WO 2018/133859 Al 26 July 2018 (26.07.2018) W ! P O PCT

(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization I International Bureau (10) International Publication Number (43) International Publication Date WO 2018/133859 Al 26 July 2018 (26.07.2018) W ! P O PCT

(51) International Patent Classification: HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, C07C 47/56 (2006.01) C07D 265/38 (2006.01) KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, C07D 493/10 (2006.01) C09K 11/06 (2006.01) MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, C07D 217/22 (2006.01) OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY,TH, TJ, TM, TN, (21) International Application Number: TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. PCT/CN2018/073585 (84) Designated States (unless otherwise indicated, for every (22) International Filing Date: kind of regional protection available): ARIPO (BW, GH, 22 January 2018 (22.01.2018) GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, (25) Filing Language: English UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, (26) Publication Language: English EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, (30) Priority Data: MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, 62/448,5 14 20 January 2017 (20.01.2017) US TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). (71) Applicant: THE UNIVERSITY OF HONG KONG [CN/CN]; Pokfulam Road, Hong Kong, Hong Kong (CN). Published: (72) Inventors: YANG, Dan; 16D, Glory Heights, 52 Lyttelton — with international search report (Art. 21(3)) Road, Mid-level, Hong Kong, Hong Kong (CN). YE, Sen; 4D, On Hing Building, 326-332 Des Voeux Road West, Sai Ying Pun, Hong Kong, Hong Kong (CN). HU, Jun; Room 10, 4/F, Joy Fat Mansion, 522-530 Queen's Road West, Sai Wan, Hong Kong, Hong Kong (CN). (74) Agent: PANAWELL & PARTNERS, LLC; 1002-1005, China Life Tower, 16 Chao Yang Men Wai Street, Chaoyang District, Beijing 100020 (CN). (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN,

(54) Title: COMPOUNDS AND METHODS FOR DETECTION OF HYDROGEN PEROXIDE

20 0 -

3 5 1 00-

00-

S00- S

. 0- 500 S50 S00 650 700 00 FIG. 2A Wavelength nm

(57) Abstract: Disclosed herein are compounds and methods for using the same for the detection of hydrogen peroxide m situ. The I compounds can include compounds of Formula (I), Formula (II), or Formula (III), or a salt thereof, wherein X is a monovalent pro- fluorophore or pro-luminophore moiety; X2 is a divalent pro-fluorophore or pro-luminophore moiety; each of A and A' is independently represented by formula (IV) or formula (V) : COMPOUNDS AND METHODS FOR DETECTION OF HYDROGEN PEROXIDE

Technical Field The present invention belongs to the field of fluorogenic or luminogenic probes, and relates to compounds and methods for the detection of hydrogen peroxide.

Background of the Invention 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 (H20 2) is a reactive oxygen species that plays a crucial role in oxidative stress and signal transduction in organisms. In order to investigate the reaction mechanisms of hydrogen peroxide, 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. There are commercially available fluorescence probes for hydrogen peroxide detection analysis, for example, Ara p ex Red reagent has been used to detect the release of hydrogen peroxide from activated human leukocytes. However, many commercially available fluorescence probes have drawbacks, such as slow response time and sensitivity, making tracking hydrogen peroxide in situ difficult.

Brief Summary of the Invention Because of the need to develop new chemiluminescence and/or fluorescence probes to monitor oxidative stress and signal transduction for various pathologies, provided herein are compounds and methods for using the same that can be used to detect hydrogen peroxide in situ. In at least one specific embodiment, the compounds can include compounds of Formula (I), Formula (II), or Formula (III), or a salt thereof:

A-X (II) A-X2-A' (III), where each of A and A is independently represented by formula (IV) or formula (V): where the linkage between the compound and the triphenylphosphonium moiety has the following formula (VI) or (VII):

where n = 1-10; or the linkage between the compound and the morpholine or N,N-disubstituted amine moiety has the following form

In another specific embodiment, 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.

Brief Description of the Drawings In the following detailed description, reference is made to the accompanying figures, depicting exemplary, non-limiting and non-exhaustive embodiments of the invention. So that the manner in which the above recited features of the present invention can be understood in detail, a more particular description of the invention, briefly summarized above, can be had by reference to the embodiments, some of which are illustrated in the appended figures. It should be noted, however, that the appended figures illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention can admit to other equally effective embodiments. 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. In FIG. 2A, the curves, from top to bottom, µΜ Υ µΜ µΜ respectively represent: 10 solution of compound 8-3-42+100 H2O2, 10 solution Υ µΜ µΜ Υ µΜ of compound 8-3-42+50 H20 2, 10 solution of compound 8-3-42+40 H20 2, 10 µΜ Υ µΜ µΜ solution of compound 8-3-42+30 H20 2, 10 solution of compound Υ µΜ µΜ Υ µΜ µΜ -3-42+20 H20 2, 10 solution of compound -3-42+ 10 H20 2, 10 solution of Υ µΜ µΜ Υ µΜ µΜ compound 8-3-42+8 H20 2, 10 solution of compound -3-42+6 H20 2, 10 Υ µΜ µΜ Υ µΜ solution of compound 8-3-42+4 H20 2, 10 solution of compound -3-42+2 H20 2, and 10 µΜ solution of compound YS-3-42 only. FIG. 2B is a graph showing the fluorescence intensity of YS-3-42 in the presence of different ROS/ RNS FIG. 3A is graph showing the florescence intensity of compound YS-4-45 with increasing concentration of hydrogen peroxide. In FIG. 3A, the curves, from top to bottom, respectively µΜ Υ µΜ µΜ represent: 10 solution of compound 8-4-45+100 H20 2, 10 solution of compound Υ µΜ µΜ Υ µΜ µΜ 8-4-45+50 H20 2, 10 solution of compound -4-45+40 H20 2, 10 solution of Υ µΜ µΜ Υ µΜ µΜ compound 8-4-45+30 H20 2, 10 solution of compound -4-45+20 H20 2, 10 Υ µΜ µΜ Υ µΜ solution of compound 8-4-45+10 H20 2, 10 solution of compound -4-45+8 µΜ Υ µΜ µΜ H20 2, 10 solution of compound -4-45+4 H20 2, and 10 solution of compound YS-4-45 only. FIG. 3B is a graph showing the fluorescence intensity of YS-4-45 in the presence of different ROS/ RNS. FIG. 4A is graph showing the florescence intensity of compound YS-2-172 with increasing concentration of hydrogen peroxide. In FIG. 4A, the curves, from top to bottom, µΜ Υ µΜ µΜ respectively represent: 10 solution of compound 8-2-172+500 H20 2, 10 solution Υ µΜ µΜ of compound 8-2-172+100 H20 2, and 10 solution of compound YS-2-172 only.e FIG. 4B is a graph showing the fluorescence intensity of YS-2-172 in the presence of different ROS/ RNS. FIG. 5 shows the confocal images {left) for RAW264.7 cells co-incubated with YS-3-42 (10 µΜ ) with or without phorbol 12-myristate 13-acetate (PMA) (200 ng/mL) and DPI (100 nM). Relative mean fluorescence intensity of cells in each group was quantified {right). Scale bars represent 10 µ . Data are mean ± s.e.m., n = 79-109 cells; ***, p < 0.001 versus untreated cells or control. FIG. 6 shows the confocal images {left) of RAW264.7 (normal cells) and MDA-MB-231 (breast cancer cells) cells co-incubated with YS-3-42 (10 µΜ ) {right). Relative mean fluorescence intensity of cells in each group was quantified. Scale bars represent 10 µ . Data are mean ± s.e.m., n = 72-109 cells; **, p < 0.01. 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 µΜ ) with or without phorbol 12-myristate 13-acetate (PMA) (200 ng/mL) and DPI (100 nM). Scale bars represent 10 µιη. FIG. 9A is graph showing the florescence intensity of compound YS-4-112 with increasing concentration of hydrogen peroxide. In FIG. 9A, the curves, from top to bottom, µΜ Υ µΜ µΜ respectively represent: 10 solution of compound 8-4-112+100 H20 2, 10 solution Υ µΜ µΜ Υ µΜ of compound 8-4-112+50 H20 2, 10 solution of compound 8-4-112+30 H20 2, 10 µΜ Υ µΜ µΜ solution of compound 8-4-112+20 H20 2, 10 solution of compound Υ µΜ µΜ Υ µΜ µΜ 8-4-112+10 H20 2, 10 solution of compound 8-4-112+8 H20 2, 10 solution Υ µΜ µΜ Υ µΜ of compound 8-4-112+6 H20 2, 10 solution of compound 8-4-112+4 H20 2, 10 µΜ Υ µΜ µΜ solution of compound 8-4-112+2 H20 2, and 10 solution of compound YS-4-112 only. FIG. 9B is a standard H20 2 calibration curve for quantification. FIG. 10A shows the confocal images of live zebrafishes at different development stages co-incubated with YS-4-112 (10 µΜ). FIG. 10B shows the confocal images of PMA treated live zebrafishes co-incubated with YS-3-42 (10 µΜ) . Scale bars represent 500 µ .

FIG.1 1A shows the H20 2 scavenging activity of ascorbic acid, and FIG. 1IB shows the

H20 2 scavenging activity of epigallocatechin gallate in a high throughput assay with YS-4-112.

Detailed Disclosure of the Invention 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 performance of these compounds in hydrogen peroxide imaging was highly robust in multiple cell types including normal cells and cancer cells. In addition, these compounds can also be employed to develop fast, accurate, or high-throughput detection methods for drug screening, cancer screening and disease diagnosis. The compounds can include, but are not limited to, compounds of Formula (I), Formula (II), or Formula (III), or a salt thereof: Α-Χ2-Α' (III), where: R , R2, R3, R4, and R5 are independently selected from the group consisting of H, F, CI, 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 (-SO 3H), sulfonate ester, sulfonamide, -C(=0)-P 1, and -C(=0)-M-P 2, where P and P2 is selected from the group consisting of hydrogen, halo, alkoxy, hydroxy, thiol, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, carbamate, amino, alkylamino, arylamino, dialkylamino, alkylarylamino, diarylamino, alkylthio, heteroalkyl, alkyltriphenylphosphonium, and a heterocyclyl having from 3 to 7 ring atoms, or R2 and R3 come together to form a 5, 6, or 7-membered ring selected from the group consisting of aryl, heterocyclic, heteroaryl and heteroaromatic, or R4 and R5 come together to form a 5, 6, or 7-membered ring which is selected from the group consisting of aryl, heterocyclic, heteroaryl and heteroaromatic; M is selected from the group consisting of alkylene, alkenylene, alkynylene, arylene, aralkylene and alkarylene; 6 R is selected from the group consisting of a hydrogen, alkyl, alkoxyalkyl, alkanoyl, -CF 3, halogen-substituted lower alkyl, and (C=0)-0-Z 1, where Z is the group selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl and arylalkyl; X1 is a monovalent pro-fluorophore or pro-luminophore moiety; X2 is a divalent pro-fluorophore or pro-luminophore moiety; each of A and A' is independently represented by formula (IV) or formula (V):

where at least one of R7 and R9 is (C=0)-W 1, where W is selected from the group consisting of hydrogen, alkyl, alkoxyalkyl, alkanoyl, CF3, halogen-substituted lower alkyl, and (C=0)-0-Z 2, where Z2 is the group selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl or arylalkyl, and the other of one ofR andR , R8, R 0, and R are independently selected from the group consisting of H, F, CI, 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, hydroxy, thiol, thioalkyl, alkoxy, alkylthio, alkoxyalkyl, aryloxy, arylalkoxy, acyloxy, nitro, carbamoyl, trifluoromethyl, phenoxy, benzyloxy, phosphonic acid, phosphate ester, sulfonic acid (-SO3H), sulfonate ester, sulfonamide, -C(=0)-P 3 and -C(=0)-M-P 4, where P3 and P4 are independently selected from the group consisting of hydrogen, halo, alkoxy, hydroxy, thiol, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, carbamate, amino, alkylamino, arylamino, dialkylamino, alkylarylamino, diarylamino, alkylthio, heteroalkyl, alkyltriphenylphosphonium, or heterocyclyl having from 3 to 7 ring atoms; M is alkylene, alkenylene, alkynylene, arylene, aralkylene or alkarylene; at least one of R8 and R 0 is preferably hydroxyl, alkoxy, or electron donating group selected from amino, alkylamino, arylamino, dialkylamino, alkylarylamino, diarylamino; or R7 and R8 come together to form a 5, 6, or 7-membered ring which is selected from aryl, heterocyclic, heteroaryl, or heteroaromatic; or R 0 and R 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 71206-95-6; 67769-47-5), monovalent laurdan (CAS Number 74515-25-6), monovalent 2-naphthylamine (CAS Number 91-59-8), monovalent resorufin (CAS Number 635-78-9) and monovalent luciferin (such as CAS Number 2591-17-5). 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 71206-95-6; 67769-47-5), divalent laurdan (CAS Number 74515-25-6), divalent 2-naphthylamine (CAS Number 91-59-8), divalent resorufin (CAS Number 635-78-9) and divalent lucifenn (such as CAS Number 2591-17-5). The compounds can include, but are not limited to, compounds 1-28 and 57: - 8 - wherein, R=H or CF3

The compounds can include, but are not limited to, compounds 29-44:

wherein, R=H or CF3 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):

where n = 1-10; or the linkage between the compound and the morpholine or N,N-disubstituted moiety has the following formula (VIII) or ( IX):

where n = 1 - 10; R or R in formula (IX) is independently is a Ci-io alkyl or alkene. The compounds can include, but are not limited to, compounds 45-56:

wherein, R=H or CF3

As used herein, the term "alkyl" includes saturated aliphatic hydrocarbons including straight chains and branched chains. In some embodiments, 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. For example, the term "Ci-6 alkyl," as well as the alkyl moieties of other groups referred to herein (e.g., Ci-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). For yet another example, the term "C 1-4 alkyl" refers to linear or branched aliphatic hydrocarbon chains of 1 to 4 carbon atoms; the term "C 1-3 alkyl" refers to linear or branched aliphatic hydrocarbon chains of 1 to 3 carbon atoms; the term "C 1-2 alkyl" refers to linear or branched aliphatic hydrocarbon chains of 1 to 2 carbon atoms; and the term "Ci alkyl" refers to methyl. The term "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. As used herein, the term "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. In some embodiments, 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. For example, as used herein, the term "C2-6 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-l-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. When the compounds contain an alkenyl group, the alkenyl group may exist as the pure E form, the pure Z form, or any mixture thereof. As used herein, the term "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. In some embodiments, the alkynyl group has 2 to 20, 2 to 10, 2 to 6, or 3 to 6 carbon atoms. For example, as used herein, the term "C2-6 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. As used herein, the term "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[l.l.l]pentanyl, bicyclo[2.2.1]heptanyl, bicyclo[3.2.1]octanyl or bicyclo[5.2.0]nonanyl, decahydronaphthalenyl, etc.). 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. For example, the term "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[l.l.l]pentanyl, or cyclodecanyl); and the term "C3-7 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[l.l.l]pentan-l-yl, or bicyclo[l.l.l]pentan-2-yl). For another example, the term "C3-6 cycloalkyl" includes saturated or unsaturated, non-aromatic, monocyclic or polycyclic (such as bicyclic) hydrocarbon rings of 3 to 6 ring-forming carbon atoms. For yet another example, the term "C3-4 cycloalkyl" refers to cyclopropyl or cyclobutyl. Also included in the term "cycloalkyl" are 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 lH-inden-2(3H)-one-l-yl). The cycloalkyl group optionally can be substituted by 1 or more (e.g., 1 to 5) suitable substituents. As used herein, the term "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. For example, as used herein, the term "C6 -io 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. The term "arylene" refers to a divalent aryl moiety. As used herein, the term "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 . In some embodiments, the heteroaryl group has 5 to 10 ring-forming atoms including one to four heteroatoms. The heteroaryl group can also contain one to three oxo or thiono (i.e. =S) groups. In some embodiments, the heteroaryl group has 5 to 8 ring forming atoms including one, two or three heteroatoms. For example, 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; and 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. For another example, 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. Examples of 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. Examples of fused bicyclic heteroaryls include two fused 5- and/or 6-membered monocyclic rings including one to four heteroatoms. As used herein, the term "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. Examples of 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. Non-limiting examples of heterocyclic radicals include 2-pyrrolinyl, 3-pyrrolinyl, pyrrolindinyl, 1,3-dioxolanyl, 2H-pyranyl, 4H-pyranyl, piped dinyl, 1,4-dioxanyl, morpholinyl, 1,4-dithianyl, thiomorpholinyl, and the like. As used herein, the term "alkoxy" or "alkyloxy" include an -O-alkyl group. For example, the term "Ci -6 alkoxy" or "Ci -6 alkyloxy" includes an -0-(Ci -6 alkyl) group; and the term "C1-4 alkoxy" or "C1-4 alkyloxy" can include an -0-(C 1-4 alkyl) group. For another example, the term "C1-2 alkoxy" or "C1-2 alkyloxy" refers to an -0-(C 1-2 alkyl) group. Examples of alkoxy 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.

As used here, the term "C6- 10 aryloxy" includes an -O-(C 6-10 aryl) group. An example of a

C6-io aryloxy group is -O-phenyl [i.e., phenoxy]. The C6-io aryloxy group optionally can be substituted by 1 or more (e.g., 1 to 5) suitable substituents. As used herein, the term "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. As used herein, the term "oxo" refers to =0. When an oxo is substituted on a carbon atom, they together form a carbonyl moiety [-C(=0)-]. When an oxo is substituted on a sulfur atom, they together form a sulfinyl moiety [-S(=0)-]; when two oxo groups are substituted on a sulfur atom, they together form a sulfonyl moiety [-S(=0)2-] . As used herein, the term "optionally substituted" means that 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., CH3) 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 f orescent 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. In some embodiments, 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.

MATERIALSAND METHODS The examples and embodiments described herein are for illustrative purposes only and various modifications or changes in light thereof will be suggested to persons skilled in the art and are included within the spirit and purview of this application. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto.

Example 1 - synthesis of green Fluorogenic Compounds YS-3-42, YS-4-45, YS-2-172, and Red Fluorogenic Compound YS-4-112

To a stirred solution of NaH (60 wt. % in mineral oil, 44 mg, 1.10 mmol) in DMF (2 mL, anhydrous) was added 2-hydroxy-4-(hydroxymethyl)benzaldehyde (152 mg, 1.00 mmol) in

DMF (2 mL, anhydrous) at 0 °C under argon. After stirred for 15 min, chloromethyl methly ether (91 1.20 mmol) was added dropwise, and then stirred for another 30 min. Then 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. Compound YS-3-1 was isolated as pale yellow oil by flash chromatography on silica gel, by δ using EtOAc:Hexane (1:1) as an eluent. Yield: 174 mg (89%). 1H NMR (300 MHz, CDC13) 10.39 (s, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.19 (s, 1H), 7.00 (d, J = 8.0 Hz, 1H), 5.27 (s, 2H), 4.69 δ (s, 2H), 3.48 (s, 3H); C NMR (75 MHz, CDC13) 189.7, 159.9, 150.1, 128.6, 124.4, 119.7, 112.6, 94.5, 64.4, 56.6.

To a stirred solution of YS-3-1 (130 mg, 0.662 mmol) and carbamoylimidazole (100 mg, 0.796 mmol) in DMF (4 mL, anhydrous) was added NaH (60% wt in mineral oil, 30 mg, 0.728 mmol). The suspension was stirred at room temperature for 18 h, and then concentrated under an air stream. Compound YS-3-2 was isolated as a white sticky solid by flash chromatography on silica gel, by using EtOAc:Hexane (1:1) as an eluent. Yield: 54 mg (32%). 1H NMR (300 δ MHz, CDCI3) 10.41 (s, 1H), 7.75 (d, J = 7.9 Hz, 1H), 7.13 (s, 1H), 6.99 (d, J = 7.9 Hz, 1H), δ 5.25 (s, 2H), 5.07 (s, 2H), 3.47 (s, 3H), 2.77 (d, J = 4.9 Hz, 3H); C NMR (75 MHz, CDC13) 189.4, 159.7, 156.7, 145.4, 128.6, 124.8, 120.6, 113.7, 94.6, 65.7, 56.6, 27.6.

To a solution of YS-3-3 (200 mg, 0.478 mmol) in methanol (4 mL), was added a solution of NaOH (191 mg, 4.78 mmol) in water (2 mL). The resulting mixture was stirred at room temperature for 1 h, and then the organic solvent was evaporated, neutralized with 1 N HCl until a large amount of precipitate was formed. The suspension was filtered, washed with water and dried in vacuo to get the crude fluorescein derivative. To a solution of the crude product in

DMF (5 mL), was added Et3N (201 , 1.43 mmol) at room temperature under argon. After stirring for 10 min, 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. To a solution of the crude product in DMF (5 mL) was added Cs2C0 3 (188 mg, 0.526 mmol) at room temperature under argon, after stirring for 30 min, Mel (60 , 0.956 mmol) was added. The resulting mixture was stirred for 2 h, and then 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. Compound YS-3-9 was isolated as a white sticky solid by flash chromatography on silica gel, by using EtOAc:Hexane (1:4) as an eluent. Yield: 77 mg (31%). δ 1H NMR (400 MHz, CDC13) 8.69 (s, 0 . 5H), 8.36 (d, = 8.0 Hz, 0 . 5H), 8.31 (d, = 8.0 Hz, 0.5H), 8.10 (d, = 8.0 Hz, 0.5H), 7.81 (s, 0.5H), 6.96 (d, = 8.8 Hz, 1H), 6.88 (d, = 8.8 Hz, 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). C NMR δ (100 MHz, CDC13) 168.02, 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. F NMR δ (376 MHz, CDC13) -72.6 (m, 3F).

An oven-dried round bottom flask was charged with Pd2(dba) 3 (12 mg, 0.013 mmol), Xantphos (23 mg, 0.039 mmol) and CS2CO3 (59 mg, 0.182 mmol), and flushed with argon for 5 min. A solution of YS-3-9 (70 mg, 0.13 mmol) and YS-3-2 (40 mg, 0.156 mmol) in anhydrous dioxane (5 mL) was added, and the resulting mixture was stirred at room temperature under argon for 30 min, heated to 100 °C and stirred for another 20 h . The reaction mixture was allowed to cool to room temperature, diluted with DCM and filtered through a pad of celite. The filtrate was then concentrated in vacuo. Compound YS-3-36 was isolated as a white sticky solid by flash chromatography on silica gel, by using EtOAc:DCM (1:5) as an δ eluent. Yield: 38 mg (46%). 1H MR (400 MHz, CDC13) 10.44 (s, 1H), 8.69 (s, 0.5H), 8.37 - 8.32 (m, 0.5H), 8.31 - 8.27 (m, 0.5H), 8.09 (d, = 8.0 Hz, 0.5H), 7.82 (s, 0.5H), 7.80 (d, = 8.0 Hz, 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, δ 3H), 3.37 (s, 3H); C MR (100 MHz, CDC13) 189.39, 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, 129.1 1, 129.07, 128.75, 128.56, 128.52, 127.1 1, 126.90, 125.43, 125.38, 124.98, 124.36, 121.29, 120.34, 116.16, 113.99, 113.45, 112.19, 110.31

To a solution of YS-3-36 ( 11 mg, 0.017 mmol) in DCM (3 mL) in ice/water bath, TFA (3 mL) was added dropwise, and then the solution was stirred at room temperature for 2 h . The reaction mixture was concentrated in vacuo, azeotroped with toluene for 3 times and then dissolved in THF (4.5 mL). A solution of LiOH (2.5 mg, 0.104 mmol) in water (1.5 mL) was added dropwise at room temperature. The resulting mixture was stirred at room temperature for 2 h . The reaction mixture was diluted with ethyl acetate, washed with 1 N HC1, water, and brine. The organic layer was dried over anhydrous magnesium sulfate, and concentrated in vacuo. Compound YS-3-42 was isolated as a white sticky solid by flash chromatography on silica gel, by using EtOH:DCM (1:9) as an eluent. Yield: 7.8 mg (79%). 1H NMR (400 MHz, Acetone-d6) δ 10.01 (s, 1H), 8.55 (s, 0.5H), 8.42 (d, = 7.9 Hz, 0.5H), 8.36 (d, = 7.5 Hz, 0.5H), 8.15 (d, = 8.2 Hz, 0.5H), 7.88 (s, 0,5H), 7.78 - 7.71 (m, 1H), 7.48 (d, = 7.5 Hz, 0.5H), 7.46 (d, = 8.6 Ηζ,ΙΗ), 7.20 (d, = 8.6 Hz, H), 7.05 (d, = 9.0 Hz, 2H), 6.98 - 6.88 (m, 3H), 6.88 - 6.82 (m, 1H), 6.74 (d, = 8.7 Hz, 1H), 5.23 (s, 1H), 3.89 (s, 3H), 3.41 (s, 3H); C δ NMR (100 MHz, CDC1 3) 196.25, 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

Yield = 45% YS-4-42

The mixture of 3-iodophenol (1.1 g, 5.0 mmol) and phthalic anhydride (370 mg, 2.50 mmol) in methanesulfonic acid (3 mL) was stirred at 135 °C for 48 h under argon. After cooling to room temperature, the reaction mixture was poured into 50 mL of an ice/water mixture and stirred to precipitate a grey solid. The solid was collected by filtration and dissolved in chloroform before passing through a pad of silica to yield colorless solution. The solution was concentrated to give YS-4-42 [817170-65-3] as a white solid. Yield: 629 mg δ (45%). 1H NMR (300 MHz, CDC13) 8.07 - 8.00 (m, 1H), 7.69 - 7.62 (m, 4H), 7.38 (d, = 1.6 Hz, = 1.6 Hz, 1H), 7.16 - 7.08 (m, 1H), 6.55 (s, 1H), 6.52 (s, 1H).

An oven-dried round bottom flask was charged with Pd2(dba) 3 (12 mg, 0.013 mmol),

Xantphos (23 mg, 0.039 mmol) and Cs2C0 3 (59 mg, 0.182 mmol), and flushed with argon for 5 min. A solution of YS-4-42 (36 mg, 0.066 mmol) and YS-3-2 (50 mg, 0.197 mmol) in dioxane (5 mL) was added, and the resulting mixture was first stirred at room temperature under argon for 30 min, heated to 100 °C and stirred for another 20 h . The reaction mixture was allowed to cool to room temperature, diluted with DCM and filtered through a pad of celite. The filtrate was then concentrated in vacuo. Compound YS-4-44 was isolated as a white sticky solid by flash chromatography on silica gel, by using EtOAc:Hexane (3:2) as an eluent. δ Yield: 18 mg (34%). 1H NMR (300 MHz, CDC13) 10.44 (s, 2H), 8.05 (d, J = 6.8 Hz, 1H), 7.79 (d, J = 8.0 Hz, 2H), 7.73 - 7.63 (m, 2H), 7.25 (d, J = 1.9 Hz, 2H), 7.19 (d, J = 7.1 Hz, 1H), 7.1 1 (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, δ 6H), 3.37 (s, 6H); C NMR (75 MHz, CDC13) 189.3, 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. YS-4-44 Yield = 99% YS-4-45

To a solution of YS-4-44 (12 mg, 0.015 mmol) in DCM (3 mL) in ice/water bath was added TFA (3 mL) dropwise, and then the solution was stirred at room temperature for 2 h . The reaction mixture was concentrated in vacuo and azeotroped with toluene for 3 times. Compound YS-4-45 was isolated as a white sticky solid by flash chromatography on silica gel, δ by using EtOAc:Hexane (3:2) as an eluent. Yield: 10 mg (99%). 1H NMR (400 MHz, CDC13) 11.04 (s, 2H), 9.87 (s, 2H), 8.05 (d, J = 7.4 Hz, 1H), 7.74 - 7.62 (m, 2H), 7.53 (d, J = 7.9 Hz, 2H), 7.22 (d, J = 7.5 Hz, 1H), 7.01 (d, J = 8.7 Hz, 2H), 6.93 (d, J = 8.0 Hz, 2H), 6.90 (s, 2H), δ 6.82 (s, 1H), 6.80 (s, 1H), 5.19 (s, 4H), 3.38 (s, 6H); C NMR (150 MHz, CDC13) 196.1, 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

YS-2-1 66

The suspension of 4-bromo-l,8-naphthalic anhydride (1.229 g, 4.40 mmol) and propylamine (0.400 mL, 4.90 mmol) in ethanol (200 mL) was stirred at 50 °C for 1 h, and then heated to reflux for another 1 h . The reaction mixture was cooled to room temperature, and then concentrated in vacuo. Compound YS-2-166 [100865-05-2] was isolated as a white sticky solid by flash chromatography on silica gel, by using Hexane:DCM (1:1) as an eluent. Yield: δ 1.268 g (90%). 1H NMR (300 MHz, CDC13) 8.65 (d, = 7.3 Hz, 1H), 8.55 (d, = 8.5 Hz, 1H), 8.40 (d, J = 7.9 Hz, 1H), 8.03 (d, J = 7.9 Hz, 1H), 7.84 (dd, J = 8.5, 7.3 Hz, 1H), 4.17 -

4.10 (m, 2H), 1.83 - 1.69 (m, 2H), 1.01 (t, J = 7.4 Hz, 3H). YS-2-1 66 YS-2-1 67

A solution of YS-2-166 (250 mg, 0.789 mmol), Cu powder (5 mg, 0.0789 mmol), ammonium hydroxide solution (32 wt. % NH 3 in water, 5 mL) and N -methyl-2-pyrrolidone (5 mL) in a sealed tube was stirred at 100 °C for 12 h . Then the reaction mixture was cooled to room temperature, 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. Compound YS-2-167 [94860-68-1] was isolated as a yellow sticky solid by flash chromatography on silica gel, by using acetone: chloroform (1:6) as an eluent. Yield: 74 mg δ (37%). 1H NMR (300 MHz, CDC1 3) 8.59 (d, = 7.3 Hz, 1H), 8.41 (d, = 8.2 Hz, 1H), 8.10 (d, J = 8.4 Hz, 1H), 7.65 (dd, J = 8.4, 7.3 Hz, 1H), 6.88 (d, J

2H), 1.81 - 1.63 (m, 2H 1.00 (t, J = 7.4 Hz, 3H).

YS-2-1 65

To a solution of 2-hydroxy-4-(hydroxymethyl)benzaldehyde (40 mg, 0.263 mmol) and

K2C0 3 (72 mg, 0.526 mmol) in THF (5 mL anhydrous) was added allyl bromide (173 , 0.526 mmol) at room temperature under argon. The solution was stirred under reflux for 12 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. Compound YS-2-165 was isolated as a colorless oil by flash chromatography on silica gel, by δ using EtOAc:Hexane (1:2) as an eluent. Yield: 3 1 mg (70%). 1H NMR (400 MHz, CDC13) 10.46 (s, 1H), 7.79 (d, = 7.9 Hz, 1H), 7.03 (s, 1H), 6.96 (d, = 7.9 Hz, 1H), 6.13 - 6.01 (m,

1H), 5.48 - 5.41 (m, 1H), 5.33 (d, J = 10.6 Hz, 1H), 4.73 (s, 2H), 4.66 (d, J = 5.1 Hz, 2H); C δ NMR (100 MHz, CDC13) 189.7, 161.4, 149.9, 132.4, 128.8, 124.3, 118.8, 118.3, 110.8, 69.3, 64.8. YS-2-167 YS-2-1 72

To a solution of YS-2-167 (46 mg, 0.182 mmol) in toluene (5 mL, anhydrous) were added DIPEA (90 , 0.546 mmol) and triphosgene (65 mg, 0.218 mmol) at room temperature under argon. The solution was heated to reflux for 1 h . After cooling to room temperature, YS-2-165 (35 mg, 0.182 mmol) in anhydrous DCM (2 mL) was added to the reaction mixture, which was stirred for another 3 h, concentrated in vacuo, and dissolved in ethanol (5 mL). The resulting solution was degassed with argon for 30 min and added Pd(PPh 3)4 (21 mg, 0.0182 mmol) under argon. The resulting mixture was heated under reflux for 12 h . The reaction mixture was cooled to room temperature, filtered over a pad of celite and concentrated in vacuo. Compound YS-2-172 was isolated as a yellow sticky solid by flash chromatography on silica gel, by using δ EtOAc:Hexane (1:2) as an eluent. Yield: 12 mg (15%). 1H NMR (300 MHz, CDC13) 11.09 (s, 1H), 9.91 (s, 1H), 8.64 (d, J = 7.1 Hz, 1H), 8.61 (d, J = 8.3 Hz, 1H), 8.36 (d, J = 8.3 Hz, 1H),

8.20 (d, J = 8.4 Hz, 1H), 7.83 - 7.75 (m, 1H), 7.60 (d, J = 8.2 Hz, 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 = 7.4 Hz, 3H); δ C NMR (125 MHz, CDC13) 196.2, 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.

resorufin Yield = 74% YS-4-1 10 To a solution of resorufin (250 mg, 1.17 mmol) in DMF (12 mL, anhydrous) in ice/water bath was added NaH (60% wt in mineral oil, 59 mg, 1.41 mmol) and the mixture was stirred for 30 min. Then, PhNTf 2 (500 mg, 1.41 mmol) was added, and the reaction mixture was allowed to warm to room temperature and stirred for another 8 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. Compound YS-4-110 [941601-75-8] was isolated as a yellow sticky solid by flash column chromatography on silica gel, by using EtOAc:Hexane (1:2) as an eluent. Yield: 300 mg (74%). 1H NMR (300 MHz, δ CDC13) 7.85 - 7.75 (m, 1H), 7.36 (d, J = 9.9 Hz, 1H), 7.23 - 7.18 (m, 2H), 6.81 (dd, J = 9.9, 2.0 Hz, 1H), 6.27 (d, = 2.0 Hz, 1H).

YS-4-1 11

An oven-dried round bottom flask was charged with Pd2(dba) 3 (18 mg, 0.020 mmol), Xantphos (34 mg, 0.059 mmol) and CS2CO3 (59 mg, 0.182 mmol), and flushed with argon for 5 min. A solution of YS-4-110 (83 mg, 0.24 mmol) and YS-3-2 (56 mg, 0.20 mmol) in dioxane (5 mL) was added, and the resulting mixture was first stirred at room temperature under argon for 30 min, then heated to 100 °C and stirred for another 24 h . The reaction mixture was allowed to cool to room temperature, diluted with DCM and filtered through a pad of celite. The filtrate was then concentrated in vacuo. Compound YS-4-111 was isolated as a red sticky solid by flash column chromatography on silica gel, by using EtOAc:DCM (1:4) as an eluent. δ Yield: 5 1 mg (57%). 1H NMR (300 MHz, CD2C 12) 7.83 - 7.71 (m, 2H), 7.43 (d, = 9.8 Hz, 1H), 7.40 - 7.32 (m, 2H), 7.17 (s, 1H), 7.04 (d, J = 7.9 Hz, 1H), 6.80 (dd, J = 9.8, 2.0 Hz, 1H), 6.27 (d, J = 2.0 Hz, 1H), 5.26 (s, 2H), 5.23 (s, 2H), 3.47 (s, 3H), 3.42 (s, 3H); C NMR (75 δ MHz, CD2C 12) 189.35, 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.

YS-4-1 11 YS-4-1 12 To a solution of YS-4-111 (12 mg, 0.027 mmol) in DCM (3 mL) in ice/water bath was added TFA (3 mL) dropwise, and then the solution was stirred at room temperature for 2 h . The reaction mixture was concentrated in vacuo and azeotroped with toluene for 3 times. Compound YS-4-112 was isolated as a red sticky solid by flash column chromatography on silica gel, by using EtOAc:DCM (1:4) as an eluent. Yield: 10 mg (92%). 1H NMR (300 MHz,

CDC13) 11.08 (s, 1H), 9.89 (s, 1H), 7.77 (d, = 9.2 Hz, 1H), 7.56 (d, J = 7.8 Hz, 1H), 7.44 (d, = 9.8 Hz, 1H), 7.36 (d, = 7.7 Hz, 2H), 6.97 (d, = 7.9 Hz, 2H), 6.86 (dd, = 9.8, 2.0 Hz, δ 1H), 6.33 (d, = 2.0 Hz, 1H), 5.24 (s, 2H), 3.46 (s, 3H); C NMR (100 MHz, CDC13) 196.2, 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.

Example 2 - Sensitive and selective Detection of HYDROGEN PEROXIDE with Green Fluorogenic Compound YS-3-42 This Example shows that green fluorogenic compound YS-3-42 sensitively and selectively detects hydrogen peroxide. Specifically, compound YS-3-42 is dissolved in 0.1 M potassium phosphate buffer at pH 7.4 to form a 10 µΜ solution (with 0.1% DMF and 100 µΜ

CCI 3CN), with excitation and emission spectra at 480 nm and 527 nm, respectively. The 10 µΜ 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). Specifically, the 10 µΜ solution of compound YS-3-42 is treated with various ROS/RNS (100 µΜ) . 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.

Example 3 - Sensitive and selective Detection of HYDROGEN PEROXIDE with Green Fluorogenic Compound YS-4-45 This Example shows that green fluorogenic compound YS-4-45 sensitively and selectively detects hydrogen peroxide. Specifically, compound YS-4-45 is dissolved in 0.1 M µΜ µΜ phosphate buffer at pH 7.4 to form a 10 solution (with 0.1% DMF and 100 CC1 3CN), with excitation and emission spectra at 520 nm and 543 nm, respectively. The 10 µΜ 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). Specifically, the 10 µΜ solution of compound YS-4-45 is treated with various ROS/RNS (100 µΜ) . 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.

Example 4 - Sensitive and selective Detection of HYDROGEN PEROXIDE with Green Fluorogenic Compound YS-2-172 This Example shows that green fluorogenic compound YS-2-172 sensitively and selectively detects hydrogen peroxide. Specifically, compound YS-2-172 is dissolved in 0.1 M µΜ phosphate buffer/CH 3CN (v/v = 1/1) at pH 7.4 to form a 10 solution (with 0.1% DMF), with excitation and emission spectra at 429 nm and 530 nm, respectively. The 10 µΜ 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). Specifically, the 10 µΜ solution of compound YS-2-172 is treated with various ROS/RNS (100 µΜ). 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.

Example 5 - application of subject compounds in cell assay 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% C0 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 χ 104 cells/mL in 35-mm confocal dish (Mat-Tek: MA, USA). For acute hydrogen peroxide induction (30 min), phorbol 12-myristate 13-acetate (PMA) 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) was added along with PMA during hydrogen peroxide induction. It is known that cancer cells, compared to normal cells, are under increased oxidative stress and increased generation of ROS. Thus, the difference in the basal level ofhydrogen peroxide between cancer and normal cells was tested with YS-3-42 (see FIG. 6). In addition, subject compounds showed no cytotoxicity in their application concentrations (see FIG. 7). 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. Similarly, YS-4-45 was also successfully applied in confocal imaging of endogenous

H20 2 in RAW264.7 cells (see FIG.8). PMA challenged H20 2 production could be robustly visualized, which could be efficiently attenuated by the addition of DPI.

Example 6 - Quantification of HYDROGEN PEROXIDE with Red Fluorogenic Compound YS-4-112 This example shows that red fluorogenic compound YS-4-112 could detect hydrogen peroxide quantitatively. Specifically, compound YS-4-112 is dissolved in 0.1 M potassium µΜ µΜ phosphate buffer at pH 7.4 to form a 10 solution (with 0.5% DMF and 100 CC1 3CN), with excitation and emission spectra at 565 nm and 602 nm, respectively. The 10 µΜ 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. 9B, a linear relationship of fluorescence intensities of YS-4-112 testing solutions at 602 nm with the concentrations of µΜ H 2O2 (0-30 ) was observed, and the detection limit was estimated to be as low as 4.8 nM

(3a/k). This standard calibration curve could be applied to quantify H20 2 concentrations in various samples and high throughput assays.

Example 7 - application of subject compounds in Live Animal assay This example shows that 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. µΜ µΜ Embryos were treated by 10 YS-3-42 or YS-4-112 (with 100 CC13CN 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 2O2 production and contribution in zebrafish at different development stages could be visualized with YS-4-112. (FIG. 10A).

PMA challenged H 2O2 production in Zebrafish was also successfully detected with YS-3-42 (FIG. 10B).

Example 8 - application of subject compounds in High ThrougHput assay This example shows the subject compounds could be applied in developing high throughput assay. Firstly, 0.125, 0.25, 0.5, 1.0, 2.0 mM (final concentrations) of antioxidant µΙ were added to 200 _, 1.0 mM H20 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. Then, 2 µΙ_, of resulting solution was added to µΜ µΜ a solution of YS-4-112 (10 in 0 .1 M phosphate buffer at pH 7.4 with 100 CC13CN, 200 in each well), and the solution was incubated for 30 min at 37 °C on a 96-well plate to assay the remaining H20 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 H20 2 scavenging percentage, which was calculated by the following equation:

H = (Fo - Fi) / (Fo - F la k) * 100% Fo, fluorescence intensity without antioxidant; F], fluorescence intensity with various concentrations of antioxidant; F anh fluorescence background of YS-4-112.

The H20 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. While the present invention is described herein with reference to illustrative embodiments for particular applications, it should be understood that the invention is not limited thereto. Those having ordinary skill in the art and access to the teachings provided herein will recognize additional modifications, applications, and embodiments within the scope thereof and additional fields in which the present invention would be of significant utility. It is therefore intended by the appended claims to cover any and all such applications, modifications and embodiments within the scope of the present invention.

Compounds having the structures 1-6, 8-21, 23-28, wherein R=H or CF3, and compounds having the structures 7 and 22, wherein R=CF 3, can be prepared with similar methods for preparing Compound YS-3-42 (i.e., compound having the structure 7, wherein R=H) and Compound YS-2-172 (i.e., compound having the structure 22, wherein R=H), in which starting materials for corresponding monovalent pro-fluorophore or pro-luminophore moiety and/or a starting material with R=CF 3 are used. Structures for these compounds are confirmed by H HMR and MS.

Compounds having the structures 30-44, wherein R=H or CF3, and compound having the structure 29, wherein R=CF 3, can be prepared with similar methods for preparing Compound YS-4-45 (i.e., compound having the structure 29, wherein R=H), in which starting materials of corresponding divalent pro-fluorophore or pro-luminophore moiety and/or a starting material with R=CF3 are used. Structures for these compounds are confirmed by 1H HMR and MS. 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. As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including", "includes", "having", "has", "with", or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising." The transitional terms/phrases (and any grammatical variations thereof) "comprising", "comprises", "comprise", "consisting essentially of, "consists essentially of, "consisting", and "consists" can be used interchangeably. All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification. References www.thermofisher.com/order/catalog/product/A22177 www.thermofisher.com/hk/en/home/references/molecular-probes-the-handbook/probes-fo r-reactive-oxygen-species-including-nitric-oxide/generating-and-detecting-reactive-oxygen-spe cies.html CLAIMS

1. A compound of Formula (I), Formula (II), or Formula (III), or a salt thereof:

(I),

A-X (II),

A-X2-A' (III), wherein:

R , R2, R3, R4, and R5 are independently selected from the group consisting of H, F, CI,

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, tnfluoromethyl, phenoxy, benzyloxy, phosphonic acid, phosphate ester, sulfonic acid (-SO3H), sulfonate ester, sulfonamide, -C(=0)-P 1, and -C(=0)-M-P 2, wherein P and P is selected from the group consisting of hydrogen, halo, alkoxy, hydroxy, thiol, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, carbamate, amino, alkylamino, arylamino, dialkylamino, alkylarylamino, diarylamino, alkylthio, heteroalkyl, alkyltriphenylphosphonium, and a heterocyclyl having from 3 to 7 ring atoms, or R2 and R3 come together to form a 5, 6, or 7-membered ring selected from the group consisting of aryl, heterocyclic, heteroaryl and heteroaromatic, or R4 and R5 come together to form a 5, 6, or 7-membered ring which is selected from the group consisting of aryl, heterocyclic, heteroaryl and heteroaromatic, M is selected from the group consisting of alkylene, alkenylene, alkynylene, arylene, aralkylene and alkarylene; R6 is selected from the group consisting of a hydrogen, alkyl, alkoxyalkyl, alkanoyl, 1 -CF 3, halogen-substituted lower alkyl, and (C=0)-0-Z , wherein Z is the group selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl and arylalkyl; X1 is a monovalent pro-fluorophore or pro-luminophore moiety; X2 is a divalent pro-fluorophore or pro-luminophore moiety; each of A and A' is independently represented by formula (IV) or formula (V):

wherein at least one of R^dR is (C - W , wherein W is selected from the group consisting of hydrogen, alkyl, alkoxyalkyl, 2 alkanoyl, CF3, halogen-substituted lower alkyl, and (C=0)-0-Z , wherein Z2 is the group selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl or arylalkyl, and the other of one of R andR 9, R8, R 0 , and R are independently selected from the group consisting of H, F, CI, 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, hydroxy, thiol, thioalkyl, alkoxy, alkylthio, alkoxyalkyl, aryloxy, arylalkoxy, acyloxy, nitro, carbamoyl, trifluoromethyl, phenoxy, benzyloxy, phosphonic acid, phosphate 3 4 ester, sulfonic acid (-S0 3H), sulfonate ester, sulfonamide, -C(=0)-P and -C(=0)-M-P , wherein P3 and P4 are independently selected from the group consisting of hydrogen, halo, alkoxy, hydroxy, thiol, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, carbamate, amino, alkylamino, arylamino, dialkylamino, alkylarylamino, diarylamino, alkylthio, heteroalkyl, alkyltriphenylphosphonium, or heterocyclyl having from 3 to 7 ring atoms; M is alkylene, alkenylene, alkynylene, arylene, aralkylene or alkarylene; at least one of R8 and R 0 is preferably hydroxyl, alkoxy, or electron donating group selected from amino, alkylamino, arylamino, dialkylamino, alkylarylamino, diarylamino; or R7 and R8 come together to form a 5, 6, or 7-membered ring which is selected from aryl, heterocyclic, heteroaryl, or heteroaromatic; or R 0 and R come together to form a 5, 6, or 7-membered ring which is selected from aryl, heterocyclic, heteroaryl, or heteroaromatic.

2 . The compound of claim 1, wherein at least one of R , R3, and R5 is hydroxyl group in Formula (I).

3 . The compound of claim 2, wherein R6 is selected from the group consisting of hydrogen, 3 3 CF3, halogen-substituted lower alkyl, or (C=0)-0-Z , wherein Z is an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl or arylalkyl in Formula (I).

4 . The compound of claim 1, wherein at least one of R8 and R 0 is a hydroxyl group in Formula (IV) and (V).

5. The compound of claim 4, wherein at least one of R7 andR 9 is (C=0)-W 2, wherein W2 is a 4 4 hydrogen, CF3, halogen-substituted lower alkyl, or (C=0)-0-Z , wherein Z is a group selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl or arylalkyl.

6 . The compound of claim 1, wherein X1 is selected from the group consisting of: monovalent pro-fluorescein, monovalent pro-coumarin, monovalent pro-amine naphthalimide, monovalent pro-dansyl, monovalent pro-bimane, monovalent pro-eosin, monovalent pro-rhodamine, monovalent pro-rhodol, monovalent pro-cyanine, monovalent nile red, monovalent xanthone, monovalent xanthene, monovalent flazo orange, monovalent SNARF-1, monovalent pro-lucifer yellow, monovalent pro-laurdan, monovalent pro-2-naphthylamine, monovalent resorufin, and monovalent luciferin.

7 . The compound of claim 1 or claim 6, wherein the compound has one of the structures 1-28 and 57:

wherein R=H or CF3.

8. The compound of claim 1, wherein X2 is selected from the group consisting of: pro-fluorescein, divalent pro-coumarin, divalent pro-amine naphthalimide, divalent pro-dansyl, divalent pro-bimane, divalent pro-eosin, divalent pro-rhodamine, divalent pro-rhodol, divalent pro-cyanine, divalent nile red, divalent xanthone, divalent xanthene, divalent flazo orange, divalent SNARF-1, and divalent resorufin, including conjugates.

The compound of claim 1 or claim 8, wherein the compound has one of the structures -44:

wherein, R=H or CF3

10. The compound of claim 1, wherein the compound comprises one or more free carboxyl groups, wherein 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, wherein the linkage between the compound and the triphenylphosphonium moiety has the following formula (VI) or (VII):

or the linkage between the compound and the morpholine or N,N-disubstituted moiety has the following formula (VIII) or ( IX):

wherein n = 1 - 10; R or R in formula (IX) is independently is a Ci-io alkyl or alkene.

The compound of claim 10, wherein the compound has one of the structures 45-56: wherein, R=H or CF3

12. A fluorogenic or luminogenic probe composition comprising the compound of any one of claims 1 to 11, and a carrier.

13. The fluorogenic or luminogenic probe composition of claim 12, wherein the fluorogenic or luminogenic probe composition further comprises a solvent, an acid, a base, a buffer solution, or a combination thereof.

14. A method for detecting the presence of, and/or determining the level of hydrogen peroxide in a sample, comprising: contacting a compound of any one of claims 1 to 11 with the sample to form a fluorescent or luminescent compound; and determining fluorescence or luminescence property of the fluorescent or luminescent compound.

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

16. The method of claim 15, wherein the sample is a biological sample comprising a microorganism, cell, tissue, organ, a part from plant or animal, whole plant or animal, or their extract.

17. The method of claim 15, wherein the sample is a pathological sample comprising blood, urine, saliva, serum, breath gas, exhaled breath condensate, joint fluid, or their extract.

18. A method for detecting the presence of, or determining the level of hydrogen peroxide in vivo in an organism, comprising: administering a compound of any one of claims 1 to 11 to the organism to form a fluorescent or luminescent compound; and determining fluorescence or luminescence property of the fluorescent or luminescent compound.

19. A method for detecting the presence of, or determining the level of hydrogen peroxide in vitro, comprising: administering a compound of any one of claims 1 to 11 to the in vitro sample to form a fluorescent or luminescent compound; and determining fluorescence or luminescence property of the fluorescent or luminescent compound.

20. A high-throughput screening method for detecting the presence of, or determining the level of, hydrogen peroxide in samples, wherein the high-throughput method comprises the steps of: contacting a compound of any one of claims 1 to 11 with the samples to form one or more fluorescent or luminescent compounds; and determining fluorescence or luminescence properties of the fluorescent or luminescent compounds to determine the presence and/or amount of hydrogen peroxide in the samples. 21. A high-throughput method for screening one or more target compounds that increase or decrease the level of hydrogen peroxide, wherein the high-throughput method comprises the steps of: contacting a compound of any one of claims 1 to 11 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.

22. Use of a compound of any one of claims 1 to 11 or a fluorogenic or luminogenic probe composition of claim 12 or 13 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 a compound of any one of claims 1 to 1 1 or a fluorogenic or luminogenic probe composition of claim 12 or 13 for screening one or more target compounds that increase or decrease the level of hydrogen peroxide.

n erna ona app ca on o. PCT/CN2018/073585

A. CLASSIFICATION OF SUBJECT MATTER C07C 47/56(2006.01)i; C07D 493/10(2006.01)i; C07D 217/22(2006.01)i; C07D 265/38(2006.0 1)i; C09K l l/06(2006.01)i

According to International Patent Classification (IPC) or to both national classification and IPC B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) C07C 47/-; C07D 493/-; C07D 217/-; C07D 265/-; C09K 11/-

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) CNPAT, CNKI, WPI, EPODOC, ISI Web of Knowledge, STN((REGISTRY,MARPAT):luminescence,fluorescence,dye,probe, hydrogen peroxide,H202,searching the formula

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

X US 6174656 Bl (FUJI PHOTO FILM CO. , LTD.) 16 January 2001 (2001-01-16) 1-5 description, column 1, lines 1-22, column 11, line 28 to column 17 , line 20, column 2 1, line 25 to column 22, line 40, column 33 , line 1 to column 34, line 30

Y US 6174656 Bl (FUJI PHOTO FILM CO. , LTD.) 16 January 2001 (2001-01-16) 6-22 description , column 7 , line 1 to column 8 , line 27 , column 34 , lines 47-65

X JP 2003075970 A (KONISHIROKU PHOTO IND.) 12 March 2003 (2003-03-12) 1-5 pages 55-59

Y JP 2003075970 A (KONISHIROKU PHOTO IND.) 12 March 2003 (2003-03-12) 6-22 claims 8 , 10

X CN 1300962 A (KONICA CORPORATION) 27 June 2001 (2001-06-27) 1-5 description , page 29

Y WO 2012074693 Al (ENZO BIOCHEM, INC. ET AL.) 07 June 2012 (2012-06-07) 6-22 description, page 3 , lines 4-10, page 9 , paragraphs 1-2, page 2 1, paragraph 2 to page 23 , paragraph 2 , page 36 , paragraph 4 to page 37 , paragraph 2

I IFurther documents are listed in the continuation of Box C. | 1See patent family annex.

Special categories of cited documents: later document published after the international filing date or priority date and not in conflict with the application but cited to understand the document defining the general state of the art which is not considered principle or theory underlying the invention to be of particular relevance document of particular relevance; the claimed invention cannot be earlier application or patent but published on or after the international considered novel or cannot be considered to involve an inventive step filing date when the document is taken alone document which may throw doubts on priority claim(s) or which is document of particular relevance; the claimed invention cannot be cited to establish the publication date of another citation or other considered to involve an inventive step when the document is special reason (as specified) combined with one or more other such documents, such combination document referring to an oral disclosure, use, exhibition or other being obvious to a person skilled in the art means document member of the same patent family document published prior to the international filing date but later than the priority date claimed Date of the actual completion of the international search Date of mailing of the international search report

08 April 2018 19 April 2018

Name and mailing address of the ISA/CN Authorized officer STATE INTELLECTUAL PROPERTY OFFICE OF THE P.R.CHINA 6, Xitucheng R ., Jimen Bridge, Haidian District, Beijing CHEN,Xi 100088 China Facsimile No. (86-10)62019451 Telephone No. (86-10)53962145 Form PCT/ISA/210 (second sheet (Jul 2009 n erna ona app ca on o. PCT/CN2018/073585

Box No. II Observations where certain claims were found unsearchable (Continuation of item 2 of first sheet)

This international search report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons:

1. 0 Claims Nos.: 14-20,22 because they relate to subject matter not required to be searched by this Authority, namely:

[1] Claims 14-20, 22 are directed to a method for diagnosis of diseases, the search has been carried out and based on the use of claimed compound for manufacturing medicaments having the alleged effects.

2. Claims Nos.: because they relate to parts of the international application that do not comply with the prescribed requirements to such an extent that no meaningful international search can be carried out, specifically:

3. Claims Nos.: because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a).

Form PCT/ISA/210 (continuation of first sheet) (July 2009) n erna ona app ca on o. Information on patent family members PCT/CN2018/073585

Patent document Publication date Publication date Patent family member(s) cited in search report (day/month/year) (day/month/year)

US 6174656 Bl 16 January 2001 US 6420610 Bl 16 July 2002 JP 2003075970 A 12 March 2003 None CN 1300962 A 27 June 2001 US 2002146650 Al 10 October 2002 EP 1109064 Al 20 June 2001 US 6455235 Bl 24 September 2002 WO 2012074693 Al 07 June 2012 US 2012122136 Al 17 May 2012 US 8828678 B2 09 September 2014 EP 3192849 Al 19 July 2017 CA 2818102 Al 07 June 2012 EP 2640805 Bl 19 April 2017 US 9574222 B2 2 1 February 2017 US 2017137863 Al 18 May 2017 US 2015191768 Al 09 July 2015 EP 2640805 Al 25 September 2013

Form PCT/ISA/210 (patent family annex) (July 2009)