Compounds and methods for the detection of superoxide

Abstract

Disclosed herein are compounds that can be used to selectively detect superoxide in a sample. Also disclosed herein are compositions that include the compounds, and methods for detecting superoxide using the compounds.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 224,087, filed July 21, 2021, which is incorporated by reference herein in its entirety.

[0002] Disclosed herein are compounds that can be used to selectively detect superoxide in a sample. Also disclosed herein are compositions that include the compounds, and methods for detecting superoxide using the compounds. [Background technology]

[0003] Superoxide is a highly labile and highly regulated reactive oxygen species that is central to cellular homeostasis. Dysregulation of superoxide can lead to disease states including cardiovascular disease, cancer, atherosclerosis, hypertension, diabetes, and endothelial dysfunction. Direct and specific detection of superoxide in a cellular context is highly desirable. The short residence time (10–20 s depending on superoxide dismutase availability) allows for rapid detection of superoxide in a cellular context. -6 ~10 -9 Due to its half-life of 1000 s, it is inherently difficult to accurately measure superoxide in cells. Current techniques for detecting superoxide often rely on chemiluminescent and fluorescent probes that lack sufficient selectivity. For example, hydroethidine has a tendency to undergo autoxidation and other non-superoxide specific oxidation reactions, which result in the production of fluorescent products with emission spectra similar to superoxide specific products. Another compound, luminol, can also react with reactive oxygen species other than superoxide, and one of the reactive intermediates in the oxidation pathway of luminol itself produces superoxide, leading to a potential overestimation of the amount of superoxide present. Summary of the Invention

[0004] In one aspect, the present disclosure provides a compound of formula (II): [ka] or a salt thereof, wherein R 1 But -CN and [ka] is selected from R 2 is selected from hydrogen and halo; n is 0, 1, 2, or 3; Each R 3 is independently selected from C1-C4 alkyl, C1-C4 alkoxy, -OC(O)-C1-C4 alkyl, hydroxy, amino, and the group-linker-X, where X is a targeting moiety; R 4a and R 4b one of which is hydroxy or -OC(O)-C1-C4 alkyl and the other is hydrogen or a group -linker-X, where X is a targeting moiety; R 5 is selected from hydrogen and C1-C4 alkyl; Z is a bond or a formula [ka] Based on R 6 is selected from C1-C4 alkyl and group-linker-Y, where Y is a targeting moiety, or a salt thereof.

[0005] In some embodiments, Z is of the formula [ka] In some embodiments, R 6 is methyl. In some embodiments, R 6 is the group-linker-Y, where Y is a mitochondrial targeting moiety. In some embodiments, Y is a triphenylphosphonium moiety.

[0006] In some embodiments, n is 1 and R 3 is selected from -OC(O)CH3 and hydroxy.

[0007] In some embodiments, R 4a is hydrogen and R 4b is hydroxy or -OC(O)CH3.

[0008] In some embodiments, the compound is a compound of formula (I): [ka] or a salt thereof, wherein R 1 But -CN and [ka] is selected from R 2 is selected from hydrogen and halo; n is 0, 1, 2, or 3; Each R 3 is independently selected from C1-C4 alkyl, C1-C4 alkoxy, amino, and the group-linker-X, where X is a targeting moiety; R 4a and R 4b one of is hydroxy and the other is hydrogen or a group-linker-X, where X is a targeting moiety; R 5 is selected from hydrogen and C1-C4 alkyl, or a salt thereof.

[0009] In some embodiments, R 1 is -CN. In some embodiments, R 1 teeth, [ka] In some embodiments, R 5 is selected from hydrogen and methyl.

[0010] In some embodiments, R 2 is hydrogen. In some embodiments, R 2 is fluoro.

[0011] In some embodiments, the compound is a compound of formula (Ia): [ka] or a salt thereof, wherein R 4a is hydrogen or a group-linker-X, where X is a targeting moiety, or a salt thereof.

[0012] In some embodiments, the compound is a compound of formula (Ib): [ka] or a salt thereof, wherein R 4b is hydrogen or a group-linker-X, where X is a targeting moiety, or a salt thereof.

[0013] In some embodiments, n is 0, 1, or 2, and each R 3 is independently selected from C1-C4 alkyl, C1-C4 alkoxy, -OC(O)-C1-C4 alkyl, and hydroxy. In some embodiments, n is 0, 1, or 2, and each R 3 is independently selected from C1-C4 alkyl and C1-C4 alkoxy. In some embodiments, at least one R 3 is the group-linker-X, where X is a mitochondrial targeting moiety. In some embodiments, X is a triphenylphosphonium moiety.

[0014] In some embodiments, the compound is [ka] [ka] [ka] and salts thereof.

[0015] In one aspect, the present disclosure provides a method for detecting superoxide in a sample, comprising: contacting the sample with a compound disclosed herein (e.g., a compound of Formula (I) or Formula (II)); contacting the sample with a luciferin-utilizing luciferase, if such a luciferin-utilizing luciferase is not already present in the sample; and detecting luminescence in the sample.

[0016] In some embodiments, the sample comprises living cells. In some embodiments, the cells express a luciferin-utilizing luciferase. In some embodiments, the method comprises adding a luciferin-utilizing luciferase to the sample. In some embodiments, the luciferin-utilizing luciferase is a firefly luciferase or a click beetle luciferase.

[0017] In one aspect, the disclosure provides a kit comprising a compound disclosed herein (e.g., a compound of Formula (I) or Formula (II)). In some embodiments, the kit further comprises a luciferin-utilizing luciferase enzyme or a nucleotide sequence encoding a luciferin-utilizing luciferase enzyme. In some embodiments, the kit further comprises a buffer reagent.

[0018] Other aspects and embodiments will become apparent in light of the following description and drawings. [Brief description of the drawings]

[0019] [Figure 1]1A-1B show schematic diagrams of a method for detecting superoxide in cells using compounds such as those disclosed herein. Unstable superoxide generated outside (A) or inside (B) a cell is detected using a proluciferin probe. After superoxide production, the proluciferin probe reacts rapidly with the highly unstable superoxide to generate a stable luciferin product. Luciferin is detected using a luciferase reaction, and the luminescence signal is proportional to superoxide production. [Diagram 2] Shown is a schematic diagram of a luminescent assay to detect superoxide in a sample of cells or other analytes; the "processing" step refers to the addition of unknown experimental components (e.g., drug candidates), which can be added before, after, or simultaneously with the superoxide probe. [Diagram 3] 1 shows data from an assay to detect superoxide in acellular samples as described in Example 2. [Figure 4] 1 shows data from an assay to detect superoxide in cells following treatment with the superoxide-generating drug compounds dimethoxynaphthoquinone (DMNQ) and antimycin A, as described in Example 3. [Diagram 5] 1 shows data from an assay to detect xanthine oxidase (XO) activity in vitro as described in Example 4. [Figure 6] 6A-6B show data from an assay detecting superoxide production by macrophages as described in Example 5. [Figure 7] 1 shows data from an assay detecting superoxide production in a kinetic mode as described in Example 6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Disclosed herein are probe compounds, particularly caged luciferin and hydroxycyanobenzothiazole compounds and their derivatives, that have high specificity for the detection of superoxide versus other reactive oxygen species. After reaction with superoxide, a stable reporter product is formed, allowing the assay to be performed in an "add and read" plate-based format or in a kinetic mode with media sampling.

[0021] definition Although any method and material similar or equivalent to those described herein can be used to practice or test the embodiments described herein, some preferred methods, compositions, devices, and materials are described herein.However, in describing the materials and methods of the present invention, it should be understood that the present invention is not limited to the specific molecules, compositions, methodologies, or protocols described herein, as these may vary through routine experimentation and optimization.It should also be understood that the terms used herein are for the purpose of describing only specific versions or embodiments, and are not intended to limit the scope of the embodiments described herein.

[0022] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. For example, any nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein and the techniques thereof are well known and commonly used in the art. The meaning and scope of the terms should be clear, however, in case of any potential ambiguity, the definitions provided herein shall take precedence over any dictionary or foreign definitions. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0023] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "peptide" is a reference to one or more peptides and equivalents thereof known to those skilled in the art, and so forth.

[0024] As used herein, the term "and / or" includes any and all combinations of the listed items, including any of the listed items individually. For example, "A, B, and / or C" includes A, B, C, AB, AC, BC, and ABC, each of which shall be considered to be separately described by the phrase "A, B, each and / or C."

[0025] As used herein, the term "comprising" and linguistic variations thereof indicate the presence of the recited feature(s), element(s), method step(s), etc., without excluding the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term "consisting of" and linguistic variations thereof indicate the presence of the recited feature(s), element(s), method step(s), etc., and excludes unrecited feature(s), element(s), method step(s), etc., except for impurities normally associated therewith. The phrase "consisting essentially of" indicates the recited feature(s), element(s), method step(s), etc., as well as any additional feature(s), element(s), method step(s), etc. that do not substantially affect the basic nature of the composition, system, or method. Many embodiments herein are described using the open "comprising" term. Such embodiments encompass embodiments in the closed form "consisting of" and / or "consisting essentially of," which may alternatively be claimed or described using such language.

[0026] For the recitation of numerical ranges herein, each intervening numerical value therebetween to the same degree of precision is expressly contemplated. For example, for the range 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0027] "Luminescence" refers to the light output of a luciferase enzyme under appropriate conditions, e.g., in the presence of a suitable substrate, such as luciferin or a hydroxycyanobenzothiazole compound (e.g., generated after reaction of superoxide with a compound of formula (I) or formula (II)). Light output can be measured as an immediate or near-instantaneous measurement of light output at the start of a luminescence reaction, which can be initiated after addition of a luciferin substrate (sometimes referred to as "T=0" luminescence or "flash"). The reaction chamber (e.g., a plate such as a 96-well plate) can be placed in a reader that can measure light output, e.g., using a luminometer or photomultiplier tube. Light output or luminescence can also be measured over time, e.g., for a period of seconds, minutes, hours, etc., in the same reaction chamber. Light output or luminescence can be reported as an average over time, a half-life of the signal decay, a sum of the signal over a period of time, or a peak output. Luminescence can be measured in relative light units (RLU).

[0028] As used herein, the term "sample" is used in its broadest sense. In one sense, it is meant to include specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples can be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, serum, and the like. Samples can also refer to cells, cell lysates, or purified forms of the enzymes, peptides, and / or polypeptides described herein (e.g., purified protein samples). Cell lysates can include cells lysed with a lysing agent or a lysate, such as rabbit reticulocyte or wheat germ lysate. Samples can also include cell-free samples, such as in vitro samples and cell-free expression systems. Environmental samples include environmental materials, such as surface materials, soil, water, crystals, and industrial samples. Samples can also include purified samples, such as purified protein samples. However, such examples should not be construed as limiting the sample types applicable to the present invention.

[0029] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, chemical elements are defined according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivities, are described in Sorrell, Organic Chemistry, 2001, pp. 111-115, 1997. nd edition,University Science Books,Sausalito,2006,Smith,March's Advanced Organic Chemistry:Reactions,Mechanism,and Structure,7 th Edition, John Wiley & Sons, Inc., New York, 2013, Larock, Comprehensive Organic Transformations, 3 rdEdition, John Wiley & Sons, Inc., New York, 2018, and Carruthers, Some Modern Methods of Organic Synthesis, 3 rd Edition, Cambridge University Press, Cambridge, 1987, the entire contents of each of which are incorporated herein by reference.

[0030] As used herein, the term "acyl" refers to the group -C(=O)R, where R is selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, and heterocyclylalkyl.

[0031] As used herein, the term "alkyl" refers to a linear or branched saturated hydrocarbon chain. An alkyl chain can be, for example, 1 to 30 carbon atoms (C1 to C2). 30 Alkyl), 1 to 24 carbon atoms (C1 to C 24 alkyl), e.g., 1 to 16 carbon atoms (C 16 Alkyl), 1 to 14 carbon atoms (C1 to C 14 Alkyl), 1 to 12 carbon atoms (C1 to C 12 Alkyl), 1 to 10 carbon atoms (C1 to C 10 The alkyl radicals may contain 1 to 8 carbon atoms (C1-C8 alkyl), 1 to 6 carbon atoms (C1-C6 alkyl), or 1 to 4 carbon atoms (C1-C4 alkyl). Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl.

[0032] As used herein, the term "alkenyl" refers to a straight or branched hydrocarbon chain containing at least one carbon-carbon double bond. The double bond(s) may be located at any position along the hydrocarbon chain. Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl.

[0033] As used herein, the term "alkynyl" refers to a straight or branched hydrocarbon chain containing at least one carbon-carbon triple bond. The triple bond(s) may be located at any position along the hydrocarbon chain. Representative examples of alkynyl include, but are not limited to, ethynyl, propynyl, and butynyl.

[0034] The term "alkoxy" as used herein refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, and tert-butoxy.

[0035] As used herein, the term "amino" refers to the group -NR x R y R x and R y is selected from hydrogen and alkyl (eg, C1-C4 alkyl).

[0036] As used herein, the term "aryl" refers to an aromatic carbocyclic ring system having a single ring (monocyclic) or multiple rings (e.g., bicyclic or tricyclic), including fused ring systems, and zero heteroatoms. As used herein, an aromatic carbocyclic ring system having 6 to 14 ring carbon atoms (C6-C8) is an aromatic ring system having 6 to 14 ring carbon atoms (C6-C8). 14 Aryl), 6 to 12 ring carbon atoms (C6 to C 12 aryl), or 6 to 10 ring carbon atoms (C 10Representative examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, and phenanthrenyl.

[0037] As used herein, the term "cycloalkyl" refers to a saturated carbocyclic ring system containing 3 to 10 carbon atoms and 0 heteroatoms. Cycloalkyls can be monocyclic, bicyclic, bridged, fused, or spirocyclic. Representative examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, adamantyl, bicyclo[2.2.1]heptanyl, bicyclo[3.2.1]octanyl, and bicyclo[5.2.0]nonanyl.

[0038] As used herein, the term "halogen" or "halo" means F, Cl, Br, or I.

[0039] As used herein, the term "heteroaryl" refers to an aromatic group having a single ring (monocyclic) or multiple rings (bicyclic or tricyclic) with one or more ring heteroatoms independently selected from O, N, and S. An aromatic monocyclic ring is a 5- or 6-membered ring containing at least one heteroatom independently selected from O, N, and S (e.g., 1, 2, 3, or 4 heteroatoms independently selected from O, N, and S). A 5-membered aromatic monocyclic ring has two double bonds, and a 6-membered aromatic monocyclic ring has three double bonds. A bicyclic heteroaryl group is exemplified by a monocyclic aryl group, as defined herein, or a monocyclic heteroaryl ring fused to a monocyclic heteroaryl group, as defined herein. A tricyclic heteroaryl group is exemplified by a monocyclic heteroaryl ring fused to two rings independently selected from a monocyclic aryl group, as defined herein, and a monocyclic heteroaryl group, as defined herein. Representative examples of monocyclic heteroaryls include, but are not limited to, pyridinyl (including pyridin-2-yl, pyridin-3-yl, pyridin-4-yl), pyrimidinyl, pyrazinyl, pyridazinyl, pyrrolyl, benzopyrazolyl, 1,2,3-triazolyl, 1,3,4-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-oxadiazolyl, imidazolyl, thiazolyl, isothiazolyl, thienyl, furanyl, oxazolyl, isoxazolyl, 1,2,4-triazinyl, and 1,3,5-triazinyl. Representative examples of bicyclic heteroaryls include, but are not limited to, benzimidazolyl, benzodioxolyl, benzofuranyl, benzoxadiazolyl, benzopyrazolyl, benzothiazolyl, benzothienyl, benzotriazolyl, benzoxadiazolyl, benzoxazolyl, chromenyl, imidazopyridine, imidazothiazolyl, indazolyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolinyl, naphthyridinyl, purinyl, pyridoimidazolyl, quinazolinyl, quinolinyl, quinoxalinyl, thiazolopyridinyl, thiazolopyrimidinyl, thienopyrrolyl, and thienothienyl.Representative examples of tricyclic heteroaryls include, but are not limited to, dibenzofuranyl and dibenzothienyl. Monocyclic, bicyclic, and tricyclic heteroaryls are connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the ring.

[0040] As used herein, the term "heterocycle" or "heterocyclic" refers to a saturated or partially unsaturated non-aromatic cyclic group having one or more ring heteroatoms independently selected from O, N, and S. A heterocycle can be a monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle. A monocyclic heterocycle is a 3-, 4-, 5-, 6-, 7-, or 8-membered ring containing at least one heteroatom independently selected from O, N, and S. A 3- or 4-membered ring contains 0 to 1 double bond and 1 heteroatom selected from O, N, and S. A 5-membered ring contains 0 or 1 double bond and 1, 2, or 3 heteroatoms selected from O, N, and S. A 6-membered ring contains 0, 1, or 2 double bonds and 1, 2, or 3 heteroatoms selected from O, N, and S. The 7- and 8-membered rings contain 0, 1, 2, or 3 double bonds and 1, 2, or 3 heteroatoms selected from O, N, and S. The heteroatoms in the ring can be oxidized (e.g., if a ring heteroatom is S, it can be oxidized to SO or SO2). Representative examples of monocyclic heterocycles include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, oxetanyl, pyridine, phenyl ... These include perazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, 1,2-thiazinanyl, 1,3-thiazinanyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidethiomorpholinyl (thiomorpholinesulfone), thiopyranyl, and trithianyl.A bicyclic heterocycle is a monocyclic heterocycle fused to a phenyl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl, or a monocyclic heterocycle fused to a monocyclic cycloalkenyl, or a monocyclic heterocycle fused to a monocyclic heterocycle, or a spiro heterocyclic group, or a bridged monocyclic heterocycle system in which two non-adjacent atoms of the rings are joined by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of 2, 3, or 4 carbon atoms. Representative examples of bicyclic heterocycles include, but are not limited to, benzopyranyl, benzothiopyranyl, chromanyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothienyl, 2,3-dihydroisoquinoline, 2-azaspiro[3.3]heptan-2-yl, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hept-2-yl), 2,3-dihydro-1H-indolyl, isoindolinyl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, and tetrahydroisoquinolinyl. Tricyclic heterocycles are exemplified by bicyclic heterocycles fused to a phenyl group, or bicyclic heterocycles fused to a monocyclic cycloalkyl, or bicyclic heterocycles fused to a monocyclic cycloalkenyl, or bicyclic heterocycles fused to a monocyclic heterocycle, or bicyclic heterocycles in which two non-adjacent atoms of the bicyclic ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of 2, 3, or 4 carbon atoms. Examples of tricyclic heterocycles include, but are not limited to, octahydro-2,5-epoxypentalene, hexahydro-2H-2,5-methanocyclopenta[b]furan, hexahydro-1H-1,4-methanocyclopenta[c]furan, aza-adamantane (1-azatricyclo[3.3.1.1. 3,7 ]decane), and oxa-adamantane (2-oxatricyclo[3.3.1.1 3,7 ]decane. The monocyclic, bicyclic, and tricyclic heterocycles are connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the ring.

[0041] As used herein, the term "hydroxy" means a --OH group.

[0042] As used herein, the term "targeting moiety" refers to a moiety that binds to or localizes to a particular locale. The moiety may be, for example, a small molecule, a peptide, a protein, a nucleic acid, a nucleic acid analog, or a carbohydrate. The locale may be an organelle, a subcellular compartment, a particular cell type, or a particular tissue.

[0043] When a group or moiety can be substituted, the term "substituted" indicates that one or more (e.g., 1, 2, 3, 4, 5, or 6, in some embodiments, 1, 2, or 3, and in other embodiments, 1 or 2) hydrogens on the indicated group in the phrase using "substituted" can be replaced with a selection of the enumerated indicated group or suitable substituents known to those of skill in the art (e.g., one or more of the groups listed below), provided that the normal valence of the specified atom is not exceeded. Substituents include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, acyl, amino, amido, amidino, aryl, azido, carbamoyl, carboxyl, carboxyl ester, cyano, cycloalkyl, cycloalkenyl, guanidino, halo, haloalkyl, haloalkoxy, heteroalkyl, heteroaryl, heterocyclyl, hydroxy, hydrazino, imino, oxo, nitro, phosphate, phosphonate, sulfonic acid, thiol, thione, or combinations thereof.

[0044] As used herein, in chemical structures, the indications: [ka] represents the point of attachment of one moiety to another (eg, a substituent to the remainder of a compound).

[0045] For the compounds described herein, groups and substituents thereof may be selected according to the allowed valences of atoms and substituents such that selection and substitution result in stable compounds that do not spontaneously undergo transformation, for example, by rearrangement, cyclization, elimination, and the like.

[0046] Where substituents are specified by their conventional chemical formula written from left to right, they optionally include the substituents resulting from writing the structure from right to left, e.g., -CHO- optionally also recites -OCH-, -OC(O)NH- optionally also recites -NHC(O)O-.

[0047] compound Compounds of formula (II): [ka] or a salt thereof, wherein R 1 But -CN and [ka] is selected from R 2 is selected from hydrogen and halo; n is 0, 1, 2, or 3; Each R 3 is independently selected from C1-C4 alkyl, C1-C4 alkoxy, -OC(O)-C1-C4 alkyl, hydroxy, amino, and the group-linker-X, where X is a targeting moiety; R 4a and R 4b one of which is hydroxy or -OC(O)-C1-C4 alkyl and the other is hydrogen or a group -linker-X, where X is a targeting moiety; R 5 is selected from hydrogen and C1-C4 alkyl; Z is a bond or a formula [ka] Based on R 6 Disclosed herein are compounds, or salts thereof, wherein: is selected from C1-C4 alkyl and group-linker-Y, and Y is a targeting moiety.

[0048] In some embodiments, R 1is -CN. In such embodiments, the compound is a compound of formula (II'): [ka] or a salt thereof, wherein R 2 , n, R 3 , R 4a , R 4b and Z are as defined and described herein.

[0049] In some embodiments, R 1 teeth, [ka] In such embodiments, the compound is a compound of formula (II″): [ka] or a salt thereof, wherein R 2 , n, R 3 , R 4a , R 4b , R 5 and Z are as defined and described herein.

[0050] In some embodiments, R 2 is hydrogen. In some embodiments, R 2 is halo. In some embodiments, R 2 is fluoro.

[0051] In some embodiments, Z is a bond. In some embodiments, Z is of the formula [ka] where R 6 is selected from C1-C4 alkyl and the group-linker-Y, where Y is a targeting moiety. In some embodiments, Z is a group of the formula [ka] where R6 is selected from methyl and the group-linker-Y, where Y is a targeting moiety. In some embodiments, Z is a group of the formula [ka] where R 6 is methyl. In some embodiments, Z is of the formula [ka] where R 6 is the group-linker-Y, where Y is a targeting moiety.

[0052] In some embodiments, R 4a is hydrogen and R 4b is hydroxy or -OC(O)-C1-C4 alkyl. In some embodiments, R 4a is hydrogen and R 4b is hydroxy. In some embodiments, R 4a is hydrogen and R 4b is -OC(O)CH3. In some embodiments, R 4a is hydroxy and R 4b is hydrogen. In some embodiments, R 4a is hydroxy and R 4b is the group-linker-X, where X is a targeting moiety.

[0053] In some embodiments, the group in formula (II) [ka] teeth, [ka] having a formula selected from:

[0054] In some embodiments, n is 0, 1, or 2. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2.

[0055] In some embodiments, each R 3 is independently selected from C1-C4 alkyl, C1-C4 alkoxy, -OC(O)-C1-C4 alkyl, and hydroxy. 3 is independently selected from C1-C4 alkyl and C1-C4 alkoxy. 3 is independently selected from C-methyl, methoxy, -OC(O)CH, and hydroxy. 3 is independently selected from methyl and methoxy. 3 is independently selected from -OC(O)CH and hydroxy. In some embodiments, n is 1 and R 3 In some embodiments, n is 1 and R 3 In some embodiments, n is 1 and R 3 is -OC(O)CH3. In some embodiments, n is 1 and R 3 is hydroxy.

[0056] In some embodiments, R 5 is hydrogen. In some embodiments, R 5 is C1-C4 alkyl (for example, methyl).

[0057] Compounds of formula (I): [ka] or a salt thereof, wherein R 1 But -CN and [ka] is selected from R 2 is selected from hydrogen and halo; n is 0, 1, 2, or 3; Each R 3 is independently selected from C1-C4 alkyl, C1-C4 alkoxy, amino, and the group-linker-X, where X is a targeting moiety; R 4a and R 4b one of is hydroxy and the other is hydrogen or a group-linker-X, where X is a targeting moiety; R 5 Also disclosed herein are compounds, or salts thereof, wherein is selected from hydrogen and C1-C4 alkyl.

[0058] In some embodiments, R 1 is -CN. In such embodiments, the compound is a compound of formula (I'): [ka] or a salt thereof, wherein R 2 , n, R 3 , R 4a , and R 4b is as defined and described herein.

[0059] In some embodiments, R 1 teeth, [ka] In such embodiments, the compound is a compound of formula (I″): [ka] or a salt thereof, wherein R 2 , n, R 3 , R 4a , R 4b , and R 5 is as defined and described herein.

[0060] In some embodiments, the compound is a compound of formula (Ia): [ka] or a salt thereof, wherein R 1 , R 2 , n, and R 3 is as defined and described herein; R 4a is hydrogen or a -linker-X group, where X is a targeting moiety.

[0061] In some embodiments, the compound is a compound of formula (Ia') or (Ia''): [ka] or a salt thereof, wherein R 2 , n, and R 3 is as defined and described herein; R 4a is hydrogen or a -linker-X group, X is a targeting moiety, R 5 is hydrogen or methyl.

[0062] In some embodiments, the compound is a compound of formula (Ib): [ka] or a salt thereof, wherein R 1 , R 2 , n, and R 3 is as defined and described herein; R 4b is hydrogen or a -linker-X group, where X is a targeting moiety.

[0063] In some embodiments, the compound is a compound of formula (Ib') or (Ib''): [ka] or a salt thereof, wherein R 2 , n, and R 3 is as defined and described herein; R 4bis hydrogen or a -linker-X group, X is a targeting moiety, R 5 is hydrogen or methyl.

[0064] In some embodiments, R 2 is hydrogen. In some embodiments, R 2 is halo. In some embodiments, R 2 is fluoro.

[0065] In some embodiments, R 4a is hydrogen and R 4b is hydroxy. In some embodiments, R 4a is hydroxy and R 4b is hydrogen. In some embodiments, R 4a is hydroxy and R 4b is the group-linker-X, where X is a targeting moiety.

[0066] In some embodiments, the group in formula (I) [ka] teeth, [ka] having a formula selected from:

[0067] In some embodiments, n is 0, 1, or 2. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2.

[0068] In some embodiments, each R 3 is independently selected from C1-C4 alkyl and C1-C4 alkoxy. 3 is independently selected from methyl and methoxy.

[0069] In some embodiments, R 5is hydrogen. In some embodiments, R 5 is C1-C4 alkyl (for example, methyl).

[0070] In some embodiments, the compound comprises at least one group that is a targeting moiety. 3 , or R 4a and R 4b In some embodiments, one of R is a group-linker-X, where X is a mitochondrial targeting moiety. 6 is a group-linker-Y, where Y is a mitochondrial targeting moiety. In such embodiments, the compound can be targeted to a specific locale, e.g., a specific organelle, such as a mitochondria. Mitochondrial targeting can be particularly useful for the compounds disclosed herein, since mitochondria produce superoxide when electrons that "leak" from the electron transport chain are captured by molecular oxygen. Specific targeting of selective superoxide probes to mitochondria can allow for direct measurement of superoxide generated in mitochondria, e.g., in living cells. For example, in some embodiments, at least one R 3 , or R 4a and R 4b In some embodiments, at least one of R 3 , or R 4a and R 4b In some embodiments, one of R is a group-linker-X, where X is a triphenylphosphonium moiety. 6 is the group-linker-Y, where Y is a triphenylphosphonium moiety or a trialkylammonium moiety. 6 is the group-linker-Y, where Y is a triphenylphosphonium moiety.

[0071] A linker can be any group that provides sufficient distance between the targeting moiety X or Y and the remainder of the compound to allow each to function unperturbed (or minimally perturbed) by binding to the other. Linkers include methylene (-CH2-), ether (-O-), amine (-NH-), alkylamine (-NR-, where R is an optionally substituted C1-C6 alkyl group), thioether (-S-), disulfide (-SS-), amide (-C(O)NH-), ester (-C(O)O-), carbamate (-OC(O)NH-), sulfonamide (-S(O)2NH-), arylene (e.g., phenylene (-CH4-), heterocyclylene (e.g., piperazinylene), etc. [ka] and any combination thereof. In some embodiments, the linker comprises one or more -(CH2CH2O)-(oxyethylene) groups. In some embodiments, the linker comprises one or more alkylene groups (e.g., -(CH2) n-, n is 1-12, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or any suitable range therebetween. In some embodiments, the linker comprises one or more branched alkylene groups. In some embodiments, the linker comprises at least one amide group (-C(O)NH-). In some embodiments, the linker comprises one or more substituents, pendants, side chains, etc., including any suitable organic functional group (e.g., -OH, -NH2, -SH, -CN, =O, =S, halogens (e.g., -F, -Cl, -Br, -I), -COOH, -CONH2, -CH3, etc.). In some embodiments, the linker comprises two or more linearly connected C, S, N, and / or O atoms. In some embodiments, the linker comprises 1 to 200 linearly connected atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, or any suitable range therebetween (e.g., 2-20, 10-50, 6-18)). In some embodiments, the linker comprises 1 to 200 linearly connected atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, or any suitable range therein (e.g., 2-20, 10-50, 6-18)).

[0072] In some embodiments, the linker is a straight chain alkylene linker, e.g., the linker has the formula -(CH) n -, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, n is 4, 5, 6, 7, or 8. In some embodiments, n is 6, i.e., the linker has the formula: [ka] has.

[0073] In some embodiments, the linker comprises a combination of methylene (-CH-), ether (-O-), and amide (-C(O)NH-) moieties. For example, in some embodiments, the linker has the formula: [ka] has.

[0074] In some embodiments, the compound is [ka] [ka] [ka] and salts thereof.

[0075] In some embodiments, the compound is in a salt form, i.e., a charged form of the parent compound associated with a counterion. The neutral form of the compound can be regenerated by contacting the salt with a base or acid and isolating the parent compound in a conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salt is equivalent to the parent form of the compound for the purposes of this disclosure.

[0076] Specifically, the compound is or can be anionic (e.g., -COOH becomes -COO - In the case of compounds having functional groups, salts may be formed with one or more suitable cations. Examples of suitable inorganic cations are Li + , Na + , and K + Alkali metal cations such as Ca 2+ and Mg 2+ Examples of suitable organic cations include, but are not limited to, alkaline earth cations such as NH4, NH5, NH6, NH7, NH8, NH9, NH10, NH11, NH20, NH30, NH40, NH41, NH42, NH43, NH44, NH45, NH46, NH47, NH48, NH49, NH50, NH51, NH52, NH53, NH54, NH55, NH56, NH57, NH58, NH59, NH60, NH61, NH62, NH63, NH64, NH65, NH66, NH67, NH68, NH69, NH70, NH71, NH72, NH73, NH74, NH75, NH76, NH77 +) and substituted ammonium ions (e.g., NH3R1 + , NH2R2 + , NHR3 + , and NR4 + ) are included, but are not limited to. Some examples of suitable substituted ammonium ions are those derived from ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids such as lysine and arginine. In some embodiments, the compound is a potassium salt. In some embodiments, the compound is a sodium salt.

[0077] The compound is or can be cationic (e.g., -NH2 becomes -NH3 +In the case of compounds having a functional group (which may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 109, 109 Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyloxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalenecarboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, phenylsulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, tetrafluoroboric acid, toluenesulfonic acid, trifluoromethanesulfonic acid, and valeric acid. In some embodiments, the compound is a halide salt, such as a chloride, bromide, or iodide salt, hi some embodiments, the compound is a tetrafluoroborate or trifluoromethanesulfonate salt.

[0078] The compounds can be prepared by several suitable methods, some of which are shown in the examples. The compounds and intermediates can be isolated and purified by methods well known to those skilled in the art of organic synthesis. Examples of conventional methods for isolating and purifying compounds can include, but are not limited to, chromatography on solid supports such as silica gel, alumina, or silica derivatized with alkylsilane groups, by recrystallization at high or low temperatures, with optional pretreatment with activated carbon, thin layer chromatography, distillation at various pressures, sublimation under vacuum, and trituration, as described, for example, in "Vogel's Textbook of Practical Organic Chemistry," 5th edition (1989), by Furniss, Hannaford, Smith, and Tatchell, pub. Longman Scientific & Technical, Essex CM20 2JE, England.

[0079] The reaction conditions and reaction times for each individual step may vary depending on the particular reactants used and the substituents present in the reactants used. The reactions may be worked up in conventional manner, for example by removing the solvent from the residue and further purified according to methods generally known in the art, such as, but not limited to, crystallization, distillation, extraction, trituration and chromatography. Unless otherwise stated, starting materials and reagents are either commercially available or can be prepared by one skilled in the art from commercially available materials using methods described in the chemical literature.

[0080] Standard experimentation, including appropriate manipulation of the reaction conditions, reagents, and sequence of the synthetic route, protection of any chemical functionality that may not be compatible with the reaction conditions, and deprotection at suitable points in the reaction sequence of the method, is within the scope of the present invention. Suitable protecting groups and methods for protecting and deprotecting different substituents using such suitable protecting groups are well known to those skilled in the art, examples of which can be found in PGM Wuts and TW Greene, in Greene's book titled Protective Groups in Organic Synthesis (4 th ed.), John Wiley & Sons, NY (2006).

[0081] When an optically active form of a disclosed compound is required, it can be obtained by carrying out one of the procedures described herein using an optically active starting material (prepared, for example, by asymmetric induction of a suitable reaction step), or by resolution of a stereoisomeric mixture of the compound or intermediate using standard procedures (such as chromatographic separation, recrystallization, or enzymatic resolution).

[0082] Similarly, when a pure geometric isomer of a compound is required, it can be obtained by carrying out one of the procedures described herein using a pure geometric isomer as a starting material, or by resolution of a mixture of geometric isomers of the compound or intermediates using standard procedures, such as chromatographic separation.

[0083] The synthetic schemes and specific examples described are illustrative and are not to be construed as limiting the scope of the disclosure or the claims. Alternatives, modifications, and equivalents of the synthetic methods and specific examples are contemplated.

[0084] The present disclosure also includes isotopically labeled compounds that are identical to those recited in Formula (I) and Formula (II), but which have one or more atoms replaced by an atom having an atomic mass or mass number different from that normally found in nature. Examples of isotopes suitable for inclusion in the compounds of the present invention are hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, e.g., 2 H, 3 H, 13 C. 14 C. 15 N, 18 O. 17 O. 31 P, 32 P, 35 S, 18 F, and 36 Isotopically labeled compounds of formula (I) and formula (II) can generally be prepared by conventional techniques known to those skilled in the art, or by processes similar to those described in the accompanying examples using appropriate isotopically labeled reagents in place of non-isotopically labeled reagents.

[0085] Methods, Systems, and Kits of Use Disclosed herein are methods for detecting superoxide using the compounds disclosed herein (e.g., compounds of formula (I) and formula (II), including compounds of formula (I'), (I"), (Ia), (Ia'), (Ia"), (Ib), (Ib'), (Ib"), (II'), and (II")). The compounds include compounds having an ortho- or para-hydroxy group, such as a moiety, specifically: [ka] This group reacts with superoxide to produce the corresponding quinone compound, releasing an uncaged luciferin or hydroxycyanobenzothiazole compound, the latter of which is a substrate for luciferin-utilizing luciferase enzymes that result in light emission. Luciferases that utilize luciferin and hydroxycyanobenzothiazole compounds to produce light emission ("luciferin-utilizing luciferases" or "luciferin-utilizing luciferase enzymes") include those found in a variety of organisms, such as beetles (e.g., Photinus pyralis and Photuris pennsylvanica (North American fireflies), Pyrophorus plagiophthalamus (Jamaica click beetle)), Renilla reniformis (sea pansies), and some bacteria (e.g., Xenorhabdus luminescens and Vibrio spp.).

[0086] Before uncaging by reaction with superoxide, the compounds of formula (I) and formula (II) are non-reactive with luciferase enzyme, so luminescence is observed only in the presence of superoxide. The fact that reactivity occurs from superoxide is confirmed in experiments in which superoxide dismutase is added, eliminating the signal. As shown in the examples, the probe compounds disclosed herein have excellent selectivity against other reactive oxygen species such as hydrogen peroxide and singlet oxygen, as well as nitrogen species such as sodium nitrite and NONOate.

[0087] Without wishing to be bound by theory, the compounds may react with superoxide in a manner similar to hydroquinone, where deprotonation followed by single-electron oxidation leads to the formation of a semiquinone intermediate, which undergoes further oxidation and hydrolysis to release the uncaged luciferin or hydroxycyanobenzothiazole compound and a quinone-type by-product. 1 is -CN and R 4a is hydrogen and R 4bSuch reactions of compounds of formula (I) where Z is hydroxy are shown in Scheme 1. In some embodiments, compounds of formula (II) may be provided with a biscarbamic acid linker (i.e., Z is of the formula [ka] In such embodiments, deprotonation followed by single electron oxidation results in the formation of a semiquinone intermediate, which ultimately undergoes further oxidation and hydrolysis, resulting in the self-immolation of the biscarbamic acid linker to release the uncaged luciferin or hydroxycyanobenzothiazole and a quinone-type by-product from the reactive moiety. 1 but, [ka] and R 4a is hydrogen, n is 1, and R 3 is hydroxy, and R 4b Such a reaction of a compound of formula (II) where is hydroxy is shown in Scheme 2. [ka]

[0088] The advantage of the compounds of formula (I) and formula (II) is that the uncaged product is detected by bioluminescence and does not require external light (as with fluorescent probes). Furthermore, superoxide species are very unstable, but after the reaction of the compound with superoxide, the uncaged compound is stable. Since superoxide species continue to be produced by the cells, the stable uncaged compound will continue to accumulate and can be detected by measuring light production (e.g., using a luciferase reaction). Light production will directly correlate with the production of superoxide species and provide a convenient quantitative approach for the detection of short-lived superoxide species. The uncaged luciferin or hydroxycyanobenzothiazole compound can be measured by adding the detection reagent directly to the sample using a homogenous "add and read" format, or by measuring the release of luciferin derivatives into the medium at different time points by medium sampling and luciferin detection.

[0089] Thus, the present disclosure provides a method for detecting superoxide in a sample, comprising contacting the sample with a compound of Formula (I) or Formula (II), contacting the sample with a luciferin-utilizing luciferase if a luciferin-utilizing luciferase is not already present in the sample, and detecting luminescence in the sample.

[0090] The method includes contacting a sample with a compound of formula (I) or formula (II). The compound of formula (I) or formula (II) can be part of a solution that can include other components, such as a solvent, a buffer, salts, detergents, additives, and the like. For example, the compound of formula (I) or formula (II) can be prepared as a solution in a solvent, such as dimethyl sulfoxide, or as a solution in a buffer, such as phosphate buffered saline. In some embodiments, the method includes first contacting the sample with a compound of formula (I) or formula (II) and incubating the sample for a period of time to allow the superoxide to react with the compound of formula (I) or formula (II). In some embodiments, this incubation step can be carried out for a period of time ranging from about 1 minute to about 4 days, from about 15 minutes to about 1 day, from about 1 hour to about 12 hours, or any range therebetween. For example, in some embodiments, the incubation step can be carried out for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours.

[0091] In certain embodiments, the luciferin-utilizing luciferase is not already present in the sample, and thus the method includes contacting the sample with a luciferin-utilizing luciferase, hi some embodiments, the luciferin-utilizing luciferase is a firefly luciferase or a click beetle luciferase.

[0092] When a luciferin-utilizing luciferase is contacted with a sample, it may be included as part of a luciferase reaction mixture. A "luciferase reaction mixture" contains a luciferin-utilizing luciferase enzyme and other materials that enable the luciferase enzyme to generate a light signal. The materials necessary to generate a luminescent signal, as well as the specific concentrations and / or amounts of the necessary materials, will vary depending on the luciferase enzyme being used. Generally, for beetle luciferase, the additional materials include ATP and magnesium (Mg), such as magnesium sulfate. 2+) salts. In some embodiments, other materials can be added to the solution including buffers to maintain the reaction at the appropriate pH, additives such as PRIONEX or bovine serum albumin (BSA) to help maintain luciferase activity, reducing agents, detergents, esterases, salts, amino acids (e.g., D-cysteine), etc. An exemplary luciferase reaction mixture contains Rhinoceros Beetle Luciferase, MgSO4, ATP, Tergitol NP-9, and Tricine.

[0093] In other embodiments, the luciferin-utilizing luciferase is already present in the sample. For example, in such embodiments, the sample may contain cells that express the luciferin-utilizing luciferase enzyme.

[0094] In some embodiments of the above methods, the method further comprises contacting the sample with another compound, such as a candidate drug compound, or any compound that would be useful in determining the amount of superoxide produced when the compound is contacted with the sample. In such embodiments, the compound can be contacted with the sample simultaneously with the compound of Formula (I) or Formula (II), or can be added to the sample after the compound of Formula (I) or Formula (II) has been added.

[0095] In one embodiment of a cell-based assay, cells may be lysed in an appropriate lysis buffer. For animal cells, a buffer containing 0.1-1.0% non-ionic detergent such as Triton X 100 or Tergitol is typically sufficient. Bacteria, plant, fungal, or yeast cells are usually more difficult to lyse. Detergents, freeze / thaw cycles, hypotonic buffers, sonication, cavitation, or combinations of these methods may be used. The method of lysis that produces a lysate is compatible with the detection of luciferase or other enzyme activity, or other molecules or conditions.

[0096] In any of the above embodiments, the sample may be contained in any suitable container. For example, the sample may be in a vial or in a well of a plate (e.g., a 96-well plate).

[0097] In some embodiments, rather than using a plate reader to detect luminescence directly in the wells of a plate (e.g., a 96-well plate), luciferin release into the medium can be monitored by removing a small amount of medium from the sample and detecting luminescence in the removed medium. Such a method can allow kinetic information regarding superoxide generation.

[0098] The present disclosure further provides a system or kit comprising a compound described herein (i.e., a compound of formula (I) or a salt thereof, or a compound of formula (II) or a salt thereof). The system or kit comprises the compound, either alone or in a solvent, such as water, DMSO, or a buffer. When the compound is provided alone, the system or kit may further comprise a solvent in which the compound can be dissolved. The system or kit may further comprise one or more reagents used to perform an assay to detect superoxide in a sample, such as those described above. In some embodiments, the kit further comprises a luciferin-utilizing luciferase enzyme or a nucleotide sequence encoding a luciferin-utilizing luciferase enzyme, such as those described herein.

[0099] The system or kit may further include at least one of a container and instructions. For example, the components of the system or kit may be supplied in any type of container. For example, a sealed glass ampoule may contain lyophilized luciferase or a buffer solution packaged under a neutral, non-reactive gas, such as nitrogen. The ampoule may be made of any suitable material, such as glass, an organic polymer, such as polycarbonate, polystyrene, ceramic, metal, or any other material typically used to hold reagents. Other examples of suitable containers include simple bottles that may be manufactured from materials similar to ampoules. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. The container may have a sterile access port, such as a bottle with a stopper that can be pierced by a hypodermic needle. Other containers may have two compartments separated by an easily removable membrane that allows the components to mix after removal. The removable membrane may be glass, plastic, rubber, and the like.

[0100] Kits may also be supplied with instructional materials. The instructions may be printed on paper or other substrate and / or supplied as an electronically readable medium such as a floppy disk, CD-ROM, DVD-ROM, Zip disk, videotape, audiotape, etc. The detailed instructions may not be physically associated with the kit; instead, the user may be directed to an internet website or the instructions may be supplied as e-mail.

[0101] The following examples further illustrate aspects of the present disclosure but, of course, should not be construed as in any way limiting its scope. EXAMPLES

[0102] The following abbreviations are used in the examples: AcOH is acetic acid, DCM is dichloromethane, DIPEA is N,N-diisopropylethylamine, DMA is N,N-dimethylacetamide, DMF is N,N-dimethylformamide, DMSO is dimethylsulfoxide, ES is electrospray, EtOAc is ethyl acetate, h is hour, HATU is 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxidohexafluorophosphate, HPLC is high performance liquid chromatography, LCMS is liquid chromatography mass spectrometry, MeCN is acetonitrile, MeOH is methanol, RB is round bottom, tBuXPhos-Pd G3 is [(2-di-tert-butylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)-2-(2'-amino-1,1'-biphenyl)]palladium(II) methanesulfonate, TFA is trifluoroacetic acid, Tf2O is trifluoromethanesulfonic anhydride, and THF is tetrahydrofuran.

[0103] Example 1: Compound synthesis Intermediate 1: 2-cyanobenzo[d]thiazol-6-yl trifluoromethanesulfonate [ka] To a 250 mL RB flask was added 6-hydroxybenzo[d]thiazole-2-carbonitrile (3.00 g, 17.0 mmol) and DCM (80 mL). The mixture was stirred. To the mixture was added triethylamine (3.31 mL, (25.5 mmol) followed by TfO (3.72 mL, 22.1 mmol) dropwise over 2 min. After 10 min, the mixture was concentrated onto Celite and purified by silica gel chromatography using 0-50% EtOAc in heptane as eluent to give intermediate 1: 2-cyanobenzo[d]thiazol-6-yl trifluoromethanesulfonate. LCMS (C9H3F3N2O3S2) (ES, m / z) 309 [M+H] + .

[0104] Intermediate 2: 2-cyano-5-fluorobenzo[d]thiazol-6-yl trifluoromethanesulfonate [ka] Intermediate 2 was prepared in a manner similar to that of Intermediate 1. LCMS (C9H2F4N2O3S2) (ES, m / z) 327 [M+H] + .

[0105] Intermediate 3: tert-Butyl 2-(3,4-dihydroxyphenoxy)acetate Step 1: tert-butyl 2-(4-formyl-3-hydroxyphenoxy)acetate [ka] To a 100 mL flask was added 2,4-dihydroxybenzaldehyde (1.00 g, 7.24 mmol), K2CO3 (1.50 g, 10.9 mmol), and MeCN (15 mL). To the stirred mixture was added 2-bromoacetate tert-butyl (1.17 mL, 7.96 mmol). The mixture was stirred and heated at 85 °C for 1 h. The mixture was cooled to room temperature, concentrated onto Celite, and purified by silica gel chromatography using 0-60% EtOAc in heptane as eluent to give 2-(4-formyl-3-hydroxyphenoxy)acetate tert-butyl. LCMS (C 13 H 16 O5)(ES,m / z)253[M+H] + .

[0106] Step 2: tert-Butyl 2-(3,4-dihydroxyphenoxy)acetate [ka] To a 20 mL vial was added tert-butyl 2-(4-formyl-3-hydroxyphenoxy)acetate (100 mg, 0.396 mmol), THF (1 mL), and water (1 mL). The solution was stirred and sparged with nitrogen for 1 min. To the vial was added sodium percarbonate (124 mg, 0.396 mmol). The mixture was stirred for 30 min. The mixture was quenched with AcOH (0.2 mL). The mixture was extracted with DCM (5 mL). The organic layer was collected on a phase separator. The solvent was evaporated and the residue was purified by silica gel chromatography using 0-70% EtOAc in heptane as eluent to give intermediate 3 (tert-butyl 2-(3,4-dihydroxyphenoxy)acetate). LCMS (C 12 H 16 O5)(ES,m / z)241[M+H] + .

[0107] Intermediate 4: tert-Butyl 2-(2,5-dihydroxyphenoxy)acetate Step 1: tert-butyl 2-(5-formyl-2-hydroxyphenoxy)acetate [ka] To a 100 mL round bottom flask was added 3,4-dihydroxybenzaldehyde (1.00 g, 7.24 mmol), a 4 M solution of NaOH in MeOH (5 mL), and DMA (10 mL). To the stirred mixture was added 2-bromoacetate tert-butyl (1.17 mL, 7.96 mmol) as a toluene solution (5 mL). After 30 seconds, the mixture was quenched with 2 M HCl (ca. 10 mL). The mixture was extracted with diethyl ether (50 mL) and the organic layer was washed with water (3×30 mL). The organic layer was dried over MgSO4, filtered, and the solvent was evaporated. The residue was purified by silica gel chromatography using 0-60% EtOAc in heptane to give 2-(5-formyl-2-hydroxyphenoxy)acetate tert-butyl. LCMS (C 13 H 16 O5)(ES,m / z)253[M+H] + .

[0108] Step 2: tert-Butyl 2-(2,5-dihydroxyphenoxy)acetate [ka] To a 20 mL vial was added tert-butyl 2-(5-formyl-2-hydroxyphenoxy)acetate (200 mg, 0.793 mmol), THF (2 mL), and water (2 mL). The solution was stirred and sparged with nitrogen for 1 min. To the vial was added sodium percarbonate (249 mg, 0.793 mmol). The mixture was stirred under nitrogen for 2 h. The mixture was quenched with AcOH (0.4 mL). The mixture was extracted with DCM (5 mL). The organic layer was collected on a phase separator. The solvent was evaporated and the residue was purified by silica gel chromatography using 0-70% EtOAc in heptane as eluent to give intermediate 4 (tert-butyl 2-(2,5-dihydroxyphenoxy)acetate). LCMS (C 12 H 16 O5)(ES,m / z)241[M+H] + .

[0109] Intermediate 5: tert-Butyl 2-(2,5-dihydroxy-3-methoxyphenoxy)acetate Step 1: tert-butyl 2-(5-formyl-2-hydroxy-3-methoxyphenoxy)acetate [ka] To a 20 mL vial was added 3,4-dihydroxy-5-methoxybenzaldehyde (100 mg, 0.595 mmol) and DMF (2.5 mL). The mixture was sparged with nitrogen for 1 min. To the stirred mixture was added sodium hydride (60 wt%, 71.4 mg, 1.78 mmol). The mixture was stirred for 15 min. To the mixture was added tert-butyl 2-bromoacetate (0.095 mL, 0.654 mmol) all at once. After 15 min, the mixture was neutralized with 2 M HCl and then diluted with water (5 mL). The mixture was extracted with diethyl ether (20 mL). The organic layer was washed with water (3 x 20 mL). The organic layer was dried over MgSO4, filtered, and the solvent was evaporated. The residue was purified by silica gel chromatography using 0-60% EtOAc in heptane to give tert-butyl 2-(5-formyl-2-hydroxy-3-methoxyphenoxy)acetate. 14 H 18 O6)(ES,m / z)281[MH] - .

[0110] Step 2: tert-butyl 2-(2,5-dihydroxy-3-methoxyphenoxy)acetate [ka] To a 20 mL vial was added tert-butyl 2-(5-formyl-2-hydroxy-3-methoxyphenoxy)acetate (80.3 mg, 0.285 mmol), THF (1 mL), and water (1 mL). The solution was stirred and sparged with nitrogen for 1 min. To the vial was added sodium percarbonate (89.3 mg, 0.285 mmol). The mixture was stirred under nitrogen for 2 h. The mixture was quenched with AcOH (0.15 mL). The mixture was extracted with diethyl ether. The organic layer was washed with water. The organic layer was dried over MgSO4, filtered, and the solvent was evaporated. The residue was purified by silica gel chromatography using 0-70% EtOAc in heptane as eluent to give intermediate 5 (tert-butyl 2-(2,5-dihydroxyphenoxy-3-methoxyphenoxy)acetate). LCMS (C 13 H 18O6)(ES,m / z)271[M+H] + .

[0111] Intermediate 6: 3,4-Bis(benzyloxy)phenol Step 1: 3,4-Bis(benzyloxy)benzaldehyde [ka] To a 100 mL RB flask was added 3,4-dihydroxybenzaldehyde (2.00 g, 14.5 mmol), K2CO3 (6.00 g, 43.4 mmol), DMF (15 mL), and benzyl bromide (4.30 mL, 36.2 mmol). The mixture was stirred and heated at 55 °C for 2 h. The mixture was cooled to room temperature, diluted in EtOAc (70 mL), and filtered through Celite. The solvent was evaporated and the residue was purified by silica gel chromatography using 0-50% EtOAc in heptane as eluent. The residue from the evaporated fraction was triturated in heptane to give 3,4-bis(benzyloxy)benzaldehyde. LCMS (C 21 H 18 O3)(ES,m / z)319[M+H] + .

[0112] Step 2: 3,4-Bis(benzyloxy)phenol [ka] To a 100 mL flask was added 3,4-bis(benzyloxy)benzaldehyde (2.00 g, 6.28 mmol), NaHCO3 (1.58 g, 18.9 mmol), m-CPBA (1.63 g, 9.42 mmol), and DCM (20 mL). The mixture was stirred at room temperature for 4 h. The mixture was diluted in DCM (20 mL) and MeOH (20 mL). The mixture was filtered. The filtrate was concentrated. To the residue was added K2CO3 (1.73 g, 12.6 mmol) and MeOH (15 mL). The mixture was stirred for 10 min. The mixture was diluted in EtOAc (150 mL) and water (150 mL). The layers were separated and the organic layer was washed with saturated aqueous K2CO3 solution (100 mL). The organic layer was dried over sodium sulfate, filtered and the solvent was evaporated to intermediate 6 (3,4-bis(benzyloxy)phenol). LCMS (C 20 H 18 O3)(ES,m / z)307[M+H] + .

[0113] Intermediate 7: (3,4-dihydroxyphenyl)ethane-1,2-diylbis(methylcarbamate) tert-butyl Step 1: 3,4-bis(benzyloxy)phenyl tert-butyl ethane-1,2-diylbis(methylcarbamate) [ka] To a 20 mL vial was added intermediate 6 (769 mg, 2.51 mmol), THF (10 mL), and DIPEA (1.75 mL, 10.0 mmol). This mixture was added dropwise over 1 min to a stirred solution of triphosgene (372 mg, 1.26 mmol) in THF (5 mL). After 5 min, tert-butyl methyl(2-(methylamino)ethyl)carbamate (0.614 mL, 3.26 mmol) was added to the mixture. After 20 min, the mixture was adsorbed onto Celite and purified by silica gel chromatography using 0-100% EtOAc in heptane as eluent to give 3,4-bis(benzyloxy)phenyl tert-butyl ethane-1,2-diylbis(methylcarbamate). LCMS (C 30 H 36N2O6)(ES,m / z)543[M+Na + .

[0114] Step 2: tert-butyl (3,4-dihydroxyphenyl)ethane-1,2-diylbis(methylcarbamate) [ka] To a 20 mL vial was added 3,4-bis(benzyloxy)phenyl tert-butyl ethane-1,2-diylbis(methylcarbamate), 10% Pd / C (76.2 mg, 0.0716 mmol), and EtOAc (3 mL). The mixture was stirred under an atmosphere of hydrogen. After 16 h, the mixture was filtered through Celite and the filtrate's solvent was evaporated to give intermediate 7 ((3,4-dihydroxyphenyl)ethane-1,2-diylbis(methylcarbamate) tert-butyl). LCMS (C 16 H 24 N2O6)(ES,m / z)285[M+H-C4H8] + .

[0115] Intermediate 8: 2,2,2-Trifluoromethyl (2-(methylamino)ethyl)carbamate 3,4-dihydroxyphenyl [ka] To a 20 mL vial was added intermediate 7 (45.0 mg, 0.132 mmol) and TFA (2 mL). The mixture was stirred for 10 min. The solvent was evaporated to give intermediate 8, 3,4-dihydroxyphenyl 2,2,2-trifluoroacetate methyl(2-(methylamino)ethyl)carbamate. LCMS (C 11 H 16 N2O4)(ES,m / z)241[M+H] + .

[0116] Intermediate 9: 2,2,2-trifluoroacetic acid diacetate 4-((methyl(2-(methylamino)ethyl)carbamoyl)oxy)-1,2-phenylene Step 1: 4-(((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-1,2-phenylene diacetate [ka] To a 20 mL vial was added intermediate 7 (46.0 mg, 0.135 mmol), acetic anhydride (1 mL), and pyridine (1 mL). The mixture was stirred at room temperature for 10 min. The solvent was evaporated and the residue was coevaporated with toluene three times to give 4-(((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-1,2-phenylene diacetate. LCMS (C 20 H 28 N2O8)(ES,m / z)447[M+Na] + .

[0117] Step 2: 2,2,2-trifluoroacetic acid diacetate 4-((methyl(2-(methylamino)ethyl)carbamoyl)oxy)-1,2-phenylene [ka] To a 20 mL vial was added 4-(((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)-(methyl)carbamoyl)oxy)-1,2-phenylene diacetate (57.0 mg, 0.134 mmol) and TFA (2 mL). The mixture was stirred for 10 min. The solvent was evaporated to give intermediate 9 (4-((methyl(2-(methylamino)ethyl)carbamoyl)oxy)-1,2-phenylene 2,2,2-trifluoroacetic acid diacetate). LCMS (C 15 H 20 N2O6)(ES,m / z)325[M+H] + .

[0118] Intermediate 10: (3,4-dihydroxyphenyl)ethane-1,2-diylbis(methylcarbamate) 2-cyanobenzo[d]thiazol-6-yl [ka] To a 20 mL vial was added 6-hydroxybenzo[d]thiazole-2-carbonitrile (19.0 mg, 0.108 mmol), THF (1.5 mL), and DIPEA (0.075 mL, 0.43 mmol). The mixture was added dropwise over 1 min to a stirred solution of triphosgene (12.8 mg, 0.0431 mmol) in THF (1 mL). After 5 min, to the mixture was added intermediate 8 (33.7 mg, 0.140 mmol) in THF (1 mL). After 20 min, the mixture was adsorbed onto Celite and purified by silica gel chromatography using 0-100% EtOAc in heptane as eluent to give intermediate 10 (3,4-dihydroxyphenyl)ethane-1,2-diylbis(methylcarbamate) 2-cyanobenzo[d]thiazol-6-yl. LCMS (C 20 H 18 N4O6S)(ES,m / z)443[M+H] + .

[0119] Intermediate 11: 2,2,2 Trifluoroacetate (6-(((3,4-diacetoxyphenoxy)carbonyl)(2-(methylamino)ethyl)amino)hexyl)triphenylphosphonium bromide Step 1: (6-((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)amino)hexyl)triphenylphosphonium bromide [ka] To a 20 mL vial was added (6-bromohexyl)triphenylphosphonium bromide (1.00 g, 1.98 mmol) and DMF (10 mL). To this mixture was added tert-butyl (2-aminoethyl)(methyl)carbamate (1.72 g, 9.88 mmol). The mixture was stirred and heated at 55° C. for 4 h. The mixture was cooled to room temperature and the solvent was evaporated. The residue was purified by amine-functionalized silica gel chromatography using 0-10% MeOH in DCM as eluent. The residue was further purified by trituration twice with diethyl ether to give (6-((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)amino)hexyl)triphenylphosphonium bromide. LCMS (C 32 H 44 N2O2P)(ES,m / z)519[M] + .

[0120] Step 2: (6-(((3,4-bis(benzyloxy)phenoxy)carbonyl)(2-((tert-butoxycarbonyl)(methyl)amino)ethyl)amino)hexyl)triphenylphosphonium bromide [ka] To a 20 mL vial was added intermediate 6 (403 mg, 1.31 mmol), THF (5 mL), and DIPEA (0.931 mL, 5.26 mmol). The mixture was added dropwise over 1 min to a stirred solution of triphosgene (195 mg, 0.657 mmol) in THF (2 mL). After 15 min, the mixture was added (6-((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)amino)hexyl)triphenylphosphonium bromide (788 mg, 1.31 mmol) in THF (2 mL). After 20 min, the mixture was adsorbed onto Celite and purified by silica gel chromatography using 0-10% MeOH in DCM as eluent. The residue was dissolved in THF (20 mL). The residual DIPEA-HCl salt was precipitated, removed by filtration, and washed with THF. The filtrate's solvent was evaporated to give (6-(((3,4-bis(benzyloxy)-phenoxy)carbonyl)(2-((tert-butoxycarbonyl)(methyl)amino)ethyl)amino)hexyl)-triphenyl-phosphonium bromide. LCMS (C 53 H 60 N2O6P)(ES,m / z)851[M] + .

[0121] Step 3: (6-((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)((3,4-dihydroxyphenoxy)carbonyl)amino)hexyl)triphenylphosphonium bromide [ka] To a 100 mL RB flask was added (6-(((3,4-bis(benzyloxy)-phenoxy)carbonyl)(2-((tert-butoxycarbonyl)(methyl)amino)ethyl)amino)hexyl)-triphenyl-phosphonium bromide (751 mg, 0.805 mmol), 10% Pd / C (600 mg, 0.564 mmol), EtOAc (3 mL), MeOH (3 mL), and AcOH (1 mL). The mixture was stirred under an atmosphere of hydrogen. After 72 h, the mixture was filtered through Celite and the solvent was evaporated to give (6-((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)((3,4-dihydroxyphenoxy)carbonyl)amino)hexyl)-triphenylphosphonium bromide. LCMS (C 39 H 48 N2O6P)(ES,m / z)671[M] + .

[0122] Step 4: (6-((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)((3,4-diacetoxyphenoxy)carbonyl)amino)hexyl)-triphenylphosphonium bromide [ka] To a 20 mL vial was added (6-((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)((3,4-dihydroxyphenoxy)carbonyl)amino)hexyl)-triphenylphosphonium bromide (590 mg, 785 mmol), acetic anhydride (2 mL), and pyridine (2 mL). The mixture was stirred at room temperature for 10 min. The solvent was evaporated and the residue was coevaporated with toluene three times. The residue was purified by silica gel chromatography using 0-10% MeOH in DCM as eluent to give (6-((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)((3,4-diacetoxyphenoxy)carbonyl)amino)hexyl)-triphenylphosphonium bromide. LCMS (C 43 H 52 N2O8P)(ES,m / z)755[M] + .

[0123] Step 5: 2,2,2 Trifluoroacetate (6-(((3,4-diacetoxyphenoxy)carbonyl)(2-(methylamino)ethyl)amino)hexyl)triphenylphosphonium bromide [ka] To a 20 mL vial was added (6-((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)((3,4-diacetoxyphenoxy)carbonyl)amino)hexyl)-triphenylphosphonium bromide (100 mg, 0.120 mmol) and TFA (2 mL). The mixture was stirred for 10 min. The solvent was evaporated to give intermediate 11 (2,2,2-trifluoroacetic acid (6-(((3,4-diacetoxyphenoxy)carbonyl)(2-(methylamino)ethyl)amino)hexyl)-triphenylphosphonium bromide). LCMS (C 38 H 44 N2O6P)(ES, m / z) 655[M] + .

[0124] Compound 1: 6-(4-hydroxy-3,5-dimethoxyphenoxy)benzo[d]thiazole-2-carbonitrile [ka] To a 20 mL vial was added intermediate 1 (100 mg, 0.324 mmol), tBuXPhos-Pd G3 (12.9 mg, 0.0162 mmol), potassium phosphate (138 mg, 0.649 mmol), 2,6-dimethoxybenzene-1,4-diol (66.2 mg, 0.389 mmol), and toluene (3 mL). The mixture was sparged with nitrogen for 1 min. The mixture was stirred and heated at 120 °C for 2 h. The mixture was diluted in EtOAc (5 mL) and filtered. The filtrate's solvent was evaporated and the residue was purified by silica gel chromatography using 0-80% EtOAc in heptane to give compound 1 (6-(4-hydroxy-3,5-dimethoxyphenoxy)benzo[d]thiazole-2-carbonitrile). LCMS (C 16 H 12N2O4S)(ES,m / z)329[M+H] + . 1 H NMR (400 MHz,DMSO-d6)δ 8.37 (d,J = 2.4 Hz,1H),8.22 (dd,J = 9.1,2.1 Hz,1H),7.77 (d,J = 2.7 Hz,1H),7.41 (dd,J = 9.3,2.7 Hz,1H),6.51 (d,J = 2.1 Hz,2H),3.74 (d,J = 2.1 Hz,6H).

[0125] Compounds 2-5 in Table 1 below were prepared from intermediate 1 or intermediate 2 and the corresponding commercially available phenols in a manner similar to that for the preparation of compound 1. [Table 1]

[0126] Compound 6: (6-(2-(5-((2-cyanobenzo[d]thiazol-6-yl)oxy)-2-hydroxy-3-methoxyphenoxy)acetamido)hexyl)triphenylphosphonium bromide Step 1: tert-butyl 2-(5-((2-cyanobenzo[d]thiazol-6-yl)oxy)-2-hydroxy-3-methoxyphenoxy)acetate [ka] To a 20 mL vial was added Intermediate 1 (49.0 mg, 0.159 mmol), Intermediate 5 (51.2 mg, 0.189 mmol), tBuXPhos-Pd G3 (6.3 mg, 0.0080 mmol), potassium phosphate (67.5 mg, 0.318 mmol), and toluene (1.5 mL). The mixture was sparged with nitrogen for 1 min. The mixture was stirred and heated at 100 °C for 1 h. The mixture was adsorbed onto Celite and purified by silica gel chromatography with 0-60% EtOAc in heptane to give tert-butyl 2-(5-((2-cyanobenzo[d]thiazol-6-yl)oxy)-2-hydroxy-3-methoxyphenoxy)acetate. LCMS (C 21 H20 N2O6S)(ES,m / z)429[M+H] + .

[0127] Step 2: 2-(5-((2-cyanobenzo[d]thiazol-6-yl)oxy)-2-hydroxy-3-methoxyphenoxy)acetic acid [ka] To a 20 mL vial was added tert-butyl 2-(5-((2-cyanobenzo[d]thiazol-6-yl)oxy)-2-hydroxy-3-methoxyphenoxy)acetate (42.5 mg, 0.0992 mmol) and TFA (1 mL). The mixture was stirred for 30 min. The solvent was evaporated to give 2-(5-((2-cyanobenzo[d]thiazol-6-yl)oxy)-2-hydroxy-3-methoxyphenoxy)acetic acid. LCMS (C 17 H 12 N2O6S)(ES,m / z)373[M+H] + .

[0128] Step 3: (6-(2-(5-((2-cyanobenzo[d]thiazol-6-yl)oxy)-2-hydroxy-3-methoxyphenoxy)acetamido)hexyl)triphenylphosphonium bromide [ka] To a 20 mL vial was added tert-butyl 2-(5-((2-cyanobenzo[d]thiazol-6-yl)oxy)-2-hydroxy-3-methoxyphenoxy)acetate (36.9 mg, 0.0991 mmol), (6-aminohexyl)triphenylphosphonium bromide hydrochloride (56.9 mg, 0.119 mmol), DMF (1 mL), and DIPEA (0.052 mL, 0.30 mmol). The mixture was stirred. To the mixture was added HATU (45.2 mg, 0.119 mmol). After 1 h, the mixture was purified by reverse phase HPLC (MeCN / water with 0.1% TFA) to give compound 6 ((6-(2-(5-((2-cyanobenzo[d]thiazol-6-yl)oxy)-2-hydroxy-3-methoxyphenoxy)acetamido)hexyl)-triphenylphosphonium bromide). LCMS(C 41 H 39 N3O5PS) + (ES, m / z) 716 [M] + . 1 H NMR (400 MHz,DMSO-d6)δ 8.66 (s,1H),8.26 (s,1H),8.21 (dd,J = 9.2,2.7 Hz,1H),7.90 (q,J = 4.6,2.9 Hz,3H),7.78 (td,J = 7.9,6.4,3.6 Hz,13H),7.38 (dt,J = 8.6,3.0 Hz,1H),6.54 (dt,J = 12.7,3.1 Hz,2H),4.42 (d,J = 2.7 Hz,2H),3.74 (d,J = 2.7 Hz,3H),3.63 - 3.47 (m,2H),3.12 (d,J = 6.8 Hz,2H),1.61 - 1.19 (m,8H).

[0129] Compounds 7-9 in Table 2 below were prepared from intermediate 1 and the corresponding phenol intermediates 3-5 in a manner similar to that for the preparation of compound 6. [Table 2]

[0130] Compound 10: (S)-2-(6-(4-hydroxy-3,5-dimethoxyphenoxy)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid [ka] To a 20 mL vial was added compound 1 and DMF (2 mL). The mixture was stirred. To the mixture was added a solution of D-cysteine ​​hydrochloride in 0.5 M aqueous phosphate buffer pH 8 buffer (2 mL). The mixture was stirred for 30 min. The mixture was purified by reverse phase HPLC (MeCN / water with 0.1% TFA) to give compound 10 ((S)-2-(6-(4-hydroxy-3,5-dimethoxyphenoxy)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid). LCMS (C 19 H 16 N2O6S2)(ES,m / z)433[M+H] + . 1 H NMR (400 MHz,DMSO-d6)δ 13.21 (s,1H),8.33 (s,1H),8.12 (dd,J = 9.0,3.1 Hz,1H),7.65 (s,1H),7.27 (dd,J = 9.3,3.8 Hz,1H),6.50 (s,2H),5.43 (t,J = 9.1 Hz,1H),3.77 (d,J = 10.7 Hz,1H),3.73 (d,J = 2.9 Hz,6H),3.68 (d,J = 10.1 Hz,1H).

[0131] Compounds 11-15 in Table 3 below were prepared from the corresponding cyanobenzothiazole compounds 2-6 and D-cysteine ​​in a manner similar to that for the preparation of compound 10. [Table 3-1] [Table 3-2]

[0132] Compound 16: (S)-(6-(2-(2-hydroxy-3-methoxy-5-((2-(4-(methoxycarbonyl)-4,5-dihydrothiazol-2-yl)benzo[d]thiazol-6-yl)oxy)phenoxy)acetamido)hexyl)triphenylphosphonium bromide [ka] To a 4 mL vial containing compound 6 ((6-(2-(5-((2-cyanobenzo[d]thiazol-6-yl)oxy)-2-hydroxy-3-methoxyphenoxy)acetamido)hexyl)triphenylphosphonium bromide) (15.8 mg, 0.0198 mmol), DMF (1 mL) was added. The mixture was stirred. To the mixture was added a solution of D-methyl cysteinate (3.2 mg, 0.024 mmol) in 0.5 M phosphate buffer at pH 8 (0.5 mL). After 30 min, the mixture was purified by reverse phase HPLC (MeCN / water with 0.1% TFA) to give compound 16 ((S)-(6-(2-(2-hydroxy-3-methoxy-5-((2-(4-(methoxycarbonyl)-4,5-dihydrothiazol-2-yl)benzo[d]thiazol-6-yl)oxy)phenoxy)acetamido)hexyl)triphenylphosphonium bromide). LCMS (C 45 H 45 N3O7PS2) + (ES,m / z)834 [M] + . 1H NMR (400 MHz,DMSO-d6)δ 8.63 (s,1H),8.26 (d,J = 6.2 Hz,1H),8.16 - 8.09 (m,1H),7.89 (d,J = 7.0 Hz,3H),7.78 (q,J = 6.1,5.0 Hz,13H),7.62 (d,J = 3.2 Hz,1H),7.32 - 7.22 (m,1H),6.53 (d,J = 11.3 Hz,2H),5.53 (t,J = 9.2 Hz,1H),4.42 (s,2H),3.82 (t,J = 10.6 Hz,1H),3.77 - 3.72 (m,6H),3.69 (d,J = 10.4 Hz,1H),3.12 (q,J = 6.9 Hz,2H),1.51 - 1.25 (m,8H).

[0133] Compound 17: (S)-2-(6-(4-hydroxyphenoxy)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid Step 1: (Z)-N-(2-bromo-4-fluorophenyl)-4-chloro-5H-1,2,3-dithiazol-5-imine [ka] 2-Bromo-4-fluoroaniline (2.00 g, 10.5 mmol) was dissolved in dichloromethane (50 mL). 5-Dichloro-1,2,3-dithiazolium chloride (Appel's salt, 2.63 g, 12.6 mmol) was added and the reaction mixture was stirred overnight. The reaction mixture was then extracted with dichloromethane / water. The organic layer was collected and concentrated to an orange oil and used directly in the next step.

[0134] Step 2: 6-Fluorobenzo[d]thiazole-2-carbonitrile [ka] (Z)-N-(2-bromo-4-fluorophenyl)-4-chloro-5H-1,2,3-dithiazol-5-imine (0.26 g, 0.80 mmol) was dissolved in anhydrous pyridine (10 mL). Copper iodide (0.18 g, 0.90 mmol) and DIPEA (0.28 mL, 1.6 mmol) were added and the reaction was heated at 90° C. for 1 h. The mixture was concentrated and the residue was extracted with ethyl acetate / water. The organic layer was collected and concentrated. The resulting residue was purified by flash chromatography on silica gel. 1 H NMR (300 MHz,CD2Cl2)δ 8.22 (m,1H),7.71 (m,1H),7.44 (m,1H); FNMR (300 MHz,CD2Cl2)δ 110.91.

[0135] Step 3: 6-(4-methoxyphenoxy)benzo[d]thiazole-2-carbonitrile [ka] 4-Methoxyphenol (60 mg, 0.48 mmol) was dissolved in equimolar aqueous potassium hydroxide solution. Water was removed by lyophilization. The resulting solid was resuspended in DMF (10 mL). 6-Fluorobenzo[d]thiazole-2-carbonitrile (10 mg, 0.056 mmol) was added and the resulting solution was transferred to a microwave tube and reacted at 90° C., 90 W for 10 min. (CEM Discovery Synthesizer). The reaction mixture was then purified by reverse phase HPLC to give 6-(4-methoxyphenoxy)benzo[d]thiazole-2-carbonitrile.

[0136] Step 4: (S)-2-(6-(4-hydroxyphenoxy)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid [ka] 6-(4-Methoxyphenoxy)benzo[d]thiazole-2-carbonitrile (40 mg, 0.14 mmol) was mixed with pyridinium hydrochloride (5 g). The mixture was heated to 160° C. for 20 min. THF was added and the resulting suspension was filtered. The filtrate was concentrated and redissolved in MeCN. D-Cysteine ​​(23.8 mg, 0.140 mmol) was dissolved in water and added to the previous solution. Triethylamine was added dropwise to adjust the reaction pH to 8. The reaction was then stirred for 20 min and purified by reverse phase HPLC to give compound 17 ((S)-2-(6-(4-hydroxyphenoxy)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid). MS(C 17 H 12 N2O4S2)(ES,m / z)373[M+H] + . 1 H NMR (300 MHz,DMSO)δ 9.37 (s,1H),8.03 (d,1H),7.52 (d,1H),7.17 (dd,1H),6.92 (m,2H),6.77 (m,2H),5.35 (t,1H),3.65 (m,2H).

[0137] Compound 18: (S)-2-(6-(((2-(((3,4-dihydroxyphenoxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid [ka] To a 20 mL vial was added intermediate 10 (21.0 mg, 0.0475 mmol) and DMF (2 mL). The mixture was stirred. To this mixture was added D-cysteine ​​(6.9 mg, 0.057 mmol) in pH 8 buffer (2 mL). The mixture was stirred for 2 h. The mixture was purified by reverse phase HPLC (MeCN / water with 0.1% TFA) to give compound 18 ((S)-2-(6-(((2-(((3,4-dihydroxyphenoxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid). LCMS (C 23 H 22 N4O8S2)(ES,m / z)547[M+H] + . 1 H NMR (400 MHz,DMF-d7)δ 13.91 (s,1H),9.51 (d,J = 3.8 Hz,1H),9.26 (d,J = 19.5 Hz,1H),8.36 (dd,J = 15.4,8.8 Hz,1H),7.73 - 7.53 (m,1H),7.02 - 6.78 (m,3H),6.63 (dddd,J = 17.9,14.8,8.5,2.6 Hz,1H),5.75 (t,J = 9.4 Hz,1H),4.14 - 3.92 (m,6H),3.39 - 3.17 (m,6H).

[0138] Compound 19: (S)-2-(6-(((2-(((3,4-dihydroxyphenoxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylate methyl [ka] To a 20 mL vial was added intermediate 10 (27.6 mg, 0.0475 mmol) and DMF (2 mL). The mixture was stirred. To the mixture was added D-cysteine ​​methyl ester (10.1 mg, 0.749 mmol) in pH 8 buffer (2 mL). The mixture was stirred for 2 h. The mixture was purified by reverse phase HPLC (MeCN / water with 0.1% TFA) to give compound 19 ((S)-2-(6-(((2-(((3,4-dihydroxyphenoxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylate methyl). LCMS (C 24 H 24 N4O8S2)(ES,m / z)561[M+H] + . 1 H NMR (400 MHz,DMSO-d6)δ 9.07 (d,J = 16.4 Hz,1H),8.84 (d,J = 9.7 Hz,1H),8.16 (dt,J = 10.2,5.8 Hz,1H),8.04 - 7.81 (m,1H),7.35 (dd,J = 25.8,8.6 Hz,1H),6.66 (dd,J = 18.3,8.4 Hz,1H),6.49 (dd,J = 20.3,9.8 Hz,1H),6.32 (td,J = 21.6,18.0,8.4 Hz,1H),5.57 (t,J = 9.6 Hz,1H),3.85 (t,J = 10.6 Hz,1H),3.77 (d,J = 2.5 Hz,3H),3.74 - 3.51 (m,5H),3.20 - 2.88 (m,6H).

[0139] Compound 20: (S)-4-(((2-((((2-(4-(methoxycarbonyl)-4,5-dihydrothiazol-2-yl)benzo[d]thiazol-6-yl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-1,2-phenylene diacetate Step 1: 4-(((2-((((2-cyanobenzo[d]thiazol-6-yl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-1,2-phenylene diacetate [ka] To a 20 mL vial was added 6-hydroxybenzo[d]thiazole-2-carbonitrile (18.0 mg, 0.102 mmol), THF (1.5 mL), and DIPEA (0.073 mL, 0.41 mmol). The mixture was added dropwise over 1 min to a stirred solution of triphosgene (12.8 mg, 0.0431 mmol) in THF (1 mL). After 5 min, to the mixture was added intermediate 9 (43.1 mg, 0.132 mmol) in THF (1 mL). After 20 min, the mixture was adsorbed onto Celite and purified by silica gel chromatography using 0-100% EtOAc in heptane as eluent to give 4-(((2-((((2-cyanobenzo[d]thiazol-6-yl)oxy)carbonyl)-(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-1,2-phenylene diacetate. LCMS(C 24 H 22 N4O8S)(ES,m / z)527[M+H] + .

[0140] Step 2: (S)-4-(((2-((((2-(4-(methoxycarbonyl)-4,5-dihydrothiazol-2-yl)benzo[d]thiazol-6-yl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-1,2-phenylene diacetate [ka] To a 20 mL vial was added -(((2-((((2-cyanobenzo[d]thiazol-6-yl)oxy)carbonyl)-(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-1,2-phenylene diacetate (38.4 mg, 0.0729 mmol) and DMF (2 mL). The mixture was stirred. To this mixture was added D-cysteine ​​methyl ester (11.8 mg, 0.875 mmol) in pH 8 buffer (2 mL). The mixture was stirred for 2 h. The mixture was purified by reverse phase HPLC (MeCN / water with 0.1% TFA) to give compound 20 ((S)-4-(((2-((((2-(4-(methoxycarbonyl)-4,5-dihydrothiazol-2-yl)benzo[d]thiazol-6-yl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-1,2-phenylene diacetate). LCMS (C 28 H 28 N4O 10 S2) (ES, m / z) 645 [M+H] + . 1 H NMR (400 MHz,DMSO-d6)δ 8.16 (td,J = 11.2,10.5,4.7 Hz,1H),7.97 (dd,J = 28.4,15.3 Hz,1H),7.52 - 7.21 (m,2H),7.19 - 6.86 (m,2H),5.58 (t,J = 8.8 Hz,1H),3.85 (t,J = 10.7 Hz,1H),3.77 (d,J = 2.9 Hz,3H),3.74 - 3.62 (m,5H),3.19 - 2.96 (m,6H),2.31 - 2.20 (m,6H).

[0141] Compound 21: (6-((2-((((2-cyanobenzo[d]thiazol-6-yl)oxy)carbonyl)(methyl)amino)ethyl)-((3,4-diacetoxyphenoxy)carbonyl)amino)hexyl)triphenylphosphonium bromide [ka] To a 20 mL vial was added 6-hydroxybenzo[d]thiazole-2-carbonitrile (20.0 mg, 0.114 mmol), THF (1.5 mL), and DIPEA (0.100 mL, 0.568 mmol). The mixture was added dropwise over 1 min to a stirred solution of triphosgene (16.8 mg, 0.0568 mmol) in THF (1 mL). After 5 min, the mixture was added to Intermediate 11 (87.7 mg, 0.119 mmol) in THF (1 mL). After 20 min, the mixture was concentrated and purified by reverse phase HPLC (MeCN / water with 0.1% TFA) to give compound 21 ((6-((2-((((2-cyanobenzo[d]thiazol-6-yl)oxy)carbonyl)(methyl)amino)ethyl)-((3,4-diacetoxyphenoxy)carbonyl)amino)hexyl)triphenylphosphonium bromide). LCMS (C 47 H 46 N4O8PS)(ES,m / z)857[M] + . 1 H NMR (400 MHz,acetonitrile-d3)δ 8.18 (td,J = 9.4,9.0,6.0 Hz,1H),7.95 - 7.78 (m,4H),7.74 - 7.65 (m,12H),7.50 - 7.31 (m,1H),7.24 - 6.82 (m,3H),3.77 - 3.29 (m,6H),3.15 (p,J = 7.4 Hz,3H),3.03 (d,J = 6.5 Hz,2H),2.29 - 2.20 (m,6H),1.69 - 1.28 (m,10H).

[0142] Compound 22: (S)-(6-((((2-(4-carboxy-4,5-dihydrothiazol-2-yl)benzo[d]thiazol-6-yl)oxy)carbonyl)(2-(((3,4-diacetoxyphenoxy)carbonyl)(methyl)amino)ethyl)amino)hexyl)triphenylphosphonium bromide [ka] To an 8 mL vial containing compound 21 (10.0 mg, 0.0107 mmol) was added DMF (1.5 mL). The mixture was stirred. To this mixture was added a solution of D-cysteine ​​(1.7 mg, 0.014 mmol) in pH 8 buffer (0.5 mL). The mixture was purified by reverse phase HPLC (MeCN / water with 0.5% TFA) to give compound 22 ((S)-(6-((((2-(4-carboxy-4,5-dihydrothiazol-2-yl)benzo[d]thiazol-6-yl)oxy)carbonyl)(2-(((3,4-diacetoxyphenoxy)carbonyl)(methyl)amino)ethyl)-amino)hexyl)triphenylphosphonium bromide). LCMS (C 50 H 50 N4O 10 PS2)(ES,m / z)961[M] + . 1 H NMR (400 MHz,DMF-d7)δ 14.0 (br s,1H),8.39 - 8.26 (m,1H),8.17 - 8.08 (m,9H),8.04 - 7.96 (m,6H),7.69 - 7.30 (m,3H),5.75 (t,J = 9.1 Hz,1H),4.16 - 4.00 (m,2H),3.51 - 3.22 (m,6H),2.54 - 2.44 (m,6H),2.01 - 1.69 (m,8H),1.68 - 1.53 (m,2H).

[0143] Compound 23: (S)-(6-((2-(((3,4-diacetoxyphenoxy)carbonyl)(methyl)amino)ethyl)(((2-(4-(methoxycarbonyl)-4,5-dihydrothiazol-2-yl)benzo[d]thiazol-6-yl)oxy)carbonyl)amino)hexyl)triphenylphosphonium bromide [ka] To an 8 mL vial containing compound 21 (19.8 mg, 0.0211 mmol) was added DMF (1.5 mL). The mixture was stirred. To this mixture was added a solution of D-cysteine ​​methyl ester (1.7 mg, 0.014 mmol) in pH 8 buffer (0.5 mL). The mixture was purified by reverse phase HPLC (MeCN / water with 0.5% TFA) to give compound 23 ((S)-(6-((2-(((3,4-diacetoxyphenoxy)carbonyl)(methyl)amino)ethyl)(((2-(4-(methoxycarbonyl)-4,5-dihydrothiazol-2-yl)benzo[d]thiazol-6-yl)oxy)carbonyl)amino)hexyl)triphenylphosphonium bromide). LCMS (C 51 H 52 N4O 10 PS2)(ES,m / z)975[M] + . 1 H NMR (400 MHz,DMSO-d6)δ 8.15 (td,J = 9.4,4.4 Hz,1H),8.01 - 7.85 (m,4H),7.75 (td,J = 9.7,8.2,5.4 Hz,12H),7.39 - 7.18 (m,2H),7.14 - 6.81 (m,2H),5.57 (t,J = 9.2 Hz,1H),3.85 (t,J = 10.6 Hz,1H),3.77 (s,3H),3.41 - 3.23 (m,4H),3.05 (dd,J = 55.3,8.7 Hz,3H),2.31 - 2.10 (m,6H),1.65 - 1.26 (m,10H).

[0144] Example 2: Cell-free detection and specificity of superoxide The superoxide probe, compound 1 (6-(4-hydroxy-3,5-dimethoxyphenoxy)benzo[d]thiazole-2-carbonitrile), was diluted in PBS to 25 μM in the wells of a 96-well white-walled assay plate. A generator of reactive oxygen and nitrogen species was then diluted in PBS and added to the assay plate. The plate was mixed for 5 minutes using an orbital plate shaker, followed by a 30 minute incubation at room temperature protected from light. For luciferin detection, a luciferin-utilizing luciferase enzyme, UltraGlo (Promega Corporation), was prepared by mixing luciferin detection reagent (Promega), reconstitution buffer (Promega), and d-cysteine ​​(Promega) and 1 volume was added to the reaction. The plate was mixed using an orbital plate shaker and luminescence was measured using a GloMax® luminometer. The data are shown in Figure 3, where the blank contained PBS only, HX = hypoxanthine, XO = xanthine oxidase, and SOD = superoxide dismutase. The data demonstrate the presence of a strong signal in the presence of HX and XO, which are known to react to produce superoxide. This signal was reduced in the presence of superoxide dismutase, confirming that superoxide is the source of the signal. Little or no signal was detected in the presence of hydrogen peroxide, rose bengal (which produces singlet oxygen), sodium nitrite, or NONOate.

[0145] Example 3: Detection of superoxide in cells Human hepatoma cells (Hep G2) were seeded into the wells of a 96-well white-walled assay tissue culture plate. The seeded cells were incubated overnight in a humidified tissue culture incubator at 37°C with 5% CO2. The next day, the superoxide probe, compound 1 (6-(4-hydroxy-3,5-dimethoxyphenoxy)benzo[d]thiazole-2-carbonitrile), was added to the cells in culture medium at a final concentration of 12.5uM. Then, dimethoxynaphthoquinone (DMNQ, 50μM) or antimycin A (10μM) diluted in culture medium was added to the reaction wells, and the assay plate(s) were incubated for 1 hour in a humidified tissue culture incubator at 37°C with 5% CO2. For luciferin detection, a luciferin-utilizing luciferase enzyme, UltraGlo (Promega Corporation), was prepared by mixing luciferin detection reagent (Promega), reconstitution buffer (Promega), and d-cysteine ​​(Promega) and 1 volume was added to the reaction. Plates were mixed using an orbital plate shaker and luminescence was measured using a GloMax® luminometer. Data are shown in Figure 4. An increase in signal was detected when cells were treated with either dimethoxynaphthoquinone (DMNQ) or antimycin A, both of which are known to induce the formation of superoxide.

[0146] Example 4: Detection of xanthine oxidase in vitro Xanthine oxidase (XO) is an enzyme that catalyzes the oxidation of hypoxanthine or xanthine to uric acid and superoxide. XO is normally found in the liver and jejunum and plays an important role in purine catabolism. During severe liver injury, xanthine oxidase is released into the blood and can be used as a marker for liver injury. Here, we provide an example of measuring xanthine oxidase activity using the proluciferin compounds described herein. Purified xanthine oxidase was serially diluted 3-fold in 0.1 M Tris, pH 7.5. 25 μl of the diluted enzyme was transferred to wells of a 96-well assay plate and the reaction was initiated by adding 25 μl of 300 μM xanthine and 25 μl of 75 μM compound 20. After 10, 20, 30, and 60 min of incubation at 37° C., 75 μl of luciferin detection reagent containing esterase was added to the samples and luminescence was read after an additional 20 min of incubation at room temperature.

[0147] The increase in luminescence signal was dependent on the presence of the xanthine oxidase substrate, xanthine, and was linear with respect to xanthine oxidase concentration and time. The data shown in Figure 5 indicate that superoxide produced by xanthine oxidase interacts with proluciferin compounds to release luciferin, which can be used to measure activity or screen inhibitors for superoxide-producing enzymes.

[0148] Example 5: Detection of superoxide production by macrophages The production of superoxide by macrophages plays an important role in immune responses and inflammation. Superoxide is produced by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (known as NOX enzyme) and is induced by PMA (4β-phorbol 13-acetate 12-myristate). Here, we show an example of measuring PMA-induced superoxide production in the RAW 247.6 macrophage cell line. RAW 247.6 cells were seeded overnight in DMEM medium containing 10% FBS. To start the experiment, the medium was removed, the cells were washed once with PBS, and 50 μl of PBS containing 5 mM glucose, 1 mM MgCl2, 0.5 mM CaCl2, and 0.05% BSA was added to the cells in the presence or absence of 20 μM PMA. To measure the production of superoxide, 50 μl of 50 μM compound 18 (Figure 6A) or 100 μM compound 20 (Figure 6B) was added to the cells. Cells were treated for 2 h at 37° C., luciferin production was measured by adding an equal volume of luciferin detection reagent containing esterase, and luminescence was read after 20 min incubation at room temperature.

[0149] For both compounds, higher luminescence signals were measured in the presence of cells compared to the medium only control. The data in Figures 6A-6B show that the luminescence signal was significantly increased with PMA treatment illustrating the utility of the methods of the present invention for measuring drug-induced changes in superoxide production by mammalian cells.

[0150] Example 6: Detection of superoxide production in kinetic mode. RAW 247.6 macrophage cells were seeded at 30,000 cells per well overnight in DMEM medium with 10% FBS. The next day, the medium was removed and the cells were washed with PBS. 100ul of PBS containing 5mM glucose, 1mM MgCl2, 0.5mM CaCl2, 0.05% BSA, and 50μM compound 20 was added to the cells in the presence or absence of 20μM PMA. The cells were returned to 37°C in a tissue culture incubator. At different time points (10, 20, 30, and 60 minutes), 10μl was removed from the sample and mixed with 10μl of luciferin detection reagent with esterase, and luminescence was read after 20 minutes of incubation at room temperature. The data in Figure 7 show that a time-dependent increase in signal was detected in cells treated with PMA, indicating increased superoxide production.

[0151] All references cited in this specification, including publications, patent applications, and patents, are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and is hereby incorporated by reference in its entirety.

[0152] In the context of the description of the invention (particularly in the context of the claims which follow), use of the terms "a," "an," and "the," as well as "at least one" and similar referents, should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Unless otherwise indicated herein or clearly contradicted by context, use of the term "at least one" followed by a list of one or more items (e.g., "at least one of A and B") is to be construed to mean one item (A or B) selected from the listed items, or any combination of two or more of the listed items (A and B). The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise indicated. The recitation of ranges of values ​​herein is intended to serve merely as a shorthand method of individually referring to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually recited herein. All methods described herein may be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. Any and all examples provided herein, or the use of exemplary language (e.g., "etc.") are intended merely to better illuminate the invention, and do not pose limitations on the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0153] Preferred embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments may become apparent to those of ordinary skill in the art after reading the foregoing description. The inventors anticipate that those of ordinary skill in the art will employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or clearly contradicted otherwise by context.

Claims

1. A compound of formula (II): 【Chemical 1】 or a salt thereof, wherein R 1 is -CN and [Chemical Formula 2] selected from R 2 is selected from hydrogen and halo, n is 0, 1, 2, or 3, Each R 3 is independently C 1 to C 4 alkyl, C 1 to C 4 alkoxy, -OC(O)-C 1 to C 4 alkyl, hydroxy, amino, and a group selected from -linker-X, where X is a targeting moiety R 4a and R 4b One of them is hydroxy or -OC(O)-C 1 ~C 4 alkyl, and the other is hydrogen or group - linker - X, where X is a targeting moiety R 5 is selected from hydrogen and C 1 -C 4 -alkyl, Z is a bond or a group of the formula 【Chemical Formula 3】

2. R 6 is C 1 to C 4 alkyl and group - linker - Y, where Y is a targeting moiety, said compound, or a salt thereof. The compound according to claim 1, or a salt thereof, wherein Z is a group of the formula

3. [Chemical Formula 4]

4.

5. R 6 The compound according to claim 2, or a salt thereof, wherein R is methyl.

6. R 6 The compound or a salt thereof according to claim 2, wherein R is a group - linker - Y and Y is a mitochondrial targeting moiety.

7.

8.

9. n is 1, and R 3 is selected from -OC(O)CH 3 and hydroxy, the compound according to claim 1, or a salt thereof.

10. R 4a is hydrogen, and R 4b is hydroxy or -OC(O)CH 3 The compound according to claim 1, or a salt thereof.

11.

12. 【Chemical Formula 5】

13. R 1 is -CN and 【Chemical Formula 6】

14. R 2 is selected from hydrogen and halogen,

15. Each R 3 is independently C 1 to C 4 alkyl, C 1 to C 4 alkoxy, amino, and group - linker - X, where X is a targeting moiety R 4a and R 4b one of which is hydroxy and the other is hydrogen or group - linker - X, where X is a targeting moiety R 5 is hydrogen and C 1 -C 4 alkyl selected from, the compound according to claim 1, or a salt thereof.

16. R 1 The compound according to claim 1, or a salt thereof, wherein R is -CN.

17. R 1 is 【Chemical Formula 7】

18.

19. R 5 The compound according to claim 10, or a salt thereof, wherein R is selected from hydrogen and methyl.

20. R 2 The compound according to claim 1, or a salt thereof, wherein R is hydrogen.

21. R 2 The compound according to claim 1, or a salt thereof, wherein R is fluoro.

22.

23. 【Chemical Formula 8】 Or a salt thereof, wherein R 4a is hydrogen or the group - linker - X, and X is a targeting moiety, the compound according to claim 1, or a salt thereof.

24.

25. 【Chemical Formula 9】 or a salt thereof, wherein R 4b is hydrogen or the group - linker - X, and X is a targeting moiety, the compound according to claim 1, or a salt thereof.

26. n is 0, 1, or 2, and each R 3 is independently C 1 to C 4 alkyl, C 1 to C 4 alkoxy, -OC(O)-C 1 to C 4 alkyl, and hydroxy, and the compound according to claim 1, or a salt thereof.

27. n is 0, 1, or 2, and each R 3 is independently C 1 to C 4 alkyl and C 1 to C 4 alkoxy, the compound according to claim 1, or a salt thereof.

28. At least one R 3 is a group-linker-X, where X is a mitochondrial targeting moiety, the compound according to claim 1, or a salt thereof.

29.

30.

20. 【Fig. 10-1】 【Chemical Formula 10-2】

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

72.

73.

74.

75.

76.

77.

78.

79.

80. ]

81. ]

82. ]

83. ]

84. ]

85. ]

86. ]

87. ]

88. ]

89. ]

90. ]

91. ]

92. ]

93. ]

94. ]

95. ]

96. ]

97. ]

98. ]

99. ]

100. ]

101. ]

102. ]

103. ]

104. ]

105. ]

106. ]

107. ]

108. ]

109. ]

110. ]

111. ]

112. ]

113. ]

114. ]

115. ]

116. ]

117. ]

118. ]

119. ]

120. ]

121. ]

122. ]

123. ]

124. ]

125. ]

126. ]

127. ]

128. ]

129. ]

130. ]

131. ]

132. ]

133. ]

134. ]

135. ]

136. ]

137. ]

138. ]

139. ]

140. ]

141. ]

142. ]

143. ]

144. ]

145. ]

146. ]

147. ]

148. ]

149. ]

150. ]

151. ]

152. ]

153. ]

154. ]

155. ]

156. ]

157. ]

158. ]

159. ]

160. ]

161. ]

162. ]

163. ]

164. ]

165. ]

166. ]

167. ]

168. ]

169. ]

170. ]

171. ]

172. ]

173. ]

174. ]

175. ]

176. ]

177. ]

178. ]

179. ]

180. ]

181. ]

182. ]

183. ]

184. ]

185. ]

186. ]

187. ]

188. ]

189. ]

190. ]

191. ]

192. ]

193. ]

194. ]

195. ]

196. ]

197. ]

198. ]

199. ]

200. ]

201. ]

202. ]

203. ]

204. ]

205. ]

206. ]

207. ]

208. ]

209. ]

210. ]

211. ]

212. ]

213. ]

214. ]

215. ]

216. ]

217. ]

218. ]

219. ]

220. ]

221. ]

222. ]

223. ]

224. ]

225. ]

226. ]

227. ]

228. ]

229. ]

230. ]

231. ]

232. ] 【Claim

Images