Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
  • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D207/30—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
  • C07D207/34—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
  • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
  • C07D493/10—Spiro-condensed systems
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/17—Nitrogen containing

Definitions

• This invention relates generally to the field of detecting and measuring peroxynitrite. More specifically, the invention relates to compounds useful as agents to specifically detect and measure peroxynitrite.
• the invention includes probe molecules, methods for their preparation and use as agents to detect and measure peroxynitrate in living cells and living tissues.
• Peroxynitrite (ONOO " ), an isomer of nitrate, has been known for about one century. During the past decade it has been extensively studied due to its potential important role in biology and medicine ; Beckman, J. S., Am. J. Physiol. Cell Physiol. 1996, 271, C1424-1437; Goldstein, S. et al., Free Radical Biol. & Med. 1996, 21, 965-974; Groves, J. T., GOT: Opin. Chem. Biol. 1999, J, 226; Radi, R. et al., Free Radical Biol. & Med. 2001, 30, 463-488; Tarpey, M. M. et al., Circ. Res.
• the reaction between nitric oxide and superoxide proceeds when the concentration of NO increases and can overcome dismutation by superoxide dismutase.
• peroxynitrite contributes to tissue injury in a number of human diseases such as ischemic reperfusion injury, rheumatoid arthritis, septic shock, multiple sclerosis, atherosclerosis, stroke, inflammatory bowl disease, cancer, and several neurodegenerative diseases (MacMillan-Crow, L. A. et al., Proc. Natl Acad. Sci. USA 1996, 93, 11853; Rodenas, J. et al., Free Radical. Biol. &Med. 2000, 28, 314; Cuzzocrea, S. et al., Pharmacol Rev. 2001, 55, 135; Szabo, C. Toxicol. Lett.
• the available analytical methods for detecting and measuring peroxynitrite can be classified into three types.
• the first type is the electrochemical sensor, which is used to estimate the amounts of peroxynitrite generated in cells under oxidative stress.
• the electrochemical sensor which is used to estimate the amounts of peroxynitrite generated in cells under oxidative stress.
• the second type relies on the employment of oxidation probes.
• DCFH (2' 1 7'-dichlordihydrofluorescein) and DHR 123 (dihydrorhodamine 123), which can be oxidized by peroxynitrite to yield highly fluorescent molecules, have been used for monitoring peroxynitrite in cells and tissues (Royall, J. A. et al., Arch. Biochem. Biophys. 1993, 302, 348-355; Kooy, N. W. et al., Free Radic. Biol. Med. 1994, 16, 149-156; Kooy, N. W. et al., Free Radic. Res. 1997, 27, 245-254; Crow, J. P.
• HPF hydroxyphenyl fluorescein
• the third type utilizes the footprinting reaction of biological molecules.
• 3-nitrotyrosine a nitration product generated after oxidation of tyrosine residues of proteins by peroxynitrite in biological systems
• NADH reduced nicotamide adenine dinucleotide
• probes or biomolecules that can directly indicate the generation of peroxynitrite in cells in an unambiguous manner. It implies that other reactive oxygen species and reactive nitrogen species present in the biological systems may compete with peroxynitrite and interfere with the results.
• This invention relates to novel compounds which are used for unambiguous detection and measurement of peroxynitrite. Specifically, this invention provides compounds, which specifically react with peroxynitrite rather than other reactive oxygen species and reactive nitrogen species, represented by the following general formula (I), (II), (HI), or a salt thereof:
• This invention also provides agents for measuring peroxynitrite comprising any of the compounds mentioned above.
• the invention also provides methods for measuring peroxynitrite in a sample comprising the steps: a) contacting any of the compounds mentioned above with the sample, and b) measuring fluorescence of a resulting compound generated by a reaction between the compound and peroxynitrite present in the sample.
• the invention also provides a high-throughput screening fluorescent method for detecting peroxynitrite comprising using an agent for measuring peroxynitrite, wherein the agent comprising any of the compounds mentioned above.
• the invention also provides a high-throughput method for screening compounds that increase or decrease the production of peroxynitrite comprising using any of the compounds mentioned above.
• FIG. 1 illustrates the reaction of the oxidation of ketone (la/lb) by peroxynitrite or Oxone ® (2KHSO 5 » KHSO 4 « K 2 SO 4 ).
• FIG. 4 to FIG.7 show the synthetic schemes of Example 1.
• FIG. 8 shows a fluorescence spectrum of a 20 ⁇ M solution of the compound
• FIG. 9 shows a fluorescence spectrum of the solution 30 min after the reaction of 15 equiv of ONOO ' with 5 mL of 20 ⁇ M ss-6.
• FIG. 10 shows an absorption spectrum of 20 ⁇ M ss-6.
• FIG. 11 shows fluorescence spectra taken 30 min after the reaction between ss-6 and ONOO ' with concentration ranging from 0 to 300 ⁇ M.
• FIG. 12 shows the linear relationship between fluorescence intensity and the concentration of ONOO " .
• FIG. 13 to FIG. 18 show the synthetic schemes of Example 5.
• FIG. 19 shows a fluorescence spectrum of a 20 ⁇ M solution of the compound
• FIG. 20 shows a fluorescence spectrum of the solution 30 min after the reaction of 15 equiv of ONOO " with 5 mL of 20 ⁇ M ss-12.
• FIG. 21 shows an absorption spectrum of 20 ⁇ M ss-12.
• FIG. 22 shows fluorescence spectra taken 30 min after the reaction between ss-6 and ONOO " with concentration ranging from 0 to 300 ⁇ M.
• FIG. 23 shows the linear relationship between fluorescence intensity and the concentration of ONOO " .
• FIG. 24 shows fluorescent microscopy results of primary cultured neuronal cells that were incubated with ss-6 and ss-12 at a concentration of 20 ⁇ M, and then treated with 10 ⁇ M and 100 ⁇ M SIN-I (3-Morpholino-sydnonimine-HCl).
• Alkyl refers to a fully saturated acyclic monovalent radical containing carbon and hydrogen, and which may be branched or a straight chain. Examples of alkyl groups are methyl, ethyl, n-butyl, t-butyl, n-heptyl, and isopropyl.
• “Lower alkyl” refers to an alkyl radical of one to six carbon atoms, as exemplified by methyl, ethyl, n-butyl, i-butyl, t-butyl, isoaniyl, n-pentyl, and isopentyl.
• Alkenyl refers to a monovalent or divalent unsaturated, preferably monounsaturated, radical containing carbon and hydrogen, and which may be cyclic, branched or a straight chain.
• Lower alkenyl refers to such a radical having one to five carbon atoms.
• Aryl refers to a substituted or unsubstituted monovalent aromatic radical, generally having a single ring (e.g., benzene) or two condensed rings (e.g., naphthyl). Monocyclic aryl groups are generally preferred.
• the term includes heteroaryl groups, which are aromatic ring groups having one or more nitrogen, oxygen, or sulfur atoms in the ring, such as furyl, pyrrole, pyridyl, and indole.
• substituted is meant that one or more ring hydrogens in the aryl group is replaced with a group or groups preferably selected from fluorine, chlorine, bromine, iodine, methyl, ethyl, hydroxyl, hydroxymethyl, nitro, amino, methylamino, dimethylamino, methoxy, halomethoxy, and halomethyl.
• Alkyl refers to an alkyl, preferably lower alkyl, substituent which is further substituted with an aryl group; examples are benzyl and phenethyl.
• Fluoropliore refers to a small molecule, or a part of a larger molecule, that can be excited by light to emit fluorescence. Preferably, fluorophores efficiently produce fluorescence upon excitation with light which has a wavelength in the range of about 200 to about 1000 nanometers, preferably in the range of about 500 to 800 nanometers.
• a fluorophore is preferably selected from acridine orange, anthracene ring, allophycocyanin, BODIPY, cyanines, coumarin, Edans, Eosin, Erythrosin, fluorescamine, fluorescein, FAM (carboxy fluorescein), HEX (hexachlorofluorescein), JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxy-fluorescein), Oregon Green, phycocyanin, phycoerythrin, rhodamine, ROX (Carboxy-X-rhodamine), TAMRA (carboxytetramethyMiodamine), TET (tetrachloro- fluorescein), Texas red, tetramethylrhodamine, and xanthines.
• Such groups are reported in the Handbook of Fluorescent Probes and Research Products, 9th Edition, Molecular Probes, Eugene, Oregon
• Inorganic ester refers to a product of the reaction of an inorganic acid and an alcohol. Inorganic esters mainly result from the condensation of an inorganic acid and an alcohol.
• salt refers to which formed by standard acid-base reactions with basic groups, such as amino groups, having a counterion derived from an organic or inorganic acid.
• counterions include chloride, sulfate, phosphate, acetate, succinate, citrate, lactate, maleate, fumarate, palmitate, cholate, glutamate, glutarate, tartrate, stearate, salicylate, methanesulfonate, benzenesulfonate, sorbate, picrate, benzoate, cinnamate, and the like.
• physiologically acceptable salt encompasses carboxylate salts having organic and inorganic cations, such as alkali and alkaline earth metal cations (e.g., lithium, sodium, potassium, magnesium, barium and calcium); ammonium; or organic cations, for example, dibenzylammonium, benzylammonium, 2- hydroxyethylammonium, bis(2-hydroxyethyl) ammonium, phenylethylbenzylammonium, and the like.
• alkali and alkaline earth metal cations e.g., lithium, sodium, potassium, magnesium, barium and calcium
• ammonium e.g., sodium, potassium, magnesium, barium and calcium
• organic cations for example, dibenzylammonium, benzylammonium, 2- hydroxyethylammonium, bis(2-hydroxyethyl) ammonium, phenylethylbenzylammonium, and the like.
• cations encompassed by the above term include the protonated form of procaine, quinine, and N-methylglucosamine, and the protonated forms of basic amino acids, such as glycine, ornithine, histidine, phenylalanine, lysine, and arginine.
• this invention provides compounds which specifically react with peroxynitrite rather than other reactive oxygen species and reactive nitrogen species.
• the compounds have the following general formula (I):
• R 1 is OR 1 ! or Mf 2 R 3 , wherein R 1 , R 2 and R 3 are independently hydrogen or a group selected from alkyl, alkenyl, alkynyl, alkoxyalkyl, alkanoyl, alkenoyl, alkynoyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, aryloyl, or polyether;
• R 2 , R 3 , R 4 , and R 5 are independently hydrogen or a group selected from halogen, alkyl, alkoxy, alkyloxy, polyether, R 2 and R 3 come together to form a 5, 6, or 7- membered ring which is selected from aryl, heterocyclic, heteroaryl or heteroaromatic, or R 4 and R 5 come together to form a 5, 6, or 7-membered ring which is selected from aryl, heterocyclic, heteroaryl or heteroaromatic;
• R 2 and R 3 come together to form a 5, 6, or 7-membered ring which is selected from aryl, heterocyclic, heteroaryl or heteroaromatic;
• R 4 and R 5 come together to form a 5, 6, or 7-membered ring which is selected from aryl, heterocyclic, heteroaryl, or heteroaromatic;
• R 1 is CH 3 or OCH 2 OZ 1 , wherein Z 1 is a group selected from alkyl, alkenyl, alkynyl, alkoxyalkyl, alkanoyl, alkenoyl, alkynoyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, aryloyl, or polyether
• This invention also provides compounds that have high specificity and selectivity in the measurement of peroxynitrite.
• the compounds have the following general formula (II):
• R 7 and R 10 are independently hydrogen or a group selected from halogen, lower alkyl, lower alkenyl, halogenated alkyl, CN, or NO 2 ;
• R 13 is OR' 4 or NR' 5 R' 6 , wherein R' 4 , R 5 and R 6 are independently hydrogen or a group selected from alkyl, alkenyl, alkynyl, alkoxyalkyl, alkanoyl, alkenoyl, alkynoyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, aryloyl, or polyether;
• R 14 and R 15 are independently hydrogen, halogen, alkyl, alkoxy, polyether, or R 14 and R 15 come together to form a 5, 6, or 7-membered ring which is selected from aryl, heterocyclic, heteroaryl, or heteroaromatic;
• R 16 is hydrogen, alkyl, alkoxy, or polyether
• CF 3 halogen-substituted lower alkyl
• n e.g., CF n H 3-11 , wherein n is 1 or 2
• W 2 is a group selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, or arylalkyl.
• R n and R 12 come together to form a ring, preferably as a 5, 6, or 7-membered ring, to form a cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic, heteroaryl or heteroaromatic; and/or
• R 4 is CH 3 or OCH 2 OZ 3 , wherein Z 3 is a group selected from alkyl, alkenyl, alkynyl, alkoxyalkyl, alkanoyl, alkenoyl, alkynoyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, aryloyl, or polyether.
• Z 3 is a group selected from alkyl, alkenyl, alkynyl, alkoxyalkyl, alkanoyl, alkenoyl, alkynoyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, aryloyl, or polyether.
• the compound represented by general formula (II) can exist as a salt. And physiologically acceptable water-soluble salts can be suitably used for the agent and the measuring method of this invention. Further, the compound represented by the general formula (II) in a free form or a salt thereof may exist as a hydrate or a solvate, and any of these substances fall within the scope of this invention.
• the types of solvents that form the solvates are not particularly limited. For example, solvents such as acetonitrile, ethanol, water, or acetonitrile-water mixture can be exemplified.
• compounds that have high specificity and selectivity in the measurement of peroxynitrile have the following general formula (III):
• R 18 and R 19 are independently hydrogen, halogen, alkyl, or alkoxy;
• R 20 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, carboxy alkyl, carboxylic ester, or aminoalkyl;
• R 21 and R 22 are independently hydrogen or a group selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, keto, carboxy alkyl, carboxylate, carboxylic ester, carbamate, amide, amino, alkylamino, poly ether, alkylthio, cyano, nitro, sulfonyl, or inorganic ester;
• R 23 is selected from below:
• R 7 , R 8 , R 9 , and R 10 are independently hydrogen or a group selected from halogen (e.g., Cl, Br, or I), alkyl (e.g., CH 3 ), alkoxy, alkyloxy, or polyether;
• halogen e.g., Cl, Br, or I
• alkyl e.g., CH 3
• alkoxy e.g., alkyloxy, or polyether
• R 25J R 26> and R 27 are independently hydrogen or a group selected from alkyl, alkenyl, alkynyl, alkoxy, alkyloxy, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, keto, aldehyde, carboxylate, carboxylic acid, carboxylic ester, carbamate, amide, amino, alkylamino, polyether, alkylthio, cyano, nitro, sulfonyl, inorganic ester, R 24 and R 25 come together to form a 5, 6, or 7-rnembered ring which is selected from cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic, heteroaryl, or hetero aromatic, R 25 and R 26 come together to form a 5, 6, or 7-membered ring which is selected from cycloalkyl, cyclo
• R' 7 and R' 8 come together to form a 5, 6, or 7-membered ring which is selected from aryl, heterocyclic, heteroaryl, or heteroaromatic;
• R 9 and R 10 come together to form a 5, 6, or 7-membered ring which is selected from aryl, heterocyclic, heteroaryl, or heteroaromatic;
• R 24 and R 25 come together to form a 5, 6, or 7-membered ring which is selected from cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic, heteroaryl, or heteroaromatic;
• R 25 and R 26 come together to form a 5, 6, or 7-membered ring which is selected from cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic, heteroaryl, or heteroaromatic; and/or
• R 26 and R 27 come together to form a 5, 6, or 7-membered ring which is selected from cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic, heteroaryl, or heteroaromatic.
• the compound represented by general formula (III) can also exist as a salt.
• physiologically acceptable water-soluble salts can be suitably used for the agent and the measuring method of this invention.
• the compound represented by the general formula (III) in a free form or a salt thereof may exist as a hydrate or a solvate, and any of these substances fall within the scope of this invention.
• the types of solvents that form the solvates are not particularly limited. For example, solvents such as acetonitrile, ethanol, water or acetonitrile-water mixture can be exemplified.
• peroxynitrite oxidizes some specific ketones represented by the general formula (I) in a way similar to the reaction with peroxymonosulfate, the commercial source of which is Oxone ® (2KHSO 5 'KHSO 4 «K 2 SO 4 ) (30-55% yield, 100% conversion) (FIG. 1).
• This reaction proceeds via a dioxirane intermediate.
• the dioxirane formation and its subsequent oxidation of phenol derivatives in an intramolecular fashion provide the basis for designing probes for the specific detection of peroxynitrite in cells.
• similar reactions do not proceed between the ketones and other reactive oxygen species or reactive nitrogen species present in the biological systems.
• fluorescent probes for peroxynitrite can be synthesized by replacing some groups in the ketones with fluorophores.
• the fluorescence properties of the BODIPY-based probes can be controlled by PET (photoinduced electron transfer) mechanism. Based on PM3 calculation method, fluorescent probes controlled by PET-dependent (photoinduced electron transfer) fluorescence off/on switching mechanism (FIG. 2) were designed.
• PET-dependent (photoinduced electron transfer) fluorescence off/on switching mechanism FOG. 2 were designed.
• the fluorophore before the oxidation with peroxynitrite, the fluorophore is masked and the probe is non-fluorescent. However, upon reaction with peroxynitrite, the fluorophore is released and become strongly fluorescent.
• This invention also provides an agent for measuring peroxynitrite comprising any of the compounds mentioned above.
• This invention also provides a method for measuring peroxynitrite in a chemical or biological sample (such as cells and tissues from animals or plants, and microorganism) comprising the steps: a) contacting any of the compound mentioned above with the chemical or biological sample, and b) measuring fluorescence of a resulting compound generated by a reaction between the compound and peroxynitrite present in the sample.
• a chemical or biological sample such as cells and tissues from animals or plants, and microorganism
• This invention also provides a high-throughput screening fluorescent method for detecting peroxynitrite comprising using the agent for measuring peroxynitrite mentioned above.
• This invention also provides a high-throughput method for screening compounds that increase or decrease the production of peroxynitrite comprising using any of the compounds mentioned above.
• the compounds of this invention may be made by one skilled in organic synthesis by known techniques as well as by the general synthetic procedures disclosed herein.
• some compounds represented by general formula (I) can be synthesized generally using procedures outlined by Yang et al (J. Org. Chem, 2000, 65, 4179-4184).
• a compound of general formula (II) can be synthesized generally by the following procedure (Nagano, T. et al., J Am. Chem. Soc. 2004, 126, 3357-3367).
• the general synthetic schemes are shown in FIG. 2.
• the corresponding pyrrole part and aldehyde part are treated with a catalytic amount of TFA (trifluoroacetic acid) in an appropriate solvent such as dichloromethane or 1,2-dichloroethane at temperatures ranging from room temperature to 80 0 C.
• TFA trifluoroacetic acid
• the corresponding pyrrole part and aldehyde part can be prepared independently, and some functional groups can be protected by protecting groups.
• the above synthetic schemes may be optimized sometimes by choosing the different protecting groups.
• Detailed explanations of protecting groups and skill of choosing a suitable protecting group can be found in, for example, a book entitled Protective Groups in Organic Synthesis, Greene, T. W, John Wiley & Sons, Inc., 1999.
• a compound of general formula (III) can be synthesized generally by the following procedure (John, E. T. et al., J. Chem. Soc, Per kin Trans I, 1995, 1993; McWatt , M. et al., Eur. J. Org. Chem.
• the corresponding fluorescein derivatives and R 23 I can be prepared independently, and some functional groups can be protected by protecting groups.
• the same as the general synthetic schemes of compound represented by general formula (II), above synthetic schemes may be optimized sometimes by choosing different protecting groups.
• the terms "work-up” and “purification” mean the combinations of techniques used in organic synthesis, e.g., washing, filtration, extraction, evaporation, distillation, crystallization, chromatography and the like.
• the intermediate may also be used in the subsequent reaction without purification.
• Compound ss-6 obtained in Example 1 was dissolved in CH 3 CN to a concentration of 2 mM, and then the solution was added a 100 mM sodium phosphate buffer (pH 7.4) for dissolution to a final concentration of 20 ⁇ M.
• the excitation spectrum and the fluorescence spectrum of the 20 ⁇ M ss-6 solution were measured using a Perkin Elmer LS50 fluorescence spectrometer. Slit width was 5 nm for both the excitation spectrum and the fluorescence spectrum, and the photomultiplier voltage was 775 V. The measurement was carried out at the excitation wavelength of 515 nm. The results are shown in FIG. 8.
• the solution was then frozen. A dark yellow solution enriched in peroxynitrite was separated out and used in all experiments.
• the peroxynitrite concentration in the stock solutions used was estimated by using an extinction coefficient of 1670 cm “1 (mol/L) "1 at 302 nm (Hughes and Nicklin;The chemistry of pernitrites. Part I. Kinetics of decomposition of pernitrious acid; J. Chem. Soc. A, 1968, 2, 450-452).
• the peroxynitrite solution prepared was usually very basic (pH 12). When larger volumes of peroxynitrite were added, part of the excess base was neutralized on the day of the experiment.
• Example 3 Comparison of Specificity nf ss-fi with Different Reactive Oxygen Species
• Compound ss-6 obtained in Example 1 was dissolved in CH 3 CN to a concentration of 2 mM, and then the solution was added with a 100 mM sodium phosphate buffer (pH 7.4) for dissolution at a final concentration of 20 ⁇ M.
• 50 ⁇ L of different reactive oxygen species (10 equiv) were added independently to 5 mL of the corresponding ss-6 solution.
• the changes in fluorescence intensity before and after the treatment were measured.
• the fluorescence intensity was measured under the same conditions as those in Example 2.
• Each concentration of the fluorescence probe was 20 ⁇ M (a 100 mM sodium phosphate buffer, pH 7.4).
• Table 1 It was verified from the result that ss-6 has very high selectivity.
• Compound ss-12 obtained in Example 5 was dissolved in CH 3 CN to a concentration of 2 niM, and then the solution was added a 100 mM sodium phosphate buffer (pH 7.4) for dissolution at a final concentration of 20 ⁇ M.
• the excitation spectrum and the fluorescence spectrum of the 20 ⁇ M ss-12 solution were measured using a Perkin Elnier® LS50 fluorescence spectrometer. Slit width was 2.5 nm for both the excitation spectrum and the fluorescence spectrum, and the photomultiplier voltage was 775 V. The measurement was carried out at the excitation wavelength of 490 nm. The results are shown in FIG. 19.
• Example 8 Specific Detection of Peroxynitrite with ss-12
• Compound ss-12 obtained in Example 5 was dissolved in CH 3 CN to a concentration of 2 mM, and then the solution was added with a 100 mM sodium phosphate buffer (pH 7.4) for dissolution at a final concentration of 20 ⁇ M. Then peroxynitrite was added to final concentrations of 0, 20, 60, 100, 200, 240 and 300 ⁇ M, and the fluorescence spectrum was measured after 30 mins. The fluorescence spectrum was measured under the same conditions as those in Example 6. The results were shown in FIG. 22. As clearly shown in FIG. 22, ss-12 gave great increase in fluorescence intensity, and the fluorescence intensity has good linear relationship with the concentration of ONOO " (shown in FIG. 23).
• primary cultured cortex neurons were prepared from embryonic day 15 Sprague-Dawley rats. Briefly, dissociated cell suspensions were plated at a density of 2 x 10 6 cells/well on poly-L-lysine-coated 6-well plates (BD Biosciences, San Diego, CA, USA) with Neurobasal/2% B27 (Gibco-BRL, Grand Island, NY) containing glutamine (0.5 mM, Sigma Chemical Company, St. Louis, MO), penicillin (100 U/mL) and streptomycin (100 ⁇ g/mL). The cells were maintained in a humidified incubator at 37 0 C, in 5% CO 2 -95% air.

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