、猩猩和人等。Such organisms include, but are not limited to, fish, mice, rats, guinea pigs, chickens, rabbits, dogs, cats, monkeys, orangutans, humans, and the like.
进一步的，所述生物分析和检测包括利用上述萤光素酶底物、荧光探针和/或荧光检测试剂盒对细胞进行荧光标记、利用流式细胞仪或荧光显微镜对荧光标记细胞进行分选和利用体内荧光成像装置观察生物体内的荧光标记的细胞。Further, the biological analysis and detection include using the above-mentioned luciferase substrates, fluorescent probes and/or fluorescent detection kits to fluorescently label cells, and use flow cytometry or fluorescence microscopy to sort fluorescently labeled cells. And use the in vivo fluorescence imaging device to observe the fluorescently labeled cells in vivo.
经试验证明，本申请中萤光素酶底物细胞毒性较低，具有良好的生物相容性，且在细胞水平上都呈现出浓度依赖性，同时d 2-cycluc相较于cycluc在低浓度时对萤光素酶有着较高的灵敏度，同时在细胞中有更强的生物发光强度；因此两者均可用于活体成像，并且由于d 2-cycluc具有更强的生物发光强度和更长的生物发光时间，在活体成像时会有更佳的成像效果。 The test proved that the present application luciferase substrate lower cytotoxicity, good biocompatibility, and emerged in a concentration-dependent cellular level, while d 2 -cycluc at low concentrations compared to cycluc It has higher sensitivity to luciferase and higher bioluminescence intensity in cells; therefore both can be used for in vivo imaging, and due to the higher bioluminescence intensity and longer duration of d 2 -cycluc Bioluminescence time, there will be better imaging results in live imaging.
上述技术方案的有益技术效果：The beneficial technical effects of the above technical solutions:
上述技术方案提供的萤光素酶底物具有选择性好、灵敏性高、检测线低及良好生物相容性等优点；进一步研究证明，在体外、细胞、体内环境中，d 2-cycluc比cycluc的生物发光强度强，生物发光时间长，且两种萤光素酶底物都具有良好的浓度依赖性，同时上述技术方案提供的制备方法简单，可操作性强，成本较低，因此具有良好的实际应用之价值。 The above technical solutions provided by the luciferase substrate has good selectivity, high sensitivity, low detection line and good biocompatibility, etc; Further studies have shown that, in vitro, cellular, in vivo environment, d 2 -cycluc ratio The bioluminescence intensity of cycluc is strong, the bioluminescence time is long, and both luciferase substrates have good concentration dependence. At the same time, the preparation method provided by the above technical scheme is simple, operability and low cost, so it has the advantages of The value of good practical application.
附图说明Description of drawings
为了更清楚地说明本发明实施例的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings used in the description of the embodiments. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained from the provided drawings without any creative effort.
图1为实施例1所制备的萤光素酶底物cycluc的核磁共振氢谱；Fig. 1 is the hydrogen nuclear magnetic resonance spectrum of the luciferase substrate cycluc prepared in Example 1;
图2为实施例1所制备的萤光素酶底物cycluc的核磁共振碳谱；Fig. 2 is the carbon nuclear magnetic resonance spectrum of the luciferase substrate cycluc prepared in Example 1;
图3为实施例1所制备的萤光素酶底物cycluc的高分辨质谱；Fig. 3 is the high-resolution mass spectrum of the luciferase substrate cycluc prepared in Example 1;
图4为实施例1所制备的萤光素酶底物cycluc的HPLC；Fig. 4 is the HPLC of the luciferase substrate cycluc prepared in Example 1;
图5为实施例2所制备的萤光素酶底物d 2-cycluc的核磁共振氢谱； Fig. 5 is the hydrogen nuclear magnetic resonance spectrum of the luciferase substrate d 2 -cycluc prepared in Example 2;
图6为实施例2所制备的萤光素酶底物d 2-cycluc的核磁共振碳谱； Fig. 6 is the carbon nuclear magnetic resonance spectrum of the luciferase substrate d 2 -cycluc prepared in Example 2;
图7为实施例2所制备的萤光素酶底物d 2-cycluc的高分辨质谱； Figure 7 is the high-resolution mass spectrum of the luciferase substrate d 2 -cycluc prepared in Example 2;
图8为实施例2所制备的萤光素酶底物d 2-cycluc的HPLC； Figure 8 is the HPLC of the luciferase substrate d 2 -cycluc prepared in Example 2;
图9为实施例3萤光素酶底物d 2-cycluc、cycluc的体外代谢研究；其中，A为0～120min内萤光素酶底物d 2-cycluc、cycluc在体外的生物发光强度变化图，B为0～30min内萤光素酶底物d 2-cycluc、cycluc在体外的生物发光强度变化图，C为30～60min内萤光素酶底物d 2-cycluc、cycluc在体外的生物发光强度变化图，D为60～120min内萤光素酶底物d 2-cycluc、cycluc在体外的生物发光强度变化图，E 为化合物cycluc和d 2-cycluc在30min、时的生物发光强度差异，F为化合物cycluc和d 2-cycluc在60min时的生物发光强度差异，G为化合物cycluc和d 2-cycluc在120min时的生物发光强度差异，H为化合物d 2-cycluc与cycluc生物发光强度比值在30min、60min、120min内的变化曲线； Example 3 FIG. 9 d is a substrate for luciferase Embodiment 2 -cycluc, in vitro metabolism studies cycluc; wherein, A is the 0 ~ 120min luciferase substrate d 2 -cycluc, cycluc change in intensity of bioluminescence in vitro FIG, B is 0 ~ 30min the luciferase substrate d 2 -cycluc, cycluc in FIG bioluminescence intensity variation in vitro, C is within 30 ~ 60min luciferase substrate d 2 -cycluc, cycluc in vitro Bioluminescence intensity change graph, D is the bioluminescence intensity change graph of luciferase substrates d 2 -cycluc and cycluc in vitro within 60-120 min, E is the bioluminescence intensity of compounds cycluc and d 2 -cycluc at 30 min, Difference, F is the bioluminescence intensity difference between compound cycluc and d 2 -cycluc at 60min, G is the bioluminescence intensity difference between compound cycluc and d 2 -cycluc at 120min, H is the bioluminescence intensity of compound d 2 -cycluc and cycluc The change curve of the ratio within 30min, 60min and 120min;
图10为实施例5萤光素酶底物d 2-cycluc、cycluc的体外浓度依赖性研究；其中，A为不同浓度d 2-cycluc、cycluc的体外成像图，B为不同浓度d 2-cycluc与生物发光强度的关系，C为不同浓度cycluc与生物发光强度的关系； Example 5 FIG. 10 d is a substrate for luciferase Embodiment 2 -cycluc, concentration-dependent in vitro studies cycluc; wherein, A d 2 -cycluc different concentrations, cycluc FIG vitro imaging, B d 2 -cycluc different concentrations The relationship with bioluminescence intensity, C is the relationship between different concentrations of cycluc and bioluminescence intensity;
图11为实施例6萤光素酶底物d 2-cycluc、cycluc的细胞毒性研究；其中，A为不同浓度d 2-cycluc对ES-2-Fluc细胞的存活率，B为不同浓度cycluc对ES-2-Fluc细胞的存活率； Figure 11 shows the cytotoxicity study of luciferase substrates d 2 -cycluc and cycluc in Example 6; wherein, A is the survival rate of ES-2-Fluc cells with different concentrations of d 2 -cycluc, and B is the effect of different concentrations of cycluc on ES-2-Fluc cells. The survival rate of ES-2-Fluc cells;
图12为实施例7萤光素酶底物d 2-cycluc、cycluc的细胞浓度依赖性研究；其中，A为不同浓度d 2-cycluc、cycluc的细胞成像，B为不同浓度d 2-cycluc与生物发光强度的关系，C为不同浓度cycluc与生物发光强度的关系，D为两种化合物浓度为5μM时，在1-60min的生物发光强度，E为两种化合物浓度为0-100μM时，d 2-cycluc与cycluc生物发光强度相对比值； Example 7 FIG. 12 is a luciferase substrate d 2 -cycluc, concentration-dependent study of cell cycluc; wherein, A d 2 -cycluc different concentrations, cycluc imaging cells, B to different concentrations and d 2 -cycluc The relationship between bioluminescence intensity, C is the relationship between different concentrations of cycluc and bioluminescence intensity, D is the bioluminescence intensity at 1-60min when the concentration of the two compounds is 5μM, E is when the concentration of the two compounds is 0-100μM, d 2 - the relative ratio of cycluc and cycluc bioluminescence intensity;
图13为实施例8萤光素酶底物d 2-cycluc的FVB-luc +小鼠体内浓度依赖性研究；其中，A为不同浓度d 2-cycluc与生物发光强度的关系，B为不同浓度d 2-cycluc的小鼠成像结果； Figure 13 is the in vivo concentration-dependent study of the luciferase substrate d 2 -cycluc of Example 8 in FVB-luc + mice; wherein, A is the relationship between different concentrations of d 2 -cycluc and bioluminescence intensity, and B is different concentrations Mouse imaging results of d 2-cycluc;
图14为实施例9萤光素酶底物d 2-cycluc、cycluc的FVB-luc +小鼠体内对比研究，其中，A为FVB-luc +小鼠腹腔注射cybluc和d 2-cycluc(100μM，100μL)在60min，B为FVB-luc +小鼠腹腔注射cybluc和d 2-cycluc(100μM，100μL)在120min，C为FVB-luc +小鼠腹腔注射cybluc和d 2-cycluc(100μM，100μL)在240min时体内光子数，D为FVB-luc +小鼠腹腔注射cybluc和d 2-cycluc(100μM，100μL)在480min时体内光子数，E为FVB-luc +小鼠腹腔注射cycluc和d 2-cycluc(100μM，100μL)在1min、5min、10min、15min、20min、25min、30min、35min、40min、45min、50min、60min、120min、240min、480min时体内光子数总和，F为在FVB-luc +小鼠腹腔注射1min、30min、60min、120min、240min、480min时，化合物d 2-cycluc与cycluc(100μM，100μL)在FVB-luc +小鼠体内的光子数比值随时间的变化趋势，G为FVB-luc +小鼠腹腔注射化合物cycluc和d 2-cycluc(100μM，100μL)在1min、60min、120min、240min、480min时成像结果。 Figure 14 is the in vivo comparative study of FVB-luc + mice of luciferase substrates d 2 -cycluc and cycluc in Example 9, wherein A is the intraperitoneal injection of cybluc and d 2 -cycluc (100 μM, A is FVB-luc + mice) 100 μL) at 60min, B is FVB-luc + mouse intraperitoneal injection of cybluc and d 2 -cycluc (100 μM, 100 μL) at 120 min, C is FVB-luc + mouse intraperitoneal injection of cybluc and d 2 -cycluc (100 μM, 100 μL) In vivo photon number at 240min, D is the in vivo photon number at 480min in FVB-luc + mouse intraperitoneal injection of cybluc and d 2 -cycluc (100 μM, 100 μL), E is FVB-luc + mouse intraperitoneal injection of cycluc and d 2 - cycluc (100μM, 100μL) in 1min, 5min, 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 60min, 120min, 240min, 480min, the sum of photons in the body, F is the sum of photons in FVB-luc + small Change trend of photon number ratio of compound d 2 -cycluc and cycluc (100μM, 100μL) in FVB-luc + mice over time when mice were injected intraperitoneally for 1min, 30min, 60min, 120min, 240min, 480min, G is FVB- luc + mice were intraperitoneally injected with compounds cycluc and d 2 -cycluc (100 μM, 100 μL) for imaging results at 1 min, 60 min, 120 min, 240 min, and 480 min.
具体实施方式detailed description
应该指出，以下详细说明都是例示性的，旨在对本发明提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。It should be noted that the following detailed description is exemplary and intended to provide further explanation of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本发明的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。It should be noted that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly dictates otherwise, the singular is intended to include the plural as well, furthermore, it is to be understood that when the terms "comprising" and/or "including" are used in this specification, it indicates that There are features, steps, operations, devices, components and/or combinations thereof. It should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments, rather than for limiting the protection scope of the present invention.
以下通过实施例对本发明做进一步解释说明，但不构成对本发明的限制。应理解这些实施例仅用于说明本发明而不用于限制本发明的范围。The present invention is further explained and illustrated by the following examples, but it does not constitute a limitation of the present invention. It should be understood that these examples are only intended to illustrate the present invention and not to limit the scope of the present invention.
实施例1：制备化合物cyclucExample 1: Preparation of compound cycluc
Figure PCTCN2020114873-appb-000006
Figure PCTCN2020114873-appb-000006
2,2,2-trifluoro-1-(5-nitroindolin-1-yl)ethan-1-one(中间体1)2,2,2-trifluoro-1-(5-nitroindolin-1-yl)ethan-1-one (Intermediate 1)
在5-硝基吲哚啉(4g，24.3mmol)和三乙胺(2.6g，26.8mmol)中加入二氯甲烷(20mL)进行搅拌，同时滴加三氟乙酸酐(5.6g，26.8mmol)。将该混合物搅拌45分钟，然后加入水(50mL)，搅拌10分钟后，将混合物用5M HCl酸化，有机层用盐水洗涤，干燥并浓缩得到中间体1，为黄色固体6g，产率为94％，mp：136-138℃。 1H NMR(400MHz,DMSO-d 6)δ8.23(d,J＝10.9Hz,3H),4.40(t,J＝8.3Hz,2H),3.36(d,J＝8.2Hz,2H). Dichloromethane (20 mL) was added to 5-nitroindoline (4 g, 24.3 mmol) and triethylamine (2.6 g, 26.8 mmol) and stirred, and trifluoroacetic anhydride (5.6 g, 26.8 mmol) was added dropwise at the same time. . The mixture was stirred for 45 minutes, then water (50 mL) was added, after stirring for 10 minutes, the mixture was acidified with 5M HCl, the organic layer was washed with brine, dried and concentrated to give Intermediate 1 as a yellow solid 6 g in 94% yield , mp: 136-138°C. 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.23 (d, J=10.9 Hz, 3H), 4.40 (t, J=8.3 Hz, 2H), 3.36 (d, J=8.2 Hz, 2H).
1-(5-aminoindolin-1-yl)-2,2,2-trifluoroethan-1-one(中间体2)1-(5-aminoindolin-1-yl)-2,2,2-trifluoroethan-1-one (Intermediate 2)
将SnCl 2·2H 2O(14.2g，63.1mmol)添加到中间体1(5.5g，21.5mmol)的乙醇(20mL)溶液中，并将反应混合物60℃加热回流4小时。冷却至室温后，减压浓缩溶剂。将得到的残余物溶于饱和NaHCO 3并用乙酸乙酯(2×100mL)萃取，然后用盐水洗涤，减压浓缩有机溶剂，并通过快速色谱法纯化(10％乙酸乙酯：石油醚)，得到中间体2，为白色固体3g，产率为61％。mp：175-176℃。 1H NMR(400MHz,DMSO-d 6)δ7.76(d,J＝8.6Hz,1H),6.54(d,J＝1.6Hz,1H),6.44(dd,J＝8.6,2.2Hz,1H),5.18(s,2H),4.19(t,J＝7.7Hz,2H),3.11(t,J＝8.1Hz,2H). The SnCl 2 · 2H 2 O (14.2g , 63.1mmol) was added to Intermediate 1 (5.5g, 21.5mmol) in ethanol (20mL), 60 deg.] C and the reaction mixture was heated at reflux for 4 hours. After cooling to room temperature, the solvent was concentrated under reduced pressure. The resulting residue was dissolved in saturated NaHCO 3 and extracted with ethyl acetate (2 × 100mL), then brine, the organic solvent was concentrated under reduced pressure and purified by flash chromatography: to give (10% ethyl acetate in petroleum ether) Intermediate 2 as a white solid 3 g in 61% yield. mp: 175-176°C. 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.76 (d, J=8.6 Hz, 1H), 6.54 (d, J=1.6 Hz, 1H), 6.44 (dd, J=8.6, 2.2 Hz, 1H) , 5.18(s, 2H), 4.19(t, J=7.7Hz, 2H), 3.11(t, J=8.1Hz, 2H).
1-(2-amino-6,7-dihydro-5H-thiazolo[4,5-f]indol-5-yl)-2,2,2-trifluoroethan-1-one(中间体3)1-(2-amino-6,7-dihydro-5H-thiazolo[4,5-f]indol-5-yl)-2,2,2-trifluoroethan-1-one (Intermediate 3)
中间体2(2g，8.695mmol)和硫氰酸钾(3.37g，34.78mmol)的冰醋酸溶液(20mL)中在20℃下搅拌10分钟。在20分钟内将液溴(1.3g，8.695mmol)缓慢滴加到上述溶液中，反应混合物在室温下进一步搅拌21小时，将反应混合物倒在碎冰上，并使用NH 4OH把pH调节至8。所得沉淀物真空过滤并干燥，得到中间体3其无需进一步纯化即可用于下一步骤，为黄色固体2.3g，产率为92％，mp：179-181℃。 1H NMR(400MHz,DMSO-d 6)δ8.33(s,1H),7.54(s,2H),7.29(s,1H),4.28(t,J＝7.9Hz,2H),3.24(t,J＝8.0Hz,2H). A solution of Intermediate 2 (2 g, 8.695 mmol) and potassium thiocyanate (3.37 g, 34.78 mmol) in glacial acetic acid (20 mL) was stirred at 20 °C for 10 min. Over 20 minutes liquid bromine (1.3g, 8.695mmol) was slowly added dropwise to the above solution, the reaction mixture was further stirred for 21 hours at room temperature the reaction mixture was poured onto crushed ice, using NH 4 OH and the pH was adjusted to 8. The resulting precipitate was vacuum filtered and dried to give Intermediate 3 which was used in the next step without further purification as a yellow solid 2.3 g, 92% yield, mp: 179-181°C. 1 H NMR (400MHz, DMSO-d 6 ) δ 8.33(s, 1H), 7.54(s, 2H), 7.29(s, 1H), 4.28(t, J=7.9Hz, 2H), 3.24(t, J=8.0Hz, 2H).
1-(2-chloro-6,7-dihydro-5H-thiazolo[4,5-f]indol-5-yl)-2,2,2-trifluoroethan-1-one(中间体4)1-(2-chloro-6,7-dihydro-5H-thiazolo[4,5-f]indol-5-yl)-2,2,2-trifluoroethan-1-one (Intermediate 4)
在1小时内向亚硝酸叔丁酯(1.45g，14.1mmol)，氯化亚铜(1.16g，11.28mmol)和乙腈(10mL)的混合物中分批加入中间体3(2.2g，7.66mmol)。在室温下搅拌2h，然后移至到65℃加热回流反应1h。将混合物过滤并将滤液倒入6M HCl中，并用乙酸乙酯(2×50mL)萃取。浓缩后，将粗产物通过快速色谱法纯化(15％乙酸乙酯：石油醚)，得到中间体4，为粉红色固体粉末1.6g，产率为68％，mp：231-233℃。 1H NMR(400MHz,DMSO-d 6)δ8.76(s,1H),8.03–7.77(m,1H),4.38(t,J＝8.0Hz,2H),3.39(t,J＝8.2Hz,2H). To a mixture of tert-butyl nitrite (1.45 g, 14.1 mmol), cuprous chloride (1.16 g, 11.28 mmol) and acetonitrile (10 mL) was added intermediate 3 (2.2 g, 7.66 mmol) portionwise over 1 hour. Stir at room temperature for 2h, then move to 65°C and heat under reflux for 1h. The mixture was filtered and the filtrate was poured into 6M HCl and extracted with ethyl acetate (2 x 50 mL). After concentration, the crude product was purified by flash chromatography (15% ethyl acetate:petroleum ether) to give Intermediate 4 as a pink solid powder 1.6 g, 68% yield, mp: 231-233°C. 1 H NMR (400MHz, DMSO-d 6 )δ8.76(s,1H),8.03-7.77(m,1H),4.38(t,J=8.0Hz,2H),3.39(t,J=8.2Hz, 2H).
2-chloro-6,7-dihydro-5H-thiazolo[4,5-f]indole(中间体5)2-chloro-6,7-dihydro-5H-thiazolo[4,5-f]indole (Intermediate 5)
将化合物4(1.5g，4.9mmol)在甲醇(5mL)中搅拌，5分钟内分批添加NaBH 4(0.725g， 19.6mmol)。将混合物搅拌15分钟，用水(50mL)稀释，并用乙酸乙酯(2×50mL)萃取。有机层用盐水洗涤并减压浓缩，得到中间体5，为白色固体粉末500mg，产率为48％，mp：207-209℃。 1H NMR(400MHz,DMSO-d 6)δ7.56(d,J＝10.0Hz,1H),6.96(s,1H),6.04(s,1H),3.51(t,J＝8.4Hz,2H),3.09–2.97(m,2H). Compound 4 (1.5g, 4.9mmol) was stirred in methanol (5mL) in added portionwise over 5 minutes NaBH 4 (0.725g, 19.6mmol). The mixture was stirred for 15 minutes, diluted with water (50 mL), and extracted with ethyl acetate (2 x 50 mL). The organic layer was washed with brine and concentrated under reduced pressure to give Intermediate 5 as a white solid powder 500 mg, 48% yield, mp: 207-209°C. 1 H NMR(400MHz, DMSO-d 6 )δ7.56(d,J=10.0Hz,1H),6.96(s,1H),6.04(s,1H),3.51(t,J=8.4Hz,2H) ,3.09–2.97(m,2H).
6,7-dihydro-5H-thiazolo[4,5-f]indole-2-carbonitrile(中间体6)6,7-dihydro-5H-thiazolo[4,5-f]indole-2-carbonitrile (Intermediate 6)
将化合物5(0.5g，1.0mmol)溶于无水乙腈中，加入氰基三甲基硅烷(1.18g，5.0mmol)和四丁基氟化铵(3.11g，5.0mmol)于90℃条件下加热回流反应，用TLC进行实时监测，反应不完全进行后处理，旋干反应液后加入适量的水用乙酸乙酯进行萃取，过滤，旋干，进行色谱柱分离纯化，选择层析液为石油醚：乙酸乙酯＝5：1，浓缩液体得中间体6，为黄色固体粉末250mg，产率为52.4％，mp：195-196℃。 1H NMR(400MHz,DMSO-d 6)δ7.76(s,1H),7.05(s,1H),6.69(s,1H),3.61(t,J＝8.1Hz,2H),3.11(t,J＝8.2Hz,2H). 13C NMR(101MHz,DMSO-d 6)δ155.01,144.90,137.95,134.26,127.44,120.06,115.00,96.85,47.06,28.58. Compound 5 (0.5 g, 1.0 mmol) was dissolved in anhydrous acetonitrile, cyanotrimethylsilane (1.18 g, 5.0 mmol) and tetrabutylammonium fluoride (3.11 g, 5.0 mmol) were added at 90 °C The reaction was heated under reflux, monitored in real time by TLC, the reaction was incomplete and post-treatment was carried out. After the reaction solution was spin-dried, an appropriate amount of water was added to extract with ethyl acetate, filtered, spin-dried, and subjected to chromatographic column separation and purification. The chromatographic liquid was selected as petroleum. Ether:ethyl acetate=5:1, the liquid was concentrated to obtain intermediate 6, which was 250 mg of yellow solid powder, the yield was 52.4%, mp: 195-196°C. 1 H NMR (400MHz, DMSO-d 6 )δ7.76(s,1H), 7.05(s,1H), 6.69(s,1H), 3.61(t, J=8.1Hz, 2H), 3.11(t, J=8.2Hz, 2H). 13 C NMR (101MHz, DMSO-d 6 )δ155.01, 144.90, 137.95, 134.26, 127.44, 120.06, 115.00, 96.85, 47.06, 28.58.
(S)-2-(6,7-dihydro-5H-thiazolo[4,5-f]indol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid(终产物cycluc)(S)-2-(6,7-dihydro-5H-thiazolo[4,5-f]indol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid (end product cycluc)
将中间体6(40mg，0.199mmol)溶于10mL二氯甲烷、10mL无水甲醇中，在氮气保护下，滴加用2mL无水甲醇、2mL蒸馏水溶解的碳酸钾(70mg)、D-半胱氨酸盐酸盐(41mg，0.238mmol)，在室温条件下反应，反应时间为1h，旋干，用1mol盐酸调pH至7析出固体，过滤，滤液为终产物，粗产物用二氯甲烷和乙酸乙酯进行打浆，过滤得到固体粉末终产物7为50mg，产率为48％，mp：145-147℃。 1H NMR(400MHz,MeOD-d 4)δ7.68(s,1H),6.99(s,1H),5.17(t,J＝9.1Hz,1H),3.79–3.55(m,4H),3.35(s,1H),3.13(t,J＝8.1Hz,2H). 13C NMR(101MHz,DMSO-d 6)δ152.43,152.19,145.03,142.89,135.96,131.19,118.44,98.76,47.16,29.17,25.78.HRMS(AP-ESI)m/z Cacld for C13H11N3O2S2[M+H] +306.0365Found:306.0368. Intermediate 6 (40 mg, 0.199 mmol) was dissolved in 10 mL of dichloromethane and 10 mL of anhydrous methanol, and under nitrogen protection, potassium carbonate (70 mg), D-cysteine dissolved in 2 mL of anhydrous methanol and 2 mL of distilled water was added dropwise. Amino acid hydrochloride (41 mg, 0.238 mmol), reacted at room temperature, the reaction time was 1 h, spin-dried, adjusted pH to 7 with 1 mol hydrochloric acid to precipitate a solid, filtered, and the filtrate was the final product, the crude product was mixed with dichloromethane and Slurry with ethyl acetate and filter to obtain a solid powder final product 7, 50 mg, with a yield of 48%, mp: 145-147°C. 1 H NMR (400MHz, MeOD-d 4 )δ7.68(s,1H),6.99(s,1H),5.17(t,J=9.1Hz,1H),3.79-3.55(m,4H),3.35( s, 1H), 3.13 (t, J=8.1Hz, 2H). 13 C NMR (101MHz, DMSO-d 6 )δ152.43, 152.19, 145.03, 142.89, 135.96, 131.19, 118.44, 98.76, 47.16, 29.17, 25.78. HRMS(AP-ESI)m/z Cacld for C13H11N3O2S2[M+H] + 306.0365Found:306.0368.
实施例2：制备化合物d 2-cycluc： Example 2: Preparation of compound d 2 -cycluc:
Figure PCTCN2020114873-appb-000007
Figure PCTCN2020114873-appb-000007
indoline-2,3-d 2(中间体8) indoline-2,3-d 2 (Intermediate 8)
1H-吲哚(20.0g，170.7mmol)加入到圆底烧瓶中，用氘代甲醇进行溶解，加入钯碳(2.0g，18.7mmol)，并且鼓入氘气在40℃下加热回流反应，并用高压反应釜施加八个大气压的压力，用TLC进行监测，大约反应72小时，仍反应不完全。进行过滤除去钯碳，旋干反应液，加入水用乙酸乙酯萃取，得黄色液体11g，产率为50％，旋干直接投下一步。1H-indole (20.0 g, 170.7 mmol) was added to a round-bottomed flask, dissolved in deuterated methanol, added with palladium on carbon (2.0 g, 18.7 mmol), and heated to reflux at 40 °C by bubbling deuterium gas, and using The pressure of 8 atmospheres was applied to the autoclave and monitored by TLC, and the reaction was still incomplete for about 72 hours. The palladium carbon was removed by filtration, the reaction solution was spin-dried, water was added and extracted with ethyl acetate to obtain 11 g of a yellow liquid with a yield of 50%, and the spin-dried solution was directly transferred to the next step.
1-(indolin-1-yl-2,3-d 2)ethan-1-one(中间体9) 1-(indolin-1-yl-2,3-d 2 )ethan-1-one (intermediate 9)
将中间体8(5g，41.3mmol)溶于冰醋酸中，在室温下将乙酰氯(19.5mL，247.6mmol)一滴滴加入到溶液中，然后将反应液移至90℃进行加热回流反应，搅拌1.5小时，TLC进行监测，反应完全。旋干反应液，加入水用乙酸乙酯萃取，拌样过柱子，得到固体粉末4g，产率为60％，mp：167-169℃。 1H NMR(400MHz,CDCl 3)δ8.20(d,J＝8.0Hz,1H),7.20–7.14(m,2H),7.02–6.97(m,1H),4.00(d,J＝7.0Hz,1H),3.16(d,J＝8.0Hz,1H),2.19(s,3H). 13C NMR(101MHz,CDCl 3)δ168.76,142.92,131.08,127.52,124.57,123.58,116.93,48.68,27.87,24.22. Intermediate 8 (5 g, 41.3 mmol) was dissolved in glacial acetic acid, acetyl chloride (19.5 mL, 247.6 mmol) was added dropwise to the solution at room temperature, and then the reaction solution was moved to 90 °C for heating and refluxing reaction, stirring 1.5 hours, TLC monitoring, the reaction is complete. The reaction solution was spin-dried, and water was added to extract with ethyl acetate. The sample was mixed and passed through a column to obtain 4 g of solid powder with a yield of 60%, mp: 167-169°C. 1 H NMR (400 MHz, CDCl 3 ) δ 8.20 (d, J=8.0 Hz, 1H), 7.20-7.14 (m, 2H), 7.02-6.97 (m, 1H), 4.00 (d, J=7.0 Hz, 1H), 3.16(d, J=8.0Hz, 1H), 2.19(s, 3H). 13 C NMR ( 101MHz, CDCl 3 )δ168.76, 142.92, 131.08, 127.52, 124.57, 123.58, 116.93, 48.68, 27.87, 24.222 .
1-(5-nitroindolin-1-yl-2,3-d 2)ethan-1-one(中间体10) 1-(5-nitroindolin-1-yl-2,3-d 2 )ethan-1-one (Intermediate 10)
将中间体9(1.4g，8.58mmol)溶于无水乙腈中，加入Fe(NO 3) 3·9H 2O(1.7g，4.29mmol)，在50℃进行加热回流反应，TLC进行监测，反应完全。减压浓缩反应液，用乙酸乙酯溶解，加入水进行萃取，加入硅胶旋干溶剂过柱子，得到白色固体粉末0.5g，产率为30％，mp：143-145℃。 1H NMR(400MHz,CDCl 3)δ8.28(d,J＝8.9Hz,1H),8.10(dt,J＝10.8,5.4Hz,1H),8.03(s,1H),4.19(dd,J＝10.2,3.3Hz,1H),3.44–3.15(m,1H),2.27(d,J＝9.7Hz,3H). 13C NMR(101MHz,CDCl 3)δ169.61,148.37,143.50,132.41,124.70,120.31,116.14,49.37,27.25,24.30. Intermediate 9 (1.4 g, 8.58 mmol) was dissolved in anhydrous acetonitrile, Fe(NO 3 ) 3 ·9H 2 O (1.7 g, 4.29 mmol) was added, and the reaction was heated and refluxed at 50° C. The reaction was monitored by TLC. completely. The reaction solution was concentrated under reduced pressure, dissolved in ethyl acetate, extracted with water, added with silica gel and spin-dried as solvent and passed through a column to obtain 0.5 g of white solid powder with a yield of 30%, mp: 143-145°C. 1 H NMR (400 MHz, CDCl 3 ) δ 8.28 (d, J=8.9 Hz, 1H), 8.10 (dt, J=10.8, 5.4 Hz, 1H), 8.03 (s, 1H), 4.19 (dd, J= 10.2, 3.3Hz, 1H), 3.44-3.15 (m, 1H), 2.27 (d, J=9.7Hz, 3H). 13 C NMR ( 101MHz, CDCl 3 )δ169.61, 148.37, 143.50, 132.41, 124.70, 120.31, 116.14, 49.37, 27.25, 24.30.
1-(5-aminoindolin-1-yl-2,3-d 2)ethan-1-one(中间体11) 1-(5-aminoindolin-1-yl-2,3-d 2 )ethan-1-one (Intermediate 11)
将中间体10(1.0g，4.8mmol)溶于无水乙醇中，加入用水溶解的氯化铵(2.6g，48mmol)，再加入活化的锌粉(6.3g，96mmol)，室温搅拌反应，反应很快，大概1小时反应完全。过滤除掉锌粉，旋干反应液，用乙酸乙酯打浆，过滤，干燥，得黄色固体产物0.9g，产率为70％，mp：156-156℃。 1H NMR(400MHz,CDCl 3)δ8.00(d,J＝8.4Hz,1H),6.55–6.49(m,2H),3.97(t,J＝6.5Hz,1H),3.08(t,J＝6.8Hz,1H),2.17(s,3H). 13C NMR(101MHz,CDCl 3)δ167.65,142.80,117.74,113.84,111.68,48.72,28.02,23.92. Intermediate 10 (1.0g, 4.8mmol) was dissolved in absolute ethanol, water-dissolved ammonium chloride (2.6g, 48mmol) was added, then activated zinc powder (6.3g, 96mmol) was added, and the reaction was stirred at room temperature. Very quickly, about 1 hour to complete the reaction. The zinc powder was removed by filtration, the reaction solution was spin-dried, slurried with ethyl acetate, filtered and dried to obtain 0.9 g of a yellow solid product with a yield of 70%, mp: 156-156°C. 1 H NMR (400 MHz, CDCl 3 ) δ 8.00 (d, J=8.4 Hz, 1H), 6.55-6.49 (m, 2H), 3.97 (t, J=6.5 Hz, 1H), 3.08 (t, J= 6.8Hz, 1H), 2.17(s, 3H). 13 C NMR ( 101MHz, CDCl 3 ) δ 167.65, 142.80, 117.74, 113.84, 111.68, 48.72, 28.02, 23.92.
1-(2-amino-6,7-dihydro-5H-thiazolo[4,5-f]indol-5-yl-6,7-d 2)ethan-1-one(中间体12) 1-(2-amino-6,7-dihydro-5H-thiazolo[4,5-f]indol-5-yl-6,7-d 2 )ethan-1-one (intermediate 12)
将中间体11(0.9g，5.05mmol)和硫氰酸钾(2.6g，20.1mmol)溶于冰醋酸中，搅拌10分钟，滴加用冰醋酸稀释的液溴(1.1g，5.05mmol)，缓慢滴加10分钟，室温搅拌反应15小时。旋干反应液，加水溶解，用氢氧化钠溶液调pH＝8，过滤，干燥，得红色固体粉末0.9g，产率为80.1％，mp：185-187℃。 1H NMR(400MHz,DMSO-d 6)δ8.29(s,1H),7.29(s,2H),7.19(s,1H),4.09(q,J＝7.0Hz,1H),3.15(t,J＝7.0Hz,1H),2.14(s,3H). Intermediate 11 (0.9 g, 5.05 mmol) and potassium thiocyanate (2.6 g, 20.1 mmol) were dissolved in glacial acetic acid, stirred for 10 minutes, and liquid bromine (1.1 g, 5.05 mmol) diluted with glacial acetic acid was added dropwise, It was slowly added dropwise for 10 minutes, and the reaction was stirred at room temperature for 15 hours. The reaction solution was spin-dried, dissolved in water, adjusted to pH=8 with sodium hydroxide solution, filtered and dried to obtain 0.9 g of a red solid powder with a yield of 80.1%, mp: 185-187°C. 1 H NMR (400MHz, DMSO-d 6 ) δ 8.29(s, 1H), 7.29(s, 2H), 7.19(s, 1H), 4.09(q, J=7.0Hz, 1H), 3.15(t, J=7.0Hz, 1H), 2.14(s, 3H).
1-(2-chloro-6,7-dihydro-5H-thiazolo[4,5-f]indol-5-yl-6,7-d 2)ethan-1-one(中间体13) 1-(2-chloro-6,7-dihydro-5H-thiazolo[4,5-f]indol-5-yl-6,7-d 2 )ethan-1-one (Intermediate 13)
将亚硝基叔丁酯(0.92g，8.97mmol)和氯化亚铜(0.7g，7.01mmol)用无水乙腈进行溶解，分批次加入中间体12(1.1g，4.67mmol)，5分钟加完，于室温反应2小时，再移至65℃继续加热回流反应1小时。将反应液过滤，滤液倒入6M HCl中，用乙酸乙酯进行萃取，干燥，浓缩，用柱色谱进行分离纯化，层析液选择石油醚：乙酸乙酯＝5：1，浓缩干燥得黄色固体粉末780.6mg，产率为76.3％，mp：156-158℃。 1H NMR(400MHz,DMSO-d 6)δ8.65(s,1H),7.78(s,1H),4.20–4.15(m,1H),3.31–3.25(m,1H),2.20(s,3H). 13C NMR(101MHz,DMSO-d 6)δ169.55,150.71,146.86,141.81,135.16,133.46,118.90,108.39,49.19,35.29,24.50. Nitroso-tert-butyl ester (0.92 g, 8.97 mmol) and cuprous chloride (0.7 g, 7.01 mmol) were dissolved in anhydrous acetonitrile, and Intermediate 12 (1.1 g, 4.67 mmol) was added in portions over 5 min After the addition, the reaction was carried out at room temperature for 2 hours, and then moved to 65° C. to continue heating and refluxing for 1 hour. The reaction solution was filtered, the filtrate was poured into 6M HCl, extracted with ethyl acetate, dried, concentrated, and separated and purified by column chromatography. Powder 780.6 mg, 76.3% yield, mp: 156-158°C. 1 H NMR (400MHz, DMSO-d 6 )δ8.65(s,1H), 7.78(s,1H), 4.20-4.15(m,1H), 3.31-3.25(m,1H), 2.20(s,3H) ). 13 C NMR (101MHz, DMSO-d 6 )δ169.55, 150.71, 146.86, 141.81, 135.16, 133.46, 118.90, 108.39, 49.19, 35.29, 24.50.
2-chloro-6,7-dihydro-5H-thiazolo[4,5-f]indole-6,7-d 2(中间体14) 2-chloro-6,7-dihydro-5H-thiazolo[4,5-f]indole-6,7-d 2 (Intermediate 14)
用干燥的THF溶解中间体13(300mg，712.2mmol)，在-70℃条件下，滴加用THF稀释的1M的LiAlH 4(27mg，712.2mmol)混悬液，搅拌反应，反应很快。用TLC进行监测，30min反应完全。(LiAlH 4的后处理过程：X g的LiAlH 4，加入X mL的水，再加入X mL 15％NaOH溶液，最后加入3X mL水，搅拌30min，加硅藻土进行过滤)，旋干，用薄层色谱板进行层析，层析液选择石油醚：乙酸乙酯＝7：1，浓缩干燥得粉红色固体粉末200mg，产率为71％，mp：207-209℃。 1H NMR(400MHz,CDCl 3)δ7.60(s,1H),6.86(s,1H),3.65(t,J＝7.2Hz,1H),3.22–3.12(m,1H). 13C NMR(101MHz,DMSO-d 6)δ152.17,145.05,142.90,135.96,131.10,118.45,98.75,47.17,29.07. Intermediate 13 (300 mg, 712.2 mmol) was dissolved in dry THF, and 1M LiAlH 4 (27 mg, 712.2 mmol) suspension diluted with THF was added dropwise at -70°C, and the reaction was stirred rapidly. Monitored by TLC, the reaction was complete in 30 min. (4 LiAIH4 post-processing: X g of LiAIH4 4, X mL of water was added, then add X mL 15% NaOH solution and finally 3X mL of water, stirred for 30min, was added Celite), spin dry, with Chromatography was performed on a thin-layer chromatography plate, and the chromatographic solution was petroleum ether:ethyl acetate=7:1, concentrated and dried to obtain 200 mg of pink solid powder, the yield was 71%, mp: 207-209°C. 1 H NMR (400MHz, CDCl 3 ) δ7.60 (s, 1H), 6.86 (s, 1H), 3.65 (t, J = 7.2Hz, 1H), 3.22-3.12 (m, 1H). 13 C NMR ( 101MHz, DMSO-d 6 )δ152.17, 145.05, 142.90, 135.96, 131.10, 118.45, 98.75, 47.17, 29.07.
6,7-dihydro-5H-thiazolo[4,5-f]indole-2-carbonitrile-6,7-d 2(中间体15) 6,7-dihydro-5H-thiazolo[4,5-f]indole-2-carbonitrile-6,7-d 2 (Intermediate 15)
将中间体14(300mg，423.9mmol)溶于无水乙腈中，加入氰基三甲基硅烷(2.13mol，211.8mg)和四丁基氟化铵(2.13mol，558.6mg)，于90℃条件下加热回流反应，用TLC进行实时监测，反应不完全，进行后处理，旋干反应液，加入适量的水用乙酸乙酯进行萃取，过滤，旋干，进行色谱柱分离纯化，选择层析液为石油醚：乙酸乙酯＝5：1，浓缩液体，得黄色固体粉末即为产物200mg，产率为64.7％，mp：195-196℃。 1H NMR(400MHz,DMSO-d 6)δ7.64(s,1H),7.11(s,1H),6.69(s,1H),3.67(t,J＝8.1Hz,1H),3.14(t,J＝8.2Hz,1H). Intermediate 14 (300 mg, 423.9 mmol) was dissolved in anhydrous acetonitrile, cyanotrimethylsilane (2.13 mol, 211.8 mg) and tetrabutylammonium fluoride (2.13 mol, 558.6 mg) were added, at 90 °C The reaction was heated under reflux, monitored in real time by TLC, the reaction was incomplete, post-treatment was performed, the reaction solution was spin-dried, an appropriate amount of water was added for extraction with ethyl acetate, filtered, spin-dried, and the chromatographic column was separated and purified, and the chromatographic solution was selected. It is petroleum ether:ethyl acetate=5:1, and the liquid is concentrated to obtain 200 mg of yellow solid powder, the yield is 64.7%, mp: 195-196°C. 1 H NMR (400MHz, DMSO-d 6 ) δ 7.64(s, 1H), 7.11(s, 1H), 6.69(s, 1H), 3.67(t, J=8.1Hz, 1H), 3.14(t, J=8.2Hz, 1H).
(4R)-2-(6,7-dihydro-5H-thiazolo[4,5-f]indol-2-yl-6,7-d 2)-4,5-dihydrothiazole-4-carboxylic acid(终产物d 2-cycluc) (4R)-2-(6,7-dihydro-5H-thiazolo[4,5-f]indol-2-yl-6,7-d 2 )-4,5-dihydrothiazole-4-carboxylic acid (final product d 2- cycluc)
将中间体15(500mg，1.0mmol)溶于10mL二氯甲烷、10mL无水甲醇中，在氮气保护下，滴加用2mL无水甲醇、2mL蒸馏水溶解的碳酸钾(370mg)、D-半胱氨酸盐酸盐(450mg，2.0mmol)，在室温条件下反应，反应时间为1h，旋干，用1mol盐酸调pH至7析出固体，过滤，滤液为终产物。粗产物用二氯甲烷和乙酸乙酯进行打浆，过滤得到目标产物黄色固体粉末544.4mg，产率为78.1％，mp：145-147℃。 1H NMR(400MHz,MeOD-d 4)δ7.68(s,1H),6.99(s,1H),5.17(t,J＝9.1Hz,1H),4.00–3.95(m,1H),3.67(m,2H),3.35(s,1H),3.13(t,J＝8.1Hz,1H). 13C NMR(101MHz,CDCl 3)δ152.46,149.17,143.94,142.58,135.82,132.77,118.39,98.43,47.44,29.17,24.30.HRMS(AP-ESI)m/z Cacld for C13H9D2N3O2S2[M+H] +308.0491Found:308.0495. Intermediate 15 (500 mg, 1.0 mmol) was dissolved in 10 mL of dichloromethane and 10 mL of anhydrous methanol, and under nitrogen protection, potassium carbonate (370 mg), D-cysteine dissolved in 2 mL of anhydrous methanol and 2 mL of distilled water was added dropwise. Amino acid hydrochloride (450 mg, 2.0 mmol) was reacted at room temperature for 1 h, spin-dried, adjusted to pH 7 with 1 mol hydrochloric acid to precipitate a solid, filtered, and the filtrate was the final product. The crude product was slurried with dichloromethane and ethyl acetate, and filtered to obtain 544.4 mg of the target product as a yellow solid powder with a yield of 78.1%, mp: 145-147°C. 1 H NMR (400MHz, MeOD-d 4 )δ7.68(s,1H),6.99(s,1H),5.17(t,J=9.1Hz,1H),4.00-3.95(m,1H),3.67( m, 2H), 3.35(s, 1H), 3.13(t, J=8.1Hz, 1H). 13 C NMR ( 101 MHz, CDCl 3 ) δ 152.46, 149.17, 143.94, 142.58, 135.82, 132.77, 118.39, 98.43, 47.44 ,29.17,24.30.HRMS(AP-ESI)m/z Cacld for C13H9D2N3O2S2[M+H] + 308.0491Found:308.0495.
实施例3：萤光素酶底物cycluc、d 2-cycluc的体外代谢实验研究 Example 3: Experimental study on in vitro metabolism of luciferase substrates cycluc and d 2 -cycluc
在全黑色96孔板中加入化合物cycluc和d 2-cycluc(20μM，50μL)，再加入50μL含2mM ATP的萤光素酶溶液，立即开始拍摄，每隔5min拍摄一次，记录光子数至120min结束，用多功能荧光酶标仪
Figure PCTCN2020114873-appb-000008
测量生物发光强度，每组实验重复三次，并用Graphpad进行统计学计算。 Compounds cycluc and d 2 -cycluc (20 μM, 50 μL) were added to an all-black 96-well plate, and then 50 μL of luciferase solution containing 2 mM ATP was added. Immediately began to photograph, every 5 min, and recorded the number of photons until the end of 120 min. , using a multifunctional fluorescence microplate reader
Figure PCTCN2020114873-appb-000008
Bioluminescence intensities were measured, and each experiment was repeated three times, and statistical calculations were performed with Graphpad.
由图9可知，萤火虫萤光素酶底物cycluc和d 2-cycluc在体外的生物发光强度随着时间的延长而减弱，并且由图9A-D可知d 2-cycluc的生物发光强度远远大于cycluc。在120min内，d 2-cycluc与cycluc的生物发光强度比值最小达到10.4倍，最大达到158.1倍，可见d 2-cycluc的生物发光强度远远大于cycluc，并且生物发光时间也有很大的改善。由图9E-H可知，在30min、60min和120min时的生物发光强度具有极显著性差异，由30min内的14.9倍增加到120min内的18.5倍，这预示着d 2-cycluc比cycluc的生物发光强度更强，时间更长，更有利于用于活体成像实验。 It can be seen from Figure 9 that the in vitro bioluminescence intensity of firefly luciferase substrates cycluc and d 2 -cycluc weakens with time, and it can be seen from Figure 9A-D that the bioluminescence intensity of d 2 -cycluc is much greater than that of d 2 -cycluc. cycluc. Within 120min, the ratio of bioluminescence intensity of d 2 -cycluc to cycluc reached a minimum of 10.4 times and a maximum of 158.1 times. It can be seen that the bioluminescence intensity of d 2 -cycluc is much greater than that of cycluc, and the bioluminescence time is also greatly improved. It can be seen from Figure 9E-H that the bioluminescence intensity at 30min, 60min and 120min has a very significant difference, from 14.9 times in 30min to 18.5 times in 120min, which indicates that the bioluminescence of d 2 -cycluc is higher than that of cycluc. The intensity is stronger and the time is longer, which is more beneficial for in vivo imaging experiments.
实施例4：萤火虫萤光素酶底物cycluc和d 2-cycluc的体外动力学研究 Example 4: In vitro kinetic studies of firefly luciferase substrates cycluc and d 2 -cycluc
将50μL不同浓度的化合物溶液(0.01μM、0.05μM、0.1μM、0.25μM、0.5μM)加入全黑色96孔板中，再加入50μL含2mM ATP的萤光素酶溶液，立即用小动物活体成像仪测生物发光强度，并用Graphpad中的Linewearver-Burk公式计算Vmax和Km。Add 50 μL of compound solutions of different concentrations (0.01 μM, 0.05 μM, 0.1 μM, 0.25 μM, 0.5 μM) into an all-black 96-well plate, and then add 50 μL of luciferase solution containing 2 mM ATP, and immediately use small animal live imaging Bioluminescence intensity was measured and Vmax and Km were calculated using the Linewearver-Burk formula in Graphpad.
由表1可见，d 2-cycluc与cycluc相比反应速度增加，亲和力明显增加，这预示着d 2-cycluc可能会在低浓度时对萤光素酶有着较高的灵敏度，更适合进行细胞和活体实验研究。 It can be seen from Table 1 that compared with cycluc, d 2 -cycluc has an increased reaction rate and a significant increase in affinity, which indicates that d 2 -cycluc may have higher sensitivity to luciferase at low concentrations and is more suitable for cell and In vivo experimental studies.
表1 生物发光性质Table 1 Bioluminescence properties
Figure PCTCN2020114873-appb-000009
Figure PCTCN2020114873-appb-000009
实施例5：萤火虫萤光素酶底物cycluc和d 2-cycluc的体外浓度依赖性研究 Example 5: firefly luciferase substrate cycluc concentration dependent in vitro studies, and d is 2 -cycluc
将50μL不同浓度的化合物溶液(0.01μM、0.05μM、0.1μM、0.25μM、0.5μM)加入全黑色96孔板中，再加入50μL含2mM ATP的萤光素酶溶液，立即用小动物活体成像仪测生物发光强度，测定90min中内生物发光强度，并用Graphpad进行数据处理。Add 50 μL of compound solutions of different concentrations (0.01 μM, 0.05 μM, 0.1 μM, 0.25 μM, 0.5 μM) into an all-black 96-well plate, and then add 50 μL of luciferase solution containing 2 mM ATP, and immediately use small animal live imaging The bioluminescence intensity was measured by the instrument, and the bioluminescence intensity within 90 min was measured, and the data was processed by Graphpad.
由图10可见，cycluc和d 2-cycluc的体外生物发光强度呈现浓度依赖性，随着萤光素酶底物浓度的增加生物发光强度越来越强，并且相同浓度的d 2-cycluc比cycluc在体外的生物发光强度强很多，说明d 2-cycluc更适合用于细胞和活体实验。 It can be seen from Figure 10 that the in vitro bioluminescence intensities of cycluc and d 2 -cycluc are concentration-dependent, and the bioluminescence intensities become stronger as the concentration of luciferase substrate increases, and the same concentration of d 2 -cycluc is stronger than cycluc The in vitro bioluminescence intensity is much stronger, indicating that d 2 -cycluc is more suitable for cell and in vivo experiments.
实施例6：萤火虫萤光素酶底物对细胞的毒性测定Example 6: Toxicity assay of firefly luciferase substrates to cells
将对数期的ES-2-Fluc细胞消化离心后，用含l0％胎牛血清的RPMI 1640培养基打散吹匀后，经过细胞计数，将细胞密度调整为4×10 4个/mL，将细胞用排枪种于透明96孔板中间的60个孔的区域中(100μL/孔)，其中96孔板周围一圈以普通培养基填充，放置在细胞培养箱中孵育。 After the ES-2-Fluc cells in log phase were digested and centrifuged, they were dispersed with RPMI 1640 medium containing 10% fetal bovine serum, and the cells were counted to adjust the cell density to 4×10 4 cells/mL. Cells were seeded in a 60-well area (100 μL/well) in the middle of a transparent 96-well plate with a row gun, and a circle around the 96-well plate was filled with common medium and placed in a cell incubator for incubation.
孵育过夜，待细胞贴壁，将萤光素酶底物的浓储液用无血清RPMI 1640培养基稀释成不同的浓度梯度(0、8μM、16μM、31μM、62μM、125μM、250μM、500μM、1000μM、2000μM)加入96孔板中，每个浓度设置三个复孔，将萤光素酶底物和细胞于细胞培养箱中孵育。After incubating overnight, after the cells adhered, the concentrated stock solution of luciferase substrate was diluted with serum-free RPMI 1640 medium into different concentration gradients (0, 8 μM, 16 μM, 31 μM, 62 μM, 125 μM, 250 μM, 500 μM, 1000 μM) , 2000 μM) into a 96-well plate, three replicate wells were set for each concentration, and the luciferase substrate and cells were incubated in a cell incubator.
24h后，加入MTT(5mg/mL)20μL/孔，(MTT需现配现用：取MTT 50mg，溶于10mL的磷酸缓冲液(PBS)中，0.22μm滤膜过滤以除去溶液里的细菌)。After 24h, add MTT (5mg/mL) 20μL/well, (MTT needs to be prepared and used immediately: take 50mg of MTT, dissolve it in 10mL of phosphate buffered saline (PBS), and filter it with a 0.22μm filter to remove the bacteria in the solution) .
4h后，将96孔板中的液体用注射器吸掉，每孔入DMSO 150μL，并置于摇床上振荡5min，使形成的结晶充分溶解，于酶标仪下扫描490nm处的吸收值。After 4 hours, the liquid in the 96-well plate was sucked off with a syringe, 150 μL of DMSO was added to each well, and placed on a shaker for 5 min to fully dissolve the formed crystals, and the absorbance at 490 nm was scanned under a microplate reader.
由图11可见，为了检测萤光素酶底物对细胞的生物毒性较小，可以进行后续细胞实验研究，我们进行了细胞MTT实验。实验结果表明，两个萤光素酶底物对细胞的毒性作用均很小，cycluc的IC 50＝855.4μM，d 2-cycluc的IC 50＞5*10 5μM，远大于细胞实验时所采用的萤光素酶底物浓度，因 此可以用于生物水平的检测。 It can be seen from Fig. 11 that in order to detect that the luciferase substrate has less biological toxicity to cells, subsequent cell experiments can be carried out, and we carried out cell MTT experiments. The experimental results show that the two luciferase substrates have little toxicity to cells. The IC 50 of cycluc = 855.4 μM, and the IC 50 of d 2 -cycluc is greater than 5*10 5 μM, which is much larger than that used in cell experiments. The luciferase substrate concentration can therefore be used for biological level detection.
实施例7：细胞生物发光强度对底物浓度依赖性的测定Example 7: Determination of Substrate Concentration Dependence of Cell Bioluminescence Intensity
(1)培养ES-2-FLuc细胞至长满细胞培养瓶底约90％后，用含EDTA的0.25％胰酶消化离心，再用含10％胎牛血清的RPMI 1640培养基将细胞吹打均匀，取少量混合均匀的细胞悬液，用细胞计数板进行计数，细胞计数后将细胞密度调整为4×10 6个/mL，用排枪在全黑色96孔板中种入细胞，每孔加100μL细胞液(4万/孔)，于细胞培养箱中孵育过夜。(96孔板最外层一圈的孔，每孔加入100μL培养基；第二排孔，每孔加入100μL培养基；第3-8排孔，每孔加入100μL细胞液) (1) After culturing ES-2-FLuc cells to about 90% of the bottom of the cell culture flask, digest and centrifuge with 0.25% trypsin containing EDTA, and then use RPMI 1640 medium containing 10% fetal bovine serum to pipette the cells evenly , take a small amount of evenly mixed cell suspension, count with a cell counting plate, adjust the cell density to 4×10 6 cells/mL after counting the cells, and seed cells in a black 96-well plate with a row gun, add 100 μL to each well Cell solution (40,000/well) was incubated overnight in a cell incubator. (For the outermost wells of the 96-well plate, add 100 μL of medium to each well; for the second row of wells, add 100 μL of medium to each well; for the third to eighth rows, add 100 μL of cell fluid to each well)
(2)次日早晨用排枪吸去培养基(除最外层一圈的孔)，每孔分别加入100μL不同浓度(0μM、5μM、10μM、20μM、50μM、100μM)的萤光素酶底物cycluc、d 2-cycluc，用小动物活体成像仪进行生物发光成像。 (2) The next morning, the medium (except the outermost wells) was sucked off with a discharge gun, and 100 μL of different concentrations (0 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM) of luciferase substrate were added to each well. cycluc, d 2 -cycluc, bioluminescence imaging with a small animal in vivo imager.
由图12可见，为证明我们设计的萤光素酶底物可以用于活体检测，我们对化合物进行了细胞水平活性检测。由图12A、B、C可知，两种化合物在细胞水平上都呈现出浓度依赖性；由图D可知，在两种化合物浓度为5μM时，在1-60min内d 2-cycluc相较于cycluc在细胞中有更强的生物发光强度；由图E可知，在0-100μM范围内d 2-cycluc相较于cycluc在细胞中有更强的生物发光强度最低可达1.98倍，最高可达4.60倍。因此两者均可用于活体成像并且d 2-cycluc具有更强的生物发光强度和更长的生物发光时间，在活体成像时可能会有更好的成像效果。 As can be seen from Figure 12, in order to prove that our designed luciferase substrate can be used for in vivo detection, we carried out the activity detection of the compound at the cellular level. It can be seen from Figure 12A, B, and C that both compounds show a concentration dependence at the cellular level; from Figure D, it can be seen from Figure D that when the concentration of the two compounds is 5 μM, d 2 -cycluc is compared to cycluc within 1-60 min. There is a stronger bioluminescence intensity in cells; as shown in Figure E, in the range of 0-100μM, d 2 -cycluc has a stronger bioluminescence intensity than cycluc in cells, the lowest is 1.98 times, and the highest is 4.60 times. Therefore, both can be used for in vivo imaging and d 2 -cycluc has stronger bioluminescence intensity and longer bioluminescence time, and may have better imaging results in in vivo imaging.
实施例8：萤火虫萤光素酶底物d 2-cycluc对FVB-luc +小鼠不同浓度腹腔注射实验 Example 8: Intraperitoneal injection experiment of firefly luciferase substrate d 2 -cycluc with different concentrations of FVB-luc + mice
将化合物d 2-cycluc用生理盐水和DMSO配置成终浓度为10mM、5mM、1mM、100μM、10μM的溶液，每只FVB-luc +小鼠(转录萤光素酶)(约20g)腹腔注射200μL水合氯醛溶液(40mg/mL)麻醉，麻醉后腹腔注射100μL化合物d 2-cycluc，立即用小动物活体成像仪进行成像，时间记为1min。在1min、5min、10min、15min、20min、25min、30min、35min、40min、45min、50min、55min、60min时用小动物活体成像仪拍摄生物发光强度。取FVB-luc +小鼠除尾部以外的其他部分计算ROI值，并用Graphpad进行统计学计算。 Compound d 2 -cycluc was formulated into solutions with final concentrations of 10 mM, 5 mM, 1 mM, 100 μM, 10 μM with normal saline and DMSO, and 200 μL was injected intraperitoneally per FVB-luc + mouse (transcriptional luciferase) (about 20 g). Chloral hydrate solution (40 mg/mL) was anesthetized. After anesthesia, 100 μL of compound d 2 -cycluc was intraperitoneally injected. Immediately, imaging was performed with a small animal in vivo imager, and the time was recorded as 1 min. At 1min, 5min, 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, and 60min, the bioluminescence intensity was photographed with a small animal in vivo imager. The ROI value was calculated from other parts of FVB-luc + mice except the tail, and statistical calculation was performed with Graphpad.
由图13可见，在FVB-luc +小鼠体内腹腔注射不同浓度的化合物d 2-cycluc，FVB-luc +小鼠体内生物发光强度随着d 2-cycluc浓度的升高而增强。 It can be seen from Fig. 13 that the bioluminescence intensity in FVB-luc + mice was enhanced with the increase of the concentration of d 2 -cycluc in FVB-luc + mice by intraperitoneal injection of different concentrations of compound d 2 -cycluc.
实施例9：萤火虫萤光素酶底物cycluc和d 2-cycluc对FVB-luc +小鼠腹腔注射实验 Example 9: Intraperitoneal injection of firefly luciferase substrates cycluc and d 2 -cycluc to FVB-luc + mice
将cycluc和d 2-cycluc用生理盐水和DMSO按照9：1的比例配置成最终浓度为100μM的溶液，取6只成年雌性FVB-luc +小鼠(转录萤光素酶)并分成2组，每只FVB-luc +小鼠(转录萤光素酶)(约20g)腹腔注射200μL水合氯醛溶液(40mg/mL)麻醉，两组小鼠分别腹腔注射100μL cycluc和d 2-cycluc，立即用小动物活体成像仪进行成像，时间记为1min。在1min、5min、10min、15min、20min、25min、30min、35min、40min、45min、50min、55min、60min、120min、240min、480min用小动物活体成像仪拍摄生物发光强度。取FVB-luc +小鼠除尾部以外的其他部分计算ROI 值，并用Graphpad进行统计学计算。 Cycluc and d 2 -cycluc were prepared into a solution with a final concentration of 100 μM in a ratio of 9:1 with normal saline and DMSO, and 6 adult female FVB-luc + mice (transcriptional luciferase) were taken and divided into 2 groups, Each FVB-luc + mouse (transcription luciferase) (about 20 g) was anesthetized by intraperitoneal injection of 200 μL of chloral hydrate solution (40 mg/mL), and the two groups of mice were intraperitoneally injected with 100 μL of cycluc and d 2 -cycluc, respectively, and immediately used The small animal in vivo imager was used for imaging, and the time was recorded as 1 min. At 1min, 5min, 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, 60min, 120min, 240min, 480min, the bioluminescence intensity was photographed with a small animal in vivo imager. The ROI values were calculated from other parts of FVB-luc + mice except the tail, and statistical calculations were performed with Graphpad.
由图14可见，从图A-D中我们可以看出，FVB-luc +小鼠体内腹腔注射化合物cycluc和d 2-cycluc在1min-480min内的发光情况，除了120min时没有显著性差异，其他时间都具有一定的差异性，并且在60min和480min时都具有极显著性差异，在240min时具有显著性差异；从图E中我们可以看出，在1min-480min内的光子数总和具有极显著性差异(P＜0.01)；从图F中我们可以看出，随着时间的延长，化合物d 2-cycluc与cycluc在FVB-luc +小鼠体内的光子数比值具有下降的趋势，由腹腔注射后1min时的5.4倍降低到腹腔注射后480min时的1.8倍，我们可以看出腹腔注射cycluc和d 2-cycluc后，1-480min内d 2-cycluc组生物发光强度始终大于cycluc组，可见d 2-cycluc更适合活体内成像研究。 As can be seen from Figure 14, from Figure AD, we can see that the luminescence of FVB-luc + mice intraperitoneally injected with compounds cycluc and d 2 -cycluc within 1min-480min, except for 120min, there is no significant difference, other time is the same. There is a certain difference, and there is a very significant difference at 60min and 480min, and a significant difference at 240min; from Figure E, we can see that the sum of the number of photons within 1min-480min has a very significant difference (P<0.01); from Figure F, we can see that the ratio of photon numbers of compound d 2 -cycluc to cycluc in FVB-luc + mice has a downward trend with the prolongation of time. 5.4 times at 480 min after intraperitoneal injection, we can see that after intraperitoneal injection of cycluc and d 2 -cycluc, the bioluminescence intensity of d 2 -cycluc group is always greater than that of cycluc group within 1-480 min, and it can be seen that d 2 - cycluc is more suitable for in vivo imaging studies.
应注意的是，以上实例仅用于说明本发明的技术方案而非对其进行限制。尽管参照所给出的实例对本发明进行了详细说明，但是本领域的普通技术人员可根据需要对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围。It should be noted that the above examples are only used to illustrate the technical solutions of the present invention but not to limit them. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or equivalently replace the technical solutions of the present invention as required without departing from the spirit and scope of the technical solutions of the present invention.

Claims (10)

  1. 一种萤光素酶底物，所述荧光素酶底物结构式为：A luciferase substrate, the luciferase substrate structural formula is:
    Figure PCTCN2020114873-appb-100001
    Figure PCTCN2020114873-appb-100001
    其中，R取氢或氘。Wherein, R takes hydrogen or deuterium.
  2. 权利要求1所述荧光素酶底物的制备方法，其特征在于，所述制备方法包括：The preparation method of the luciferase substrate of claim 1, wherein the preparation method comprises:
    Figure PCTCN2020114873-appb-100002
    Figure PCTCN2020114873-appb-100002
  3. 如权利要求2所述的制备方法，其特征在于，The preparation method of claim 2, wherein,
    所述cycluc的合成步骤为：The synthetic steps of described cycluc are:
    (1)以5-硝基吲哚啉为原料和三乙胺反应，同时滴加三氟乙酸酐，将该混合物搅拌反应得到中间体1；(1) with 5-nitroindoline as raw material and triethylamine reaction, drip trifluoroacetic anhydride simultaneously, this mixture is stirred and reacted to obtain intermediate 1;
    (2)中间体1和SnCl 2·2H 2O进行反应，得到中间体2； (2) Intermediate 1 reacts with SnCl 2 ·2H 2 O to obtain Intermediate 2;
    (3)中间体2、硫氰酸钾的冰醋酸溶液和液溴进行反应，得到中间体3；(3) the glacial acetic acid solution of intermediate 2, potassium thiocyanate reacts with liquid bromine to obtain intermediate 3;
    (4)中间体3、亚硝酸叔丁酯和氯化亚铜进行反应，得到中间体4；(4) intermediate 3, tert-butyl nitrite and cuprous chloride react to obtain intermediate 4;
    (5)中间体4和NaBH 4进行反应，得到中间体5； (5) Intermediate 4 and reaction of NaBH 4, to give intermediate 5;
    (6)中间体5、氰基三甲基硅烷和四丁基氟化铵进行反应，得到中间体6；(6) intermediate 5, cyanotrimethylsilane and tetrabutylammonium fluoride react to obtain intermediate 6;
    (7)中间体6、无机碱和D-半胱氨酸盐酸盐进行反应，得到cycluc；(7) react intermediate 6, inorganic base and D-cysteine hydrochloride to obtain cycluc;
    所述d 2-cycluc的具体合成步骤为： The specific synthesis steps of the d 2 -cycluc are:
    (8)1H-吲哚和钯碳在氘气条件下进行反应，得到中间体8；(8) 1H-indole and palladium carbon react under deuterium gas conditions to obtain intermediate 8;
    (9)中间体8和乙酰氯进行反应，得到中间体9；(9) intermediate 8 reacts with acetyl chloride to obtain intermediate 9;
    (10)中间体9和Fe(NO 3) 3·9H 2O进行反应，得到中间体10； (10) Intermediate 9 reacts with Fe(NO 3 ) 3 ·9H 2 O to obtain Intermediate 10;
    (11)中间体10和氯化铵、锌粉进行反应，得到中间体11；(11) intermediate 10 reacts with ammonium chloride and zinc powder to obtain intermediate 11;
    (12)中间体11和硫氰酸钾进行反应，得到中间体12；(12) intermediate 11 reacts with potassium thiocyanate to obtain intermediate 12;
    (13)中间体12、亚硝基叔丁酯和氯化亚铜进行反应，得到中间体13；(13) intermediate 12, nitroso-tert-butyl ester and cuprous chloride react to obtain intermediate 13;
    (14)中间体13和LiAlH 4进行反应，得到中间体14； (14) and the intermediate 13 is reacted LiAlH 4 to give intermediate 14;
    (15)中间体14、氰基三甲基硅烷和四丁基氟化铵进行反应，得到中间体15；(15) Intermediate 14, cyanotrimethylsilane and tetrabutylammonium fluoride react to obtain Intermediate 15;
    (16)中间体15、碳酸钾和D-半胱氨酸盐酸盐进行反应，得到中间体15。(16) Intermediate 15, potassium carbonate and D-cysteine hydrochloride are reacted to obtain intermediate 15.
  4. 如权利要求3所述的制备方法，其特征在于，各反应步骤均在溶剂条件下进行。The preparation method of claim 3, wherein each reaction step is carried out under solvent conditions.
  5. 如权利要求4所述的制备方法，其特征在于，The preparation method of claim 4, wherein,
    所述步骤(1)中，In the step (1),
    所述溶剂可以为二氯甲烷、乙腈、甲醇或乙醇；进一步优选为二氯甲烷；The solvent can be dichloromethane, acetonitrile, methanol or ethanol; more preferably dichloromethane;
    反应温度为10～30℃，反应时间为1～3h；The reaction temperature is 10～30℃, and the reaction time is 1～3h;
    所述5-硝基吲哚啉、三乙胺、三氟乙酸酐的摩尔比为1：(1-1.5)：(1-1.5)；The molar ratio of the 5-nitroindoline, triethylamine and trifluoroacetic anhydride is 1:(1-1.5):(1-1.5);
    所述步骤(2)中，In the step (2),
    所述溶剂为二氯甲烷、乙腈、甲醇或乙醇，进一步优选为乙醇；The solvent is dichloromethane, acetonitrile, methanol or ethanol, more preferably ethanol;
    反应温度为50～70℃，反应时间为3～5h；The reaction temperature is 50～70℃, and the reaction time is 3～5h;
    所述中间体1、SnCl 2·2H 2O的摩尔比为1：(1-1.5)； The molar ratio of the intermediate 1 and SnCl 2 ·2H 2 O is 1: (1-1.5);
    所述步骤(3)中，In the step (3),
    所述溶剂为冰醋酸或盐酸，进一步优选为冰醋酸；Described solvent is glacial acetic acid or hydrochloric acid, more preferably glacial acetic acid;
    反应温度为10～30℃，反应时间为15～25h；The reaction temperature is 10～30℃, and the reaction time is 15～25h;
    所述中间体2、液溴和硫氰酸钾的摩尔比为1：1:(2-3)；The mol ratio of described intermediate 2, liquid bromine and potassium thiocyanate is 1:1:(2-3);
    所述步骤(4)中，In the step (4),
    所述溶剂为二氯甲烷、乙腈、甲醇或乙醇；进一步优选为乙腈；The solvent is dichloromethane, acetonitrile, methanol or ethanol; more preferably acetonitrile;
    反应温度为40～70℃，反应时间为1～3h；The reaction temperature is 40～70℃, and the reaction time is 1～3h;
    所述中间体3、亚硝酸叔丁酯和氯化亚铜的摩尔比为1：2:(1-3)；The mol ratio of the intermediate 3, tert-butyl nitrite and cuprous chloride is 1:2:(1-3);
    所述步骤(5)中，In the step (5),
    所述溶剂为甲醇、二氯甲烷或乙腈；进一步优选为甲醇；The solvent is methanol, dichloromethane or acetonitrile; more preferably methanol;
    反应温度为10～30℃，反应时间为5～30min；The reaction temperature is 10～30 ℃, and the reaction time is 5～30min;
    所述化合物4、NaBH 4的摩尔比为1：(3-5)； The molar ratio of compound 4 and NaBH 4 is 1:(3-5);
    所述步骤(6)中，In the step (6),
    所述溶剂为二氯甲烷、乙腈、甲醇或乙醇；进一步优选为乙腈；The solvent is dichloromethane, acetonitrile, methanol or ethanol; more preferably acetonitrile;
    反应温度为50～100℃，反应时间为1～3h；The reaction temperature is 50～100℃, and the reaction time is 1～3h;
    所述化合物5、氰基三甲基硅烷、四丁基氟化铵的摩尔比为1：(3-5)：(3-5)；The molar ratio of the compound 5, cyanotrimethylsilane and tetrabutylammonium fluoride is 1:(3-5):(3-5);
    所述步骤(7)中，In the step (7),
    所述溶剂为甲醇、二氯甲烷与水以任意比例互溶的有机溶剂；The solvent is an organic solvent that is mutually soluble in methanol, dichloromethane and water in any proportion;
    所述无机碱为碳酸钾、碳酸铯、碳酸钠、碳酸氢钠；进一步优选碳酸钾；Described inorganic base is potassium carbonate, cesium carbonate, sodium carbonate, sodium bicarbonate; further preferably potassium carbonate;
    反应温度为20～50℃，反应时间为1～5h；The reaction temperature is 20～50℃, and the reaction time is 1～5h;
    所述中间体6、碳酸钾、D-半胱氨酸盐酸盐的摩尔比为1：(1-3)：(1-3)。The molar ratio of the intermediate 6, potassium carbonate and D-cysteine hydrochloride is 1:(1-3):(1-3).
  6. 如权利要求4所述的制备方法，其特征在于，The preparation method of claim 4, wherein,
    所述步骤(8)中，In the step (8),
    所述溶剂为氘代甲醇、氘代乙腈；进一步优选为氘代甲醇；The solvent is deuterated methanol, deuterated acetonitrile; more preferably deuterated methanol;
    反应温度为30～70℃，反应时间为50～80h；The reaction temperature is 30～70℃, and the reaction time is 50～80h;
    所述1H-吲哚、钯碳的摩尔比为10：1；The mol ratio of the 1H-indole to palladium carbon is 10:1;
    所述步骤(9)中，In the step (9),
    所述溶剂为冰醋酸或盐酸；进一步优选为冰醋酸；Described solvent is glacial acetic acid or hydrochloric acid; It is further preferably glacial acetic acid;
    反应温度为50～100℃，反应时间为1～3h；The reaction temperature is 50～100℃, and the reaction time is 1～3h;
    所述中间体8、乙酰氯的摩尔比为1：(4-6)；The mol ratio of the intermediate 8 and acetyl chloride is 1: (4-6);
    所述步骤(10)中，In the step (10),
    所述溶剂为二氯甲烷、乙腈、甲醇或乙醇；进一步优选为乙腈；The solvent is dichloromethane, acetonitrile, methanol or ethanol; more preferably acetonitrile;
    反应温度为40～70℃，反应时间为1～3h；The reaction temperature is 40～70℃, and the reaction time is 1～3h;
    所述中间体9、Fe(NO 3) 3·9H 2O的摩尔比为2：1； The molar ratio of the intermediate 9 and Fe(NO 3 ) 3 ·9H 2 O is 2:1;
    优选的，所述步骤(11)中，Preferably, in the step (11),
    所述溶剂为二氯甲烷、乙腈或乙醇；进一步优选为乙醇；The solvent is dichloromethane, acetonitrile or ethanol; more preferably ethanol;
    反应温度为10～30℃，反应时间为1～3h；The reaction temperature is 10～30℃, and the reaction time is 1～3h;
    所述中间体10、氯化铵、锌粉的摩尔比为1：10：20；The molar ratio of the intermediate 10, ammonium chloride and zinc powder is 1:10:20;
    优选的，所述步骤(12)中，Preferably, in the step (12),
    所述溶剂为二氯甲烷、乙腈、甲醇或乙醇；进一步优选为乙腈；The solvent is dichloromethane, acetonitrile, methanol or ethanol; more preferably acetonitrile;
    反应温度为10～30℃，反应时间为10～30h；The reaction temperature is 10～30℃, and the reaction time is 10～30h;
    所述中间体11、硫氰酸钾、液溴的摩尔比为1：3～4：1；The molar ratio of the intermediate 11, potassium thiocyanate and liquid bromine is 1:3 to 4:1;
    优选的，所述步骤(13)中，Preferably, in the step (13),
    所述溶剂为二氯甲烷、无水乙腈、甲醇或乙醇；进一步优选为无水乙腈；The solvent is dichloromethane, anhydrous acetonitrile, methanol or ethanol; more preferably anhydrous acetonitrile;
    反应温度为40～70℃，反应时间为1～3h；The reaction temperature is 40～70℃, and the reaction time is 1～3h;
    所述中间体12、亚硝基叔丁酯、氯化亚铜的摩尔比为1：1～2：1.5；The molar ratio of the intermediate 12, nitroso-tert-butyl ester and cuprous chloride is 1:1-2:1.5;
    优选的，所述步骤(14)中，Preferably, in the step (14),
    所述溶剂为四氢呋喃、二氯甲烷、DMF、甲醇或乙醇；进一步优选为四氢呋喃；The solvent is tetrahydrofuran, dichloromethane, DMF, methanol or ethanol; more preferably tetrahydrofuran;
    反应温度为-40～-100℃，反应时间为1～2h；The reaction temperature is -40～-100℃, and the reaction time is 1～2h;
    所述中间体13、LiAlH 4的摩尔比为1～2：1； The molar ratio of the intermediate 13 and LiAlH 4 is 1-2:1;
    优选的，所述步骤(15)中，Preferably, in the step (15),
    所述溶剂为无水乙腈、二氯甲烷、DMF、甲醇或乙醇；进一步优选为无水乙腈；The solvent is anhydrous acetonitrile, dichloromethane, DMF, methanol or ethanol; more preferably anhydrous acetonitrile;
    反应温度为40～100℃，反应时间为1～2h；The reaction temperature is 40～100℃, and the reaction time is 1～2h;
    所述中间体14、氰基三甲基硅烷、四丁基氟化铵的摩尔比为1～2：1：2.5；The molar ratio of the intermediate 14, cyanotrimethylsilane, and tetrabutylammonium fluoride is 1-2:1:2.5;
    优选的，步骤(16)中，Preferably, in step (16),
    所述溶剂优选为甲醇、二氯甲烷与水以任意比例互溶的有机溶剂，The solvent is preferably an organic solvent that is mutually soluble in methanol, dichloromethane and water in any proportion,
    所述无机碱为碳酸钾、碳酸铯、碳酸钠或碳酸氢钠；进一步优选为碳酸钾；The inorganic base is potassium carbonate, cesium carbonate, sodium carbonate or sodium bicarbonate; further preferably potassium carbonate;
    反应温度为20～50℃，反应时间为1～5h；The reaction temperature is 20～50℃, and the reaction time is 1～5h;
    所述中间体15、碳酸钾、D-半胱氨酸盐酸盐的摩尔比为1：1～2：2.5。The molar ratio of the intermediate 15, potassium carbonate and D-cysteine hydrochloride is 1:1-2:2.5.
  7. 权利要求1所述萤光素酶底物在荧光产品和/或制备荧光产品中的应用；The application of the luciferase substrate of claim 1 in fluorescent products and/or preparing fluorescent products;
    优选的，所述荧光产品包括但不限于荧光探针和荧光检测试剂盒。Preferably, the fluorescent products include but are not limited to fluorescent probes and fluorescent detection kits.
  8. 一种荧光探针，其特征在于，所述荧光探针包含权利要求1所述萤光素酶底物。A fluorescent probe, characterized in that, the fluorescent probe comprises the luciferase substrate of claim 1.
  9. 一种荧光检测试剂盒，其特征在于，所述荧光检测试剂盒包含权利要求1所述萤光素酶底物或权利要求8所述荧光探针。A fluorescence detection kit, characterized in that, the fluorescence detection kit comprises the luciferase substrate of claim 1 or the fluorescent probe of claim 8.
  10. 权利要求1所述萤光素酶底物、权利要求8所述荧光探针和/或权利要求9所述荧光检测试剂盒在如下中的应用：The application of the luciferase substrate of claim 1, the fluorescent probe of claim 8 and/or the fluorescence detection kit of claim 9 in the following:
    1)环境检测；1) Environmental testing;
    2)分析化学；2) Analytical chemistry;
    3)生物分析和检测；3) Biological analysis and detection;
    优选的，所述生物分析和检测包括对生物个体水平、器官水平、组织水平和细胞水平的分析与检测；进一步优选为细胞水平；Preferably, the biological analysis and detection includes the analysis and detection of the biological individual level, the organ level, the tissue level and the cellular level; more preferably, the cellular level;
    优选的，所述生物包括鱼、小鼠、大鼠、豚鼠、鸡、兔、狗、猫、猴、猩猩和人；Preferably, the organisms include fish, mice, rats, guinea pigs, chickens, rabbits, dogs, cats, monkeys, orangutans and humans;
    进一步优选的，所述生物分析和检测包括利用所述萤光素酶底物、荧光探针和/或荧光检测试剂盒对细胞进行荧光标记、利用流式细胞仪或荧光显微镜对荧光标记细胞进行分选和利用体内荧光成像装置观察生物体内的荧光标记的细胞。Further preferably, the biological analysis and detection include using the luciferase substrate, fluorescent probe and/or fluorescence detection kit to perform fluorescent labeling on cells, and using flow cytometry or fluorescence microscope to perform fluorescent labeling on cells. Sort and visualize fluorescently labeled cells in vivo using an in vivo fluorescence imaging device.
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