CN110325502A

CN110325502A - Compound and method for detecting hydrogen peroxide

Info

Publication number: CN110325502A
Application number: CN201880011943.0A
Authority: CN
Inventors: 杨丹; 叶森; 胡俊
Original assignee: University of Hong Kong HKU
Current assignee: Versitech Ltd
Priority date: 2017-01-20
Filing date: 2018-01-22
Publication date: 2019-10-11
Anticipated expiration: 2038-01-22
Also published as: WO2018133859A1; CN110325502B

Abstract

Method disclosed herein is compound and for using the compound in situ detection hydrogen peroxide.The compound may include the compound or its salt of formula (I), formula (II) or formula (III), wherein X¹For fluorogen before unit price or preceding illuminophore part；X²It is fluorogen or preceding illuminophore part before divalent；Each of A and A' are independently indicated by formula (IV) or formula (V):

Description

Compound and method for detecting hydrogen peroxide

Technical field

The invention belongs to fluorescence or luminescence probe fields, and are related to compound and the method for detecting hydrogen peroxide.

Background of invention

Reactive oxygen species (ROS) and reactive nitrogen substance (RNS) and aging, inflammation and a variety of disorders such as cancers and glycosuria The development of disease is related.Hydrogen peroxide (H₂O₂) it is to play a crucial role in oxidative stress and in terms of signal transduction in organism anti- Answering property oxygen species.In order to study the reaction mechanism of hydrogen peroxide, the analysis side using chemiluminescence and fluorescence probe has been developed Method, to detect its intracellular generation.These probes can be effectively used for the signal transduction of research oxidative stress and various pathology.

There is the commercially available fluorescence probe tested and analyzed for hydrogen peroxide, such as Amplex Red reagent has been used for detecting Release of the hydrogen peroxide from the human leukocytes of activation.However, many commercially available fluorescence probes have such as slow response time And the disadvantages of sensitivity, so that in-situ tracking hydrogen peroxide is difficult.

Summary of the invention

Since it is desired that developing new chemiluminescence and/or fluorescence probe to monitor the oxidative stress of various pathology and signal and pass It leads, there is provided herein compound and for using the method that can be used for the compound of in situ detection hydrogen peroxide.At at least one In specific embodiment, compound may include the compound or its salt of formula (I), formula (II) or formula (III):

Wherein, each of A and A' are independently indicated by formula (IV) or formula (V):

Wherein, the connection between the compound and triphenyl phosphonium part has lower formula (VI) or (VII):

Wherein, n=1-10；Or compound and morpholine or N, N- bis- replace the connection between amine moiety to have lower formula (VIII) Or (IX):

It in another embodiment, the use of the method for chemiluminescence and/or fluorescent chemicals may include making institute Compound is stated to be contacted with sample to form fluorescence or luminophor；With the fluorescence or hair of the determination fluorescence or luminophor Light property.

Brief description

In following detailed description, exemplary, non-limiting and exhaustive embodiment party of the invention is depicted with reference to attached drawing Case.Therefore, it in a manner of features described above of the invention can be understood in detail, can be obtained by reference to embodiment simple above The more specific description of the invention to be summarized, some of embodiments are shown in the accompanying drawings.It is noted, however, that Attached drawing illustrates only typical embodiments of the invention, and is therefore not considered limiting of its scope, because of the invention It can recognize other equally valid embodiments.

Fig. 1 is the reaction mechanism between the YS-4-45 and hydrogen peroxide proposed.

Fig. 2A is the figure for showing the fluorescence intensity of compound YS-3-42 and changing with the concentration increase of hydrogen peroxide.In Fig. 2A In, curve respectively indicates from top to bottom :+100 μM of H of 10 μM of solution of compound YS-3-42₂O₂, 10 μM of compound YS-3-42 + 50 μM of H of solution₂O₂, compound YS-3-42+40 μM of H of 10 μM of solution₂O₂, 10 μM+30 μM of solution of compound YS-3-42 H₂O₂, compound YS-3-42+20 μM of H of 10 μM of solution₂O₂, compound YS-3-42+10 μM of H of 10 μM of solution₂O₂, compound + 8 μM of H of 10 μM of solution of YS-3-42₂O₂, compound YS-3-42+6 μM of H of 10 μM of solution₂O₂, compound YS-3-42 10 μ + 4 μM of H of M solution₂O₂, compound YS-3-42+2 μM of H of 10 μM of solution₂O₂Only 10 μM of solution of compound YS-3-42.Figure 2B is the figure for showing the fluorescence intensity of YS-3-42 in the presence of different ROS/RNS.

Fig. 3 A is the figure for showing the fluorescence intensity of compound YS-4-45 and changing with the concentration increase of hydrogen peroxide.In Fig. 3 A In, curve respectively indicates from top to bottom :+100 μM of H of 10 μM of solution of compound YS-4-45₂O₂, 10 μM of compound YS-4-45 + 50 μM of H of solution₂O₂, compound YS-4-45+40 μM of H of 10 μM of solution₂O₂, 10 μM+30 μM of solution of compound YS-4-45 H₂O₂, compound YS-4-45+20 μM of H of 10 μM of solution₂O₂, compound YS-4-45+10 μM of H of 10 μM of solution₂O₂, compound + 8 μM of H of 10 μM of solution of YS-4-45₂O₂, compound YS-4-45+4 μM of H of 10 μM of solution₂O₂Only compound YS-4-45 10 μM of solution.Fig. 3 B is the figure for showing the fluorescence intensity of YS-4-45 in the presence of different ROS/RNS.

The fluorescence intensity that Fig. 4 A shows compound YS-2-172 increases the figure of variation with the concentration of hydrogen peroxide.In Fig. 4 A In, curve respectively indicates from top to bottom :+500 μM of H of 10 μM of solution of compound YS-2-172₂O₂, compound YS-2-172 10 μM+100 μM of H of solution₂O₂Only 10 μM of solution of compound YS-2-172.Fig. 4 B is the presence shown in different ROS/RNS The figure of the fluorescence intensity of lower YS-2-172.

Fig. 5, which is shown, is being with or without phorbol 12-myristinate 13- acetic acid esters (PMA) (200ng/mL) and DPI In the case where (100nM) with the total focused image (left side) of YS-3-42 (10 μM) the RAW264.7 cell being incubated for altogether.To in each group The relative mean fluorescent intensity of cell carry out quantitative (right side).Scale bar represents 10 μm.Data are average value ± s.e.m., n= 79-109 cell；* *, relative to untreated cell or control P < 0.001.

Fig. 6 shows the Confocal Images (left side) of RAW264.7 (normal cell) and is incubated for altogether with YS-3-42 (10 μM) The Confocal Images (right side) of MDA-MB-231 (breast cancer cell) cell.To the relative mean fluorescent intensity of the cell in each group It is quantified.Scale bar represents 10 μm.Data are average value ± s.e.m., n=72-109 cell；*, P < 0.01.

Fig. 7 shows the cytotoxicity of probe YS-3-42 and YS-4-45.

Fig. 8, which is shown, is being with or without phorbol 12-myristinate 13- acetic acid esters (PMA) (200ng/mL) and DPI In the case where (100nM) with the total focused image of YS-4-45 (10 μM) the RAW264.7 cell being incubated for altogether.Scale bar represents 10 μ m。

Fig. 9 A is the figure for showing the fluorescence intensity of compound YS-4-112 and changing with the concentration increase of hydrogen peroxide.In Fig. 9 A In, curve respectively indicates from top to bottom :+100 μM of H of 10 μM of solution of compound YS-4-112₂O₂, compound YS-4-112 10 μM+50 μM of H of solution₂O₂, compound YS-4-112+30 μM of H of 10 μM of solution₂O₂, compound YS-4-112 10 μM of solution+ 20μM H₂O₂, compound YS-4-112+10 μM of H of 10 μM of solution₂O₂, compound YS-4-112+8 μM of H of 10 μM of solution₂O₂、 + 6 μM of H of 10 μM of solution of compound YS-4-112₂O₂, compound YS-4-112+4 μM of H of 10 μM of solution₂O₂, compound YS- + 2 μM of H of 10 μM of solution of 4-112₂O₂Only 10 μM of solution of compound YS-4-112.Fig. 9 B is for quantitative standard H₂O₂ Calibration curve.

Figure 10 A is shown and the total focused view of YS-4-112 (10 μM) the work zebra fish in the different stages of development being incubated for altogether Picture.Figure 10 B shows the Confocal Images of the work zebra fish for the PMA processing being incubated for altogether with YS-3-42 (10 μM).Scale bar represents 500μm。

Figure 11 A shows the H of ascorbic acid₂O₂Scavenging capacity.Figure 11 B is shown in the high throughput test using YS-4-112 The H of Epigallo-catechin gallate (EGCG)₂O₂Scavenging capacity.

Detailed description of the invention

Compound disclosed herein can have quick response, and be to be used for original position, detected in vivo and in vitro The probe of the high selectivity of hydrogen oxide.The compound can have the machine that new sum is effectively used for hydrogen peroxide detection Reason.Sensitivity and selectivity to hydrogen peroxide can be continuously improved in they, and avoids the interference from cell ROS/RNS.

Performance of these compounds in terms of hydrogen peroxide imaging includes normal cell and cancer cell in various kinds of cell type In be height it is steady.In addition, these compounds can be used for exploitation for drug screening, screening for cancer and medical diagnosis on disease Quickly, accurately or high-throughput detection method.

The compound can include but is not limited to the compound or its salt of formula (I), formula (II) or formula (III):

Wherein:

R¹、R²、R³、R⁴And R⁵Independently selected from H, F, Cl, Br, I, CN, alkyl, halogenated alkyl, miscellaneous alkyl, alkenyl, alkynes Base, aralkyl, aryl, alkaryl, heterocycle, naphthenic base, cycloalkenyl, cycloalkynyl radical, hydroxyalkyl, aminoalkyl, amino, alkyl ammonia Base, arylamino, dialkyl amido, alkyl aryl amino, ammonia diaryl base, acyl amino, hydroxyl, mercaptan, alkylthio, alkane Oxygroup, alkylthio group, alkoxyalkyl, aryloxy group, alkoxy aryl, acyloxy, nitro, carbamoyl, trifluoromethyl, benzene oxygen Base, benzyloxy, phosphonic acids, phosphate, sulfonic acid (- SO₃H), sulphonic acid ester, sulfonamide ,-C (=O)-P¹With-C (=O)-M-P², wherein P¹And P²Selected from hydrogen, halogen, alkoxy, hydroxyl, mercaptan, alkyl, alkenyl, alkynyl, naphthenic base, cycloalkenyl, cycloalkynyl radical, aryl, Alkaryl, aralkyl, carbamate groups, amino, alkyl amino, arylamino, dialkyl amido, alkyl aryl amino, two Arylamino, alkylthio group, miscellaneous alkyl, alkyl triphenyl phosphonium and heterocycle or R with 3 to 7 annular atoms²And R³It is formed together 5,6 or 7 member rings or R selected from aryl, heterocycle, heteroaryl and heteroaromatic group⁴And R⁵It is formed together selected from aryl, heterocycle 5,6 or 7 member rings of base, heteroaryl and heteroaromatic group,

M is selected from alkylidene, alkenylene, alkynylene, arlydene, sub- aralkyl and alkarylene；

R⁶Selected from hydrogen, alkyl, alkoxyalkyl, alkanoyl ,-CF₃, halogen replace low alkyl group and (C=O)-O-Z¹, Wherein, Z¹It is the base for being selected from alkyl, alkenyl, alkynyl, naphthenic base, cycloalkenyl, cycloalkynyl radical, aryl, alkaryl and aryl alkyl Group；

X¹It is fluorogen or preceding illuminophore part before unit price；

X²It is fluorogen or preceding illuminophore part before divalent；

Each of A and A' are independently indicated by formula (IV) or formula (V):

Wherein, R⁷And R⁹At least one of be (C=O)-W¹, wherein W¹Selected from hydrogen, alkyl, alkoxyalkyl, alkane acyl Base, CF₃, halogen replace low alkyl group and (C=O)-O-Z², wherein Z²It is selected from alkyl, alkenyl, alkynyl, naphthenic base, ring Alkenyl, cycloalkynyl radical, aryl, alkaryl or aryl alkyl group and R⁷And R⁹Another, R⁸、R¹⁰And R¹¹Independently selected from H, F, Cl, Br, I, CN, alkyl, halogenated alkyl, miscellaneous alkyl, alkenyl, alkynyl, aralkyl, aryl, alkaryl, heterocycle, ring Alkyl, cycloalkenyl, cycloalkynyl radical, hydroxyalkyl, aminoalkyl, amino, alkyl amino, arylamino, dialkyl amido, alkylaryl Amino, ammonia diaryl base, acyl amino, hydroxyl, mercaptan, alkylthio, alkoxy, alkylthio group, alkoxyalkyl, aryloxy group, Alkoxy aryl, acyloxy, nitro, carbamoyl, trifluoromethyl, phenoxy group, benzyloxy, phosphonic acids, phosphate, sulfonic acid (- SO₃H), sulphonic acid ester, sulfonamide ,-C (=O)-P³With-C (=O)-M-P⁴, wherein P³And P⁴Independently selected from hydrogen, halogen, alcoxyl Base, hydroxyl, mercaptan, alkyl, alkenyl, alkynyl, naphthenic base, cycloalkenyl, cycloalkynyl radical, aryl, alkaryl, aryl alkyl, amino Formic acid esters, amino, alkyl amino, arylamino, dialkyl amido, alkyl aryl amino, ammonia diaryl base, alkylthio group, miscellaneous alkane Base, alkyl triphenyl phosphonium or the heterocycle with 3 to 7 annular atoms；

M is alkylidene, alkenylene, alkynylene, arlydene, sub- aralkyl or alkarylene；

R⁸And R¹⁰At least one of preferably hydroxyl, alkoxy or be selected from amino, alkyl amino, arylamino, dioxane Base amino, alkyl aryl amino, ammonia diaryl base electron donating group；

Or R⁷And R⁸It is formed together 5,6 or 7 member rings selected from aryl, heterocycle, heteroaryl or heteroaromatic group；

Or R¹⁰And R¹¹It is formed together 5,6 or 7 member rings selected from aryl, heterocycle, heteroaryl or heteroaromatic group.

Fluorogen and/or preceding illuminophore part can include but is not limited to before unit price: monovalent fluorescein (fluorescein) (CAS 2321-07-5), monovalent cumarin (CAS 91-64-5), monovalent amine naphthalimide, monovalent dansyl, unit price two Amine (bimane) (CAS 79769-56-5), monovalent eosin, monovalent rhodamine (CAS 81-88-9；989-38-8；62669- 70-9), monovalent paramethylaminophenol (CAS 3086-44-0), monovalent cyanine, monovalent Nile red (CAS 7385-67-3), unit price Xanthone (CAS 90-47-1), monovalent xanthene (CAS 92-83-1), unit price flazo orange (CAS 3566-94-7), unit price SNARF-1, monovalent fluorescein (CAS 71206-95-6；67769-47-5), unit price 6- dodecanoyl-N, N- dimethyl -2- naphthalene Amine (laurdan) (CAS 74515-25-6), unit price 2- naphthylamines (CAS 91-59-8), monovalent resorufin (CAS 635-78- And monovalent fluorescein (luciferin) (such as CAS 2591-17-5) 9).

Fluorogen or/and preceding illuminophore part can include but is not limited to before divalent: divalent fluorescein (CAS 2321- 07-5), divalent cumarin (CAS 91-64-5), divalent amines naphthalimide, divalent dansyl, divalent diamines (No. CAS 79769-56-5), divalent eosin, divalent rhodamine (CAS 81-88-9；989-38-8；62669-70-9), divalent is to first ammonia Base phenol (CAS 3086-44-0), divalent cyanine, divalent Nile red (CAS 7385-67-3), divalent xanthone (CAS 90- 47-1), divalent xanthene (CAS 92-83-1), divalent flazo orange (CAS 3566-94-7), divalent SNARF-1, divalent fluorescence Huang (CAS 71206-95-6；67769-47-5), divalent 6- dodecanoyl-N, N- dimethyl -2- naphthylamines (CAS 74515- 25-6), divalent 2- naphthylamines (CAS 91-59-8), divalent resorufin (CAS 635-78-9) and divalent fluorescein (such as No. CAS 2591-17-5)。

The compound can include but is not limited to compound 1-28 and 57:

Wherein, R=H or CF₃。

The compound can include but is not limited to compound 29-44:

Wherein, R=H or CF₃。

The compound may include but be not limited to one or more free carboxyl groups, wherein at least one carboxyl base Morpholine or N of the group by amide key connection and positively charged Mitochondrially targeted triphenyl phosphonium part or lysosome targeting, N- Disubstituted amine moiety conjugation, wherein the connection between the compound and triphenyl phosphonium part have lower formula (VI) or (VII):

Wherein n=1-10；

Or the compound and morpholine or N, N- bis- replace the connection between amine moiety to have lower formula (VIII) or (IX):

Wherein, n=1-10；R in formula (IX)¹²Or R¹³It independently is C_1-10Alkyl or alkene.

The compound can include but is not limited to compound 45-56:

Wherein, R=H or CF₃。

As used herein, term " alkyl " includes the saturated aliphatic hydrocarbon containing straight chain and branch.In some realities It applies in scheme, alkyl group has 1 to 20 carbon atom, 1 to 10 carbon atom, 1 to 6 carbon atom or 1 to 4 carbon atom. For example, term " C_1-6Alkyl ", and the moieties of other groups being mentioned above are (for example, C_1-6Alkoxy) refer to 1 to 6 The linear chain or branched chain group of carbon atom is (for example, methyl, ethyl, n-propyl, isopropyl, normal-butyl, isobutyl group, sec-butyl, tertiary fourth Base, n-pentyl or n-hexyl).For another example, term " C_1-4Alkyl " refers to the linear chain or branched chain of 1 to 4 carbon atom Aliphatic hydrocarbon chain；Term " C_1-3Alkyl " refers to the aliphatic hydrocarbon chain of the linear chain or branched chain of 1 to 3 carbon atom；Term " C_1-2Alkane Base " refers to the aliphatic hydrocarbon chain of the linear chain or branched chain of 1 to 2 carbon atom；And term " C₁Alkyl " refers to methyl.Term is " low Grade alkyl " refers to the linear chain or branched chain group of 1 to 6 carbon atom.Alkyl group optionally can be by one or more (such as 1 to 5 It is a) suitably substituent group substitution.

As it is used herein, term " alkenyl " includes the aliphatic hydrocarbon at least one carbon-carbon double bond, including tool There are the straight chain and branch of at least one carbon-carbon double bond.In some embodiments, kiki alkenyl group has 2 to 20 carbon atoms, 2 To 10 carbon atoms, 2 to 6 carbon atoms, 3 to 6 carbon atoms or 2 to 4 carbon atoms.For example, as used herein, term “C_2-6Alkenyl " means the unsaturated group (having at least one carbon-carbon double bond) of the linear chain or branched chain of 2 to 6 carbon atoms, packet It includes but is not limited to: vinyl, 1- acrylic, 2- acrylic (allyl), isopropenyl, 2- methyl-1-propylene base, 1- butylene Base, 2- cyclobutenyl etc..Kiki alkenyl group can optionally be replaced by one or more (such as 1 to 5) suitable substituent group.When When compound contains kiki alkenyl group, which, which can be used as pure E type, pure Z-type or its any mixture, exists.

As used herein, term " alkynyl " includes the aliphatic hydrocarbon at least one triple carbon-carbon bonds, including tool There are the straight chain and branch of at least one triple carbon-carbon bonds.In some embodiments, alkynyl group has 2 to 20,2 to 10,2 To 6 or 3 to 6 carbon atoms.For example, as used herein, term " C_2-6Alkynyl " refer to 2 to 6 carbon atoms as above Defined linear chain or branched chain hydrocarbon chain alkynyl group.Alkynyl group can be optionally suitable by one or more (such as 1 to 5) Substituent group replace.

As used herein, term " naphthenic base " includes saturated or unsaturated non-aromatic monocyclic or polycyclic (as bicyclic) Hydrocarbon ring is (for example, monocycle such as cyclopropyl, cyclobutyl, cyclopenta, cyclohexyl, suberyl, cyclooctyl, cyclononyl or bicyclic including spiral shell Ring, condensed or bridge joint system (such as bicyclic [1.1.1] amyl, bicyclic [2.2.1] heptane base, bicyclic [3.2.1] octyl or bicyclic [5.2.0] nonyl, decahydro naphthalene etc.).The group of naphthene base can have 3 to 15 carbon atoms.In some embodiments, ring Alkyl is optionally containing the non-accumulated non-aromatic double or triple bonds of one, two or more and/or 1 to 3 oxo group. In some embodiments, bicycloalkyl radicals have 6 to 14 carbon atoms.For example, term " C_3-14Naphthenic base " includes 3 to 14 The saturation of ring carbons or unsaturated non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon ring are (for example, cyclopropyl, cyclobutyl, ring Amyl, cyclohexyl, bicyclic [1.1.1] amyl or cyclodecyl)；With term " C_3-7Naphthenic base " includes 3 to 7 ring carbons Saturated or unsaturated non-aromatic monocyclic is polycyclic (such as bicyclic) (for example, cyclopropyl, cyclobutyl, cyclopenta, cyclohexyl, bicyclic [1.1.1] amyl- 1- base or bicyclic [1.1.1] amyl- 2- yl).For another example, term " C_3-6Naphthenic base " includes 3 to 6 The saturation of ring carbons or unsaturated non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon ring.For another example, term " C_3-4 Naphthenic base " is cyclopropyl or cyclobutyl.Being also included in term " naphthenic base " is to be fused to cycloalkanes with one or more The part of the aromatic ring (including aryl and heteroaryl) of basic ring, for example, the benzo or thiophene of pentamethylene, cyclopentene, hexamethylene etc. Radical derivative (for example, 2,3- dihydro -1H- indenes -1- bases or 1H- indenes -2 (3H) -one -1- base).Group of naphthene base can be optionally Replaced by one or more (for example, 1 to 5) suitable substituent group.

As used herein, term " aryl " may include all carbon monocycles or thick with conjugated pi electron system Ring polycyclic aromatic group.Aryl group has 6 or 10 carbon atoms in ring (multiple rings).Most commonly, aryl group is in ring In have 6 carbon atoms.For example, as used herein, term " C_6-10Aryl " refers to the virtue containing 6 to 10 carbon atoms Race's group, such as phenyl or naphthyl.Aryl group is optionally taken by one or more (for example, 1 to 5) suitable substituent group Generation.Term " arlydene " refers to divalent aryl part.

As used herein, term " heteroaryl " includes having one or more heteroatom ring members' (ring member nitrogen atoms) Monocycle or condensed ring polycyclic aromatic heterocyclic group, the one or more heteroatom ring members described at least one ring independently select From O, S and N.Heteroaryl groups have 5 to 14 ring member nitrogen atoms, including 1 to 13 carbon atom and 1 to 8 selected from O, S and N Hetero atom.In some embodiments, heteroaryl groups have 5 to 10 ring member nitrogen atoms, including one to four hetero atom.Heteroaryl Base group can also contain one to three oxo or thion (i.e.=S) group.In some embodiments, heteroaryl groups have 5 to 8 ring member nitrogen atoms include one, two or three hetero atom.For example, term " 5 unit's heteroaryl " refers to as described above in list With the single ring heteroaryl group of 5 ring member nitrogen atoms in ring heteroaryl ring；Term " 6 unit's heteroaryl " is included in bicyclic heteroaryl ring In with 6 ring member nitrogen atoms single ring heteroaryl group as defined above；And term " 5 or 6 unit's heteroaryl " is included in monocycle With the single ring heteroaryl group as defined above of 5 or 6 ring member nitrogen atoms in heteroaryl ring.For another example, term " 5 or 10 unit's heteroaryl " includes the institute as above in monocycle or bicyclic heteroaryl ring with 5,6,7,8,9 or 10 ring member nitrogen atoms The monocycle or bicyclic heteroaryl group of definition.Heteroaryl groups optionally can suitably be taken by 1 or multiple (for example, 1 to 5) Replace for base.The example of bicyclic heteroaryl includes with those of 5 ring member nitrogen atoms (including 1-3 hetero atom) or with 6 Those of ring member nitrogen atoms (including one, two or three nitrogen heteroatom).The example of condensed bicyclic heteroaryl includes two thick The 5 and/or 6 unit monocycle rings (including one to four hetero atom) closed.

As used herein, term " heterocycle " include saturation and fractional saturation have 5 to 15 selected from carbon, The ring members of nitrogen, sulphur and oxygen contain heteroatomic annular group, and wherein at least one annular atom is hetero atom.Heterocyclyl groups can To contain one, two or three ring, wherein this ring can be connected in a manner of pendency or can be condensed.Saturated heterocyclyl The example of group includes 3 to 6 containing 1 to 4 nitrogen-atoms yuan heteromonocyclic group group [such as pyrrolidinyl, imidazolidinyl, the piperazine of saturation Piperidinyl, piperazinyl etc.]；Saturation containing 1 to 2 oxygen atom and 1 to 3 nitrogen-atoms 3 to 6 yuan of heteromonocyclic group groups [such as Quinoline base etc.]；3 to 6 yuan of heteromonocyclic group groups of the saturation containing 1 to 2 sulphur atom and 1 to 3 nitrogen-atoms are [for example, thiazolidinyl Deng].The example of the heterocyclyl groups of fractional saturation includes dihydro-thiophene, dihydropyran, dihydrofuran and thiazoline.Heterocycle The non-limiting example of group includes 2- pyrrolinyl, 3- pyrrolinyl, pyrrolidinyl, 1,3-dioxolane base, 2H- pyrans Base, 4H- pyranose, piperidyl, 1,4- dioxanes base, morpholinyl, 1,4- dithianyl, thio-morpholinyl etc..

As used herein, term " alkoxy " or " alkyl oxy " include-O- alkyl group.For example, term " C_1-6Alcoxyl Base " or " C_1-6Alkyl oxy " includes-O- (C_1-6Alkyl) group；With term " C_1-4Alkoxy " or " C_1-4Alkyl oxy " can wrap Include-O- (C_1-4Alkyl) group.For another example, term " C_1-2Alkoxy " or " C_1-2Alkyl oxy " refers to-O- (C_1-2Alkane Base) group.The example of alkoxy includes methoxyl group, ethyoxyl, propoxyl group (such as positive propoxy and isopropoxy), tert-butoxy Deng.The alkoxy or alkyl oxy group can optionally be replaced by the suitable substituent group of 1 or multiple (for example, 1 to 5).

As used herein, term " C_6-10Aryloxy group " includes-O- (C_6-10Aryl) group.C_6-10Aryloxy group Example is-O- phenyl [that is, phenoxy group].C_6-10Aryloxy group can be closed optionally by one or more (for example, 1 to 5) Suitable substituent group replaces.

As used herein, term " aminoalkyl " includes straight chain and/or branched alkyl base with 1 to about 10 carbon atom Group, any one of described 1 to about 10 carbon atom can be replaced by one or more amino groups.The example packet of this kind of group Include amino methyl, amino-ethyl, aminopropyl, aminobutyl and Aminohexyl.

As it is used herein, " oxo " refers to=O.When oxo replaces on carbon atom, they are formed together carbonyl Partially [- C (=O) -].When oxo replaces on sulphur atom, they are formed together sulfinyl moiety [- S (=O) -]；When two For a oxo group when replacing on sulphur atom, they are formed together sulfonyl moieties [- S (=O)₂-]。

As used herein, term " optionally replace " refers to that substitution is optional, and therefore includes unsubstituted and take The atom in generation and part.The atom of " substitution " or part indicate that any hydrogen on specified atom or part can be with selected from finger (each hydrogen atom at most on specified atom or part is taken with selected from specified substituent group for fixed substituent group substitution Generation), condition is no more than specified atom or partial normal chemical valence, and replaces and obtain stable compound.For example, such as Fruit methyl group is (that is, CH₃) optionally replace, then for up to 3 hydrogen atoms on carbon atom can be taken with substituent group Generation.

The compound is used as the reagent for measuring, detecting and/or screening hydrogen peroxide.These compounds can be with Generate fluorescence or luminous color, such as blue, green, yellow, red or remote red.These compounds can be used for directly or The amount of the presence of hydrogen peroxide and/or hydrogen peroxide in earthmeter chemical example, biological sample and pathological sample.The chemical combination Object can be used for the level of presence that is in situ, detecting hydrogen peroxide in vivo and in vitro or determining hydrogen peroxide.

Use the horizontal side of hydrogen peroxide in the presence of hydrogen peroxide in the compound test sample and/or determining sample Method can include but is not limited to: contact the compound or its salt of formula (I), formula (II), formula (III) with sample to form fluorescence And/or luminophor；With the fluorescence and/or luminosity of the determination fluorescence or luminophor.

It is used to detect the presence of the internal hydrogen peroxide of organism using the compound or determines the internal mistake of organism The horizontal method of hydrogen oxide can include but is not limited to: give the compound or its salt of formula (I), formula (II), formula (III) to life Object is to form fluorescence and/or luminophor；With the fluorescence and/or photism of the determination fluorescence and/or luminophor Matter.

Using the compound for detecting the presence of hydrogen peroxide in vitro or determining the horizontal method of hydrogen peroxide It can include but is not limited to: giving the compound or its salt of formula (I), formula (II), formula (III) to external sample to form fluorescence And/or luminophor；With the fluorescence of the determination fluorescence and/or luminophor and/or shine.

Use the horizontal high pass of hydrogen peroxide in the presence of hydrogen peroxide in the compound test sample or measurement sample Amount method can include but is not limited to: contact the compound or its salt of formula (I), formula (II), formula (III) with sample to form one Kind or a variety of fluorescence or luminophor；With the fluorescence and/or luminosity of the determination fluorescence and/or luminophor with true The amount of the presence of hydrogen peroxide and/or hydrogen peroxide in random sample product.

It is used to screen the one or more target compounds for increasing or decreasing hydrogen peroxide level using the compound High throughput method can include: contact the compound or its salt of formula (I), formula (II), formula (III) with target compound to form one Kind or a variety of fluorescence or luminophor；With the fluorescence or luminosity for measuring the fluorescence or luminophor, to determine mesh Mark the presence of compound and/or the amount of target compound.

Sample for any method using the compound can include but is not limited to chemical example, biological sample and Pathological sample.Biological sample can include but is not limited to microorganism, cell, tissue, organ, the part from plant or animal, Entire plant or animal and their extract.Pathological sample can include but is not limited to blood, urine, saliva, serum, breathing Gas, Exhaled breath condensate, joint fluid and their extract.

Method using compound may include being used for compound in fluorescence and/or luminescence probe composition.Fluorescence and/ Or luminescence probe composition can include but is not limited to one or more carriers, one or more solvents, one or more acid, one Kind or a variety of alkali, one or more buffers and its mixture.

In some embodiments, the present invention relates to the compound of the present invention or fluorescence of the invention or luminescence probe to combine Object is for the presence of hydrogen peroxide in the internal of organism or in vitro test sample and/or the water of determining wherein hydrogen peroxide Flat purposes；Or the compound of the present invention or fluorescence of the invention or luminescence probe composition increase or decrease peroxide for screening Change the purposes of horizontal one or more target compounds of hydrogen.

Material and method

Embodiment described herein being for illustration purposes only with embodiment, and it will suggest it to those skilled in the art Various modifications or change and various modifications or change include in spirit and scope.In addition, disclosed herein Any element or limitation of what invention or its embodiment can be with disclosed herein any and/or every other element or limitations (individually or with any combination) or its any other invention or combination of embodiment, and all these combinations are it is contemplated that this hair Bright range but not limited to this range.

Embodiment 1- green fluorescence compound YS-3-42, YS-4-45, YS-2-172 and red fluorescence compound YS-4- 112 synthesis

It is stirred under argon gas at 0 DEG C to the DMF (2mL, anhydrous) of NaH (60wt%, 44mg, 1.10mmol in mineral oil) DMF (2mL, the anhydrous) solution of 2- hydroxyl -4- (methylol) benzaldehyde (152mg, 1.00mmol) is added in solution.15 points of stirring Zhong Hou is added dropwise chloromethyl methyl ether (91 μ L, 1.20mmol), is then stirred for 30 minutes.Then it is diluted with ethyl acetate anti- Mixture is answered, with 1N HCl, water and salt water washing.Organic layer is dry with anhydrous magnesium sulfate, and be concentrated in a vacuum.By fast Compound YS-3-1 is separated into light yellow oil on silica gel by fast chromatography, uses ethyl acetate: hexane (1:1) is as washing De- agent.Yield: 174mg (89%).¹H NMR(300MHz,CDCl₃) δ 10.39 (s, 1H), 7.75 (d, J=8.0Hz, 1H), 7.19 (s, 1H), 7.00 (d, J=8.0Hz, 1H), 5.27 (s, 2H), 4.69 (s, 2H), 3.48 (s, 3H)；¹³C NMR(75MHz, CDCl₃)δ189.7,159.9,150.1,128.6,124.4,119.7,112.6,94.5,64.4,56.6。

To YS-3-1 (130mg, 0.662mmol) and carbamoylimidazole (100mg, 0.796mmol) DMF (4mL, It is anhydrous) NaH (60wt%, 30mg, 0.728mmol in mineral oil) is added in agitating solution.Suspension is stirred at room temperature It 18 hours, is then concentrated under air stream.Compound YS-3-2 is separated into white sticky on silica gel by flash chromatography Solid uses ethyl acetate: hexane (1:1) is as eluant, eluent.Yield: 54mg (32%).

¹H NMR(300MHz,CDCl₃) δ 10.41 (s, 1H), 7.75 (d, J=7.9Hz, 1H), 7.13 (s, 1H), 6.99 (d, J=7.9Hz, 1H), 5.25 (s, 2H), 5.07 (s, 2H), 3.47 (s, 3H), 2.77 (d, J=4.9Hz, 3H)；¹³C NMR (75MHz,CDCl₃)δ189.4,159.7,156.7,145.4,128.6,124.8,120.6,113.7,94.6,65.7,56.6, 27.6。

It is added into methanol (4mL) solution of YS-3-3 (200mg, 0.478mmol) NaOH (191mg, 4.78mmol) Water (2mL) solution.Obtained mixture is stirred at room temperature 1 hour, organic solvent is then evaporated, with 1N HCl neutralize until Form a large amount of precipitatings.Suspension is filtered, be washed with water and is dried in a vacuum, to obtain thick fluorescein derivative.In argon gas Under at room temperature to the DMF of crude product (5mL) solution be added Et₃N (201 μ L, 1.43mmol).It stirs after ten minutes, by PhNTf₂ In the mixture that (205mg, 0.574mmol) is added to, it is stirred at room temperature for 2 hours.It is anti-with ethyl acetate dilution Mixture is answered, with 1N HCl, water and salt water washing.Organic layer is dry with anhydrous magnesium sulfate, and be concentrated in a vacuum, to obtain Crude product fluorescein trifluoromethayl sulfonic acid ester derivant.It is added at room temperature into the DMF of crude product (5mL) solution under argon gas Cs₂CO₃(188mg, 0.526mmol) is added MeI (60 μ L, 0.956mmol) after stirring 30 minutes.Obtained mixture is stirred It mixes 2 hours, is then diluted with ethyl acetate, with 1N HCl, water and salt water washing.Organic layer is dry with anhydrous magnesium sulfate, and It is concentrated in a vacuum.Compound YS-3-9 is separated into white sticky solid on silica gel by flash chromatography, uses acetic acid Ethyl ester: hexane (1:4) is used as eluant, eluent.Yield: 77mg (31%).

¹H NMR(400MHz,CDCl₃) δ 8.69 (s, 0.5H), 8.36 (d, J=8.0Hz, 0.5H), 8.31 (d, J= 8.0Hz, 0.5H), 8.10 (d, J=8.0Hz, 0.5H), 7.81 (s, 0.5H), 6.96 (d, J=8.8Hz, 1H), 6.88 (d, J= 8.8Hz,1H),6.81(s,1H),6.72–6.63(m,2H),3.99(s,1.5H),3.89(s,1.5H),3.84(s,3H)。

¹³C NMR(100MHz,CDCl₃)δ168.02,165.42,161.97,156.46,152.91,151.89, 150.26,136.86,136.53,132.80,131.54,130.12,130.08,129.75,129.03,128.98,127.06, 126.82,125.58,125.29,124.27,119.18,116.98,112.77,110.78,101.19,101.16,82.06, 81.78,55.80,52.93。¹⁹F NMR(376MHz,CDCl₃)δ–72.6(m,3F)。

To being packed into Pd in the round-bottomed flask of oven drying₂(dba)₃(12mg, 0.013mmol), Xantphos (23mg, 0.039mmol) and Cs₂CO₃(59mg, 0.182mmol), and with argon cleaning 5 minutes.It is added YS-3-9 (70mg, 0.13mmol) With anhydrous dioxanes (5mL) solution of YS-3-2 (40mg, 0.156mmol), and gained mixture is stirred under argon gas in room temperature It mixes 30 minutes, be heated to 100 DEG C and be stirred for 20 hours.It is cooled to room temperature reaction mixture, diluted with DCM and passes through diatom Soil pad filtering.Then filtrate is concentrated in a vacuum.Compound YS-3-36 is separated on silica gel by flash chromatography white Color viscous solid is used as eluant, eluent by using EtOAc:DCM (1:5).Yield: 38mg (46%).

¹H NMR(400MHz,CDCl₃)δ10.44(s,1H),8.69(s,0.5H),8.37–8.32(m,0.5H),8.31– 8.27 (m, 0.5H), 8.09 (d, J=8.0Hz, 0.5H), 7.82 (s, 0.5H), 7.80 (d, J=8.0Hz, 1H), 7.25-7.23 (m,1H),7.11(s,1H),7.02–6.96(m,2H),6.80–6.75(m,2H),6.70–6.60(m,2H),5.22(s,2H), 5.20(s,2H),4.00(s,1.5H),3.89(s,1.5H),3.84(s,3H),3.46(s,3H),3.37(s,3H)；¹³C NMR (100MHz,CDCl₃)δ189.39,168.42,168.35,165.59,161.83,159.91,156.92,154.77, 153.34,152.41,152.34,151.61,151.54,145.23,145.18,144.82,136.62,136.28,132.53, 131.25,130.04,129.11,129.07,128.75,128.56,128.52,127.11,126.90,125.43,125.38, 124.98,124.36,121.29,120.34,116.16,113.99,113.45,112.19,110.31,110.28,101.15, 101.12,94.64,82.94,82.74,66.89,56.63,55.77,52.89,37.69。

TFA (3mL) is added dropwise into DCM (3mL) solution of YS-3-36 (11mg, 0.017mmol) in ice water bath, Then solution is stirred at room temperature 2 hours.Reaction mixture is concentrated, in a vacuum and toluene azeotropic for 3 times, is then dissolved in In THF (4.5mL).Water (1.5mL) solution of LiOH (2.5mg, 0.104mmol) is added dropwise at room temperature.It is mixed by what is obtained Object is closed to be stirred at room temperature 2 hours.Reaction mixture is diluted with ethyl acetate, with 1N HCl, water and salt water washing.It will be organic Layer is dry with anhydrous magnesium sulfate, and is concentrated in a vacuum.Compound YS-3-42 is separated on silica gel by flash chromatography White sticky solid uses ethyl alcohol: DCM (1:9) is as eluant, eluent.Yield: 7.8mg (79%)¹H NMR (400MHz, acetone- D6) δ 10.01 (s, 1H), 8.55 (s, 0.5H), 8.42 (d, J=7.9Hz, 0.5H), 8.36 (d, J=7.5Hz, 0.5H), 8.15 (d, J=8.2Hz, 0.5H), 7.88 (s, 0,5H), 7.78-7.71 (m, 1H), 7.48 (d, J=7.5Hz, 0.5H), 7.46 (d, J =8.6Hz, 1H), 7.20 (d, J=8.6Hz, H), 7.05 (d, J=9.0Hz, 2H), 6.98-6.88 (m, 3H), 6.88-6.82 (m, 1H), 6.74 (d, J=8.7Hz, 1H), 5.23 (s, 1H), 3.89 (s, 3H), 3.41 (s, 3H)；¹³C NMR(100MHz, CDCl₃)δ196.25,168.46,165.64,161.95,161.85,156.94,154.81,153.31,152.47,152.40, 151.65,146.36,145.18,136.65,136.32,134.14,132.54,131.27,130.08,129.12,129.08, 128.56,128.51,127.12,126.93,125.49,125.40,124.43,120.26,118.50,115.96,113.95, 112.24,110.34,101.18,101.15,66.68,55.78,52.91。

By 3- iodophenol (1.1g, 5.0mmol) and phthalic anhydride (370mg, 2.50mmol) in methanesulfonic acid (3mL) Mixture 135 DEG C under argon gas stir 48 hours.After cooling to room temperature, reaction mixture 50mL ice/water is poured into mix It closes in object, and stirs to be settled out gray solid.Solid by filtration is collected and is dissolved in chloroform, silicagel pad is then passed through To obtain colourless solution.Solution is concentrated, is white solid to obtain YS-4-42 [817170-65-3].Yield: 629mg (45%).

¹H NMR(300MHz,CDCl₃) δ 8.07-8.00 (m, 1H), 7.69-7.62 (m, 4H), 7.38 (d, J=1.6Hz, 1H), 7.36 (d, J=1.6Hz, 1H), 7.16-7.08 (m, 1H), 6.55 (s, 1H), 6.52 (s, 1H).

Pd is packed into the round-bottomed flask of oven drying₂(dba)₃(12mg,0.013mmol)、Xantphos(23mg, 0.039mmol) and Cs₂CO₃(59mg, 0.182mmol), and with argon cleaning 5 minutes.Addition YS-4-42 (36mg, 0.066mmol) and dioxanes (5mL) solution of YS-3-2 (50mg, 0.197mmol), and by obtained mixture first in argon It is stirred at room temperature 30 minutes, is heated to 100 DEG C and is stirred for 20 hours under gas.It is cooled to room temperature reaction mixture, uses DCM It dilutes and is filtered by Celite pad.Then filtrate is concentrated in a vacuum.By flash chromatography on silica gel by compound YS-4-44 is separated into white sticky solid, uses ethyl acetate: hexane (3:2) is as eluant, eluent.Yield: 18mg (34%).

¹H NMR(300MHz,CDCl₃) δ 10.44 (s, 2H), 8.05 (d, J=6.8Hz, 1H), 7.79 (d, J=8.0Hz, 2H), 7.73-7.63 (m, 2H), 7.25 (d, J=1.9Hz, 2H), 7.19 (d, J=7.1Hz, 1H), 7.11 (s, 2H), 7.05- 6.96(m,4H),6.84(s,1H),6.81(s,1H),5.22(s,4H),5.20(s,4H),3.46(s,6H),3.37(s,6H)；

¹³C NMR(75MHz,CDCl₃)δ189.3,169.2,159.8,154.7,152.9,151.3,145.1,144.7, 135.3,130.1,128.7,128.5,126.3,125.3,124.9,124.0,121.3,120.3,116.6,113.8, 113.4,94.6,81.8,66.8,56.5,37.6。

TFA (3mL) is added dropwise into DCM (3mL) solution of YS-4-44 (12mg, 0.015mmol) in ice water bath, Then solution is stirred at room temperature 2 hours.Reaction mixture is concentrated in a vacuum, and with toluene azeotropic 3 times.By quick Compound YS-4-45 is separated into white sticky solid on silica gel by chromatography, uses ethyl acetate: hexane (3:2) is as washing De- agent.Yield: 10mg (99%).

¹H NMR(400MHz,CDCl₃) δ 11.04 (s, 2H), 9.87 (s, 2H), 8.05 (d, J=7.4Hz, 1H), 7.74- 7.62 (m, 2H), 7.53 (d, J=7.9Hz, 2H), 7.22 (d, J=7.5Hz, 1H), 7.01 (d, J=8.7Hz, 2H), 6.93 (d, J=8.0Hz, 2H), 6.90 (s, 2H), 6.82 (s, 1H), 6.80 (s, 1H), 5.19 (s, 4H), 3.38 (s, 6H)；¹³C NMR (150MHz,CDCl3)δ196.1,169.2,161.8,154.71,152.8,151.4,146.2,145.0,135.3,134.0, 130.1,128.5,126.3,125.3,124.1,121.1,120.2,118.4,116.6,115.9,113.8,81.9,66.6, 37.6。

By the bromo- 1,8- naphthalic anhydride (1.229g, 4.40mmol) of 4- and propylamine (0.400mL, 4.90mmol) in ethyl alcohol Suspension in (200mL) stirs 1 hour at 50 DEG C, is then heated to reflux and continues 1 hour again.Reaction mixture is cooling To room temperature, then it is concentrated in a vacuum.By flash chromatography on silica gel by compound YS-2-166 [100865-05-2] point From for white sticky solid, use hexane: DCM (1:1) is as eluant, eluent.Yield: 1.268g (90%).

¹H NMR(300MHz,CDCl₃) δ 8.65 (d, J=7.3Hz, 1H), 8.55 (d, J=8.5Hz, 1H), 8.40 (d, J =7.9Hz, 1H), 8.03 (d, J=7.9Hz, 1H), 7.84 (dd, J=8.5,7.3Hz, 1H), 4.17-4.10 (m, 2H), 1.83-1.69 (m, 2H), 1.01 (t, J=7.4Hz, 3H).

By YS-2-166 solution (250mg, 0.789mmol), Cu powder (5mg, 0.0789mmol), Ammonia (32wt.%NH₃Aqueous solution, 5mL) and n-methyl-2-pyrrolidone (5mL) stirred 12 hours at 100 DEG C in seal pipe. Then reaction mixture is cooled to room temperature, is diluted with ethyl acetate, with 1N HCl, water and salt water washing.By organic layer nothing Water magnesium sulfate is dry, and is concentrated in a vacuum.By flash chromatography on silica gel by compound YS-2-167 [94860-68- 1] be separated into tacky yellowish solid, use acetone: chloroform (1:6) is as eluent.Yield: 74mg (37%).

¹H NMR(300MHz,CDCl₃) δ 8.59 (d, J=7.3Hz, 1H), 8.41 (d, J=8.2Hz, 1H), 8.10 (d, J =8.4Hz, 1H), 7.65 (dd, J=8.4,7.3Hz, 1H), 6.88 (d, J=8.2Hz, 1H), 4.17-4.10 (m, 2H), 1.81-1.63 (m, 2H), 1.00 (t, J=7.4Hz, 3H).

At room temperature under argon gas to 2- hydroxyl -4- (methylol) benzaldehyde (40mg, 0.263mmol) and K₂CO₃(72mg, Allyl bromide, bromoallylene (173 μ L, 0.526mmol) is added in THF (5mL is anhydrous) solution 0.526mmol).Under reflux by the solution Stirring 12 hours.With ethyl acetate diluted reaction mixture, with 1N HCl, water and salt water washing.By organic layer anhydrous slufuric acid Magnesium is dry, and is concentrated in a vacuum.Compound YS-2-165 is separated into colorless oil on silica gel by flash chromatography, Use ethyl acetate: hexane (1:2) is as eluant, eluent.Yield: 31mg (70%).

¹H NMR(400MHz,CDCl₃) δ 10.46 (s, 1H), 7.79 (d, J=7.9Hz, 1H), 7.03 (s, 1H), 6.96 (d, J=7.9Hz, 1H), 6.13-6.01 (m, 1H), 5.48-5.41 (m, 1H), 5.33 (d, J=10.6Hz, 1H), 4.73 (s, 2H), 4.66 (d, J=5.1Hz, 2H)；

¹³C NMR(100MHz,CDCl₃)δ189.7,161.4,149.9,132.4,128.8,124.3,118.8,118.3, 110.8,69.3,64.8。

It is added under argon gas into the toluene of YS-2-167 (46mg, 0.182mmol) (5mL, anhydrous) solution at room temperature DIPEA (90 μ L, 0.546mmol) and triphosgene (65mg, 0.218mmol).Solution is heated to reflux and continues 1 hour.It is cooling To room temperature, the solution of the anhydrous DCM (2mL) of YS-2-165 (35mg, 0.182mmol) is added into reaction mixture, then It is stirred for 3 hours, is concentrated in a vacuum, and be dissolved in ethyl alcohol (5mL).By acquired solution with argon-degassed 30 minutes, and Pd (PPh is added under argon gas₃)₄(21mg,0.0182mmol).Gained mixture is heated 12 hours under reflux.Reaction is mixed It closes object to be cooled to room temperature, be filtered by Celite pad, and be concentrated in a vacuum.By flash chromatography on silica gel by compound YS-2-172 is separated into tacky yellowish solid, uses ethyl acetate: hexane (1:2) is as eluant, eluent.Yield: 12mg (15%).

¹H NMR(300MHz,CDCl₃) δ 11.09 (s, 1H), 9.91 (s, 1H), 8.64 (d, J=7.1Hz, 1H), 8.61 (d, J=8.3Hz, 1H), 8.36 (d, J=8.3Hz, 1H), 8.20 (d, J=8.4Hz, 1H), 7.83-7.75 (m, 1H), 7.60 (d, J=8.2Hz, 1H), 7.55 (s, 1H), 7.09-7.01 (m, 2H), 5.31 (s, 2H), 4.17-4.10 (m, 2H), 1.81- 1.72 (m, 2H), 1.01 (t, J=7.4Hz, 3H)；

¹³C NMR(125MHz,CDCl₃)δ196.2,164.2,163.8,152.8,145.3,138.6,134.3,132.6, 131.5,129.1,127.0,125.8,123.8,120.6,119.0,117.2,116.6,66.8,42.1,21.5,11.7。

NaH is added into the DMF of resorufin (250mg, 1.17mmol) (12mL, anhydrous) solution in ice water bath (in mine 60wt%, 59mg, 1.41mmol in object oil), and the mixture is stirred 30 minutes.Then, PhNTf is added₂(500mg, 1.41mmol), and reaction mixture is warmed to room temperature, and be stirred for 8 hours.With ethyl acetate diluted reaction mixture, use 1N HCl, water and salt water washing.Organic layer is dry with anhydrous magnesium sulfate, and be concentrated in a vacuum.Pass through flash column chromatography Compound YS-4-110 [941601-75-8] is separated into tacky yellowish solid on silica gel, uses ethyl acetate: hexane (1: 2) it is used as eluant, eluent.Yield: 300mg (74%).

¹H NMR(300MHz,CDCl₃) δ 7.85-7.75 (m, 1H), 7.36 (d, J=9.9Hz, 1H), 7.23-7.18 (m, 2H), 6.81 (dd, J=9.9,2.0Hz, 1H), 6.27 (d, J=2.0Hz, 1H).

Pd is packed into the round-bottomed flask of oven drying₂(dba)₃(18mg,0.020mmol)、Xantphos(34mg, 0.059mmol) and Cs₂CO₃(59mg, 0.182mmol), and with argon cleaning 5 minutes.Addition YS-4-110 (83mg, 0.24mmol) and dioxanes (5mL) solution of YS-3-2 (56mg, 0.20mmol), and obtained mixture is existed under argon gas It stirs 30 minutes first at room temperature, is then heated to 100 DEG C and is stirred for 24 hours.It is cooled to room temperature reaction mixture, is used DCM is diluted and is filtered by Celite pad.Then filtrate is concentrated in a vacuum.It will be changed on silica gel by flash column chromatography It closes object YS-4-111 and is separated into Red viscous solid, use EtOAc:DCM (1:4) as eluant, eluent.Yield: 51mg (57%).

¹H NMR(300MHz,CD₂Cl₂) δ 7.83-7.71 (m, 2H), 7.43 (d, J=9.8Hz, 1H), 7.40-7.32 (m, 2H), 7.17 (s, 1H), 7.04 (d, J=7.9Hz, 1H), 6.80 (dd, J=9.8,2.0Hz, 1H), 6.27 (d, J=2.0Hz, 1H),5.26(s,2H),5.23(s,2H),3.47(s,3H),3.42(s,3H)；

¹³C NMR(75MHz,CD₂Cl₂)δ189.35,186.41,160.19,154.70,150.07,148.64, 147.28,144.96,144.49,135.23,135.19,131.43,130.70,128.67,125.40,122.20,120.68, 113.98,112.39,107.14,95.05,67.39,56.79,37.56。

TFA is added dropwise into DCM (3mL) solution of YS-4-111 (12mg, 0.027mmol) in ice water bath (3mL) then solution is stirred at room temperature 2 hours.Reaction mixture is concentrated in a vacuum, and with toluene azeotropic 3 times.It is logical Cross flash column chromatography and compound YS-4-112 be separated into Red viscous solid on silica gel, use EtOAc:DCM (1:4) as Eluant, eluent.Yield: 10mg (92%).

¹H NMR(300MHz,CDCl₃) δ 11.08 (s, 1H), 9.89 (s, 1H), 7.77 (d, J=9.2Hz, 1H), 7.56 (d, J=7.8Hz, 1H), 7.44 (d, J=9.8Hz, 1H), 7.36 (d, J=7.7Hz, 2H), 6.97 (d, J=7.9Hz, 2H), 6.86 (dd, J=9.8,2.0Hz, 1H), 6.33 (d, J=2.0Hz, 1H), 5.24 (s, 2H), 3.46 (s, 3H)；

¹³C NMR(100MHz,CDCl₃)δ196.2,186.5,162.0,154.5,149.7,148.3,146.8,145.8, 135.2,134.9,134.2,131.1,130.6,121.8,120.4,118.6,116.1,112.0,107.2,67.1,37.4。

Embodiment 2- is with green fluorescence compound YS-3-42 to the sensitivity and selective enumeration method of hydrogen peroxide

The present embodiment shows that green fluorescence compound YS-3-42 delicately and selectively detects hydrogen peroxide.Specifically Compound YS-3-42 is dissolved in the 0.1M kaliumphosphate buffer that pH is 7.4 and (contains 0.1% with the solution for forming 10 μM by ground DMF and 100 μM of CCl₃CN), wherein excitation spectrum and emission spectrum respectively at 480nm and 527nm.By compound YS-3-42 10 μM of various concentration of solution hydrogen peroxide treatment.Fig. 2A is shown, and the fluorescence intensity of compound YS-3-42 is with hydrogen peroxide The increase of concentration and increase.

Different reactive oxygen species (ROS) and reactive nitrogen substance (RNS) are compared with the reaction of compound YS-3-42 Property.Specifically, 10 μM of solution of compound YS-3-42 are handled with (100 μM) of various ROS/RNS.Fig. 2 B is shown, and uses peroxidating Hydrogen processing with other ROS and RNS processing than causing the much higher fluorescence intensity of compound YS-3-42 to increase.

Embodiment 3- is with green fluorescence compound YS-4-45 to the sensitivity and selective enumeration method of hydrogen peroxide

The present embodiment shows that green fluorescence compound YS-4-45 delicately and selectively detects hydrogen peroxide.Specifically Compound YS-4-45 is dissolved in the 0.1M phosphate buffer that pH is 7.4 by ground, (is contained with the solution for forming 10 μM 0.1%DMF and 100 μM of CCl₃CN), wherein excitation spectrum and emission spectrum respectively at 520nm and 543nm.By compound The hydrogen peroxide treatment of 10 μM of solution various concentrations of YS-4-45 30 minutes.Fig. 3 A is shown, the fluorescence of compound YS-4-45 Intensity increases with the concentration of hydrogen peroxide and is increased.

Different reactive oxygen species (ROS) and reactive nitrogen substance (RNS) are compared with the reaction of compound YS-4-45 Property.Specifically, 10 μM of solution of compound YS-4-45 are handled with (100 μM) of various ROS/RNS.Fig. 3 B is shown, and uses peroxidating Hydrogen processing 30min (left side histogram in Fig. 3 B) or 60min (the right histogram in Fig. 3 B) with other ROS and RNS processing than being led The much higher fluorescence intensity of compound YS-4-45 is caused to increase.

Embodiment 4- is with green fluorescence compound YS-2-172 to the sensitivity and selective enumeration method of hydrogen peroxide

The present embodiment shows that green fluorescence compound YS-2-172 delicately and selectively detects hydrogen peroxide.Specifically Compound YS-2-172 is dissolved in 0.1M phosphate buffer/CH that pH is 7.4 by ground₃In CN (v/v=1/1), to form 10 μM solution (containing 0.1%DMF), wherein excitation spectrum and emission spectrum are respectively at 429nm and 530nm.By compound YS- The hydrogen peroxide treatment of 10 μM of solution various concentrations of 2-172 60 minutes.Fig. 4 A is shown, and the fluorescence of compound YS-2-172 is strong Degree increases with the concentration of hydrogen peroxide and is increased.

Different reactive oxygen species (ROS) and reactive nitrogen substance (RNS) are compared with the reaction of compound YS-2-172 Property.Specifically, 10 μM of solution of compound YS-2-172 are handled with (100 μM) of various ROS/RNS.Fig. 4 B is shown, and uses peroxide Change hydrogen processing 30min (left side histogram in Fig. 4 B) or 60min (the right histogram in Fig. 4 B) than being handled with other ROS and RNS The much higher fluorescence intensity of compound YS-2-172 is caused to increase.

Embodiment 5- applies motif compound in test cell line

RAW264.7 cell, a kind of mouse monokaryon granulomacrophage system obtain from ATCC (American Type Tissue Culture) It obtains and contains 5%CO at 37 DEG C₂Under be maintained at and be supplemented with 10% heat-inactivated fetal calf serum (Gibco) and 1% penicillin/streptomycin DMEM (the improvement Eagle culture medium of Dulbecco) in.It updates within growth medium every two to three days.In 80% meet, lead to It crosses scraping to remove cell, is washed with fresh culture and be centrifuged (spin down) (500rpm Eppendorf microcentrifugation Machine) it is used for cell count.For co-focusing imaging, by cell with 2 × 10⁴The density of a cell/mL is typically seeded in 35mm It is copolymerized in burnt culture dish (Mat-Tek:MA, USA).

For acute hydrogen peroxide-induced (30min), with specified dosage by phorbol 12-myristinate 13- acetic acid esters (PMA) it is added to HBSS (Hank balanced salt solution) and is incubated for altogether with YS-3-42 until imaging (see Fig. 5).It is lured in hydrogen peroxide Enzyme inhibitor (NOX inhibitor DPI) is added together with PMA during leading.Be known that cancer cell is compared with normal cell In the case where increased oxidative stress and increased ROS generate.Therefore, the foundation level of the hydrogen peroxide between cancer and normal cell Difference tested with YS-3-42 (see Fig. 6).In addition, motif compound its using show under concentration no cytotoxicity (see Fig. 7).Fig. 7 shows the cytotoxicity of RAW264.7 cell middle probe YS-3-42 and YS-4-45.Make RAW264.7 cell with increasing The concentration and probe concentration added is incubated for 24 hours.Probe shows negligible toxicity or no toxicity after being incubated for for 24 hours.Data indicate Average value ± s.e.m, it is triplicate to carry out Cell-Titer Glo measurement.

Similarly, YS-4-45 is also successfully applied to endogenous H in RAW264.7 cell₂O₂Co-focusing imaging (see figure 8).The H of PMA challenge₂O₂Generation can steadily show, this can be actively damped by adding DPI.

Embodiment 6- quantifies hydrogen peroxide with red fluorescence compound YS-4-112

The embodiment shows that red fluorescence compound YS-4-112 can quantitatively detect hydrogen peroxide.Specifically, by chemical combination Object YS-4-112 is dissolved in the 0.1M kaliumphosphate buffer that pH is 7.4 to form 10 μM of solution (containing 0.5%DMF and 100 μM CCl₃CN), wherein excitation spectrum and emission spectrum are respectively at 565nm and 602nm.By 10 μM of compound YS-4-112 The hydrogen peroxide treatment of the various concentration of solution.Fig. 9 A is shown, and the fluorescence intensity of compound YS-4-112 is with concentration of hydrogen peroxide Increase and increase.As depicted in FIG. 9 B, observe the fluorescence intensity of YS-4-112 test solution at 602nm and H₂O₂ The linear relationship of the concentration of (0-30 μM), and estimate detection limit down to 4.8nM (3 σ/K).The standard calibration curve can be used for H in quantitative various samples and high throughput test₂O₂Concentration.

Embodiment 7- applies motif compound in living animal test

The embodiment shows that YS-3-42 and YS-4-112 can detect the hydrogen peroxide in zebra fish living.Adult fish (HKWT) Mating and the selection of zebrafish embryo carry out in Hong Kong University's zebra fish core facility.It collects ovum and has placed it in E-3 training In the 90mm culture dish for supporting base, and it is incubated at 28 DEG C until embryonic development is to desired stages (such as pf for 24 hours, 48hpf, 72hpf； Hpf: after fertilization hour).Under the microscope by Dumont Tweezers carefully by the chorion (chorion) of pf embryo for 24 hours It removes to discharge embryo.With or without PMA (500ng/mL) by embryo at room temperature through 10 μM YS-3-42 or YS-4-112 (contains 100 μM of CCl in 1mL E3 buffer₃CN it) handles 30 minutes, is then washed with 1mL E-3 culture medium Twice, it is then imaged on LSM 710.The generation and contribution of hydrogen peroxide can be used in the zebra fish of different stages of development YS-4-112 visualizes (Figure 10 A).The H that PMA is challenged in zebra fish₂O₂Generation also successfully detects (Figure 10 B) with YS-3-42.

Application of the embodiment 8- motif compound in high throughput test

The embodiment shows that motif compound can be used for developing high throughput test.Firstly, by 0.125,0.25,0.5,1.0, The antioxidant of 2.0mM (final concentration) is added to the H of the 1.0mM of 200 μ L₂O₂PH be 7.4 0.1M phosphate buffer In, and the solution is incubated for 30 minutes at 37 DEG C on 96 orifice plates.Then, the obtained solution of 2 μ L is added to YS-4- (10 μM, in the 0.1M phosphate buffer that pH is 7.4, contain 100 μM of CCl to 112 solution₃CN, each 200 μ L of hole) in, And solution is incubated for 30 minutes at 37 DEG C on 96 orifice plates, to measure remaining H₂O₂Concentration.96 orifice plate can be placed In plate reader, to determine the fluorescent emission in each hole excited with 565nm at 602nm.Oxidation resistance can pass through H₂O₂Clearance rate assessment, is calculate by the following formula:

H=(F₀-F₁)/(F₀-F_Blank) * 100%

F₀, the not no fluorescence intensity of antioxidant；F₁, the fluorescence intensity of the antioxidant with various concentration；F_Blank, YS- The fluorescence background of 4-112.

The H of ascorbic acid and Epigallo-catechin gallate (EGCG) (EGCG)₂O₂Scavenging capacity passes through the high throughput test (Figure 11 A and Figure 11 B) is determined.Those data are highly useful for drug screening and assessment.

Although herein by reference to the illustrative embodiment for specific application, present invention is described, should manage Solution, the invention is not limited thereto.Those will be recognized separately with ordinary skill with the people that can obtain introduction provided herein Outer modification, using and embodiment and other field within the scope of its, the present invention will have significant effectiveness wherein.Cause This, is intended to cover any and all this applications, modification and embodiment in the scope of the present invention by appended claims.

Compound with structure 1-6,8-21,23-28, wherein R=H or CF₃And the compound with structure 7 and 22, Wherein R=CF₃, can be with being used to prepare compound YS-3-42 (that is, the compound with structure 7, wherein R=H) and compound The similar approach preparation of YS-2-172 (that is, the compound with structure 22, wherein R=H), wherein using for corresponding unit price Preceding fluorogen or the starting material and/or R=CF of preceding illuminophore part₃Starting material.The structure quilt of these compounds¹HHMR It is confirmed with MS.

Compound with structure 30-44, wherein R=H or CF₃And the compound with structure 29, wherein R=CF₃, can With with the similar approach preparation for being used to prepare compound YS-4-45 (that is, the compound with structure 29, wherein R=H), wherein Use fluorogen before corresponding divalent or the starting material and/or R=CF of preceding illuminophore part₃Starting material.These compounds Structure quilt¹HHMR and MS confirmation.

The compound of the present invention can have such as proposed in Fig. 1 for same or like with reacting for hydrogen peroxide Mechanism.Therefore, similar technical effect and application, including but not limited to peroxide can also be obtained for the compound of the present invention Change the sensitivity and selective enumeration method of hydrogen.

As it is used herein, singular " one (a) ", " one (an) " and " (the) " are intended to also include plural number Form, unless the context clearly indicates otherwise.In addition, with regard to term " including (including) ", " including (includes) ", " tool Have (having) ", " having (has) ", " containing (with) " or its modification scope used in detailed description and/or claims and Speech, such term be intended in a manner of being similar to term " comprising (comprising) " include.Transitional term/phrase (and its any grammatical variants), " including (comprising) ", " including (comprises) ", " including (comprise) ", " base This by ... form (consisting essentially of) ", " substantially by ... form (consists Essentially of) " and " by ... form (consisting) " and " by ... form (consists) " it can be interchanged It uses.

All patents for being mentioned herein or quoting, patent application, provisional application and openly, including all attached drawings and table are logical It crosses reference to be integrally incorporated, until the degree that they are not contradicted with the clearly teaching of this specification.

Bibliography

www.thermofisher.com/order/catalog/product/A22177

www.thermofisher.com/hk/en/home/references/molecular-probes-the- handbook/probes-for-reactive-oxygen-species-including-nitric-oxide/ generating-and-detecting-reactive-oxygen-species.html

Claims

1. the compound or its salt of a kind of formula (I), formula (II) or formula (III):

A-X¹(II),

A-X²- A ' (III),

Wherein:

R¹、R²、R³、R⁴And R⁵Independently selected from H, F, Cl, Br, I, CN, alkyl, halogenated alkyl, miscellaneous alkyl, alkenyl, alkynyl, virtue Alkyl, aryl, alkaryl, heterocycle, naphthenic base, cycloalkenyl, cycloalkynyl radical, hydroxyalkyl, aminoalkyl, amino, alkyl amino, virtue Base amino, dialkyl amido, alkyl aryl amino, ammonia diaryl base, acyl amino, hydroxyl, mercaptan, alkylthio, alkoxy, Alkylthio group, alkoxyalkyl, aryloxy group, alkoxy aryl, acyloxy, nitro, carbamoyl, trifluoromethyl, phenoxy group, benzyl Oxygroup, phosphonic acids, phosphate, sulfonic acid (- SO₃H), sulphonic acid ester, sulfonamide ,-C (=O)-P¹With-C (=O)-M-P²,

Wherein, P¹And P²Selected from hydrogen, halogen, alkoxy, hydroxyl, mercaptan, alkyl, alkenyl, alkynyl, naphthenic base, cycloalkenyl, ring Alkynyl, aryl, alkaryl, aralkyl, carbamate groups, amino, alkyl amino, arylamino, dialkyl amido, alkyl virtue Base amino, ammonia diaryl base, alkylthio group, miscellaneous alkyl, alkyl triphenyl phosphonium and the heterocycle with 3 to 7 annular atoms,

Or R²And R³5,6 or 7 member rings selected from aryl, heterocycle, heteroaryl and heteroaromatic group are formed together,

Or R⁴And R⁵5,6 or 7 member rings selected from aryl, heterocycle, heteroaryl and heteroaromatic group are formed together,

R⁶Selected from hydrogen, alkyl, alkoxyalkyl, alkanoyl ,-CF₃, halogen replace low alkyl group and (C=O)-O-Z¹,

Wherein, Z¹It is selected from alkyl, alkenyl, alkynyl, naphthenic base, cycloalkenyl, cycloalkynyl radical, aryl, alkaryl and aryl alkyl Group；

X¹It is fluorogen or preceding illuminophore part before unit price；

X²It is fluorogen or preceding illuminophore part before divalent；

Each of A and A' are independently indicated by formula (IV) or formula (V):

Wherein, R⁷And R⁹At least one of be (C=O)-W¹,

Wherein, W¹Selected from hydrogen, alkyl, alkoxyalkyl, alkanoyl, CF₃, halogen replace low alkyl group and (C=O)-O-Z²,

Wherein Z²It is selected from alkyl, alkenyl, alkynyl, naphthenic base, cycloalkenyl, cycloalkynyl radical, aryl, alkaryl or aryl alkyl Group,

And R⁷And R⁹Another, R⁸、R¹⁰And R¹¹Independently selected from H, F, Cl, Br, I, CN, alkyl, halogenated alkyl, miscellaneous alkyl, chain Alkenyl, alkynyl, aralkyl, aryl, alkaryl, heterocycle, naphthenic base, cycloalkenyl, cycloalkynyl radical, hydroxyalkyl, aminoalkyl, ammonia Base, alkyl amino, arylamino, dialkyl amido, alkyl aryl amino, ammonia diaryl base, acyl amino, hydroxyl, mercaptan, sulphur Substituted alkyl, alkoxy, alkylthio group, alkoxyalkyl, aryloxy group, alkoxy aryl, acyloxy, nitro, carbamoyl, trifluoro Methyl, phenoxy group, benzyloxy, phosphonic acids, phosphate, sulfonic acid (- SO₃H), sulphonic acid ester, sulfonamide ,-C (=O)-P³With-C (=O)- M-P⁴,

Wherein P³And P⁴Independently selected from hydrogen, halogen, alkoxy, hydroxyl, mercaptan, alkyl, alkenyl, alkynyl, naphthenic base, cyclenes Base, cycloalkynyl radical, aryl, alkaryl, aryl alkyl, carbamate, amino, alkyl amino, arylamino, dialkyl amido, Alkyl aryl amino, ammonia diaryl base, alkylthio group, miscellaneous alkyl, alkyl triphenyl phosphonium or the heterocycle with 3 to 7 annular atoms； M is alkylidene, alkenylene, alkynylene, arlydene, sub- aralkyl or alkarylene；

R⁸And R¹⁰At least one of preferably hydroxyl, alkoxy or be selected from amino, alkyl amino, arylamino, dialkyl amino Base, alkyl aryl amino, ammonia diaryl base electron donating group；

2. compound described in claim 1, wherein R in formula (I)¹、R³And R⁵At least one of be hydroxyl group.

3. compound as claimed in claim 2, wherein R in formula (I)⁶Selected from hydrogen, CF₃, halogen replace low alkyl group or (C= O)-O-Z³, wherein Z³It is alkyl, alkenyl, alkynyl, naphthenic base, cycloalkenyl, cycloalkynyl radical, aryl, alkaryl or aryl alkyl.

4. compound described in claim 1, wherein R in formula (IV) and formula (V)⁸And R¹⁰At least one of be hydroxyl group.

5. compound as claimed in claim 4, wherein R⁷And R⁹At least one of be (C=O)-W², wherein W²For hydrogen, CF₃、 The low alkyl group or (C=O)-O-Z that halogen replaces⁴, wherein Z⁴It is selected from alkyl, alkenyl, alkynyl, naphthenic base, cycloalkenyl, ring Alkynyl, aryl, alkaryl or aryl alkyl group.

6. compound described in claim 1, wherein X¹The amine naphthalene two before cumarin, unit price before fluorescein, unit price before unit price Carboximide, unit price before dansyl, unit price before diamines (pro-bimane), unit price before eosin, unit price before rhodamine, unit price before to first Cyanine, monovalent Nile red, monovalent xanthone, monovalent xanthene, unit price flazo orange, unit price SNARF-1, unit price before amino phenols, unit price 2- naphthylamines, monovalent resorufin and monovalent fluorescence before 6- dodecanoyl-N, N- dimethyl -2- naphthylamines, unit price before preceding fluorescein, unit price Element.

7. claim 1 or compound as claimed in claim 6, wherein the compound has one of structure 1-28 and 57:

Wherein, R=H or CF₃。

8. compound described in claim 1, wherein X²Two formyl of amine naphthalene is sub- before cumarin, divalent before preceding fluorescein, divalent Cyanine before paramethylaminophenol, divalent before rhodamine, divalent before eosin, divalent before diamines, divalent before dansyl, divalent before amine, divalent, Divalent Nile red, divalent xanthone, divalent xanthene, divalent flazo orange, divalent SNARF-1 and divalent resorufin, including conjugation Object.

9. claim 1 or compound according to any one of claims 8, wherein the compound has one of structure 29-44:

Wherein, R=H or CF₃。

10. compound described in claim 1, wherein the compound includes one or more free carboxyl groups, wherein At least one carboxylic group passes through amide key connection and positively charged Mitochondrially targeted triphenyl phosphonium part or lysosome target To morpholine or N, N- disubstituted amine moiety conjugation, wherein the connection between the compound and triphenyl phosphonium part has Lower formula (VI) or (VII):

Wherein, n=1-10；

11. compound described in any one of claim 10, wherein the compound has one of structure 45-56:

Wherein, R=H or CF₃。

12. a kind of fluorescence or luminescence probe composition, it includes the compounds and load described in any one of claims 1 to 11 Body.

13. fluorescence described in claim 12 or luminescence probe composition, wherein the fluorescence or luminescence probe composition also wrap Containing solvent, acid, alkali, buffer solution or combinations thereof.

14. a kind of horizontal method of hydrogen peroxide in presence for hydrogen peroxide in test sample and/or determining sample, Include:

Contact the compound of any one of claims 1 to 11 with sample, to form fluorescence or luminophor；With

Determine the fluorescence or luminosity of fluorescence or luminophor.

15. method of claim 14, wherein the sample is chemical example or biological sample or pathological sample.

16. method of claim 15, wherein the sample be include microorganism, cell, tissue, organ, from plant Animal part, entire plant or animal or their extract biological sample.

17. method of claim 15, wherein the sample be include blood, urine, saliva, serum, breathing gas, exhale The pathological sample of outlet condensate, joint fluid or their extract.

18. a kind of presence for detecting the hydrogen peroxide of organism in vivo or the hydrogen peroxide in vivo determining organism Horizontal method comprising:

The compound of any one of claims 1 to 11 is given to the organism to form fluorescence or luminophor；With

Determine the fluorescence or luminosity of fluorescence or luminophor.

19. a kind of method of presence or the level for determining hydrogen peroxide in vitro for detecting hydrogen peroxide in vitro, packet It includes:

The compound of any one of claims 1 to 11 is given to external sample to form fluorescence or luminophor；With

Determine the fluorescence or luminosity of fluorescence or luminophor.

20. the horizontal high flux screening of hydrogen peroxide in a kind of presence for hydrogen peroxide in test sample or determining sample Method, wherein the high throughput method the following steps are included:

The compound of any one of claims 1 to 11 is contacted with sample to form one or more fluorescence or light emitting compound Object；With

The fluorescence or luminosity of fluorescence or luminophor are determined, to determine the presence of hydrogen peroxide and/or peroxide in sample Change the amount of hydrogen.

21. it is a kind of for screening the high throughput method for increasing or decreasing one or more target compounds of hydrogen peroxide level, Wherein, the high throughput method the following steps are included:

The compound of any one of claims 1 to 11 is contacted with target compound to form one or more fluorescence or shine Compound；With

Fluorescence or the luminosity of fluorescence or luminophor are measured to determine presence and/or the target compound of target compound Amount.

22. the compound of any one of claims 1 to 11 or the fluorescence of claim 12 or 13 or luminescence probe composition are used The presence of hydrogen peroxide and/or the internal of organism or in vitro is determined in sample in detection organism internal or in vitro Sample in hydrogen peroxide horizontal purposes；Or the compound or claim 12 or 13 of any one of claims 1 to 11 Fluorescence or luminescence probe composition be used for screen increase or decrease hydrogen peroxide level one or more target compounds Purposes.