Compounds and methods for detecting 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 of related applications This application claims priority and benefits of U.S. Provisional Patent Application No. 63 / 224,087, filed on 21 July 2021, which is incorporated herein by reference in its entirety.

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

[0003] Superoxide is a highly unstable, highly regulated reactive oxygen species 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. Short residence time (10 depending on superoxide dismutase availability) -6 ~10 -9 Due to its half-life of only a few seconds, accurately measuring superoxide in cells is inherently difficult. Current techniques for detecting superoxide often rely on chemiluminescent and fluorescent probes that lack sufficient selectivity. For example, hydroethidine is prone to auto-oxidation and other non-superoxide-specific oxidation reactions, which results in the production of fluorescent products with emission spectra similar to those of 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 generates superoxide, leading to a potential overestimation of the amount of superoxide present. [Overview of the Initiative]

[0004] In one embodiment, the present disclosure relates to a compound of formula (II): [Chemical formula] or a salt thereof, wherein R 1 is -CN and [Chemical formula] 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 the group - linker - X, where X is a targeting moiety, R 5 is selected from hydrogen and C1-C4 alkyl, Z is a bond or the formula [Chemical formula] is a group of R 6 is selected from C1-C4 alkyl and the group - linker - Y, where Y is a targeting moiety, to provide a compound, or a salt thereof.

[0005] In some embodiments, Z is the formula [Chemical formula] is a group of. 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 The compound is selected from -OC(O)CH3 and hydroxyl.

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

[0008] In some embodiments, the compound is a compound of formula (I): [ka] or a salt thereof, in the formula, R 1 However, -CN and [ka] Selected from, R 2 However, hydrogen and halo are selected, n is 0, 1, 2, or 3, Each R 3 However, independently selected from C1-C4 alkyl, C1-C4 alkoxy, amino, and group-linker-X, where X is the targeting moiety. R 4a and R 4b One of them is hydroxyl, and the other is hydrogen or group-linker-X, where X is the targeting moiety. R 5 However, it is a compound, or a salt thereof, selected from hydrogen and C1-C4 alkyl groups.

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

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

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

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

[0013] In some embodiments, n is 0, 1, or 2, and each R 3 The elements are 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 These are independently selected from C1-C4 alkyl and C1-C4 alkoxy. In some embodiments, at least one R 3 is a base-linker-X, where X is the mitochondrial targeting moiety. In some embodiments, X is a triphenylphosphonium moiety.

[0014] In some embodiments, the compound is [ka] [ka] [ka] Selected from the group consisting of salts thereof.

[0015] In one embodiment, the present disclosure relates to a method for detecting superoxide in a sample, Contacting the sample with a compound disclosed herein (e.g., a compound of formula (I) or formula (II)), If luciferin-utilizing luciferase is not yet present in the sample, the sample is brought into contact with luciferin-utilizing luciferase. The present invention provides a method that includes detecting luminescence in a sample.

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

[0017] In one embodiment, the present 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 explanation of the drawing]

[0019] [Figure 1]Figures 1A-1B show schematic diagrams of a method for detecting superoxide in cells using compounds such as those disclosed herein. Unstable superoxide produced extracellularly (A) or intracellularly (B) is detected using a proluciferin probe. After superoxide production, the proluciferin probe rapidly reacts with the highly unstable superoxide to produce a stable luciferin product. Luciferin is detected using a luciferase reaction, and the luminescence signal is proportional to the superoxide production. [Figure 2] This diagram shows a schematic representation of a luminescence assay for detecting superoxide in a sample of cells or other analytes. The "processing" step refers to the addition of an unknown experimental component (e.g., a candidate drug), which can be added before, after, or simultaneously with the superoxide probe. [Figure 3] The data from the assay for detecting superoxide in cell-free samples, as described in Example 2, is shown below. [Figure 4] As described in Example 3, data from an assay detecting superoxide in cells after treatment with the superoxide-generating drug compound dimethoxynaphthoquinone (DMNQ) and antimycin A are shown. [Figure 5] The data from the assay for detecting xanthine oxidase (XO) activity in vitro, as described in Example 4, is shown. [Figure 6] Figures 6A-6B show data from an assay for detecting superoxide production by macrophages, as described in Example 5. [Figure 7] The data from the assay for detecting superoxide production in kinetic mode, as described in Example 6, is shown. [Modes for carrying out the invention]

[0020] Probe compounds, particularly caged luciferin and hydroxycyanobenzothiazole compounds and their derivatives, are disclosed herein, which have high specificity for the detection of superoxide compared to other reactive oxygen species. A stable reporter product is formed after reaction with superoxide, allowing the assay to be carried out in a "add and read" plate-based format or in kinetic mode by culture medium sampling.

[0021] definition Any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the embodiments described herein, although several preferred methods, compositions, apparatus, 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 can be varied 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 meanings generally understood by those skilled in the art. For example, any nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. The meaning and scope of 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 plural forms and plural terms shall include singular forms.

[0023] As used herein and in the appended claims, the singular forms "a," "an," and "the" refer to multiple subjects unless the context otherwise clearly indicates. For example, a reference to "peptides" refers to one or more peptides and their equivalents known to those skilled in the art.

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

[0025] Where used herein, the term “contains” and its linguistic variations indicate the presence of described features, elements, method steps, etc., without excluding the presence of additional features, elements, method steps, etc. Conversely, the term “consisting of” and its linguistic variations indicate the presence of described features, elements, method steps, etc., excluding undescribed features, elements, method steps, etc., except for impurities that are usually associated with them. The phrase “essentially consisting of” indicates described features, elements, method steps, etc., as well as any additional features, elements, method steps, etc., that do not substantially affect the fundamental nature of the composition, system, or method. Many embodiments herein are described using the open phrase “comprising.” Such embodiments include multiple embodiments in the closed form of “consisting of” and / or “essentially consisting of,” which may alternatively be claimed or described using such language.

[0026] For the purposes of enumerating numerical ranges in this specification, each numerical value that intersects them with the same degree of precision is explicitly intended. For example, for the range 6–9, the digits 7 and 8 are intended in addition to 6 and 9, and for the range 6.0–7.0, the digits 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly intended.

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

[0028] As used herein, the term “sample” is used in its broadest sense. In a sense, it means specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and include fluids, solids, tissues, and gases. Biological samples include blood products such as plasma and serum. Samples may also refer to cells, cell lysates, or purified forms of enzymes, peptides, and / or polypeptides as described herein (e.g., purified protein samples). Cell lysates may include cells lysed with a solvent or lysate such as rabbit reticulocyte or wheat germ lysate. Samples may 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 may also include purified samples such as purified protein samples. However, such examples should not be construed as limiting the types of samples to which the present invention is applicable.

[0029] Specific definitions of functional groups and chemical terms are described in more detail below. For the purposes of this disclosure, chemical elements are as follows: Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Ed., identified according to the inside of the cover, specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional parts and reactivity, are described in Sorrell, Organic Chemistry, 2 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 This is described in Edition, Cambridge University Press, Cambridge, 1987, and the entire contents of each of these 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" means a straight-chain or branched saturated hydrocarbon chain. An alkyl chain is, for example, a chain with 1 to 30 carbon atoms (C1 to C2). 30 Alkyl), 1 to 24 carbon atoms (C1 to C 24 Alkyl), for example, 1 to 16 carbon atoms (C1 to 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 Alkyl groups may include alkyl groups, 1 to 8 carbon atoms (C1-C8 alkyl groups), 1 to 6 carbon atoms (C1-C6 alkyl groups), or 1 to 4 carbon atoms (C1-C4 alkyl groups). Typical examples of alkyl groups, though not limited to them, include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, 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” means a straight or branched hydrocarbon chain containing at least one carbon-carbon double bond. The double bond(s) may be located at any position on the hydrocarbon chain. Representative examples of alkenyls 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" means a straight or branched hydrocarbon chain containing at least one carbon-carbon triple bond. Triple bonds may be located at any position within the hydrocarbon chain. Typical examples of alkynyls include, but are not limited to, ethynyl, propynyl, and butynyl.

[0034] As used herein, the term "alkoxy" refers to an alkyl group, as defined herein, that is added to the parent molecule via an oxygen atom. Typical examples of alkoxys 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 It refers to R x and R y This is selected from hydrogen and alkyl (e.g., C1-C4 alkyl).

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

[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, crosslinked, condensed, 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 terms "halogen" or "halo" mean 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) having one or more ring heteroatoms independently selected from O, N, and S. An aromatic monocyclic ring is a five-membered or six-membered ring containing at least one heteroatom independently selected from O, N, and S (e.g., one, two, three, or four heteroatoms independently selected from O, N, and S). A five-membered aromatic monocyclic ring has two double bonds, and a six-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 obtained by addition-condensation to a monocyclic heteroaryl group as defined herein. A tricyclic heteroaryl group is exemplified by a monocyclic heteroaryl ring obtained by addition to a monocyclic aryl group as defined herein, or two rings independently selected from a monocyclic heteroaryl group as defined herein. Representative examples of monocyclic heteroaryls include, but are not limited to, pyridinyl (including pyridine-2-yl, pyridine-3-yl, and pyridine-4-yl), pyrimidinyl, pyrazinyl, pyridadinyl, 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, benzofuranil, benzoxadiazolyl, benzopyrazolyl, benzothienyl, benzotriazolyl, benzoxadiazolyl, benzoxazolyl, clomenyl, imidazopyridine, imidazothiazolyl, indazolyl, indolyl, isobenzofuranil, isoindolyl, isoquinolinyl, naphthilidinyl, purinyl, pyridoimidazolyl, quinazolinyl, quinolinyl, quinoxalinyl, thiazolopyridinyl, thiazolopyrimidinyl, thienopyrrolyl, and thienothienyl.Typical examples of tricyclic heteroaryls include, but are not limited to, dibenzofuranyl and dibenzothienyl. Monocyclic, bicyclic, and tricyclic heteroaryls are linked to the parent molecule through any carbon or nitrogen atom contained within the ring.

[0040] As used herein, the terms “heterocycle” or “heterocyclic” refer to a saturated or partially unsaturated non-aromatic cyclic group having one or more ring heteroatoms independently selected from O, N, and S. Heterocycles can be monocyclic, bicyclic, or tricyclic. Monocyclic heterocycles are 3, 4, 5, 6, 7, or 8-membered rings containing at least one heteroatom independently selected from O, N, and S. 3 or 4-membered rings contain 0 to 1 double bonds and one heteroatom selected from O, N, and S. 5-membered rings contain 0 or 1 double bonds and 1, 2, or 3 heteroatoms selected from O, N, and S. 6-membered rings contain 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 (for example, if the ring heteroatom is S, it can be oxidized to SO or SO2). Representative examples of monocyclic heterocycles, though not limited to these, include azetidinyl, azepanyl, azilidinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolidinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolidinyl, oxazolidinyl, oxetanyl, and pi Examples include perazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, 1,2-thiadinyl, 1,3-thiadinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidethiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianil.A bicyclic heterocycle is a bridged monocyclic heterocycle system in which two non-adjacent atoms of a 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. Representative examples of bicyclic heterocycles, though not limited to these, include benzopyranil, benzothiopyranil, chromanil, 2,3-dihydrobenzofuranil, 2,3-dihydrobenzothienyl, 2,3-dihydroisoquinoline, 2-azaspiro[3.3]heptan-2-yl, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hepta-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 group, or bicyclic heterocycles fused to a monocyclic cycloalkenyl group, or bicyclic heterocycles fused to a monocyclic heterocycle, or bicyclic heterocycles in which two non-adjacent atoms of a 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, but not limited to these, include 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 Examples include decane. Monocyclic, bicyclic, and tricyclic heterocyclic rings are connected to the parent molecule through any carbon or nitrogen atom contained within the ring.

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

[0042] As used herein, the term “targeted moiety” refers to a moiety that binds to or is localized to a particular locale. A moiety may be, for example, a small molecule, a peptide, a protein, a nucleic acid, a nucleic acid analog, or a carbohydrate. A locale may be an organelle, an intracellular compartment, a particular cell type, or a particular tissue.

[0043] When a group or part can be substituted, the term “substituted” means that one or more hydrogens on the group indicated in the expression using “substituted” (e.g., 1, 2, 3, 4, 5, or 6; in some embodiments, 1, 2, or 3; in other embodiments, 1 or 2) can be replaced with a selection of the listed indicated groups or a suitable substituent known to those skilled 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, amide, amidino, aryl, azide, carbamoyl, carboxyl, carboxyl ester, cyano, cycloalkyl, cycloalkenyl, guanidino, halo, haloalkyl, haloalkoxy, heteroalkyl, heteroaryl, heterocyclyl, hydroxy, hydrazino, imino, oxo, nitro, phosphate, phosphonate, sulfonic acid, thiol, thion, or combinations thereof.

[0044] When used herein, the chemical structure is indicated as follows: [ka] This represents a bond point between one part and another part (for example, a substituent on the rest of the compound).

[0045] For the compounds described herein, their groups and substituents may be selected according to the allowable valencies of the atoms and substituents, and as a result, the selection and substitution result in stable compounds that do not spontaneously undergo transformations such as rearrangement, cyclization, or removal.

[0046] If substituents are identified by their conventional chemical formulas written from left to right, they optionally include substituents resulting from writing the structure from right to left; for example, -CH2O- optionally also includes -OCH2-, and -OC(O)NH- optionally includes -NHC(O)O-.

[0047] compound Compound of formula (II): [ka] or a salt thereof, in the formula, R 1 However, -CN and [ka] Selected from, R 2 However, hydrogen and halo are selected, n is 0, 1, 2, or 3, Each R 3 However, independently selected from C1-C4 alkyl, C1-C4 alkoxy, -OC(O)-C1-C4 alkyl, hydroxy, amino, and group-linker-X, where X is the targeting moiety. R 4a and R 4b One of them is a hydroxyl group or -OC(O)-C1~C4 alkyl group, and the other is a hydrogen group or a linker-X, where X is the targeting moiety. R 5 However, hydrogen and C1-C4 alkyl are selected, Z is a combination or expression [ka] It is the basis of, R 6 A compound or a salt thereof is disclosed herein, selected from C1-C4 alkyl groups and a group-linker-Y, where Y is the targeting moiety.

[0048] In some embodiments, R 1is -CN. In such embodiments, the compound is the compound of formula (II'): [ka] or a salt thereof, where 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 the compound of formula (II''): [ka] or a salt thereof, where 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 a halo. In some embodiments, R 2 It is fluoro.

[0051] In some embodiments, Z is a combination. In some embodiments, Z is an expression. [ka] It is the basis of, and in the formula, R 6 Z is selected from C1-C4 alkyl groups and group-linker-Y, where Y is the targeting moiety. In some embodiments, Z is a formula [ka] It is the basis of, and in the formula, R6 Z is selected from methyl and group-linker-Y, where Y is the targeting moiety. In some embodiments, Z is a formula [ka] It is the basis of, and in the formula, R 6 is methyl. In some embodiments, Z is of the formula [ka] It is the basis of, and in the formula, R 6 is the base-linker-Y, where Y is the targeting portion.

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

[0053] In some embodiments, the base in formula (II) [ka] teeth, [ka] It has an expression that is 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. In some embodiments, each R 3 is independently selected from C1-C4 alkyl and C1-C4 alkoxy. In some embodiments, each R 3 is independently selected from C1-methyl, methoxy, -OC(O)CH3, and hydroxy. In some embodiments, each R 3 is independently selected from methyl and methoxy. In some embodiments, each R 3 is independently selected from -OC(O)CH3 and hydroxy. In some embodiments, n is 1 and R 3 is methyl. In some embodiments, n is 1 and R 3 is methoxy. 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 (e.g., methyl).

[0057] The compound of formula (I):

Chemical formula

Chemical formula

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

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

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

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

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

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

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

[0065] In some embodiments, R 4a is hydrogen, and R 4b is hydroxyl. In some embodiments, R 4a It is hydroxyl, and R 4b is hydrogen. In some embodiments, R 4a It is hydroxyl, and R 4b is a base-linker-X, where X is the targeting portion.

[0066] In some embodiments, the base of formula (I) [ka] teeth, [ka] It has an expression that is 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] Several embodiments, each R 3 R is independently selected from C1-C4 alkyl and C1-C4 alkoxy. In some embodiments, each R 3 This is independently selected from methyl and methoxy.

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

[0070] In some embodiments, the compound comprises at least one group which is the targeting moiety. In some embodiments, at least one R 3 , or R 4a and R 4b One of these is the base-linker-X, where X is the mitochondrial targeting moiety. In some embodiments, R 6 is a group-linker-Y, where Y is the mitochondrial targeting moiety. In such embodiments, the compound can be targeted to a specific locale, such as a specific organelle, such as mitochondria. Mitochondrial targeting may be particularly useful for the compounds disclosed herein because mitochondria produce superoxide when electrons "leaking" from the electron transport chain are captured by molecular oxygen. Specific targeting of a selective superoxide probe to mitochondria can enable the direct measurement of superoxide produced in mitochondria, for example, in living cells. For example, in some embodiments, at least one R 3 , or R 4a and R 4b One of these is a group-linker-X, where X is a triphenylphosphonium moiety or a trialkylammonium moiety. In some embodiments, at least one R 3 , or R 4a and R 4b One of them is the group-linker-X, where X is the triphenylphosphonium moiety. In some embodiments, R 6 R is a group-linker-Y, where Y is a triphenylphosphonium moiety or a trialkylammonium moiety. In some embodiments, R 6 The base is the linker Y, and Y is the triphenylphosphonium moiety.

[0071] A linker can be any group that provides sufficient distance between the targeted moiety X or Y and the rest of the compound, allowing each to function without (or with minimal) disruption by bonding to the other. Linkers can be 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 (-C6H4-)), heterocyclylene (e.g., piperazine). [ka] and may include one or more groups independently selected from any combination thereof. In some embodiments, the linker includes one or more -(CH2CH2O)-(oxyethylene) groups. In some embodiments, the linker includes one or more alkylene groups (e.g., -(CH2) n-, n is 1 to 12, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or any preferred range between them). 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., comprising any preferred 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 linked C, S, N, and / or O atoms. In some embodiments, the linker includes 1 to 200 linearly linked 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 preferred range between them (e.g., 2 to 20, 10 to 50, 6 to 18)). In some embodiments, the linker includes 1 to 200 linearly linked 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 preferred range among them (e.g., 2 to 20, 10 to 50, 6 to 18)).

[0072] In some embodiments, the linker is a linear alkylene linker, for example, the linker is of formula (CH2) n - comprises the formula, 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 is of formula: [ka] It holds.

[0073] In some embodiments, the linker comprises a combination of methylene (-CH2-), ether (-O-), and amide (-C(O)NH-) moieties. For example, in some embodiments, the linker is of the formula: [ka] It holds.

[0074] In some embodiments, the compound is [ka] [ka] [ka] Selected from the group consisting of salts thereof.

[0075] In some embodiments, the compound is in salt form, i.e., the charged form of the parent compound related to the 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 the 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 in other respects, the salt is equivalent to the parent form of the compound for the purposes of this disclosure.

[0076] Specifically, a compound is anionic or can be anionic (for example, -COOH is -COO - If it has a functional group (which may be), the salt may be formed with one or more suitable cations. Examples of suitable inorganic cations include Li + kaNa + , and K + Alkali metal cations such as Ca 2+ and Mg 2+ This includes, but is not limited to, alkaline earth cations such as , and other cations. Potassium and sodium salts may be particularly preferred. Examples of preferred organic cations include ammonium ions (i.e., NH4). +) and substituted ammonium ions (e.g., NH3R1 + NH2R2 + NHR3 + , and NR4 + This includes, but is not limited to, ) . Some suitable examples of substituted ammonium ions are 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] A compound is cationic or can be cationic (for example, -NH2 is -NH3) +If the functional group (which may be) is present, the salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrogen chloride, hydrogen bromide, hydrogen iodide, sulfuric acid, sulfurous acid, nitric acid, nitrite, phosphoric acid, and phosphorous acid. 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, camphor sulfonic 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, mucinic 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. In some embodiments, the compound is a tetrafluoroborate or trifluoromethanesulfonate.

[0078] The compounds can be prepared by several preferred 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 include, but are not limited to, chromatography on a solid support such as silica gel, alumina, or silica derivatized with alkylsilane groups, recrystallization at high or low temperatures with optional pretreatment on activated carbon, thin-layer chromatography, distillation at various pressures, sublimation under vacuum, and grinding, as described in, for example, “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 step may vary depending on the specific reactants used and the substituents present in them. The reaction may be post-treated by conventional methods, for example, by removing the solvent from the residue, and may also be purified by methods generally known in the art, such as crystallization, distillation, extraction, grinding, and chromatography, but not limited to these. Unless otherwise stated, the starting materials and reagents may be commercially available or prepared by those skilled in the art from commercially available materials using methods described in the chemical literature.

[0080] Standard experiments, including the appropriate handling of reaction conditions, reagents, and synthesis pathway sequences, protection of any chemical functionalities that may not be compatible with the reaction conditions, and deprotection at a suitable point in the reaction sequence of the method, are 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, and examples thereof can be found in PGM Wuts and TW Greene, in Greene's book titled Protective Groups in Organic Synthesis (4 th This can be found in (ed.), John Wiley & Sons, NY (2006).

[0081] When an optically active form of the disclosed compound is required, it can be obtained by performing one of the procedures described herein using an optically active starting material (e.g., prepared by asymmetric induction of a preferred reaction step), or by decomposition of a mixture of stereoisomers of the compound or intermediate using standard procedures (such as chromatographic separation, recrystallization, or enzymatic degradation).

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

[0083] The synthesis schemes and specific examples described herein are illustrative and should not be construed as limiting the scope of this disclosure or the claims. Substitutions, modifications, and equivalents of the synthesis methods and specific examples are intended.

[0084] This disclosure also includes isotope-labeled compounds, which are identical to those enumerated in formulas (I) and (II), but in which one or more atoms are replaced by atoms having atomic weights or mass numbers different from those 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, for example, respectively. 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F, and 36 The isotope-labeled compounds of formulas (I) and (II) can generally be prepared by conventional techniques known to those skilled in the art, or by processes similar to those described in the attached examples, using appropriate isotope-labeled reagents instead of non-isotope-labeled reagents.

[0085] Instructions for use, system, and kit A method for detecting superoxide using compounds disclosed herein (for example, compounds of formula (I) and formula (II), including compounds of formula (I'), (I''), (Ia), (Ia'), (Ia''), (Ib), (Ib'), (Ib''), (II'), and (II'')) is disclosed herein. The compounds are partially, specifically: having an ortho- or para-hydroxyl group: [ka] This group is characterized by reacting with superoxide to produce a corresponding quinone compound, releasing uncaged luciferin or hydroxycyanobenzothiazole compounds, the latter of which are substrates for luciferin-utilizing luciferase enzymes that produce luminescence. Luciferases that produce luminescence using luciferin and hydroxycyanobenzothiazole compounds ("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 (Jamaican click beetle)), Renilla reniformis (sea pansies), and several bacteria (e.g., Xenorhabdus luminescens and Vibrio spp.).

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

[0087] While not wishing to be bound by theory, the compound may react with superoxide in a manner similar to that of hydroquinone, deprotonation followed by single-electron oxidation, leading to the formation of a semiquinone intermediate, which undergoes further oxidation and hydrolysis to release uncaged luciferin or hydroxycyanobenzothiazole compounds and quinone-type byproducts. In the formula, R 1 However, it is -CN, and R 4a However, it is hydrogen, R 4bSuch a reaction of a compound of formula (I), where hydroxyl is present, is shown in Scheme 1. In some embodiments, a compound of formula (II) is a biscarbamate linker (i.e., Z is of formula (II)). [ka] It may include (when it is the group of), 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-sacrifice of the biscarbamate linker and the release of uncaged luciferin or hydroxycyanobenzothiazole and quinone-type byproducts from the reactive moiety. In the formula, R 1 but, [ka] And R 4a However, it is hydrogen, n is 1, and R 3 However, it is hydroxyl, and R 4b However, such a reaction of the compound of formula (II), which is hydroxyl, is shown in Scheme 2. [ka]

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

[0089] Accordingly, 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 luciferin-utilizing luciferase if luciferin-utilizing luciferase is not yet present in the sample, and detecting luminescence in the sample.

[0090] The method includes the step of contacting a sample with a compound of formula (I) or formula (II). The compound of formula (I) or formula (II) may be part of a solution that may contain other components, such as a solvent, buffer, salt, detergent, or additive. For example, the compound of formula (I) or formula (II) may 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 the compound of formula (I) or formula (II), and then incubating the sample for a certain period of time to allow the superoxide to react with the compound of formula (I) or formula (II). In some embodiments, this incubation step may be carried out over a period of about 1 minute to about 4 days, about 15 minutes to about 1 day, about 1 hour to about 12 hours, or any range in between. For example, in some embodiments, the incubation step can be carried out for approximately 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours.

[0091] In certain embodiments, the luciferin-utilizing luciferase is not already present in the sample, and therefore the method includes the step of contacting the sample with the luciferin-utilizing luciferase. In some embodiments, the luciferin-utilizing luciferase is firefly luciferase or click mushroom luciferase.

[0092] When luciferin-utilizing luciferase is brought into contact with a sample, it may be included as part of the luciferase reaction mixture. The "luciferase reaction mixture" contains the luciferin-utilizing luciferase enzyme and other materials that enable the luciferase enzyme to generate a light signal. The materials required to generate the light signal, as well as the specific concentrations and / or amounts of the required materials, will vary depending on the luciferase enzyme being used. Generally, for beetle luciferase, additional materials include ATP and magnesium (Mg) such as magnesium sulfate. 2+)It can contain salts. In some embodiments, other materials can be added to a solution containing a buffer for maintaining the reaction at an appropriate pH, additives such as PRIONEX or bovine serum albumin (BSA) useful for maintaining luciferase activity, a reducing agent, a detergent, an esterase, a salt, an amino acid (e.g., D-cysteine), etc. An exemplary luciferase reaction mixture contains firefly 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 can contain cells that express the luciferin-utilizing luciferase enzyme.

[0094] In some embodiments of the above method, the method further includes contacting the sample with another compound, e.g., a candidate drug compound, or any compound that would be useful for 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) is added.

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

[0096] In any of the embodiments described above, 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, luciferin release into the culture medium can be monitored by taking a small amount of culture medium from the sample and detecting the luminescence in the removed medium, rather than directly detecting the luminescence in the wells of a plate (e.g., a 96-well plate) using a plate reader. Such a method can provide dynamic information regarding superoxide generation.

[0098] This disclosure further provides systems or kits comprising the compounds described herein (i.e., the compound of formula (I) or a salt thereof, or the compound of formula (II) or a salt thereof). The systems or kits comprise the compounds alone or in a solvent such as water, DMSO, or a buffer. When the compounds are provided alone, the systems or kits may further comprise a solvent capable of dissolving the compounds. The systems or kits may further comprise one or more reagents used to carry out an assay for detecting superoxide in a sample, e.g., the reagents described above. In some embodiments, the kits further comprise a luciferin-utilizing luciferase enzyme or a nucleotide sequence encoding a luciferin-utilizing luciferase enzyme, e.g., those described herein.

[0099] A system or kit may further include at least one of a container and instructions. For example, the components of a system or kit may be supplied in any type of container. For example, a sealed glass ampoule may contain lyophilized luciferase or buffer packaged under a neutral, non-reactive gas such as nitrogen. The ampoule may consist of any suitable material, e.g., glass, organic polymers such as polycarbonate, polystyrene, ceramics, metals, or any other material typically used to hold reagents. Another example of a suitable container is a simple bottle that may be manufactured from a substance similar to that of an ampoule. Other containers include test tubes, vials, flasks, bottles, syringes, etc. Containers may have a sterile access port, such as a bottle with a stopper that can be pierced by a subcutaneous needle. Other containers may have two compartments separated by an easily removable membrane that allows the components to be mixed after removal. The removable membrane may be glass, plastic, rubber, etc.

[0100] The kit may also be supplied with instructional materials. These instructions may be printed on paper or other substrates and / or supplied on electronically readable media such as floppy disks, CD-ROMs, DVD-ROMs, Zip disks, videotapes, audiotapes, etc. Detailed instructions may not be physically attached to the kit; instead, users may be directed to an internet website, or the instructions may be supplied via email.

[0101] The following examples further illustrate aspects of this disclosure, but should not be construed as limiting its scope in any way. [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 dimethyl sulfoxide, ES is electrospray, ƒ is ethyl acetate, h is time, HATU is 3-oxide hexafluorophosphate 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium, HPLC is high-performance liquid chromatography, LCMS is liquid chromatography-mass spectrometry, MeCN is acetonitrile, MeOH is methanol, RB is round-bottom, and tBuXPhos-Pd G3 is methanesulfonic acid [(2-di-tert-butylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)-2-(2'-amino-1,1'-biphenyl)]palladium(II), TFA is trifluoroacetic acid, Tf2O is trifluoromethanesulfonic anhydride, and THF is tetrahydrofuran.

[0103] Example 1: Compound Synthesis Intermediate 1: 2-cyanobenzo[d]thiazole-6-yl trifluoromethanesulfonic acid [ka] 6-hydroxybenzo[d]thiazole-2-carbonitrile (3.00 g, 17.0 mmol) and DCM (80 mL) were added to a 250 mL RB flask. The mixture was stirred. Triethylamine (3.31 mL, 25.5 mmol) was added to the mixture, followed by the dropwise addition of Tf2O (3.72 mL, 22.1 mmol) over 2 minutes. After 10 minutes, the mixture was concentrated on Celite and purified by silica gel chromatography using 0-50% RINKAN in heptane to obtain intermediate 1: 2-cyanobenzo[d]thiazole-6-yl trifluoromethanesulfonic acid. LCMS(C9H3F3N2O3S2)(ES,m / z)309[M+H] + .

[0104] Intermediate 2: 2-cyano-5-fluorobenzo[d]thiazole-6-yl trifluoromethanesulfonic acid [ka] Intermediate 2 was prepared using a method similar to that used for intermediate 1. LCMS(C9H2F4N2O3S2)(ES,m / z)327[M+H] + .

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

[0106] Step 2: 2-(3,4-dihydroxyphenoxy)tert-butyl acetate [ka] To a 20 mL vial, 2-(4-formyl-3-hydroxyphenoxy)acetic acid tert-butyl (100 mg, 0.396 mmol), THF (1 mL), and water (1 mL) were added. The solution was stirred and sparged with nitrogen for 1 minute. Sodium percarbonate (124 mg, 0.396 mmol) was added to the vial. The mixture was stirred for 30 minutes. The mixture was quenched with AcOH (0.2 mL). The mixture was extracted with DCM (5 mL). The organic layer was collected by a phase separator. The solvent was evaporated, and the residue was purified by silica gel chromatography using 0-70% EtOAc in heptane as the eluent to obtain 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 [Chemical formula] To a 100 mL round-bottom flask, 3,4-dihydroxybenzaldehyde (1.00 g, 7.24 mmol), a 4 M solution of NaOH in MeOH (5 mL), and DMA (10 mL) were added. To the stirred mixture, tert-butyl 2-bromoacetate (1.17 mL, 7.96 mmol) was added as a toluene solution (5 mL). After 30 seconds, the mixture was quenched with 2 M HCl (about 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 and filtered, and the solvent was evaporated. The residue was purified by silica gel chromatography using 0-60% EtOAc in heptane to obtain tert-butyl 2-(5-formyl-2-hydroxyphenoxy)acetate. LCMS (C 13 H 16 O5)(ES, m / z) 253 [M+H] + .

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

[0109] Intermediate 5: 2-(2,5-dihydroxy-3-methoxyphenoxy) acetate tert-butyl Step 1: 2-(5-formyl-2-hydroxy-3-methoxyphenoxy) tert-butyl acetate [ka] 3,4-dihydroxy-5-methoxybenzaldehyde (100 mg, 0.595 mmol) and DMF (2.5 mL) were added to a 20 mL vial. The mixture was sparged with nitrogen for 1 minute. Sodium hydride (60% by weight, 71.4 mg, 1.78 mmol) was added to the stirred mixture. The mixture was stirred for 15 minutes. 2-tert-butyl bromoacetate (0.095 mL, 0.654 mmol) was added to the mixture all at once. After 15 minutes, 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 × 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% ethyl acetate in heptane to obtain tert-butyl 2-(5-formyl-2-hydroxy-3-methoxyphenoxy)acetate. LCMS(C 14 H 18 O6)(ES,m / z)281[MH] - .

[0110] Step 2: 2-(2,5-dihydroxy-3-methoxyphenoxy) tert-butyl acetate [ka] 80.3 mg, 0.285 mmol of 2-(5-formyl-2-hydroxy-3-methoxyphenoxy) acetate tert-butyl, 1 mL of THF, and 1 mL of water were added to a 20 mL vial. The solution was stirred and sparged with nitrogen for 1 minute. 89.3 mg, 0.285 mmol of sodium percarbonate was added to the vial. The mixture was stirred under nitrogen for 2 hours. 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% ethyl phosphate in heptane as the eluent to obtain intermediate 5 (2-(2,5-dihydroxyphenoxy-3-methoxyphenoxy) acetate tert-butyl). 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] 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) were added to a 100 mL RB flask. The mixture was stirred and heated at 55 °C for 2 hours. The mixture was cooled to room temperature, diluted in siRNA (70 mL), and filtered through Celite. The solvent was evaporated, and the residue was purified by silica gel chromatography using 0-50% siRNA in heptane as the eluent. The residue from the evaporated fraction was ground in heptane to obtain 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] 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) were added to a 100 mL flask. The mixture was stirred at room temperature for 4 hours. The mixture was diluted with DCM (20 mL) and MeOH (20 mL). The mixture was filtered. The filtrate was concentrated. K2CO3 (1.73 g, 12.6 mmol) and MeOH (15 mL) were added to the residue. The mixture was stirred for 10 minutes. The mixture was diluted with toluene (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: Ethane-1,2-diylbis(methylcarbamate)3,4-bis(benzyloxy)phenyl tert-butyl [ka] Intermediate 6 (769 mg, 2.51 mmol), THF (10 mL), and DIPEA (1.75 mL, 10.0 mmol) were added to a 20 mL vial. This mixture was added dropwise over 1 minute to a stirred solution of triphosgene (372 mg, 1.26 mmol) in THF (5 mL). After 5 minutes, methyl(2-(methylamino)ethyl)carbamate tert-butyl (0.614 mL, 3.26 mmol) was added to the mixture. After 20 minutes, the mixture was adsorbed onto Celite and purified by silica gel chromatography using 0-100% ethyl ethyl ester in heptane to obtain ethane-1,2-diylbis(methylcarbamate)3,4-bis(benzyloxy)phenyl tert-butyl. LCMS(C 30 H 36N2O6)(ES,m / z)543[M+Na + .

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

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

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

[0117] Step 2: 4-((methyl(2-(methylamino)ethyl)carbamoyl)oxy)-1,2-phenylene diacetic acid 2,2,2-trifluoroacetate diacetic acid [ka] 57.0 mg, 0.134 mmol of 4-(((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)-(methyl)carbamoyl)oxy)-1,2-phenylene (20 mL) diacetic acid and TFA (2 mL) were added to a 20 mL vial. The mixture was stirred for 10 minutes. The solvent was evaporated to obtain intermediate 9 (2,2,2-trifluoroacetic acid 4-((methyl(2-(methylamino)ethyl)carbamoyl)oxy)-1,2-phenylene). 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]thiazole-6-yl [ka] 6-hydroxybenzo[d]thiazole-2-carbonitrile (19.0 mg, 0.108 mmol), THF (1.5 mL), and DIPEA (0.075 mL, 0.43 mmol) were added to a 20 mL vial. The mixture was added dropwise over 1 minute to a stirred solution of triphosgene (12.8 mg, 0.0431 mmol) in THF (1 mL). After 5 minutes, intermediate 8 (33.7 mg, 0.140 mmol) in THF (1 mL) was added to the mixture. After 20 minutes, the mixture was adsorbed onto Celite and purified by silica gel chromatography using 0-100% siRNA in heptane as the eluate to obtain intermediate 10 (3,4-dihydroxyphenyl)ethane-1,2-diylbis(methylcarbamate)2-cyanobenzo[d]thiazole-6-yl. LCMS(C 20 H 18 N4O6S)(ES,m / z)443[M+H] + .

[0119] Intermediate 11: 2,2,2-trifluoroacetic acid bromide (6-(((3,4-diacetoxyphenoxy)carbonyl)(2-(methylamino)ethyl)amino)hexyl)triphenylphosphonium Step 1: Bromide (6-((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)amino)hexyl)triphenylphosphonium [ka] (6-bromohexyl)triphenylphosphonium bromide (1.00 g, 1.98 mmol) and DMF (10 mL) were added to a 20 mL vial. To this mixture, tert-butyl (2-aminoethyl)(methyl)carbamate (1.72 g, 9.88 mmol) was added. The mixture was stirred and heated at 55°C for 4 hours. 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 the eluent. The residue was further purified by pulverizing twice with diethyl ether to obtain (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: Bromide (6-(((3,4-bis(benzyloxy)phenoxy)carbonyl)(2-((tert-butoxycarbonyl)(methyl)amino)ethyl)amino)hexyl)triphenylphosphonium [ka] Intermediate 6 (403 mg, 1.31 mmol), THF (5 mL), and DIPEA (0.931 mL, 5.26 mmol) were added to a 20 mL vial. The mixture was added dropwise over 1 minute to a stirred solution of triphosgene (195 mg, 0.657 mmol) in THF (2 mL). After 15 minutes, bromide (6-((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)amino)hexyl)triphenylphosphonium (788 mg, 1.31 mmol) in THF (2 mL) was added to the mixture. After 20 minutes, the mixture was adsorbed onto Celite and purified by silica gel chromatography using 0-10% MeOH in DCM as the eluate. The residue was dissolved in THF (20 mL). The residual DIPEA-HCl salt was precipitated, removed by filtration, and washed with THF. The solvent in the filtrate was evaporated to obtain bromide (6-(((3,4-bis(benzyloxy)-phenoxy)carbonyl)(2-((tert-butoxycarbonyl)(methyl)amino)ethyl)amino)hexyl)-triphenyl-phosphonium. LCMS(C 53 H 60 N2O6P)(ES,m / z)851[M] + .

[0121] Step 3: Bromide (6-((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)((3,4-dihydroxyphenoxy)carbonyl)amino)hexyl)triphenylphosphonium [ka] To a 100 mL RB flask, (6-(((3,4-bis(benzyloxy)-phenoxy)carbonyl)(2-((tert-butoxycarbonyl)(methyl)amino)ethyl)amino)hexyl)triphenylphosphonium bromide (751 mg, 0.805 mmol), 10% Pd / C (600 mg, 0.564 mmol), ethyl phosphate (3 mL), meopropyl alcohol (3 mL), and acetic acid (1 mL) were added. The mixture was stirred under a hydrogen atmosphere. After 72 hours, the mixture was filtered through Celite, and the solvent was evaporated to obtain (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: Bromide (6-((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)((3,4-diacetoxyphenoxy)carbonyl)amino)hexyl)-triphenylphosphonium [ka] (6-((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)((3,4-dihydroxyphenoxy)carbonyl)amino)hexyl)-triphenylphosphonium bromide (590 mg, 785 mmol), anhydride acetate (2 mL), and pyridine (2 mL) were added to a 20 mL vial. The mixture was stirred at room temperature for 10 minutes. The solvent was evaporated, and the residue was evaporated three times with toluene. The residue was purified by silica gel chromatography using 0-10% MeOH in DCM as the eluent to obtain (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 Trifluoroacetic acid bromide (6-(((3,4-diacetoxyphenoxy)carbonyl)(2-(methylamino)ethyl)amino)hexyl)triphenylphosphonium [ka] (6-((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)((3,4-diacetoxyphenoxy)carbonyl)amino)hexyl)-triphenylphosphonium bromide (100 mg, 0.120 mmol) and TFA (2 mL) were added to a 20 mL vial. The mixture was stirred for 10 minutes. The solvent was evaporated to obtain intermediate 11 (2,2,2-trifluoroacetic acid bromide (6-(((3,4-diacetoxyphenoxy)carbonyl)(2-(methylamino)ethyl)amino)hexyl)-triphenylphosphonium). 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] 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) were added to a 20 mL vial. The mixture was sparged with nitrogen for 1 minute. The mixture was stirred and heated at 120 °C for 2 hours. The mixture was diluted in RINKAN (5 mL) and filtered. The solvent of the filtrate was evaporated, and the residue was purified by silica gel chromatography using 0-80% RINKAN in heptane to obtain compound 1 (6-(4-hydroxy-3,5-dimethoxyphenoxy)benzo[d]thiazole-2-carbonitride). 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 phenol using a method similar to that used for the preparation of compound 1. [Table 1]

[0126] Compound 6: Bromide (6-(2-(5-((2-cyanobenzo[d]thiazole-6-yl)oxy)-2-hydroxy-3-methoxyphenoxy)acetamide)hexyl)triphenylphosphonium Step 1: 2-(5-((2-cyanobenzo[d]thiazole-6-yl)oxy)-2-hydroxy-3-methoxyphenoxy)tert-butyl acetate [ka] 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) were added to a 20 mL vial. The mixture was sparged with nitrogen for 1 minute. The mixture was stirred and heated at 100 °C for 1 hour. The mixture was adsorbed onto Celite and purified by silica gel chromatography using 0-60% siRNA in heptane to obtain tert-butyl 2-(5-((2-cyanobenzo[d]thiazole-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]thiazole-6-yl)oxy)-2-hydroxy-3-methoxyphenoxy)acetic acid [ka] 2-(5-((2-cyanobenzo[d]thiazole-6-yl)oxy)-2-hydroxy-3-methoxyphenoxy)acetic acid tert-butyl (42.5 mg, 0.0992 mmol) and TFA (1 mL) were added to a 20 mL vial. The mixture was stirred for 30 minutes. The solvent was evaporated to obtain 2-(5-((2-cyanobenzo[d]thiazole-6-yl)oxy)-2-hydroxy-3-methoxyphenoxy)acetic acid. LCMS(C 17 H 12 N2O6S)(ES,m / z)373[M+H] + .

[0128] Step 3: Bromide (6-(2-(5-((2-cyanobenzo[d]thiazole-6-yl)oxy)-2-hydroxy-3-methoxyphenoxy)acetamide)hexyl)triphenylphosphonium [ka] 2-(5-((2-cyanobenzo[d]thiazole-6-yl)oxy)-2-hydroxy-3-methoxyphenoxy) tert-butyl acetate (36.9 mg, 0.0991 mmol), bromide (6-aminohexyl)triphenylphosphonium hydrochloride (56.9 mg, 0.119 mmol), DMF (1 mL), and DIPEA (0.052 mL, 0.30 mmol) were added to a 20 mL vial. The mixture was stirred. HATU (45.2 mg, 0.119 mmol) was added to the mixture. After 1 hour, the mixture was purified by reverse-phase HPLC (MeCN / water containing 0.1% TFA) to obtain compound 6 (bromide (6-(2-(5-((2-cyanobenzo[d]thiazole-6-yl)oxy)-2-hydroxy-3-methoxyphenoxy)acetamide)hexyl)-triphenylphosphonium). 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 using a method similar to that used for the preparation of compound 6. [Table 2]

[0130] Compound 10: (S)-2-(6-(4-hydroxy-3,5-dimethoxyphenoxy)benzo[d]thiazole-2-yl)-4,5-dihydrothiazole-4-carboxylic acid [ka] Compound 1 and DMF (2 mL) were added to a 20 mL vial. The mixture was stirred. A solution of D-cysteine ​​hydrochloride in 0.5 M aqueous phosphate buffer pH 8 buffer (2 mL) was added to the mixture. The mixture was stirred for 30 minutes. The mixture was purified by reverse-phase HPLC (MeCN / water containing 0.1% TFA) to obtain compound 10 ((S)-2-(6-(4-hydroxy-3,5-dimethoxyphenoxy)benzo[d]thiazole-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 ​​using a method similar to that used for preparing compound 10. [Table 3-1] [Table 3-2]

[0132] Compound 16: Bromide (S)-(6-(2-(2-hydroxy-3-methoxy-5-((2-(4-(methoxycarbonyl)-4,5-dihydrothiazole-2-yl)benzo[d]thiazole-6-yl)oxy)phenoxy)acetamide)hexyl)triphenylphosphonium [ka] A 4 mL vial containing compound 6 (bromide (6-(2-(5-((2-cyanobenzo[d]thiazole-6-yl)oxy)-2-hydroxy-3-methoxyphenoxy)acetamide)hexyl)triphenylphosphonium) (15.8 mg, 0.0198 mmol) was mixed with DMF (1 mL). The mixture was stirred. A solution of methyl D-cysteine ​​(3.2 mg, 0.024 mmol) in 0.5 M phosphate buffer (0.5 mL) at pH 8 was added to the mixture. After 30 minutes, the mixture was purified by reverse-phase HPLC (MeCN / water containing 0.1% TFA) to obtain compound 16 (bromide (S)-(6-(2-(2-hydroxy-3-methoxy-5-((2-(4-(methoxycarbonyl)-4,5-dihydrothiazole-2-yl)benzo[d]thiazole-6-yl)oxy)phenoxy)acetamide)hexyl)triphenylphosphonium). 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]thiazole-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 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, which was used directly in the next step.

[0134] Step 2: 6-Fluorobenzo[d]thiazole-2-carbonitri [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 mixture was heated at 90°C for 1 hour. 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 using 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-carbonitri [ka] 4-Methoxyphenol (60 mg, 0.48 mmol) was dissolved in an equimolar aqueous solution of potassium hydroxide. Water was removed by freeze-drying. 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 and 90 W for 10 minutes (CEM Discovery Synthesizer). The reaction mixture was then purified by reverse-phase HPLC to obtain 6-(4-methoxyphenoxy)benzo[d]thiazole-2-carbonitrile.

[0136] Step 4: (S)-2-(6-(4-hydroxyphenoxy)benzo[d]thiazole-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 at 160°C for 20 minutes. 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 product pH to 8. The reaction product was then stirred for 20 minutes and purified by reverse-phase HPLC to obtain compound 17((S)-2-(6-(4-hydroxyphenoxy)benzo[d]thiazole-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]thiazole-2-yl)-4,5-dihydrothiazole-4-carboxylic acid [ka] Intermediate 10 (21.0 mg, 0.0475 mmol) and DMF (2 mL) were added to a 20 mL vial. The mixture was stirred. D-cysteine ​​(6.9 mg, 0.057 mmol) in pH 8 buffer (2 mL) was added to this mixture. The mixture was stirred for 2 hours. The mixture was purified by reverse-phase HPLC (MeCN / water containing 0.1% TFA) to obtain compound 18 ((S)-2-(6-(((2-(((3,4-dihydroxyphenoxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)benzo[d]thiazole-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]thiazole-2-yl)-4,5-dihydrothiazole-4-carboxylate methyl [ka] Intermediate 10 (27.6 mg, 0.0475 mmol) and DMF (2 mL) were added to a 20 mL vial. The mixture was stirred. D-cysteine ​​methyl ester (10.1 mg, 0.749 mmol) in pH 8 buffer (2 mL) was added to the mixture. The mixture was stirred for 2 hours. The mixture was purified by reverse-phase HPLC (MeCN / water containing 0.1% TFA) to obtain compound 19 ((S)-2-(6-(((2-(((3,4-dihydroxyphenoxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)benzo[d]thiazole-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: Diacetate (S)-4-(((2-((((2-(4-(methoxycarbonyl)-4,5-dihydrothiazole-2-yl)benzo[d]thiazole-6-yl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-1,2-phenylene Step 1: 4-(((2-((((2-cyanobenzo[d]thiazole-6-yl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-1,2-phenylene diacetate [ka] 6-hydroxybenzo[d]thiazole-2-carbonitrile (18.0 mg, 0.102 mmol), THF (1.5 mL), and DIPEA (0.073 mL, 0.41 mmol) were added to a 20 mL vial. The mixture was added dropwise over 1 minute to a stirred solution of triphosgene (12.8 mg, 0.0431 mmol) in THF (1 mL). After 5 minutes, intermediate 9 (43.1 mg, 0.132 mmol) in THF (1 mL) was added to the mixture. After 20 minutes, the mixture was adsorbed onto Celite and purified by silica gel chromatography using 0-100% siRNA in heptane as the eluate to obtain 4-(((2-((((2-cyanobenzo[d]thiazole-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: Diacetate (S)-4-(((2-((((2-(4-(methoxycarbonyl)-4,5-dihydrothiazole-2-yl)benzo[d]thiazole-6-yl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-1,2-phenylene [ka] Diacetate-(((2-((((2-cyanobenzo[d]thiazole-6-yl)oxy)carbonyl)-(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-1,2-phenylene (38.4 mg, 0.0729 mmol) and DMF (2 mL) were added to a 20 mL vial. The mixture was stirred. D-cysteine ​​methyl ester (11.8 mg, 0.875 mmol) in pH 8 buffer (2 mL) was added to this mixture. The mixture was stirred for 2 hours. The mixture was purified by reverse-phase HPLC (MeCN / water containing 0.1% TFA) to obtain compound 20 ((S) diacetic acid-4-(((2-((((2-(4-(methoxycarbonyl)-4,5-dihydrothiazole-2-yl)benzo[d]thiazole-6-yl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-1,2-phenylene). 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: Bromide (6-((2-((((2-cyanobenzo[d]thiazole-6-yl)oxy)carbonyl)(methyl)amino)ethyl)-((3,4-diacetoxyphenoxy)carbonyl)amino)hexyl)triphenylphosphonium [ka] 6-hydroxybenzo[d]thiazole-2-carbonitrile (20.0 mg, 0.114 mmol), THF (1.5 mL), and DIPEA (0.100 mL, 0.568 mmol) were added to a 20 mL vial. The mixture was added dropwise over 1 minute to a stirred solution of triphosgene (16.8 mg, 0.0568 mmol) in THF (1 mL). After 5 minutes, intermediate 11 (87.7 mg, 0.119 mmol) in THF (1 mL) was added to the mixture. After 20 minutes, the mixture was concentrated and purified by reverse-phase HPLC (MeCN / water containing 0.1% TFA) to obtain compound 21 (bromide (6-((2-((((2-cyanobenzo[d]thiazole-6-yl)oxy)carbonyl)(methyl)amino)ethyl)-((3,4-diacetoxyphenoxy)carbonyl)amino)hexyl)triphenylphosphonium). 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: Bromide (S)-(6-((((2-(4-carboxy-4,5-dihydrothiazole-2-yl)benzo[d]thiazole-6-yl)oxy)carbonyl)(2-(((3,4-diacetoxyphenoxy)carbonyl)(methyl)amino)ethyl)amino)hexyl)triphenylphosphonium [ka] 1.5 mL of DMF was added to an 8 mL vial containing compound 21 (10.0 mg, 0.0107 mmol). The mixture was stirred. A solution of D-cysteine ​​(1.7 mg, 0.014 mmol) in pH 8 buffer (0.5 mL) was added to this mixture. The mixture was purified by reverse-phase HPLC (MeCN / water containing 0.5% TFA) to obtain compound 22 (bromide(S)-(6-((((2-(4-carboxy-4,5-dihydrothiazole-2-yl)benzo[d]thiazole-6-yl)oxy)carbonyl)(2-(((3,4-diacetoxyphenoxy)carbonyl)(methyl)amino)ethyl)-amino)hexyl)triphenylphosphonium). 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: Bromide (S)-(6-((2-(((3,4-diacetoxyphenoxy)carbonyl)(methyl)amino)ethyl)(((2-(4-(methoxycarbonyl)-4,5-dihydrothiazole-2-yl)benzo[d]thiazole-6-yl)oxy)carbonyl)amino)hexyl)triphenylphosphonium [ka] 1.5 mL of DMF was added to an 8 mL vial containing compound 21 (19.8 mg, 0.0211 mmol). The mixture was stirred. A solution of D-cysteine ​​methyl ester (1.7 mg, 0.014 mmol) in pH 8 buffer (0.5 mL) was added to this mixture. The mixture was purified by reverse-phase HPLC (MeCN / water containing 0.5% TFA) to obtain compound 23 (bromide ((S)-(6-((2-(((3,4-diacetoxyphenoxy)carbonyl)(methyl)amino)ethyl)(((2-(4-(methoxycarbonyl)-4,5-dihydrothiazole-2-yl)benzo[d]thiazole-6-yl)oxy)carbonyl)amino)hexyl)triphenylphosphonium). 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-carbonitride), was diluted to 25 μM in PBS in the wells of a 96-well white-walled assay plate. Then, the sources of reactive oxygen species and nitrogen species were diluted in PBS and added to the assay plate. The plate was mixed for 5 minutes using an orbital plate shaker, followed by incubation at room temperature for 30 minutes under light protection. For luciferin detection, the luciferin-utilizing luciferase enzyme, UltraGlo (Promega Corporation), was prepared by mixing luciferin detection reagent (Promega), reconstituted 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, with the blank containing only PBS, 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 decreased in the presence of superoxide dismutase, confirming that superoxide is the source of the signal. In the presence of hydrogen peroxide, rose bengal (which produces singlet oxygen), sodium nitrite, or NONOate, the signal was barely or completely undetectable.

[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 following day, superoxide probe, compound 1 (6-(4-hydroxy-3,5-dimethoxyphenoxy)benzo[d]thiazole-2-carbonitrile), was added to the cells in the culture medium at a final concentration of 12.5 μM. Subsequently, dimethoxynaphthoquinone (DMNQ, 50 μM) or antimycin A (10 μM), diluted in the 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, the luciferin-utilizing luciferase enzyme, UltraGlo (Promega Corporation), was prepared by mixing luciferin detection reagent (Promega), reconstituted 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 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 superoxide formation.

[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 typically found in the liver and jejunum and plays a crucial role in purine catabolism. During severe liver injury, xanthine oxidase is released into the bloodstream and can be used as a marker of liver damage. Herein lies an example of measuring xanthine oxidase activity using the proluciferin compound 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 a well 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 incubation at 37°C for 10, 20, 30, and 60 minutes, 75 μl of luciferin detection reagent containing esterase was added to the sample, and the luminescence was measured after an additional 20 minutes of incubation at room temperature.

[0147] The increase in the 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 demonstrate that superoxide produced by xanthine oxidase interacts with proluciferin compounds to release luciferin, and that this can be used to measure the activity of superoxide-producing enzymes or to screen for inhibitors.

[0148] Example 5: Detection of superoxide production by macrophages Superoxide production by macrophages plays a crucial role in immune responses and inflammation. Superoxide is produced by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (known as the NOX enzyme) and is induced by PMA (13-12-myristate 4β-phorbol acetate). Here, we present 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 begin 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 superoxide production, 50 μl of 50 μM compound 18 (Figure 6A) or 100 μM compound 20 (Figure 6B) was added to the cells. Cells were treated at 37°C for 2 hours, and luciferin production was measured by adding an equal volume of luciferin detection reagent containing esterase. Luminescence was read after incubation at room temperature for 20 minutes.

[0149] Both compounds showed higher luminescence signals in the presence of cells compared to the control in culture medium alone. The data in Figures 6A–6B show that the luminescence signal was significantly increased with PMA treatment, illustrating the usefulness of the present invention's method 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 overnight in DMEM medium containing 10% FBS at a rate of 30,000 cells per well. The following day, the medium was removed and the cells were washed with PBS. 100 μl of PBS containing 5 mM glucose, 1 mM MgCl2, 0.5 mM CaCl2, 0.05% BSA, and 50 μM compound 20 was added to the cells in the presence or absence of 20 μM PMA. The cells were brought back to 37°C in a tissue culture incubator. At different time points (10, 20, 30, and 60 minutes), 10 μl was taken from the sample and mixed with 10 μl of luciferin detection reagent containing esterase, and the luminescence was read after incubation at room temperature for 20 minutes. The data in Figure 7 show that a time-dependent increase in the signal was detected in cells treated with PMA, which showed increased superoxide production.

[0151] All references cited herein, including publications, patent applications, and patents, are incorporated herein by reference to the same extent as they are incorporated herein by reference, with each reference indicated to be incorporated individually and specifically.

[0152] In the context of the description of this invention (in particular in the context of the following claims), the terms “a,” “an,” and “the,” as well as “at least one,” and similar referents, should be interpreted as encompassing both singular and plural forms, unless otherwise indicated herein or unless clearly contradicted by the context. Unless otherwise indicated herein or unless clearly contradicted by the context, the 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”), should be interpreted as meaning one item (A or B) selected from the enumerated items, or any combination of two or more enumerated items (A and B). The terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., “including, but not limited to.”) unless otherwise indicated herein. The enumeration of value ranges in this specification is intended, unless otherwise indicated herein, merely as a shorthand method for referring individually to each distinct value that falls within the range, and each distinct value is incorporated herein as if it were individually enumerated herein. All methods described herein may be performed in any preferred order unless otherwise indicated herein, or unless the difference would be clearly inconsistent with the context. Any and all examples provided herein, or the use of exemplary language (e.g., "etc."), are intended merely to better illustrate the invention and, unless otherwise requested, do not impose any limitation on the scope of the invention. Nothing expressed herein should be construed as indicating that an unclaimed element is essential to the practice of the invention.

[0153] Preferred embodiments of the Invention, including the best mode known to the inventors for carrying out the Invention, are described herein. Variations of these preferred embodiments may become apparent to those skilled in the art after reading the foregoing description. The inventors anticipate that those skilled in the art will use such variations appropriately, and the inventors intend that the Invention may be practiced in ways other than those specifically described herein. Accordingly, the Invention includes all modifications and equivalents of the subject matter enumerated in the claims appended herein, as permitted by applicable law. Furthermore, any combination of the above elements in all possible variations thereof is covered by the Invention unless otherwise shown herein or unless the alternative is clearly inconsistent with the context. Another aspect of the present invention may be as follows: [1] Compound of formula (II): [ka] or a salt thereof, in the formula, R 1 However, -CN and [ka] Selected from, R 2 However, hydrogen and halo are selected, n is 0, 1, 2, or 3, Each R 3 However, independently, C 1 ~C 4 Alkyl, C 1 ~C 4 Alkoxy, -OC(O)-C 1 ~C 4 Selected from alkyl, hydroxy, amino, and group-linker-X, where X is the targeting moiety. R 4a and R 4b One of them is hydroxy or -OC(O)-C 1 ~C 4 One is an alkyl group, and the other is a hydrogen group or a linker-X, where X is the targeting moiety. R 5 However, hydrogen and C 1 ~C 4 Selected from alkyl groups, Z is a combination or expression

change

change

change

change

change

[10] above, or a salt thereof, selected from hydrogen and methyl. 〔12〕R 2 A compound or salt thereof described in any one of the above items [1] to

[11] , wherein the compound is hydrogen. 〔13〕R 2 A compound or salt thereof described in any one of the above items [1] to

[11] , which is fluoro.

[14] The compound is a compound of formula (Ia):

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[13] , wherein is hydrogen or group-linker-X, where X is the targeting moiety.

[15] The compound is a compound of formula (Ib):

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[13] , wherein is hydrogen or group-linker-X, where X is the targeting moiety.

[16] n is 0, 1, or 2, and each R 3 However, independently, C 1 ~C 4 Alkyl, C 1 ~C 4 Alkoxy, -OC(O)-C 1 ~C 4 A compound selected from alkyl and hydroxy, as described in any one of items [1] to

[15] above, or a salt thereof.

[17] n is 0, 1, or 2, and each R 3 However, independently, C 1 ~C 4 Alkyl and C 1 ~C 4 A compound selected from alkoxys, as described in any one of items [1] to

[16] above, or a salt thereof.

[18] at least one R 3 A compound according to any one of the above [1] to

[15] , wherein the group-linker-X is a mitochondrial targeting moiety, or a salt thereof.

[19] The compound described in

[18] above, or a salt thereof, wherein X is the triphenylphosphonium moiety. 〔20〕

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[21] A method for detecting superoxide in a sample, The above sample is brought into contact with a compound or a salt thereof described in any one of items [1] to

[20] above, If luciferin-utilizing luciferase is not yet present in the sample, the sample is brought into contact with the luciferin-utilizing luciferase. The method comprising detecting the emission of light in the sample.

[22] The method according to

[21] , wherein the sample contains living cells.

[23] The method according to

[22] , wherein the cells express the luciferin-utilizing luciferase.

[24] The method according to

[21] , comprising adding the luciferin-utilizing luciferase to the sample.

[25] The method according to any one of the above

[21] to

[24] , wherein the luciferin-utilizing luciferase is firefly luciferase or click mushroom luciferase.

[26] A kit comprising a compound or a salt thereof as described in any one of the items [1] to

[20] above.

[27] The kit according to

[26] , further comprising a luciferin-utilizing luciferase enzyme or a nucleotide sequence encoding a luciferin-utilizing luciferase enzyme.

[28] The kit according to

[26] or

[27] , further comprising a buffer reagent.

Claims

1. Compound of formula (II): 【Chemistry 1】 or a salt thereof, in the formula, R 1 However, -CN and 【Chemistry 2】 Selected from, R 2 However, hydrogen and halo are selected, 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 It is an alkyl group, and the other is a hydrogen group or a linker-X, where X is the targeting moiety. R 5 However, hydrogen and C 1 ~C 4 Selected from alkyl groups, Z is a combination or expression 【Transformation 3】 It is the basis of, R 6 However, C 1 ~C 4 A compound or a salt thereof, selected from alkyl and group-linker-Y, where Y is the targeting moiety.

2. Z is, 【Chemistry 4】 The compound according to claim 1, or a salt thereof, which is the base of the compound.

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

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

5. The compound according to claim 4, or a salt thereof, wherein Y is a triphenylphosphonium moiety.

6. n is 1, R 3 However, -OC(O)CH 3 A compound according to claim 1, or a salt thereof, selected from and hydroxyl.

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

8. The compound is a compound of formula (I): 【Transformation 5】 or a salt thereof, in the formula, R 1 However, -CN and 【Transformation 6】 Selected from, R 2 However, hydrogen and halo are selected, n is 0, 1, 2, or 3, Each R 3 However, independently, C 1 ~C 4 Alkyl, C 1 ~C 4 Selected from alkoxy, amino, and group-linker-X, where X is the targeting moiety. R 4a and R 4b One of them is hydroxyl, and the other is hydrogen or a group-linker-X, where X is the targeting moiety. R 5 However, hydrogen and C 1 ~C 4 A compound according to claim 1, or a salt thereof, selected from alkyl groups.

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

10. R 1 but, 【Transformation 7】 The compound according to claim 1, or a salt thereof.

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

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

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

14. The aforementioned compound is a compound of formula (Ia): 【Transformation 8】 or a salt thereof, in the formula R 4a The compound according to claim 1, or a salt thereof, wherein is hydrogen or a group-linker-X, where X is the targeted moiety.

15. The aforementioned compound is the compound of formula (Ib): 【Chemistry 9】 or a salt thereof, in the formula R 4b The compound according to claim 1, or a salt thereof, wherein is hydrogen or a group-linker-X, where X is the targeted moiety.

16. n is 0, 1, or 2, and each R 3 However, independently, C 1 ~C 4 Alkyl, C 1 ~C 4 Alkoxy, -OC(O)-C 1 ~C 4 A compound according to claim 1, or a salt thereof, selected from alkyl and hydroxyl.

17. n is 0, 1, or 2, and each R 3 However, independently, C 1 ~C 4 Alkyl and C 1 ~C 4 A compound according to claim 1, or a salt thereof, selected from alkoxys.

18. at least one R 3 The compound according to claim 1, or a salt thereof, wherein the group is a linker X, and X is a mitochondrial targeting moiety.

19. The compound according to claim 18, or a salt thereof, wherein X is a triphenylphosphonium moiety. 【Request Item 20】 【Chemistry 10-1】 【Chemistry 10-2】 A compound according to claim 1, selected from the group consisting of the following: or its salt.

21. A method for detecting superoxide in a sample, The above sample is brought into contact with the compound or salt thereof described in any one of claims 1 to 20, If luciferin-utilizing luciferase is not yet present in the sample, the sample is brought into contact with the luciferin-utilizing luciferase. The method comprising detecting the emission of light in the sample.

22. The method according to claim 21, wherein the sample includes living cells.

23. The method according to claim 22, wherein the cells express the luciferin-utilizing luciferase.

24. The method according to claim 21, comprising adding the luciferin-utilizing luciferase to the sample.

25. The method according to claim 21, wherein the luciferin-utilizing luciferase is firefly luciferase or click mushroom luciferase.

26. A kit comprising a compound according to any one of claims 1 to 20, or a salt thereof.

27. The kit according to claim 26, further comprising a luciferin-utilizing luciferase enzyme or a nucleotide sequence encoding a luciferin-utilizing luciferase enzyme.

28. The kit according to claim 26, further comprising a buffer reagent.

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