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WO2018133859A1 - Composés et procédés de détection de peroxyde d'hydrogène - Google Patents

Composés et procédés de détection de peroxyde d'hydrogène Download PDF

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WO2018133859A1
WO2018133859A1 PCT/CN2018/073585 CN2018073585W WO2018133859A1 WO 2018133859 A1 WO2018133859 A1 WO 2018133859A1 CN 2018073585 W CN2018073585 W CN 2018073585W WO 2018133859 A1 WO2018133859 A1 WO 2018133859A1
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compound
pro
monovalent
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divalent
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Dan Yang
Sen YE
Jun Hu
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University of Hong Kong HKU
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University of Hong Kong HKU
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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • C09K11/07—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials having chemically interreactive components, e.g. reactive chemiluminescent compositions
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C47/00—Compounds having —CHO groups
    • C07C47/52—Compounds having —CHO groups bound to carbon atoms of six—membered aromatic rings
    • C07C47/575—Compounds having —CHO groups bound to carbon atoms of six—membered aromatic rings containing ether groups, groups, groups, or groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
    • C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
    • C07D221/04—Ortho- or peri-condensed ring systems
    • C07D221/06—Ring systems of three rings
    • C07D221/14—Aza-phenalenes, e.g. 1,8-naphthalimide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D265/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one oxygen atom as the only ring hetero atoms
    • C07D265/28—1,4-Oxazines; Hydrogenated 1,4-oxazines
    • C07D265/34—1,4-Oxazines; Hydrogenated 1,4-oxazines condensed with carbocyclic rings
    • C07D265/38—[b, e]-condensed with two six-membered rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/10—Spiro-condensed systems

Definitions

  • the present invention belongs to the field of fluorogenic or luminogenic probes, and relates to compounds and methods for the detection of hydrogen peroxide.
  • Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are associated with aging, inflammation, and the progression of several diseases like cancer and diabetes.
  • Hydrogen peroxide (H 2 O 2 ) is a reactive oxygen species that plays a crucial role in oxidative stress and signal transduction in organisms.
  • analytical approaches using chemiluminescence and fluorescence probes have been developed to detect its intracellular generation. These probes can be effective for studying the oxidative stress and signal transduction for various pathologies.
  • the compounds can include compounds of Formula (I) , Formula (II) , or Formula (III) , or a salt thereof:
  • n 1-10; or the linkage between the compound and the morpholine or N, N-disubstituted amine moiety has the following formula (VIII) or (IX) :
  • the method of using the chemiluminescence and/or fluorescence compound can include contacting the compound with a sample to form a fluorescent or luminescent compound; and determining fluorescence or luminescence property of the fluorescent or luminescent compound.
  • FIG. 1 is a proposed mechanism for the reaction between YS-4-45 and hydrogen peroxide.
  • FIG. 2A is a graph showing the florescence intensity of compound YS-3-42 with increasing concentration of hydrogen peroxide.
  • the curves, from top to bottom, respectively represent: 10 ⁇ M solution of compound YS-3-42+100 ⁇ M H 2 O 2 , 10 ⁇ M solution of compound YS-3-42+50 ⁇ M H 2 O 2 , 10 ⁇ M solution of compound YS-3-42+40 ⁇ M H 2 O 2 , 10 ⁇ M solution of compound YS-3-42+30 ⁇ M H 2 O 2 , 10 ⁇ M solution of compound YS-3-42+20 ⁇ M H 2 O 2 , 10 ⁇ M solution of compound YS-3-42+10 ⁇ M H 2 O 2 , 10 ⁇ M solution of compound YS-3-42+8 ⁇ M H 2 O 2 , 10 ⁇ M solution of compound YS-3-42+6 ⁇ M H 2 O 2 , 10 ⁇ M solution of compound YS-3-42+4 ⁇ M H 2 O 2
  • FIG. 3A is graph showing the florescence intensity of compound YS-4-45 with increasing concentration of hydrogen peroxide.
  • the curves, from top to bottom, respectively represent: 10 ⁇ M solution of compound YS-4-45+100 ⁇ M H 2 O 2 , 10 ⁇ M solution of compound YS-4-45+50 ⁇ M H 2 O 2 , 10 ⁇ M solution of compound YS-4-45+40 ⁇ M H 2 O 2 , 10 ⁇ M solution of compound YS-4-45+30 ⁇ M H 2 O 2 , 10 ⁇ M solution of compound YS-4-45+20 ⁇ M H 2 O 2 , 10 ⁇ M solution of compound YS-4-45+10 ⁇ M H 2 O 2 , 10 ⁇ M solution of compound YS-4-45+8 ⁇ M H 2 O 2 , 10 ⁇ M solution of compound YS-4-45+4 ⁇ M H 2 O 2 , and 10 ⁇ M solution of compound YS-4-45 only.
  • FIG. 3B is a
  • FIG. 4A is graph showing the florescence intensity of compound YS-2-172 with increasing concentration of hydrogen peroxide.
  • the curves, from top to bottom, respectively represent: 10 ⁇ M solution of compound YS-2-172+500 ⁇ M H 2 O 2 , 10 ⁇ M solution of compound YS-2-172+100 ⁇ M H 2 O 2 , and 10 ⁇ M solution of compound YS-2-172 only.
  • FIG. 4B is a graph showing the fluorescence intensity of YS-2-172 in the presence of different ROS/RNS.
  • FIG. 7 shows the cytotoxicity of probes YS-3-42 and YS-4-45.
  • FIG. 8 shows the confocal images for RAW264.7 cells co-incubated with YS-4-45 (10 ⁇ M) with or without phorbol 12-myristate 13-acetate (PMA) (200 ng/mL) and DPI (100 nM) . Scale bars represent 10 ⁇ m.
  • FIG. 9A is graph showing the florescence intensity of compound YS-4-112 with increasing concentration of hydrogen peroxide.
  • the curves, from top to bottom, respectively represent: 10 ⁇ M solution of compound YS-4-112+100 ⁇ M H 2 O 2 , 10 ⁇ M solution of compound YS-4-112+50 ⁇ M H 2 O 2 , 10 ⁇ M solution of compound YS-4-112+30 ⁇ M H 2 O 2 , 10 ⁇ M solution of compound YS-4-112+20 ⁇ M H 2 O 2 , 10 ⁇ M solution of compound YS-4-112+10 ⁇ M H 2 O 2 , 10 ⁇ M solution of compound YS-4-112+8 ⁇ M H 2 O 2 , 10 ⁇ M solution of compound YS-4-112+6 ⁇ M H 2 O 2 , 10 ⁇ M solution of compound YS-4-112+4 ⁇ M H 2 O 2 , 10 ⁇ M solution of compound YS-4-112+2 ⁇ M H 2 O 2 ,
  • FIG. 10A shows the confocal images of live zebrafishes at different development stages co-incubated with YS-4-112 (10 ⁇ M) .
  • FIG. 10B shows the confocal images of PMA treated live zebrafishes co-incubated with YS-3-42 (10 ⁇ M) .
  • Scale bars represent 500 ⁇ m.
  • FIG. 11A shows the H 2 O 2 scavenging activity of ascorbic acid
  • FIG. 11B shows the H 2 O 2 scavenging activity of epigallocatechin gallate in a high throughput assay with YS-4-112.
  • the compounds disclosed herein can have a rapid-response and be highly selective probes for the detection of hydrogen peroxide in situ, in vivo, and in vitro.
  • the compounds can have new and efficient mechanisms for hydrogen peroxide detection. They can escalate the sensitivity and selectivity toward hydrogen peroxide and avoid the interference from cellular ROS/RNS.
  • the compounds can include, but are not limited to, compounds of Formula (I) , Formula (II) , or Formula (III) , or a salt thereof:
  • R 1 , R 2 , R 3 , R 4 , and R 5 are independently selected from the group consisting of H, F, Cl, Br, I, CN, alkyl, halogenated alkyl, heteroalkyl, alkenyl, alkynyl, aralkyl, aryl, alkaryl, heterocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl, hydroxyalkyl, aminoalkyl, amino, alkylamino, arylamino, dialkylamino, alkylarylamino, diarylamino, acylamino, hydroxyl, thiol, thioalkyl, alkoxy, alkylthio, alkoxyalkyl, aryloxy, arylalkoxy, acyloxy, nitro, carbamoyl, trifluoromethyl, phenoxy, benzyloxy, phosphonic acid, phosphate ester, sulfonic acid
  • M is selected from the group consisting of alkylene, alkenylene, alkynylene, arylene, aralkylene and alkarylene;
  • X 1 is a monovalent pro-fluorophore or pro-luminophore moiety
  • X 2 is a divalent pro-fluorophore or pro-luminophore moiety
  • each of A and A' is independently represented by formula (IV) or formula (V) :
  • M is alkylene, alkenylene, alkynylene, arylene, aralkylene or alkarylene;
  • R 8 and R 10 is preferably hydroxyl, alkoxy, or electron donating group selected from amino, alkylamino, arylamino, dialkylamino, alkylarylamino, diarylamino;
  • R 7 and R 8 come together to form a 5, 6, or 7-membered ring which is selected from aryl, heterocyclic, heteroaryl, or heteroaromatic;
  • R 10 and R 11 come together to form a 5, 6, or 7-membered ring which is selected from aryl, heterocyclic, heteroaryl, or heteroaromatic.
  • the monovalent pro-fluorophore and/or pro-luminophore moiety can include, but is not limited to, monovalent fluorescein (CAS Number 2321-07-5) , monovalent coumarin (CAS Number 91-64-5) , monovalent amine naphthalimide, monovalent dansyl, monovalent bimane (CAS Number 79769-56-5) , monovalent eosin, monovalent rhodamine (CAS Numbers 81-88-9; 989-38-8; 62669-70-9) , monovalent rhodol (CAS Number 3086-44-0) , monovalent cyanine, monovalent nile red (CAS Number 7385-67-3) , monovalent xanthone (CAS Number 90-47-1) , monovalent xanthene (CAS Number 92-83-1) , monovalent flazo orange (CAS Number 3566-94-7) , monovalent SNARF-1, monovalent lucifer yellow (CAS Numbers 71206-95
  • the divalent pro-fluorophore or/and pro-luminophore moiety can include, but is not limited to, divalent fluorescein (CAS Number 2321-07-5) , divalent coumarin (CAS Number 91-64-5) , divalent amine naphthalimide, divalent dansyl, divalent bimane (CAS Number 79769-56-5) , divalent eosin, divalent rhodamine (CAS Numbers 81-88-9; 989-38-8; 62669-70-9) , divalent rhodol (CAS Number 3086-44-0) , divalent cyanine, divalent nile red (CAS Number 7385-67-3) , divalent xanthone (CAS Number 90-47-1) , divalent xanthene (CAS Number 92-83-1) , divalent flazo orange (CAS Number 3566-94-7) , divalent SNARF-1, divalent lucifer yellow (CAS Numbers 71206-95
  • the compounds can include, but are not limited to, compounds 1–28 and 57:
  • the compounds can include, but are not limited to, compounds 29–44:
  • R H or CF 3 .
  • the compounds can include, but are not limited to, one or more free carboxyl groups, where at least one of the carboxyl groups is conjugated with a positively charged mitochondria-targeted triphenylphosphonium moiety or lysosome-targeted morpholine or N, N-disubstituted amine moiety through an amide bond linkage, where the linkage between the compound and the triphenylphosphonium moiety has the following formula (VI) or (VII) :
  • n 1-10;
  • R 12 or R 13 in formula (IX) is independently is a C 1-10 alkyl or alkene.
  • the compounds can include, but are not limited to, compounds 45–56:
  • alkyl includes saturated aliphatic hydrocarbons including straight chains and branched chains.
  • the alkyl group has 1 to 20 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms.
  • C 1-6 alkyl, ” as well as the alkyl moieties of other groups referred to herein (e.g., C 1-6 alkoxy) refers to linear or branched radicals of 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, or n-hexyl) .
  • C 1-4 alkyl refers to linear or branched aliphatic hydrocarbon chains of 1 to 4 carbon atoms
  • C 1-3 alkyl refers to linear or branched aliphatic hydrocarbon chains of 1 to 3 carbon atoms
  • C 1-2 alkyl refers to linear or branched aliphatic hydrocarbon chains of 1 to 2 carbon atoms
  • C 1 alkyl refers to methyl.
  • lower alkyl refers to linear or branched radicals of 1 to 6 carbon atoms.
  • An alkyl group optionally can be substituted by one or more (e.g. 1 to 5) suitable substituents.
  • alkenyl includes aliphatic hydrocarbons having at least one carbon carbon double bond, including straight chains and branched chains having at least one carbon-carbon double bond.
  • the 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.
  • C 2-6 alkenyl means straight or branched chain unsaturated radicals (having at least one carbon-carbon double bond) of 2 to 6 carbon atoms, including, but not limited to, ethenyl, 1-propenyl, 2-propenyl (allyl) , isopropenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and the like.
  • An alkenyl group optionally can be substituted by one or more (e.g. 1 to 5) suitable substituents.
  • the alkenyl group may exist as the pure E form, the pure Z form, or any mixture thereof.
  • alkynyl includes to aliphatic hydrocarbons having at least one carbon-carbon triple bond, including straight chains and branched chains having at least one carbon-carbon triple bond.
  • the alkynyl group has 2 to 20, 2 to 10, 2 to 6, or 3 to 6 carbon atoms.
  • C 2-6 alkynyl refers to straight or branched hydrocarbon chain alkynyl radicals as defined above, having 2 to 6 carbon atoms.
  • An alkynyl group optionally can be substituted by one or more (e.g. 1 to 5) suitable substituents.
  • cycloalkyl includes saturated or unsaturated, non-aromatic, monocyclic or polycyclic (such as bicyclic) hydrocarbon rings (e.g., monocyclics such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, or bicyclics including spiro, fused, or bridged systems (such as bicyclo [1.1.1] pentanyl, bicyclo [2.2.1] heptanyl, bicyclo [3.2.1] octanyl or bicyclo [5.2.0] nonanyl, decahydronaphthalenyl, etc.
  • monocyclics such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl
  • bicyclics including spir
  • the cycloalkyl group can have 3 to 15 carbon atoms. In some embodiments the cycloalkyl may optionally contain one, two or more noncumulative non-aromatic double or triple bonds and/or one to three oxo groups. In some embodiments, the bicycloalkyl group has 6 to 14 carbon atoms.
  • C 3-14 cycloalkyl includes saturated or unsaturated, non-aromatic, monocyclic or polycyclic (such as bicyclic) hydrocarbon rings of 3 to 14 ring-forming carbon atoms (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo [1.1.1] pentanyl, or cyclodecanyl) ; and the term “C 3-7 cycloalkyl” includes saturated or unsaturated, nonaromatic, monocyclic or polycyclic (such as bicyclic) hydrocarbon rings of 3 to 7 ring forming carbon atoms (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo [1.1.1] pentan-1-yl, or bicyclo [1.1.1] pentan-2-yl) .
  • C 3-6 cycloalkyl includes saturated or unsaturated, non-aromatic, monocyclic or polycyclic (such as bicyclic) hydrocarbon rings of 3 to 6 ring-forming carbon atoms.
  • C 3-4 cycloalkyl refers to cyclopropyl or cyclobutyl.
  • cycloalkyl moieties that have one or more aromatic rings (including aryl and heteroaryl) fused to the cycloalkyl ring, for example, benzo or thienyl derivatives of cyclopentane, cyclopentene, cyclohexane, and the like (e.g., 2, 3-dihydro-lH-indene-l-yl, or 1H-inden-2 (3H) -one-1-yl) .
  • the cycloalkyl group optionally can be substituted by 1 or more (e.g., 1 to 5) suitable substituents.
  • aryl can include all-carbon monocyclic or fused-ring polycyclic aromatic groups having a conjugated pi-electron system.
  • the aryl group has 6 or 10 carbon atoms in the ring (s) . Most commonly, the aryl group has 6 carbon atoms in the ring.
  • C 6-10 aryl means aromatic radicals containing from 6 to 10 carbon atoms such as phenyl or naphthyl.
  • the aryl group optionally can be substituted by 1 or more (e.g., 1 to 5) suitable substituents.
  • arylene refers to a divalent aryl moiety.
  • heteroaryl includes monocyclic or fused-ring polycyclic aromatic heterocyclic groups with one or more heteroatom ring members (ring forming atoms) each independently selected from 0, S and N in at least one ring.
  • the heteroaryl group has 5 to 14 ring forming atoms, including 1 to 13 carbon atoms, and 1 to 8 heteroatoms selected from 0, S, and N.
  • the heteroaryl group has 5 to 10 ring-forming atoms including one to four heteroatoms.
  • the heteroaryl group has 5 to 8 ring forming atoms including one, two or three heteroatoms.
  • the term “5-memberedheteroaryl” refers to a monocyclic heteroaryl group as defined above with 5 ring-forming atoms in the monocyclic heteroaryl ring;
  • the term “6-membered heteroaryl” includes to a monocyclic heteroaryl group as defined above with 6 ring-forming atoms in the monocyclic heteroaryl ring;
  • the term “5-or 6-membered heteroaryl” includes a monocyclic heteroaryl group as defined above with 5 or 6 ring-forming atoms in the monocyclic heteroaryl ring.
  • term “5-or 10-membered heteroaryl” includes a monocyclic or bicyclic heteroaryl group as defined above with 5, 6, 7, 8, 9 or 10 ring-forming atoms in the monocyclic or bicyclic heteroaryl ring.
  • a heteroaryl group optionally can be substituted by 1 or more (e.g., 1 to 5) suitable substituents.
  • monocyclic heteroaryls include those with 5 ring-forming atoms including one to three heteroatoms or those with 6 ring-forming atoms including one, two or three nitrogen heteroatoms.
  • fused bicyclic heteroaryls include two fused 5-and/or 6-membered monocyclic rings including one to four heteroatoms.
  • heterocyclyl includes saturated and partially saturated heteroatom-containing ring-shaped radicals having from 5 through 15 ring members selected from carbon, nitrogen, sulfur and oxygen, wherein at least one ring atom is a heteroatom.
  • Heterocyclyl radicals may contain one, two or three rings wherein such rings may be attached in a pendant manner or may be fused.
  • saturated heterocyclic radicals include saturated 3 to 6-membered heteromonocylic group containing 1 to 4 nitrogen atoms [e.g. pyrrolidinyl, imidazolidinyl, piperidino, piperazinyl, etc.
  • saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g. morpholinyl, etc. ]
  • saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms [e.g., thiazolidinyl, etc. ]
  • Examples of partially saturated heterocyclyl radicals include dihydrothiophene, dihydropyran, dihydrofuran and dihydrothiazole.
  • heterocyclic radicals include 2-pyrrolinyl, 3-pyrrolinyl, pyrrolindinyl, 1, 3-dioxolanyl, 2H-pyranyl, 4H-pyranyl, piperidinyl, 1, 4-dioxanyl, morpholinyl, 1, 4-dithianyl, thiomorpholinyl, and the like.
  • alkoxy or “alkyloxy” include an –O-alkyl group.
  • C 1-6 alkoxy or “C 1-6 alkyloxy” includes an –O– (C 1-6 alkyl) group; and the term “C 1-4 alkoxy” or “C 1-4 alkyloxy” can include an –O– (C 1-4 alkyl) group.
  • C 1-2 alkoxy or “C 1-2 alkyloxy” refers to an –O– (C 1-2 alkyl) group.
  • alkoxy examples include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy) , tert-butoxy, and the like.
  • the alkoxy or alkyloxy group optionally can be substituted by 1 or more (e.g., 1 to 5) suitable substituents.
  • C 6-10 aryloxy includes an –O– (C 6-10 aryl) group.
  • An example of a C 6-10 aryloxy group is –O–phenyl [i.e., phenoxy] .
  • the C 6-10 aryloxy group optionally can be substituted by 1 or more (e.g., 1 to 5) suitable substituents.
  • aminoalkyl includes linear and/or branched alkyl radicals having one to about ten carbon atoms any one of which may be substituted with one or more amino radicals.
  • examples of such radicals include aminomethyl, aminoethyl, aminopropyl, aminobutyl and aminohexyl.
  • substitution is optional and therefore includes both unsubstituted and substituted atoms and moieties.
  • a “substituted” atom or moiety indicates that any hydrogen on the designated atom or moiety can be replaced with a selection from the indicated substituent group (up to that every hydrogen atom on the designated atom or moiety is replaced with a selection from the indicated substituent group) , provided that the normal valency of the designated atom or moiety is not exceeded, and that the substitution results in a stable compound. For example, if a methyl group (i.e., CH 3 ) is optionally substituted, then up to 3 hydrogen atoms on the carbon atom can be replaced with substituent groups.
  • the compounds can be used as reagents for measuring, detecting and/or screening hydrogen peroxide.
  • the compounds can produce fluorescence or luminescence colors, such as blue, green, yellow, red, or far-red.
  • the compounds can be used to measure, directly or indirectly, the presence and/or amount of hydrogen peroxide in chemical samples, biological samples, and pathological samples.
  • the compounds can be used to detect the presence of, or determining the level of hydrogen peroxide in situ, in vivo and in vitro.
  • a method of using the compounds detecting the presence of, and/or determining the level of hydrogen peroxide in a sample can include, but is not limited to: contacting a compound of Formula (I) , Formula (II) , Formula (III) , or a salt thereof, with the sample to form a fluorescent and/or luminescent compound; and determining fluorescence and/or luminescence property of the fluorescent or luminescent compound.
  • a method of using the compounds for detecting the presence of, or determining the level of hydrogen peroxide in vivo in an organism can include, but is not limited to: administering a compound of Formula (I) , Formula (II) , Formula (III) , or a salt thereof, to the organism to form a fluorescent and/or luminescent compound; and determining fluorescence and/or luminescence property of the fluorescent and/or luminescent compound.
  • a method of using the compounds for detecting the presence of, or determining the level of hydrogen peroxide in vitro can include, but is not limited to: administering a compound of Formula (I) , Formula (II) , Formula (III) , or a salt thereof, to the in vitro sample to form a fluorescent and/or luminescent compound; and determining fluorescent and/or luminescent of the fluorescent and/or luminescent compound.
  • a high-throughput method of using the compounds for detecting the presence of, or determining the level of, hydrogen peroxide in samples can include, but is not limited to: contacting a compound of Formula (I) , Formula (II) , Formula (III) , or a salt thereof, with the samples to form one or more fluorescent or luminescent compounds; and determining fluorescence and/or luminescence properties of the fluorescent and/or luminescent compounds to determine the presence and/or amount of hydrogen peroxide in the samples.
  • a high-throughput method of using the compounds for screening one or more target compounds that increase or decrease the level of hydrogen peroxide can include: contacting a compound of Formula (I) , Formula (II) , Formula (III) , or a salt thereof, with target compounds to form one or more fluorescent or luminescent compounds; and measuring fluorescence or luminescence properties of the florescent or luminescent compounds to determine the presence and/or amount of the target compounds.
  • the sample for any of the methods of using the compounds can include, but is not limited to, a chemical sample, biological sample, and pathological sample.
  • the biological sample can include but is not limited to, a microorganism, cell, tissue, organ, a part from plant or animal, whole plant or animal, and their extract.
  • the pathological sample can include, but is not limited to, blood, urine, saliva, serum, breath gas, exhaled breath condensate, joint fluid, and their extract.
  • the methods of using the compounds can include the compounds being used in a fluorogenic and/or luminogenic probe composition.
  • the fluorogenic and/or luminogenic probe composition can include, but is not limited to, one or more carriers, one or more solvents, one or more acids, one or more bases, one or more buffers, and mixtures thereof.
  • the present invention relates to use of the compounds of the present invention or the fluorogenic or luminogenic probe compositions of the present invention for detecting the presence of, and/or determining the level of hydrogen peroxide in a sample, in vivo in an organism, or in vitro; or use of the compounds of the present invention or the fluorogenic or luminogenic probe compositions of the present invention for screening one or more target compounds that increase or decrease the level of hydrogen peroxide.
  • PhNTf 2 (205 mg, 0.574 mmol) was added to the resulting mixture, which was stirred at room temperature for 2 h.
  • the reaction mixture was diluted with ethyl acetate, washed with 1 N HCl, water, and brine. The organic layer was dried over anhydrous magnesium sulfate, and concentrated in vacuo to get crude triflated fluorescein derivative.
  • Cs 2 CO 3 (188 mg, 0.526 mmol) at room temperature under argon, after stirring for 30 min, MeI (60 ⁇ L, 0.956 mmol) was added.
  • This Example shows that green fluorogenic compound YS-3-42 sensitively and selectively detects hydrogen peroxide.
  • compound YS-3-42 is dissolved in 0.1 M potassium phosphate buffer at pH 7.4 to form a 10 ⁇ M solution (with 0.1%DMF and 100 ⁇ M CCl 3 CN) , with excitation and emission spectra at 480 nm and 527 nm, respectively.
  • the 10 ⁇ M solution of compound YS-3-42 is treated with hydrogen peroxide at various concentrations.
  • FIG. 2A shows that the florescence intensity of compound YS-3-42 increases with increasing concentration of hydrogen peroxide.
  • the reactivity of compound YS-3-42 is compared toward different reactive oxygen species (ROS) and reactive nitrogen species (RNS) .
  • ROS reactive oxygen species
  • RNS reactive nitrogen species
  • the 10 ⁇ M solution of compound YS-3-42 is treated with various ROS/RNS (100 ⁇ M) .
  • FIG. 2B shows that treatment with hydrogen peroxide results in a much higher increase in fluorescence intensity of compound YS-3-42 than treatment with other ROS and RNS.
  • This Example shows that green fluorogenic compound YS-4-45 sensitively and selectively detects hydrogen peroxide.
  • compound YS-4-45 is dissolved in 0.1 M phosphate buffer at pH 7.4 to form a 10 ⁇ M solution (with 0.1%DMF and 100 ⁇ M CCl 3 CN) , with excitation and emission spectra at 520 nm and 543 nm, respectively.
  • the 10 ⁇ M solution of Compound YS-4-45 is treated with hydrogen peroxide at various concentrations for 30 min.
  • FIG. 3A shows that the florescence intensity of Compound YS-4-45 increases with increasing concentration of hydrogen peroxide.
  • the reactivity of compound YS-4-45 is compared toward different reactive oxygen species (ROS) and reactive nitrogen species (RNS) .
  • ROS reactive oxygen species
  • RNS reactive nitrogen species
  • the 10 ⁇ M solution of compound YS-4-45 is treated with various ROS/RNS (100 ⁇ M) .
  • FIG. 3B shows that treatment with hydrogen peroxide for 30 min (left bars in FIG. 3B) or 60 min (right bars in FIG. 3B) results in a much higher increase in fluorescence intensity of compound YS-4-45 than treatment with other ROS and RNS.
  • This Example shows that green fluorogenic compound YS-2-172 sensitively and selectively detects hydrogen peroxide.
  • the 10 ⁇ M solution of Compound YS-2-172 is treated with hydrogen peroxide at various concentrations for 60 min.
  • FIG. 4A shows that the florescence intensity of Compound YS-2-172 increases with increasing concentration of hydrogen peroxide.
  • the reactivity of compound YS-2-172 is compared toward different reactive oxygen species (ROS) and reactive nitrogen species (RNS) .
  • ROS reactive oxygen species
  • RNS reactive nitrogen species
  • the 10 ⁇ M solution of compound YS-2-172 is treated with various ROS/RNS (100 ⁇ M) .
  • FIG. 4B shows that treatment with hydrogen peroxide for 30 min (left bars in FIG. 4B) or 60 min (right bars in FIG. 4B) results in a much higher increase in fluorescence intensity of compound YS-2-172 than treatment with other ROS and RNS.
  • RAW264.7 cells a mouse monocytic macrophage line, were acquired from ATCC (American Type Culture Collection) and maintained in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10%heat-inactivated fetal bovine serum (Gibco) and 1%penicillin/streptomycin, at 37 °C with 5%CO 2 . The growth medium was renewed every two to three days. At 80%confluence, the cells were detached by scraping, washed with fresh medium and spun down (500 rpm in Eppendorf microfuge) for cell counting. For confocal imaging, cells were typically seeded at a density of 2 ⁇ 10 4 cells/mL in 35-mm confocal dish (Mat-Tek: MA, USA) .
  • phorbol 12-myristate 13-acetate was added at specified doses to HBSS (Hank’s balanced salt solution) and co-incubated with YS-3-42 until imaging (see FIG. 5) .
  • Enzyme inhibitor NOX inhibitor DPI
  • NOX inhibitor DPI Enzyme inhibitor
  • FIG. 7 shows cytotoxicity of probes YS-3-42 and YS-4-45 in RAW 264.7 cells.
  • RAW 264.7 cells were allowed to incubate with increasing probe concentrations for 24h.
  • the probes showed negligible or no cytotoxicity after 24h incubation.
  • Data represent mean ⁇ s.e.m. for Cell-Titer Glo assays performed in triplicates.
  • YS-4-45 was also successfully applied in confocal imaging of endogenous H 2 O 2 in RAW264.7 cells (see FIG. 8) .
  • PMA challenged H 2 O 2 production could be robustly visualized, which could be efficiently attenuated by the addition of DPI.
  • red fluorogenic compound YS-4-112 could detect hydrogen peroxide quantitatively.
  • compound YS-4-112 is dissolved in 0.1 M potassium phosphate buffer at pH 7.4 to form a 10 ⁇ M solution (with 0.5%DMF and 100 ⁇ M CCl 3 CN) , with excitation and emission spectra at 565 nm and 602 nm, respectively.
  • the 10 ⁇ M solution of compound YS-4-112 is treated with hydrogen peroxide at various concentrations.
  • FIG. 9A shows that the florescence intensity of compound YS-4-112 increases with increasing concentration of hydrogen peroxide. As depicted in FIG.
  • YS-3-42 and YS-4-112 could detect hydrogen peroxide in live zebrafish.
  • Mating of adult fishes (HKWT) and selection of zebrafish embryos were done in Zebrafish Core Facility at The University of Hong Kong. Eggs were collected and placed in a 90-mm dish with E-3 medium, and incubated at 28 °C until embryos developed to the desired stage (eg 24 hpf, 48 hpf, 72 hpf; hpf: hours post fertilization) .
  • the chorion (eggshell) of the 24 hpf embryo was carefully removed by Dumont Tweezers under microscope to free the embryo.
  • Embryos were treated by 10 ⁇ M YS-3-42 or YS-4-112 (with 100 ⁇ M CCl 3 CN in 1 mL E3 buffer) with or without PMA (500 ng/mL) for 30 min at room temperature, then washed with 1 mL E-3 medium twice before imaging on LSM 710. H 2 O 2 production and contribution in zebrafish at different development stages could be visualized with YS-4-112. (FIG. 10A) . PMA challenged H 2 O 2 production in Zebrafish was also successfully detected with YS-3-42 (FIG. 10B) .
  • This example shows the subject compounds could be applied in developing high throughput assay.
  • 0.125, 0.25, 0.5, 1.0, 2.0 mM (final concentrations) of antioxidant were added to 200 ⁇ L 1.0 mM H 2 O 2 in 0.1 M phosphate buffer at pH 7.4, and the solution was incubated for 30 min at 37 °C on a 96-well plate.
  • 2 ⁇ L of resulting solution was added to a solution of YS-4-112 (10 ⁇ M in 0.1 M phosphate buffer at pH 7.4 with 100 ⁇ M CCl 3 CN, 200 ⁇ L in each well) , and the solution was incubated for 30 min at 37 °C on a 96-well plate to assay the remaining H 2 O 2 concentrations.
  • This 96-well plate could be placed on a plate reader to determine the fluorescence emission at 602 nm at each well with an excitation at 565 nm.
  • the antioxidant capacity could be evaluated by the H 2 O 2 scavenging percentage, which was calculated by the following equation:
  • F 0 fluorescence intensity without antioxidant
  • F 1 fluorescence intensity with various concentrations of antioxidant
  • F blank fluorescence background of YS-4-112.
  • H 2 O 2 scavenging activities of ascorbic acid and epigallocatechin gallate (EGCG) were determined by this high throughput assay (FIG. 11A and FIG. 11B) . Those data are very useful for drug screening and evaluation.
  • the compounds of the present invention can have the same or similar mechanism for the reaction with hydrogen peroxide as proposed in FIG. 1. Therefore, similar technical effects and applications, including but not limited to sensitive and selective detection of hydrogen peroxide, can also be obtained for the compounds of the present invention.

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Abstract

L'invention concerne des composés et des procédés de leur utilisation pour la détection in situ de peroxyde d'hydrogène. Les composés de l'invention peuvent comprendre des composés de formule (I), de formule (II), ou de formule (III), ou un sel de ceux-ci, X1 étant un pro-fluorophore monovalent ou un fragment pro-luminophore ; X2 étant un pro-fluorophore divalent ou un fragment pro-luminophore ; chacun de A et A' étant indépendamment représenté par la formule (IV) ou la formule (V).
PCT/CN2018/073585 2017-01-20 2018-01-22 Composés et procédés de détection de peroxyde d'hydrogène Ceased WO2018133859A1 (fr)

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EP3493855A4 (fr) * 2016-08-02 2020-04-01 ISI Life Sciences, Inc. Méthode de détection de cellules cancéreuses.
EP3960817A1 (fr) * 2020-08-28 2022-03-02 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Colorants fluorescents photoactivables avec des groupes d'encagement hydrophiles et leur utilisation
CN115368307A (zh) * 2022-07-12 2022-11-22 重庆医科大学 肼类化合物及其制备方法和用途
WO2025196201A1 (fr) * 2024-03-21 2025-09-25 Sony Semiconductor Solutions Corporation Composés de fluorane substitués par amide utilisés en tant que teintures thermochromiques jaunes et leur utilisation dans des systèmes d'imagerie

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CN117986288B (zh) * 2024-03-04 2025-06-03 南京工业大学 一种基于硅罗丹明的过氧化氢(h2o2)检测荧光探针的合成与应用

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CN1300962A (zh) * 1999-12-17 2001-06-27 柯尼卡株式会社 照相处理元件和使用它的成象方法
JP2003075970A (ja) * 2001-08-31 2003-03-12 Konica Corp ハロゲン化銀カラー写真感光材料、カラー写真感光材料、その画像形成方法及びデジタル画像情報作製方法
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EP3960817A1 (fr) * 2020-08-28 2022-03-02 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Colorants fluorescents photoactivables avec des groupes d'encagement hydrophiles et leur utilisation
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WO2025196201A1 (fr) * 2024-03-21 2025-09-25 Sony Semiconductor Solutions Corporation Composés de fluorane substitués par amide utilisés en tant que teintures thermochromiques jaunes et leur utilisation dans des systèmes d'imagerie

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