WO1993015219A1 - PROCEDE DE DETERMINATION QUANTITATIVE D'UNE SUBSTANCE A l'AIDE D'UN DERIVE DE COUMARINE - Google Patents

PROCEDE DE DETERMINATION QUANTITATIVE D'UNE SUBSTANCE A l'AIDE D'UN DERIVE DE COUMARINE Download PDF

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Publication number
WO1993015219A1
WO1993015219A1 PCT/JP1993/000128 JP9300128W WO9315219A1 WO 1993015219 A1 WO1993015219 A1 WO 1993015219A1 JP 9300128 W JP9300128 W JP 9300128W WO 9315219 A1 WO9315219 A1 WO 9315219A1
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WIPO (PCT)
Prior art keywords
substituted
compound
hydrogen peroxide
formula
peroxidase
Prior art date
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PCT/JP1993/000128
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English (en)
Japanese (ja)
Inventor
Norihito Aoyama
Hideki Takenaka
Akira Miike
Original Assignee
Kyowa Medex Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kyowa Medex Co., Ltd. filed Critical Kyowa Medex Co., Ltd.
Priority to JP5513100A priority Critical patent/JP2980681B2/ja
Publication of WO1993015219A1 publication Critical patent/WO1993015219A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2326/00—Chromogens for determinations of oxidoreductase enzymes

Definitions

  • the present invention relates to a method for quantifying a peroxide active substance, hydrogen peroxide and a coumarin derivative represented by the following formula (I) or ( ⁇ ) by a chemiluminescence method using a coumarin derivative.
  • the antigen, antibody or DNA is labeled by labeling the antigen, antibody or DNA with peroxidase, an oxidase other than peroxidase or a luminescent compound, and quantifying peroxidase activity or hydrogen peroxide or a luminescent compound generated by the reaction of the oxidase. There is a known method for quantifying the amount.
  • the coumarin derivatives represented by the following formulas (I) and ( ⁇ ) are known as fluorescent compounds and are commercially available [Fluorescence Analytical Chemistry, pp. 159-161 (1987) published by Baifukan; Japan Chemical Society Journal, Volume 3, p. 644 (1972); Heterocycles, Volume 7, p. 933 (1977); Industry Chemical Magazine, Vol. 71, p. 1010 (1968); Eastman Kodak Co., Ltd., Wako Pure Chemical Industries' power tag, etc.].
  • the present invention relates to hydrogen peroxide and a compound of the formula (I)
  • R 1 , R 2 , R :! , R, R s and R 6 are the same or different and each represents a hydrogen atom, substituted or unsubstituted lower alkyl, lower alkoxy, substituted or unsubstituted Aralkyl, substituted or unsubstituted aryl, halogen atom, cyano, nitro, sulfo, carboxy, alkoxycarbonyl, alkyl rubamoyl, substituted or unsubstituted aryl carbamoyl, carbamoyl, hydroxy, substituted Or an unsubstituted amino, lower alkanoyl, lower alkanoyloxy or heterocyclic group, or R 1 and R 2 together form an alkylene or alkenylene, or a R 5 together form connexion - CH 2 CH 2 CH 2 NH - the Kumari down derivative or a salt thereof to form), is reacted in the presence of a peroxidative substance, the reaction solution Meas
  • the principle of the present invention is based on the fact that the above reaction proceeds stoichiometrically, and the luminescence amount or light intensity of the reaction solution is proportional to the amount of the peroxide active substance, hydrogen peroxide or coumarin derivative. I have.
  • the structure of the coumarin derivative has not been changed after the light emission due to the reaction, it was not confirmed by chromatography that the presence of a substance that was distinguished from the coumarin derivative before the reaction was confirmed. It is clear that a compound that emits light is generated by the reaction.
  • the alkyl moiety in lower alkyl, lower alkoxy, lower alkanoyl, lower alkanoyloxy, alkoxyl-rubonyl and alkyl-rubamoyl is preferably a straight-chain or 1-8 carbon atom.
  • Branched alkyl for example, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, etc., aralkyl having 7 to 15 carbon atoms, for example, benzyl , Phenyl, etc .; aryl in aryl, aryl carbamoyl represents phenyl, naphthyl, etc .; halogen atom represents iodine, bromine, chlorine, fluorine, etc., and heterocyclic ring.
  • the group represents pyridyl, benzothiazolinyl and the like.
  • Substituents in the substituted alkyl, substituted aralkyl, substituted aryl and substituted aryl are the same or different and are substituted with 1 to 5 cyano, halogen atoms, substituted or unsubstituted amino.
  • the alkyl moiety and the halogen atom in lower alkyl, lower alkoxy and alkoxycarbonyl are as defined above.
  • the substituents in the substituted amino are the same or different and represent a substituted or unsubstituted lower alkyl, substituted or unsubstituted heterocyclic group.
  • Lower alkyl is as defined above, and the heterocyclic group is, for example, triazinyl, virazinyl Represents pyridyl, pyrimidinyl, etc.
  • Substituents in the substituted heterocyclic group may be the same or different and each represents a substituted or unsubstituted cyano, halogen atom, amino, lower alkoxy, hydroxy, dialkylamino, or the like.
  • Acceptable salts of compound (I) or (II) include acid addition salts, for example, inorganic acid salts such as hydrochloride, sulfate, phosphate and the like, acetate, maleate, fumarate, citrate Organic acid salts.
  • inorganic acid salts such as hydrochloride, sulfate, phosphate and the like, acetate, maleate, fumarate, citrate Organic acid salts.
  • Table 1 shows specific compounds used in the present invention.
  • peroxidase active substance used in the method of the present invention is a general term for compounds having peroxidase activity, and includes, for example, peroxidase derived from plants, animals or microorganisms [EC. 1. 11.1. 7), hemoglobin, heme, iron oxide, iron chloride, sodium iodide, ammonium iodide, molybdate and the like.
  • the reactions generally performed by the following are hydrochloric acid, acetic acid, acetate, succinate, oxalate, borate, phthalate, glycine, valpital salt, In a buffer solution ( ⁇ . ⁇ ) adjusted to pH 2 to 8 by combining a buffering agent such as GOOD.
  • the components other than the component to be measured are added to the buffer solution to make a reagent solution, and the sample containing the component to be measured is added to this solution at 110 to 90 ° C, preferably 20 to 90 ° C. React at 50 ° C.
  • the total luminescence of the reaction solution or the luminescence during a certain period of time is measured with a luminescence photometer or the like in the wavelength range of 190 to 750 nm, and the calibration curve is prepared using a known amount of the sample containing the component to be measured.
  • the components to be quantified in the sample can be quantified.
  • the luminescence amount corresponds to the amount of the component to be measured, it can be quantified from the integrated luminescence amount measured at the end of the reaction, but is generally calculated from the integrated luminescence amount for a certain period of time.
  • the object can be achieved by measuring the amount of change in the amount of luminescence per unit time.
  • a surfactant for example, Triton X-100 (manufactured by Yoneyama Pharmaceutical Co., Ltd.) having a concentration of 0. It can be added so as to be 01 to 5 wt%.
  • a protein, a polyalkyl quaternary amine, a fluorescent agent, dimethyl sulfoxide or the like can be used as necessary. If necessary, an alkaline solution can be added.
  • proteins include human serum albumin (BSA), human serum albumin (HSA), human immunoglobulin, and ovalbumin.
  • polyalkyl quaternary amines include: POLYGEAR LILIM CYLAMMONIUM CHROMIDE, POLY
  • fluorescent agent examples include fluorescein, 4-fluorene 7-nitole benzofurazan, and ⁇ -fluoro-4-nitrobenzenezoxa.
  • a conjugate of diazole and amide, amino acid, peptide or protein, or a derivative thereof can be used.
  • aqueous solution for example, an aqueous solution of sodium hydroxide, aqueous hydroxide or the like can be used.
  • luminescence enhancers are used at a concentration of 0.0001 to 10 wt% of the reaction solution.
  • the hydrogen peroxide that can be quantified according to the present invention can quantify not only hydrogen peroxide dissolved in a sample but also hydrogen peroxide that is quantitatively generated by an enzymatic reaction.
  • any of the substances used in the above-mentioned reaction can be quantified.
  • the present invention can be applied to the measurement of hydrogen peroxide or peroxidase activity conventionally performed in the field of diagnostic agents.
  • stoichiometry is performed using enzymes from a substrate in a biological sample.
  • Substrate quantification is performed by generating hydrogen oxide and quantifying it.
  • the enzyme activity in a biological sample is measured by measuring the rate of hydrogen peroxide generated by performing an enzymatic reaction by adding an appropriate enzyme or substrate in order to measure the enzyme activity in the sample. ing.
  • Specific examples thereof include, for example, specific oxidase (eg, glucose oxidase, galactose oxidase, cholesterol oxidase, pericase, etc.). Measurement of the activities of enzymes such as oxidase and cholinesterase can be mentioned.
  • specific oxidase eg, glucose oxidase, galactose oxidase, cholesterol oxidase, pericase, etc.
  • peroxidase is used as a labeling substance, and the enzyme is produced by the peroxidase activity after the antigen-antibody reaction or by the action of this activity.
  • Hydrogen peroxide has been quantified.
  • each method described in the enzyme immunoassay (Eiji Ishikawa et al., 1987, Medical College), for example, an antigen is reacted with an immobilized antibody, and an enzyme such as peroxidase or glucose oxidase is used as the antigen.
  • a method of reacting a labeled antibody and quantifying the activity of the enzyme itself or hydrogen peroxide generated by the action of the enzyme can be mentioned.
  • drugs contained in serum, urine, etc. Hormones or carcinoembryonic antigens (CEA), ⁇ -proteins used as indicators of clinical tests It is possible to measure trace components in the body, such as protein (AFP) and prostatic acid phosphatase (PAP).
  • CEA carcinoembryonic antigens
  • AFP protein
  • PAP prostatic acid phosphatase
  • trace components can be quantified.
  • the compound (I) or (II) is used as a labeling substance, the reaction is carried out by the method of the present invention using the antigen-antibody reactant, and the amount of the antigen or the luminescence is measured by measuring the amount or intensity of luminescence. Antibody can be quantified.
  • the present invention can be applied to a polynucleotide measurement method.
  • the polynucleotide measurement method is complementary to the tested polynucleotide.
  • Labeling a polynucleotide that binds to an enzyme or a compound (I) or (H) such as peroxidase or dalcosoxidase, and labeling the enzyme or the compound that binds to the test polynucleotide complementarily Examples include a method of measuring the enzymatic activity of a polynucleotide or quantifying a labeled compound to determine the amount of a test polynucleotide. The method is described in “DNA Probe” (disc, 1988 edition), “DNA Probe II” (disc, 1990 edition) and the like.
  • the feature of the present invention using the coumarin derivative represented by the formula (I) or ( ⁇ ) is that the range of pH at which light is emitted is as wide as ⁇ 1 to 10; In the acidic to neutral range, a strong luminescence is obtained. Luminol, isolminol, lucigenin, acridinium ester, etc., which have been used in the past, cannot satisfactorily produce luminescence under strong alkaline conditions, and are optimal for enzymes such as peroxidase. When H was less than neutral, detection was difficult. By using the method of the present invention, even when the optimum pH of an enzyme is neutral or lower, it is possible to perform measurement at the optimum pH of the enzyme.
  • Figure 1 shows the changes in the emission intensity of compounds 1, 59 and luminol when using various concentrations of hydrogen peroxide.
  • X—Nore Figure 2 shows the changes in the emission intensity of compounds 10, 12, 14, 23 and luminol when various concentrations of hydrogen peroxide were used.
  • Figure 3 shows the changes in the emission intensity of Compound 1 and luminol at various pH values.
  • Figure 4 shows the changes in the emission intensity of Compound 1 and luminol when various concentrations of peroxidase were used.
  • Figure 5 shows the change in the emission intensity of Compound 1 when various concentrations of CEA were used.
  • FIG. 6 shows the change in the luminescence intensity of Compound 35 when various concentrations of AFP were used.
  • FIG. 7 shows the change in the luminescence intensity of compound 35 when various concentrations of PAP were used. Explanation of reference numerals
  • Figure 8 shows the results obtained for compounds 31 and 35 and acridinium ester (acridinium I: Dojin Chemical Laboratories) using various concentrations of BSA.
  • Example 1 chemiluminescence using a coumarin derivative by peroxidase reaction
  • the coumarin derivative used in the present example showed an emission intensity at pH 6.0 equal to or higher than that of luminol by reacting with peroxidase in the presence of hydrogen peroxide. .
  • the coumarin derivative used showed an emission intensity equal to or higher than that of luminol.
  • Example 3 Intensity of chemiluminescence using coumarin derivative by peroxidase reaction at various pH
  • FIG. 3 shows the ratio of the amount of luminescence at each pH when the luminescence at pH showing the maximum luminescence was 100%.
  • the optimal luminescence pH of Compound 1 is in the acidic region, It was shown to be extremely wide compared to
  • the G0D labeling for the anti-CEA antibody was performed according to the method of Ishikawa et al. (Oxygen immunoassay, page 82, edited by Eiji Ishikawa et al., 1987, published by The Medical College).
  • C—N-succinimidyl 4 -— (N-Maleimide methyl) was used to introduce a maleimide group into the GOD using hexane-111-carboxylate [manufactured by PIERCE].
  • the F (ab ') z fraction obtained by digesting the anti-CEA antibody with pepsin was reduced to prepare F (ab') from which the SH group was released. Both were mixed and reacted to obtain a GOD-labeled anti-CEA antibody.
  • Magnosphere (Magnosphere: trade name) manufactured by Stelogy Bioseparation Co., Ltd .; same hereafter) was used as the magnetic particles for the magnetic particles immobilized with the anti-CEA antibody.
  • An equal amount of a CEA antibody solution was added to the fine particles, sodium borohydride as a coupling reagent was added to a final concentration of 0.1 mol, and the mixture was stirred and reacted for 2 hours. After collecting the particles with a magnet and removing the supernatant by suction, 500 ⁇ l of a phosphate buffer containing 0.1% BSA and 0.1% NaN was added to obtain anti-CEA antibody-immobilized magnetic particles. And saved.
  • the particles were collected by a magnet, the supernatant was removed by suction, and glucose was dissolved at a concentration of 54 mgZml in 1 O miol citrate buffer (01115.0) as a substrate for G0D bound to CEA. After 100 1 was added and the mixture was reacted at 37 ° C. for 30 minutes, 50 mmol of glycine buffer (pH 11) was added as a stop solution. The particles were separated by a magnet, the supernatant 101 of this solution was added to the reagent solution attached to the luminescence photometer, and the integrated luminescence amount for one minute was measured. Figure 5 shows the results.
  • a labeled compound of compound 35 against the anti-AFP antibody was prepared as follows. That is, the carboxyl group of compound 35 and the amino group of the anti-AFP antibody react with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (hereinafter referred to as EDC) at pH 7.0. Then, compound 35 was recognized as anti-AFP antibody.
  • EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide
  • Magnospher was used as the magnetic particles for the anti-AFP antibody-immobilized magnetic particles.
  • An equal volume of an anti-AFP antibody solution dissolved in 0.1 niol phosphate buffer (pH 7.0) is added to the microparticles, and sodium borohydride, a coupling reagent, is added to a final concentration of 0.1.
  • the reaction mixture was stirred to react for 2 hours.
  • the particles were collected with a magnet, the supernatant stored after aspirated off, the-phosphate buffer containing 0. 1% BSA and 0. 1% N a N 3 as 5 0 0 1
  • anti-AFP antibody-immobilized magnetic particles did.
  • a labeled compound 35 against anti-PAP antibody was prepared as follows. That is, 50 mg of compound 35 was dissolved in 10 ml of dioxane, 75 mg of EDC and 50 mg of N-hydroxysuccinic acid imid were added, and the mixture was stirred at room temperature for 24 hours. Concentrated to dryness. The solid was extracted with ethyl acetate water, and the organic layer was concentrated to dryness. This was redissolved in 10 ml of dioxane, 50 mg of -alanine was added, and the mixture was reacted at room temperature for 4 hours, and concentrated to dryness to obtain a solid.
  • the solid was reacted with goat anti-human PAPP antibody at pH 7.0 using EDC and allowed to bind to obtain a compound 35-labeled anti-PAPP antibody.
  • Magnosphere was used as the magnetic particles for the anti-PAP antibody-immobilized magnetic particles.
  • An equal volume of an anti-PAP antibody solution dissolved in 0.1 mol phosphate buffer (pH 7.0) is added to the microparticles, and the final concentration of sodium borohydride, a coupling reagent, is added. 0.1 mol was added, and the mixture was stirred and reacted for 2 hours. The particles were collected with a magnet, the supernatant was removed by suction, and then the anti-PAP solid immobilized magnetic particles were added with 501 in phosphate buffer containing 0.1% BSA and 0.1% NaN. saved.
  • phosphate buffer pH 6.0
  • acridinium ester acridinium I: Dojindo Chemical
  • a slight amount of hydrogen peroxide, a peroxide active substance or a coumarin derivative, or a peroxide is used.
  • Active substances, oxidases, or substances chemically coupled with coumarin derivatives eg, CEA, AFP, PAP, etc.

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Abstract

Procédé de détermination quantitative d'une substance présentant une activité de peroxydation, de peroxyde d'hydrogène ou d'un dérivé de coumarine représentée par les formules générales (I) ou (II), consistant à faire réagir du peroxyde d'hydrogène avec le dérivé ou un sel de celui-ci en présence de la substance active, et à déterminer la quantité ou l'intensité de luminescence du mélange de réaction. Dans ladite formule R?1, R2, R3, R4, R5 et R6¿ peuvent être identiques ou différents et représentent chacun de l'hydrogène, akyle inférieur substitué ou non substitué, arakyle substitué ou non substitué, aryle substitué ou non substitué, halogène ou analogue.
PCT/JP1993/000128 1992-02-04 1993-02-03 PROCEDE DE DETERMINATION QUANTITATIVE D'UNE SUBSTANCE A l'AIDE D'UN DERIVE DE COUMARINE WO1993015219A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP5513100A JP2980681B2 (ja) 1992-02-04 1993-02-03 クマリン誘導体を用いた物質の定量方法

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JP4/19043 1992-02-04
JP1904392 1992-02-04

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000004008A1 (fr) * 1998-07-16 2000-01-27 Gentest Corporation Nouveaux reactifs fluorescents cyp2d a essayer
US6207404B1 (en) 1999-09-30 2001-03-27 Gentest Corporation Coumarin-based CYP3A fluorescent assay reagents
DE102008049675A1 (de) 2008-09-30 2010-04-01 Markus Dr. Heinrich Verfahren zur Herstellung von 3-Aminobiphenylen
US8475776B2 (en) 2005-04-28 2013-07-02 Paloma Pharmaceuticals, Inc. Compositions and methods to treat diseases characterized by cellular proliferation and angiogenesis
US20130171664A1 (en) * 2010-01-29 2013-07-04 Dalibor Sames Ph-responsive fluorescent false neurotransmitters and their use
US9381187B2 (en) 2011-02-16 2016-07-05 Paloma Pharmaceuticals, Inc. Radiation countermeasure agents
USRE46558E1 (en) 2005-04-28 2017-09-26 Paloma Pharmaceuticals, Inc. Compositions and methods to treat diseases characterized by cellular proliferation and angiogenesis
CN111039910A (zh) * 2019-12-31 2020-04-21 云南大学 一种光引发的合成3-芳基黄酮或香豆素类化合物的方法及应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FENXI HUAXUE, Vol. 19, No. 7, (1991), G. XIE et al., "Optimization of Determining Hydrogen Peroxide by Chemiluminescence Detection", pages 823-825. *
J. BIOLUMIN CHEMILUMIN, Vol. 4, No. 1, (1989), D. SLAWINSKA et al., "Hydroxycoumarins as Sensitizers and Reactants of Chemiluminescent Oxidative Reactions", pages 226-230. *
J. CHROMATOGR., Vol. 542, No. 2, (1991), M. TOD et al., "Luminarin 4 as a Labelling Reagent for Carboxylic Acids in Liquid Chromatography With Peroxyoxalate Chemiluminescence Detection", pages 295-306. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000004008A1 (fr) * 1998-07-16 2000-01-27 Gentest Corporation Nouveaux reactifs fluorescents cyp2d a essayer
US6130342A (en) * 1998-07-16 2000-10-10 Gentest Corporation CYP2D fluorescent assay reagents
US6207404B1 (en) 1999-09-30 2001-03-27 Gentest Corporation Coumarin-based CYP3A fluorescent assay reagents
US8475776B2 (en) 2005-04-28 2013-07-02 Paloma Pharmaceuticals, Inc. Compositions and methods to treat diseases characterized by cellular proliferation and angiogenesis
USRE46558E1 (en) 2005-04-28 2017-09-26 Paloma Pharmaceuticals, Inc. Compositions and methods to treat diseases characterized by cellular proliferation and angiogenesis
DE102008049675A1 (de) 2008-09-30 2010-04-01 Markus Dr. Heinrich Verfahren zur Herstellung von 3-Aminobiphenylen
US20130171664A1 (en) * 2010-01-29 2013-07-04 Dalibor Sames Ph-responsive fluorescent false neurotransmitters and their use
US9075014B2 (en) * 2010-01-29 2015-07-07 The Trustees Of Columbia University In The City Of New York pH-responsive fluorescent false neurotransmitters and their use
US9381187B2 (en) 2011-02-16 2016-07-05 Paloma Pharmaceuticals, Inc. Radiation countermeasure agents
CN111039910A (zh) * 2019-12-31 2020-04-21 云南大学 一种光引发的合成3-芳基黄酮或香豆素类化合物的方法及应用

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