WO2014063033A2

WO2014063033A2 - Novel probes and targeting comounds for mitochondria

Info

Publication number: WO2014063033A2
Application number: PCT/US2013/065649
Authority: WO
Inventors: Lanrong Bi
Original assignee: Michigan Technological University
Priority date: 2012-10-19
Filing date: 2013-10-18
Publication date: 2014-04-24
Also published as: WO2014063033A3

Abstract

The present invention provides novel compounds which are fluorescent and target the mitochondria and methods of using them.

Description

NOVEL PROBES AND TARGETING COMOUNDS FOR MITOCHONDRIA

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Patent Application No. 61/716,260, filed on October 19, 2012, and United States Provisional Patent Application No. 61/778,602, filed March 13, 2013, the entire contents of which are hereby incorporated by reference.

BACKGROUND

[0002] Mitochondrial oxidative stress is implicated in aging and many disorders including neurodegenerative diseases, diabetes, stroke, ischemia/reperfusion injury, age- related macular degeneration and cancer. New tools for diagnosis and drug development are needed.

SUMMARY

[0003] In one embodiment, the invention provides A compound according to Formula (I):

(I)

wherein

A is a fluorophore;

W is a direct bond, -C(0)NH-, or

;

L is a linker;

n 1 or 2.

[0004] In one embodiment the invention provides a method of detecting, determining identifying oxidative stress in cells.

[0005] In one embodiment, the invention provides a method of detecting a disease disorder related to oxidative stress in a subject. [0006] In one embodiment, the invention provides a method of screening for an anti- oxidative stress agent.

[0007] In one embodiment, the invention provides a method of in vivo imaging of oxidative stress.

[0008] In one embodiment, the present invention provides a kit including a compound according to the invention.

[0009] Other aspects of the invention will become apparent by consideration of the detailed description and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 shows the chemical structure of compounds according to the invention.

[0011] FIG. 2 shows EPR spectra of MitoProbe I/II (100 μΜ) before (A, B) and after addition of Sodium L-ascorbate (ImM) (C, D).

[0012] FIG. 3 shows confocal laser-scanning fluorescent images of HeLa cells incubated with MitoProbe I (30 nM, green, 45min) or MitoProbe II (50nM, 45 min), and stained with MitoTracker (80nM, 45 min), and Hoechst 33242 (0.1 μΕ/ιηΕ, 30 min) and overlay images. Imaged with confocal laser scanning fluorescent microscope using 40X objective lens in non- FBS, non-phenol red media.

[0013] FIG. 4 shows ARPE-19 cells incubated with MitoProbe I (15nM)/II (25nM) for 45 min. after stimulated at varying concentrations of PMA (E-P) for 2h. Fluorescence and their corresponding DIC images are shown. Stimulation concentration of PMA were 0 μΜ (A, C), 0.13 μΜ (E, G), 0.19 μΜ (Ι,Κ), 0.26 μΜ (Μ,Ο). Images were obtained Zeiss Axiovert 200M with Apotome by using 20X objective lens. Graphs showed fluorescence intensity enhancement of images.

[0014] FIG. 5 shows the mitochondria morphology of ARPE-19 cells visualized by MitoProbes were classified into three categories: (A): tubular; (B) intermediate; and (C) fragmented.

[0015] FIG. 6 shows mitochondria undergoing distinct morphological changes under oxidative stress. MitoProbe I (30nM, 45 min., column 1) was incubated with ARPE-19 cells and counterstained with Hoechst 33342 (0.1 μΕ/ιηΕ, 30 min, column 2) and MitoTracker (30 nM, 45 min., column 3) and overlay images (column 4). ARPE-19 cells were incubated with varying concentrations of PMA 0 μΜ (row 1), 0.5 μΜ (row 2), 1 μΜ (row 3), 2 μΜ (row 4), 3 μΜ (row 5), 4 μΜ (row 6). All images were acquired with 40 X objective. [0016] FIG. 7 shows mitochondria undergoing distinct morphological changes under oxidative stress. MitoProbe II (30nM, 45 min., column 1) was incubated with ARPE-19 cells and counterstained with Hoechst 33342 (0.1 μΐνΓ ί, 30 min, column 2) and MitoTracker (30 nM, 45 min., column 3), and overlay images (column 4). ARPE-19 cells were incubated with varying concentrations of PMA 0 μΜ (row 1), 0.5 μΜ (row 2), 1 μΜ (row 3), 2 μΜ (row 4), 3 μΜ (row 5), 4 μΜ (row 6). All images were acquired with 40 X objective.

[0017] FIG. 8 shows MitoProbe I (30nM, green,45 min., column A), which was incubated in ARPE19 cells and counterstained with Hoechst 33342 (0.1 μΕ/ηιΕ, 30 min, column B) and MitoTracker (30 nM, 45 min., column C). Column D and E show overlay and DIC images. ARPE19 cells were incubated with varying concentrations of PMA (0 μΜ (row I), 0.5 μΜ (row II), 1 μΜ (row III), 2 μΜ (row IV), 3 μΜ (row V), 4 μΜ (row VI).

[0018] FIG. 9 shows MitoProbe II (30nM, green,45 min.,column A), which was incubated in ARPE19 cells and counterstained with Hoechst 33342 (0.1 μΕ/ηιΕ, 30 min, column B) and MitoTracker (30 nM, 45 min., column C). Columns D and E show overlay and DIC images, respectivelly. ARPE19 cells were incubated with varying concentrations of PMA (0 μΜ (row I), 0.5 μΜ (row II), 1 μΜ (row III), 2 μΜ (row IV), 3 μΜ (row V), 4 μΜ (row VI

[0019] FIG. 10 shows that exposure to low concentration of PMA induced pronounced but reversible morphology changes in the mitochondria of ARPE-19 cells. Images are representative confocal laser-scanning fluorescence images of ARPE-19 cells incubated with MitoProbe I (30 nM, 45 min., column 1) after treatment with 1 μΜ PMA (except row I ), and co-stained with Hoechst 33342 (0.1 μΕ/ηιΕ, 30 min, column 2), Mitotracker (30nM, 45 min, column 3) and overlay images (column 4). Cells were imaged after lh (row 2), 16h (row 3) and 24h (row 4). All images were acquired with 40 X objective.

[0020] FIG. 11 shows that exposure to low concentration of PMA induced pronounced but reversible morphology changes in the mitochondria of ARPE-19 cells. Images are representative confocal laser-scanning fluorescence images of ARPE-19 cells incubated with MitoProbe II (30 nM, 45 min., column 1) after treatment with 1 μΜ PMA (except row I ), and co-stained with Hoechst 33342 (0.1 μΕ/ηιΕ, 30 min, column 2), Mitotracker (30nM, 45 min, column 3), and overlay images (column 4). Cells were imaged after lh (row 2), 16h (row 3) and 24h (row 4). All images were acquired with 40 X objective.

[0021] FIG. 12 shows that exposure to low concentration of PMA induced pronounced but reversible morphology changes in the mitochondria of ARPE-19 cells. Images are representative LSM of ARPE- 19 cells incubated with MitoProbe I (30 nM) (45 min., column A) after treatment with 1 μΜ PMA (except row I), and co-stained with Hoechst 33342 (0.1 μΙ7ηιί, 30 min, B), Mitotracker (30nM, 45 min, C). Columns D and E show overlay and DIC images, respectively. Cells were imaged after lh (row II), 16h (row III) and 24h (row IV). Cells were kept in non-FBS media. Obtained with 40X objective lens.

[0022] FIG. 13 shows that exposure to low concentration of PMA induced pronounced but reversible morphology changes in the mitochondria of ARPE- 19 cells. Images are representative LSM of ARPE-19 cells incubated with MitoProbe II (30 nM) (45 min., column A) after treatment with 1 μΜ PMA (except row I), and co-stained with Hoechst 33342 (0.1 μΕ/mL, 30 min, B), Mitotracker (30nM, 45 min, C). Columns D and E show overlay and DIC images, respectively. Cells were imaged after lh (row II), 16h (row III) and 24h (row IV). Cells were kept in non-FBS media. Obtained with 60X objective lens.

[0023] FIG. 14 shows confocal laser-scanning fluorescence images of freshly frozen muscle tissue slices from the sham and limb ischemia/reperfusion group rats following incubation of MitoProbe I (80 nM)/II (90 nM) for 45min, respectively. MitoProbe I II fluorescence images are displayed in row 1, with nuclei counterstained by Hoechst 33342 and displayed in row 2, and overlay images (row 3). All images were acquired with 40 X objective.

[0024] FIG. 15 shows confocal laser-scanning fluorescence images of freshly frozen liver tissue slices from the sham and limb ischemia/reperfusion group rats following incubation of MitoProbe I (80 nM)/II (90 nM) for 45min, respectively. MitoProbe I/II fluorescence images are displayed in row 1 , with nuclei counterstained by Hoechst 33342 and displayed in row 2, and overlay images (row 3). All images were acquired with 40 X objective.

[0025] FIG. 16 shows confocal laser-scanning fluorescence images of freshly frozen kidney tissue slices from the sham and limb ischemia/reperfusion group rats following incubation of MitoProbe I (80 nM)/II (90 nM) for 45min, respectively. MitoProbe I/II fluorescence images are displayed in row 1, with nuclei counterstained by Hoechst 33342 and displayed in row 2, and overlay images (row 3). All images were acquired with 40 X objective.

[0026] FIG. 17 shows confocal laser-scanning fluorescence images of spinal cord of SOD 1 G93A mice following incubation of MitoProbe VII (250 nM) for 45min, respectively. MitoProbe I/II fluorescence images are displayed in row I, with nuclei counterstained by Hoechst 33342 and displayed in row ), and overlay images (row 3). All images were acquired with 40 X objective.

[0027] FIG. 18 shows confocal laser-scanning fluorescence images of skeletal muscles taken from the end-stage of SOD1G93A mice following incubation of MitoProbe I / II (250 nM) for lh, respectively. MitoProbe VII fluorescence images are displayed in column 1, with nuclei counterstained by Hoechst 33342 (column 2) and overlay images (column 3). All images were acquired with 40 X objective.

[0028] FIG. 19 shows confocal laser-scanning fluorescence images of freshly frozen colon/breast tumor tissue slices from the patients following incubation of MitoProbe I/II (1 iM) for lh, respectively. MitoProbe I/II fluorescence images are displayed in row 1, with nuclei counterstained by Hoechst 33342 and displayed in row 2, and overlay images (row 3). All images were acquired with 40 X objective.

[0029] FIG. 20 shows confocal laser-scanning fluorescence images of HeLa cells visualizing with MitoProbe I/II (0.5 μΜ) after fixation with formaldehyde and acetone treatment. All images were acquired with 40 X objective.

[0030] FIG. 21 shows the proposed acting mechanism of MitoProbe.

[0031] FIG. 22 shows the predicted lowest energy conformations of MitoProbe I/II.

[0032] FIG. 23 shows confocal laser-scanning fluorescent images of HeLa cells incubated with MitoProbe VI (30 nM, 45min), and stained with MitoTracker (80nM, 45 min), and Hoechst 33242 (0.1 μΙ7ιηί, 30 min) and overlay images. Imaged with confocal laser scanning fluorescent microscope using 60X objective lens in non-FBS, non-phenol red media.

[0033] FIG. 24 shows confocal laser-scanning fluorescent images of HeLa cells incubated with MitoProbe VII (30 nM, 45min), and stained with MitoTracker (80nM, 45 min), and Hoechst 33242 (0.1 μΕ/ιηΕ, 30 min) and overlay images. Imaged with confocal laser scanning fluorescent microscope using 60X objective lens in non-FBS, non-phenol red media.

[0034] FIG. 25 is a schematic showing the synthesis of MitoProbe I.

[0035] FIG. 26 is a schematic showing the synthesis of MitoProbe II.

[0036] FIG. 27 is a schematic showing the synthesis of Rhodamine B-PEG2-diTEMPO and Rhodamine B-PEG4-diTEMPO derviatives.

[0037] FIG. 28 is a schematic showing the synthesis of a Rhodamine B diTEMPO derivative. [0038] FIG. 29 is a schematic showing the synthesis of Rhodamine 101-based MitoProbe VI.

[0039] FIG. 30 is a schematic showing the synthesis of a Rhodamine 101 diTEMPO derivative (MitoProbe VII).

[0040] FIG. 31 is a schematic showing the synthesis of a Rhodamine 101-PEG2- diTEMPO derivative (MitoProbe VIII) and a Rhodamine 101-PEG4-diTEMPO derivative (MitoProbe IX).

[0041] FIG. 32 is a schematic showing the synthesis of MitoProbes 10 and 1 1.

[0042] FIG. 33 is a schematic showing the synthesis of MitoProbes 12 and 13.

[0043] FIG. 34 is a schematic showing the synthesis of MitoProbes 14 and 15.

[0044] FIG. 35 is a schematic showing the synthesis of MitoProbes 16 and 17.

[0045] FIG. 36 is a schematic showing the synthesis of MitoProbes 18 and 19.

DETAILED DESCRIPTION

[0046] Mitochondrial oxidative stress is implicated in aging and many diseases including neurodegenerative diseases, diabetes, cardiovascular disease, ischemia/reperfusion injury, age-related macular degeneration and cancer. Reactive oxygen species (ROS) production by mitochondria is critical for normal cell functioning; however, abnormal ROS production can have damaging side effects. Electron spin resonance (ESR) spectroscopy is frequently used for ROS detection, but cannot be used for real-time imaging of ROS at a single cell level. Fluorescent probes for ROS detection such as dihydrorhodamine are not preferentially localized in cells before oxidation and conventional mitochondrial dyes are limited by their instability in light. Thus, additional real-time probes at the single cell level are needed.

[0047] The present invention provides novel probes. In one embodiment, the compounds are real-time probes. Mitochondrial oxidative stress is suitable analyzed using fluorogenic spin probes that can be detected by both fluorescence and ESR spectroscopy. In the compounds of the present invention, the combination of both nitroxide and fluorescent moieties in one molecule yields a non-fluorescent compound. Without wishing to be bound by theory, this is due to the quenching of the excited singlet state of aromatic fluorescent compound by the nitroxide moiety. Suitably, free radicals are able to react efficiently with the nitroxide moiety of the compounds of the present invention leading to a diamagnetic compound, thereby eliminating intramolecular quenching and resulting in enhanced fluorescence. The compounds of the present invention suitably contain cationic residues, and are able to pass through outer membrane. The inner membrane of the mitochrondria is much more hydrophobic than the plasma membrane; thus, in order to allow partitioning of the fluorescent probe through the lipid bilayer, the compounds of the present invention suitably have a high degree of lipophilicity.

[0048] In one embodiment, the invention provides compounds of Formula (I):

(I)

wherein

A is a fluorophore;

W is a direct bond, -C(0)NH-, or

L is a linker;

n 1 or 2.

[0049] The present invention further provides methods of using the compounds of Formula (I) and kits containing compounds of Formula (I).

Definitions

[0050] The term "acyl," as used herein, refers to an alkylcarbonyl, cycloalkylcarbonyl, heterocyclylcarbonyl, arylcarbonyl or heteroarylcarbonyl substituent, any of which may be further substituted (e.g., with one or more substituents).

[0051] The term "alkyl," as used herein, refers to a straight or branched saturated hydrocarbon chain. Alkyl groups may include a specified number of carbon atoms. For example, C1-C12 alkyl indicates that the alkyl group may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 carbon atoms. An alkyl group may be, e.g., a C1-C12 alkyl group, a C1-C1₀ alkyl group, a Ci-Cs alkyl group, a C1-C6 alkyl group or a C1-C4 alkyl group. For example, exemplary Ci- C₄ alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec -butyl, isobutyl and tert-butyl groups. An alkyl group may be optionally substituted with one or more substituents.

[0052] The term "alkenyl," as used herein, refers to a straight or branched hydrocarbon chain having one or more double bonds. Alkenyl groups may include a specified number of carbon atoms. For example, C2-C12 alkenyl indicates that the alkenyl group may have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. An alkenyl group may be, e.g., a C2-C12 alkenyl group, a C2-C1₀ alkenyl group, a C2-C₈ alkenyl group, a C2-C6 alkenyl group or a C2-C4 alkenyl group. Examples of alkenyl groups include but are not limited to allyl, propenyl, 2- butenyl, 3-hexenyl and 3-octenyl groups. One of the double bond carbons may optionally be the point of attachment of the alkenyl substituent. An alkenyl group may be optionally substituted with one or more substituents.

[0053] The term "alkynyl," as used herein, refers to a straight or branched hydrocarbon chain having one or more triple bonds. Alkynyl groups may include a specified number of carbon atoms. For example, C2-C12 alkynyl indicates that the alkynyl group may have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. An alkynyl group may be, e.g., a C2-C12 alkynyl group, a C2-C1₀ alkynyl group, a C2-C₈ alkynyl group, a C2-C6 alkynyl group or a C2-C4 alkynyl group. Examples of alkynyl groups include but are not limited to ethynyl, propargyl, and 3-hexynyl. One of the triple bond carbons may optionally be the point of attachment of the alkynyl substituent. An alkynyl group may be optionally substituted with one or more substituents.

[0054] The term "aryl," as used herein, refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system, wherein any ring atom capable of substitution can be substituted (e.g., with one or more substituents). Examples of aryl moieties include but are not limited to phenyl, naphthyl, and anthracenyl. Aryl groups may be optionally substituted with one or more substituents.

[0055] The term "azide," as used herein, refers to a group of the formula -N3.

[0056] The term "cycloalkyl," as used herein, refers to non-aromatic, saturated or partially unsaturated monocyclic, bicyclic, tricyclic or polycyclic hydrocarbon groups having 3 to 12 carbons. Any ring atom can be substituted (e.g., with one or more substituents). Cycloalkyl groups can contain fused rings. Fused rings are rings that share one or more common carbon atoms. Examples of cycloalkyl groups include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, methylcyclohexyl, adamantyl, norbornyl, norbornenyl, tetrahydronaphthalenyl and dihydroindenyl. Cycloalkyl groups may be optionally substituted with one or more substituents. "Cycloalkenyl," as used herein, refers to a non-aromatic monocyclic, bicyclic, tricyclic or poly cyclic hydrocarbon group having one or more double bonds (e.g., cyclohexenyl or cyclohexadienyl). "Cycloalkynyl," as used herein, refers to a non-aromatic monocyclic, bicyclic, tricyclic or polycyclic hydrocarbon group having one or more triple bonds (e.g., cyclooctynyl), which may be optionally substituted with one or more substituents (e.g., with one or more halo groups, e.g., to generate a difluorcyclooctynyl group).

[0057] The term "halo" or "halogen," as used herein, refers to any radical of fluorine, chlorine, bromine or iodine.

[0058] The term "haloalkyl," as used herein, refers to an alkyl group as defined herein, in which one or more hydrogen atoms are replaced with halogen atoms, and includes alkyl moieties in which all hydrogens have been replaced with halogens (e.g., perfluoroalkyl such as CF3).

[0059] The term "heteroalkyl," as used herein, refers to an alkyl, alkenyl or alkynyl group as defined herein, wherein at least one carbon atom of the alkyl group is replaced with a heteroatom. Heteroalkyl groups may contain from 1 to 18 non-hydrogen atoms (carbon and heteroatoms) in the chain, or 1 to 12 atoms, or 1 to 6 atoms, or 1 to 4 atoms. Heteroalkyl groups may be straight or branched, and saturated or unsaturated. Unsaturated heteroalkyl groups have one or more double bonds and/or one or more triple bonds. Heteroalkyl groups may be unsubstituted or substituted. Exemplary heteroalkyl groups include but are not limited to alkoxyalkyl (e.g., methoxymethyl), and aminoalkyl (e.g., alkylaminoalkyl and dialkylaminoalkyl). Heteroalkyl groups may be optionally substituted with one or more substituents.

[0060] The term "heteroaryl," as used herein, refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 1 1-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms independently selected from O, N, S, P and Si (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms independently selected from O, N, S, P and Si if monocyclic, bicyclic, or tricyclic, respectively). Any ring atom can be substituted (e.g., with one or more substituents). Heteroaryl groups can contain fused rings, which are rings that share one or more common atoms. Examples of heteroaryl groups include, but are not limited, to radicals of pyridine, pyrimidine, pyrazine, pyridazine, pyrrole, imidazole, pyrazole, triazole, oxazole, isoxazole, furan, thiazole, isothiazole, thiophene, quinoline, isoquinoline, quinoxaline, quinazoline, cinnoline, indole, isoindole, indolizine, indazole, benzimidazole, phthalazine, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, phenazine, naphthyridines and purines. Heteroaryl groups may be optionally substituted with one or more substituents.

[0061] The term "heteroatom," as used herein, refers to a non-carbon or hydrogen atom such as a nitrogen, sulfur, oxygen, silicon or phosphorus atom. Groups containing more than one heteroatom may contain different heteroatoms.

[0062] The term "heterocyclyl," as used herein, refers to a nonaromatic, saturated or partially unsaturated 3-10 membered monocyclic, 8-12 membered bicyclic, or 1 1-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, S, Si and P (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, S, Si and P if monocyclic, bicyclic, or tricyclic, respectively). Any ring atom may be substituted (e.g., with one or more substituents). Heterocyclyl groups can contain fused rings, which are rings that share one or more common atoms. Examples of heterocyclyl groups include, but are not limited to, radicals of tetrahydrofuran, tetrahydrothiophene, tetrahydropyran, oxetane, piperidine, piperazine, morpholine, pyrroline, pyrimidine, pyrrolidine, indoline, tetrahydropyridine, dihydropyran, thianthrene, pyran, benzopyran, xanthene, phenoxathiin, phenothiazine, furazan, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. Heterocyclyl groups may be optionally substituted with one or more substituents.

[0063] The term "hydroxy," as used herein, refers to an -OH radical. The term "alkoxy," as used herein, refers to an -O-alkyl radical. The term "aryloxy," as used herein, refers to an - O-aryl radical.

[0064] The term "oxo," as used herein, refers to an oxygen atom, which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur (i.e. =0).

[0065] The terms "mercapto" or "thiol,"as used herein, each refer to an -SH radical. The terms "thioalkoxy" or "thioether," as used herein, each refer to an -S-alkyl radical. The term "thioaryloxy," as used herein, refers to an -S-aryl radical.

[0066] The term "substituents," as used herein, refers to a group "substituted" on an alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl group at any atom of that group. Any atom can be substituted. Suitable substituents include, without limitation: acyl, acylamido, acyloxy, alkoxy, alkyl, alkenyl, alkynyl, amido, amino, carboxy, cyano, ester, halo, hydroxy, imino, nitro, oxo (e.g., C=0), phosphonate, sulfinyl, sulfonyl, sulfonate, sulfonamino, sulfonamido, thioamido, thiol, thioxo (e.g., C=S), and ureido. In some embodiments, substituents on a group are independently any one single, or any combination of the aforementioned substituents. In some embodiments, a substituent may itself be substituted with any one of the above substituents.

[0067] The above substituents may be abbreviated herein. For example, the abbreviations Me, Et, Ph and Bn represent methyl, ethyl, phenyl and benzyl, respectively. A more comprehensive list of standard abbreviations used by organic chemists appears in a table entitled Standard List of Abbreviations of the Journal of Organic Chemistry. The abbreviations contained in this list are hereby incorporated by reference.

[0068] For compounds described herein, groups and substituents thereof may be selected in accordance with permitted valence of the atoms and the substituents, and such that the selections and substitutions result in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

[0069] Where substituent groups are specified by their conventional chemical formulae, written from left to right, they optionally encompass substituents resulting from writing the structure from right to left, e.g., -CH₂0- optionally also recites -OCH₂-.

[0070] In accordance with a convention n the art, the group:

is used in structural formulae herein to depict the bond that is the point of attachment of the moiety or substituent to the core or backbone structure.

[0071] The term "interrupted" indicates that another group is inserted between two adjacent carbon atoms (and the hydrogen atoms to which they are attached (e.g., methyl (CH₃), methylene (CH₂) or methine (CH))) of a particular carbon chain being referred to in the expression using the term "interrupted, provided that each of the indicated atoms' normal valency is not exceeded, and that the interruption results in a stable compound. Suitable groups that can interrupt a carbon chain include, e.g., with one or more non-peroxide oxy (- 0-), thio (-S-), imino (-N(H)-), methylene dioxy (-OCH₂0-), carbonyl (-C(=0)-), carboxy (- C(=0)0-), carbonyldioxy (-OC(=0)0-), carboxylato (-OC(=0)-), imine (C=NH), sulfinyl (SO) and sulfonyl (SO₂). Alkyl groups can be interrupted by one or more (e.g., 1, 2, 3, 4, 5, or about 6) of the aforementioned suitable groups. The site of interruption can also be between a carbon atom of an alkyl group and a carbon atom to which the alkyl group is attached.

[0072] The terms "fluorophore," "fluorescent moiety," "fluorescent label" and "fluorescent dye," are used interchangeably herein and refer to a molecule or a portion thereof that absorbs a quantum of electromagnetic radiation at one wavelength, and emits one or more photons at a different, typically longer, wavelength. Numerous fluorescent dyes of a wide variety of structures and characteristics are suitable for use in the compounds of the present disclosure. Suitable fluorophores are described in the Handbook of Fluorescent Probes and Research Chemicals (6th Ed., Molecular Probes, Inc., Eugene Oreg.).

[0073] The term "member atom," as used herein, refers to a polyvalent atom (e.g., a C, O, N, P or S atom) in a chain or ring system that constitutes a part of the chain or ring. For example, in a triazole ring, two carbon atoms and three nitrogen atoms are member atoms of the ring. In an exemplary linker -(CH₂CH₂0)₂-, four carbon atoms and two oxygen atoms are member atoms of the linker. Member atoms will be substituted up to their normal valence. For example, in pyridine, the five carbon atoms will each be further substituted with a hydrogen or another substituent (e.g., an alkyl group).

Compounds

[0074] In one embodiment, the invention provides compounds of Formula (I):

(I) wherein

A is a fluorophore;

W is a direct bond, -C(0)NH-, or

L is a linker;

n 1 or 2.

[0075] Suitable fluorophores include rhodamines, fluoresceins, coumarins, cyanines, and boron-dipyrromethenes (also known as BODIPYs), as well as derivatives thereof. For example, known rhodamine derivatives include amine-conjugated rhodamine compounds.

[0076] Fluorophores can be selected by one of ordinary skill in the art for incorporation into compounds of formula (I), based on the particular application of interest. For example, fluorophores having particular excitation/emission profiles may be selected, and may, for example, be orthogonal to other fluorophores being used in a particular application. In an exemplary embodiment, certain live-cell imaging experiments may be conducted using DAPI as a nuclear stain, which has an absorption maximum of about 350 nm and an emission maximum of about 460 nm. For applications employing DAPI stain, a fluorescent moiety for a compound of formula (I) may be selected to have excitation and emission properties that are different from those of the DAPI stain. For example, rhodamine B has an absorption maximum of about 540 nm and an emission maximum of about 625 nm. (As those skilled in the art appreciate, these absorption and emission values are approximate and depend on the particular environment including solvent, pH, etc.)

[0077] Suitable fluorophores include, but are not limited to, rhodamines, coumarins, fluorescein, and cyanine dyes, including Cy2, Cy 3, Cy3B, Cy 3.5, Cy 5, Cy 5.5, Cy 7 and Cy 7.5.

[0078] In the compounds of Formula (I), L is a linker. A "linker," as used herein, refers to a moiety containing from 1 to about 50 member atoms, not including substituents. For example, a linker may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 atoms, or any range therebetween.

[0079] In one embodiment, a linker suitably contains one or more triazole moieties. In exemplary linkers, a linker may include a 1,2, 3 -triazole, which may be a product of the reaction between a reagent comprising a fluorophore and an alkyne, and a molecule comprising an azide. Alternatively, a 1,2,3-triazole may be a product of the reaction between a reagent comprising a fluorophore and an azide, and a molecule comprising an alkynyl group.

[0080] In addition, a linker may comprise one or more groups selected from -CH₂-, - CH=, -C≡, -NH-, -N=, 0-, -S-, -C(O)-, -C(S)-, -S(O)-, -S(0)₂-, or any combination thereof. Any of the H atoms may be replaced by a suitable substituent, such as an alkyl group. One of ordinary skill in the art will appreciate that a linker comprising more than one of the above groups will be selected such that the linker is stable; for example, a linker may not include two adjacent -O- groups, which would generate an unstable peroxide linkage. A linker may be a straight chain, a branched chain, or may include one or more ring systems. Non-limiting exemplary linkers include those containing polyethylene glycol moieties.

[0081] Illustrative linkers include moieties such as -(CH₂)_c-D_e-(CH₂)_f- and -(CH₂)_P-M_r- C(0)-K_s-(CH₂)_q- where c is 0 to 8; D is O, NH, or S; e is 0 or 1; f is 0 to 8; p is 0 to 8; M is NH or O; K is NH or O; q is 0 to 8, and r and s are each independently 0 or 1. Other moieties which may be part of a linker include -0-, -S-,-ΝΗ-, -N(alkyl)-, -C(O)-, -S(O)-, -S(0)₂-, - S(0)₂-NH-, -CH=CH-, -OCH=CH-, -C(0)-NH-, -NH-C(0)-NH-, -NH-C(S)-NH-, -O-C(O)- NH-, -C(S)-, -CH₂-, -CH₂-CH₂-, -CH₂-CH₂-CH₂-, -CH₂-CH₂-CH₂-CH₂-, -0-CH₂-, -0-CH₂- CH₂-, -CH₂-0-CH₂-, -0-CH₂-CH₂-CH₂-, -CH₂-0-CH₂-CH₂-, -0-CH₂-CH₂-CH₂-CH₂-, -CH₂- 0-CH₂-CH₂-CH₂-, -CH₂-CH₂-0-CH₂-CH₂-, -S-CH₂-, -S-CH₂-CH₂-, -CH₂-S-CH₂-, -S-CH₂- CH₂-CH₂-, -CH₂-S-CH₂-CH₂-, -S-CH₂-CH₂-CH₂-CH₂-, -CH₂-S-CH₂-CH₂-CH₂-, -CH₂-CH₂- S-CH₂-CH₂-, -C(0)-NH-CH₂-, -C(0)-NH-CH₂-CH₂-, -CH₂-C(0)-NH-CH₂-, -C(0)-NH-CH₂- CH₂-CH₂-, -CH₂-C(0)-NH-CH₂-CH₂-, -C(0)-NH-CH₂-CH₂-CH₂-CH₂-, -CH₂-C(0)-NH-CH₂- CH₂-CH₂-, -CH₂-CH₂-C(0)-NH-CH₂-CH₂-, -CH₂-CH₂-CH₂-C(0)-NH-CH₂-CH₂-, -CH₂-CH₂- CH₂-CH₂-C(0)-NH-, -NH-C(0)-CH₂-C(0)-NH-, -NH-C(0)-CH₂-CH₂-C(0)-NH-, -NH- C(0)-CH₂-CH₂-CH₂-C(0)-NH-, -NH-C(0)-CH₂-CH₂-CH₂-CH₂-C(0)-NH-, -NH-C(O)- CH=CH-C(0)-NH-C(0)-0-CH₂-, -CH₂-C(0)-0-CH₂-, -CH₂-CH₂-C(0)-0-CH₂-, -C(0)-0- CH₂-CH₂-, -NH-C(0)-CH₂-, -NH-C(0)-CH₂-CH₂-, -CH₂-NH-C(0)-CH₂-CH₂-, -0-C(0)-NH- CH₂-, -0-C(0)-NH-CH₂-CH₂-, -NH-CH₂-, -NH-CH₂-CH₂-, -CH₂-NH-CH₂-, -CH₂-CH₂-NH- CH₂-, -C(0)-CH₂-, -C(0)-CH₂-CH₂-, -CH₂-C(0)-CH₂-, -CH₂-CH₂-C(0)-CH₂-, -CH₂-CH₂- C(0)-CH₂-CH₂-, -CH₂-CH₂-CH₂-C(0)-NH-CH₂-CH₂-NH-, -CH₂-CH₂-CH₂-C(0)-NH-CH₂- CH₂-NH-C(0)-, -CH₂-CH₂-CH₂-C(0)-NH-CH₂-CH₂-NH-C(0)-CH₂-, and -CH₂-CH₂-CH₂- C(0)-NH-CH₂-CH₂-NH-C(0)-CH₂-CH₂-, or any combination thereof.

[0082] Illustrative linkers also include those having additional ring structures, such as aryl, heteroaryl, cycloalkyl or heterocyclyl rings.

[0083] Exemplary linkers include, but are not limited to, the following:

[0084] In some embodiments, the linker may be hydrolytically stable.

[0085] It will be understood by those skilled in the art that compounds described herein may be present in multiple forms. When a compound described herein is illustrated in one form, it is expressly understood that this reference includes all forms of the compound such as tautomeric forms, prototropic forms, salt forms, and the like.

[0086] For example, it is well appreciated that fluorescein may exist in neutral, monoanionic, dianionic and lactone forms, as illustrated below. Compounds described herein that include fluorescein moieties encompass all of these forms and mixtures thereof

monoanionic dianionic lactone

[0087] For example, it is also well appreciated that rhodamine B may exist in cationic, zwitterionic and lactone forms, as illustrated below. Compounds described herein that include rhodamine moieties encompass all of these forms and mixtures thereof

cationic zwitterionic lactone

[0088] When compounds described herein bear one or more charges, it will be understood that they will also include one or more associated anions or cations to balance the charges. For example, if the compound is anionic, or has a functional group which may be anionic (e.g., -COOH may be -COO-), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na+ and K+, alkaline earth cations such as Ca²⁺ and Mg²⁺, and other cations. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH⁴⁺) and substituted ammonium ions (e.g., NH₃R , H₂R₂ , NHR₃ , NR4 ). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine.

[0089] If the compound is cationic, or has a functional group that may be cationic (e.g., - NH₂ may be -ΝΙ¾⁺), then a 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: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.

[0090] Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric.

[0091] Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diastereomeric, epimeric, atropic, stereoisomer, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and 1 -forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal- forms; a- and β-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and half chair-forms; and combinations thereof, hereinafter collectively referred to as "isomers" (or "isomeric forms").

[0092] Suitable compounds according to the present invention include, but are not limited to:





21

Synthesis

[0094] Compounds of Formula (I) may be synthesized using commercially available starting materials. Exemplary syntheses are illustrated in the Examples and described below.

[0095] For example, a fluorescent moiety may be incorporated into a compound of formula (I), for example, by using a reagent that comprises a fluorophore and a reactive group such as a carboxylic acid, an isothiocyanate, a maleimide, an alkynyl group, an azide, an amine, a thiol, or an ester such as a succinimidyl, sulfodichlorophenol, pentafluorophenyl or tetrafluorophenyl ester. Such groups may react with a complementary group, such as one present on a linker precursor compound, to attach the fluorophore to the remainder of the molecule of formula (I). Reagents comprising fluorophores and reactive groups may be commercially available, or may be synthesized according to methods described herein or other methods known to those skilled in the art.

[0096] Suitable reagents comprising fluorophores, which may be used to prepare compounds of formula (I), are known in the art. For example, reagents comprising fluorophores that are commercially available include, but are not limited to: 5- and 6- carboxyfluoresceins and esters thereof; fluorescein-5-isothiocyanate and fluorescein-6- isothiocyanate; BODIPY® dyes commercially available from Life Technologies™, such as BODIPY® succinimidyl esters; Alexa Fluor® dyes commercially available from Life Technologies™, such as Alexa Fluor® succinimidyl, tetrafluorphenyl and sulfodichlorophenol esters; CyDye fluors commercially available from GE Healthcare Biosciences, such as CyDye succinimidyl esters and maleimides; and VivoTag™ fluorophores available from PerkinElmer, such as VivoTag™ succinimidyl esters and maleimides.

[0097] It will be understood by the skilled artisan that the fluorescent moiety may include the fluorophore itself, as well as additional atoms or groups of atoms, such as atoms or groups of atoms derived from reactive groups or complementary groups that serve to link the fluorescent moiety to the remainder of the compound of formula (I).

[0098] Suitably, compounds of the present invention may be synthesized as follows. Starting from rhodamine B, a tertiary amide is prepared by coupling with alkyne derivatized piperazine. Formation of a tertiary amide bond between rhodamine B and piperazine as a linker moiety prevented cyclization of rhodamine derivative into a non-fluorescent lactam form. Thus, a compound of Formula (I) can be prepared via "click reaction" by reacting tertiary amide with 4-azido-tempo, which was synthesized from 4-hydroxyl-2, 2, 6, 6- tetramethyl-piperidine 1-oxyl. In order to avoid reduction of the nitroxide radical by sodium L-ascorbate to the nonparamagnetic hydroxylamine derivative during the click reaction, copper (I) iodide may be used as the copper (I) source instead of the Cu(II)S04/sodium L- ascorbate system.

[0099] Other methods of synthesizing the compounds of the formulae herein will be evident to those of ordinary skill in the art. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof. Methods of Use

[00100] In one embodiment, the present invention provides methods of detecting, determining or identifying oxidative stress in cells using the compounds described above. The method includes (a) contacting a sample containing cells with an effective amount of a compound of Formula (I), (b) washing away excess compound to generate a washed sample, (c) exposing the washed sample to a wavelength of light to generate fluorescence, (d) detecting the fluorescence of the washed sample, and (e) comparing the fluorescence level of the washed sample to the fluorescence level of a control sample, wherein the fluorescence level of the washed sample that is higher than the fluorescence level of the control sample indicates that that the cells have oxidative stress.

[00101] In one embodiment, the present invention provides methods of early detection of disease or disorders related to oxidative stress in a subject. The method includes (a) contacting a biological sample from a subject with an effective amount of a compound of Formula (I), (b) washing away excess compound to generate a washed sample, (c) exposing the washed sample to a wavelength of light to generate fluorescence, (d) detecting the fluorescence level of the washed sample, and (e) comparing the fluorescence level of the washed sample to the fluorescence level of a control sample, wherein the fluorescence level of the washed sample that is higher than the fluorescence level of the control sample indicates that that the subject has a disease or disorder related to oxidative stress. The control sample may be a sample taken from a subject who is healthy.

[00102] In one embodiment, a method of in vivo imaging of oxidative stress is provided. The method comprises administering an effective amount of a compound of Formula (I) to a subject and detecting fluorescence, wherein the fluorescence indicates the presence of oxidative stress. The subject may have a disease or disorder related to oxidative stress. The in vivo imaging may take place during a surgical procedure. The presence of fluorescence may indicate the presence of diseased tissue.

[00103] The disease or disorders may be cancer, diabetes, arteriosclerosis, obesity, hepatitis, AIDS, neurological diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and motor neuron diseases, including Lou Gehrig's disease (Amyotrophic Lateral Sclerosis (ALS)), apoptosis, inflammatory diseases, shock, ischemia/reperfusion injury, asthma, eczema, high bone mass syndrome, osteopetrosis, osteoporosis-pseudoglioma syndrome, digestive diseases such as gastric ulcer, irritable bowel syndrome, and ulcerative colitis, hypertension, angina pectoris, myocardial infarction, cardiomyopathy, chronic rheumatoid arthritis, Friedreich's Ataxia, musculoskeletal diseases such as migraine and tension headache, respiratory diseases such as bronchial asthma and hyperventilation syndrome, various diabetes complications, cranial nerve disease, Leber's hereditary optic neuropathy (LHON), optic neuritis, multiple sclerosis, systemic lupus erythematosis, stroke, traumatic hemorrhage, spinal cord trauma, Crohn's disease, autoimmune diseases, cataract formation, age-related macular degeneration, uveitis, emphysema, oxygen toxicity, neoplasia, and radiation sickness.

[00104] In one embodiment, the present invention provides methods for screening for an anti-oxidative stress agent. The method includes (a) administering an anti-oxidative stress candidate agent to a sample containing cells under oxidative stress conditions to generate a treated sample, (b) contacting the treated sample with an effective amount of compound of Formula (I), (c) contacting a control sample with a compound of Formula (I) described above, (d) washing away excess compound to generate a washed sample and washed control sample, (e) exposing the washed sample and washed control sample to a wavelength of light to generate fluorescence, (f) detecting the fluorescence level of the washed sample and the washed control sample, and (g) comparing the fluorescence level of the washed sample to the fluorescence level of a washed control sample, wherein the fluorescence level of the washed sample that is lower than the fluorescence level of the washed control sample indicates that the anti-oxidative stress candidate agent is an anti-oxidative stress agent. The control sample may be a sample containing cells under oxidative stress conditions but has not been treated with the anti-oxidative stress candidate agent.

[00105] A biological sample may be a sample of blood, tissue, urine, serum, plasma, amniotic fluid, cerebrospinal fluid, placental cells or tissue, endothelial cells, leukocytes, or monocytes. The sample can be used directly as obtained from a subject or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art.

[00106] Any cell type, tissue, or bodily fluid may be utilized to obtain a sample. Such cell types, tissues, and fluid may include sections of tissues such as biopsy and autopsy samples, frozen sections taken for histologic purposes, blood (such as whole blood), plasma, serum, sputum, stool, tears, mucus, saliva, bronchoalveolar lavage (BAL) fluid, hair, skin, red blood cells, platelets, interstitial fluid, ocular lens fluid, cerebral spinal fluid, sweat, nasal fluid, synovial fluid, menses, amniotic fluid, semen, etc. Cell types and tissues may also include lymph fluid, ascetic fluid, gynecological fluid, urine, peritoneal fluid, cerebrospinal fluid, a fluid collected by vaginal rinsing, or a fluid collected by vaginal flushing. A tissue or cell type may be provided by removing a sample of cells from an animal, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose). Archival tissues, such as those having treatment or outcome history, may also be used.

[00107] The term "contacting," as used herein, e.g., as in "contacting a sample" or "contacting a cell" refers to contacting a sample or cell directly or indirectly, in vitro or ex vivo. Contacting a sample may include addition of a compound to a sample (e.g., a culture of cells).

[00108] The term "effective amount," as used herein, refers to an amount of a compound or a composition effective for eliciting a desired effect. For example, in methods of selectively detecting mitochondria in a cell, an "effective amount" of a compound may be an amount that allows for visualization of a fluorescent signal that is localized to mitchondria, using a method such as fluorescence microscopy.

[00109] The term "subject," as used herein refers to a mammal, such as a mouse, dog, cat, rat, monkey, or human.

Kits

[00110] In some aspects, the present invention provides a kit, which may be used for selectively staining or detecting mitochondria in a cell.

[00111] A kit will include a compound of formula (I) as described herein. A kit may also include instructions for use of the compound of formula (I). Instructions included in kits can be affixed to packaging material or can be included as a package insert. While the instructions are typically written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD, DVD), and the like. As used herein, the term "instructions" includes the address of an internet site that provides the instructions.

[00112] For example, the kit may comprise instructions for selectively detecting a lysosome in a cell by fluorescence detection, e.g., using a fluorescence microscope, a flow cytometer, a fluorometer, a fluorescence plate reader, or a combination thereof. The kit may further comprise a calibrator or control, and/or at least one container (e.g., a tube, a microtiter plate and/or a strip) for conducting the assay, and/or a buffer, such as an assay buffer or a wash buffer, either one of which can be provided as a concentrated solution. Suitably, the kit comprises all components, i.e., reagents, standards, buffers, diluents, etc., which are necessary for conducting a particular experiment. The instructions also may include instructions for generating a standard curve or a reference standard for purposes of quantification.

[00113] The kit also may optionally include other reagents required to conduct an assay or facilitate quality control evaluations, such as buffers, salts, enzymes, enzyme co-factors, substrates, detection reagents, and the like. Other components, such as buffers and solutions for the isolation and/or treatment of a test sample (e.g., pretreatment reagents), also may be included in the kit. The kit additionally may include one or more other controls. One or more of the components of the kit may be lyophilized, in which case the kit further may comprise reagents suitable for the reconstitution of the lyophilized components.

[00114] The various components of the kit optionally are provided in suitable containers as necessary, e.g., a microtiter plate. The kit further may include containers for holding or storing a sample (e.g., a container or cartridge for a sample). Where appropriate, the kit optionally also may contain reaction vessels, mixing vessels, and other components that facilitate the preparation of reagents or the test sample. The kit also may include one or more instrument for assisting with handling a sample, such as a syringe, pipette, or the like.

EXAMPLES

[00115] Synthesis Scheme. The synthesis scheme for MitoProbe I is depicted in FIG. 25.

[00116] Reagents and Conditions. Reagents and solvents available from commercial sources were used as received unless otherwise noted. Thin layer chromatography (TLC) was performed using Sigma-Aldrich TLC plates, silica gel 60F-254 over glass support, 0.25 μιη thickness. Flash column chromatography was performed using Alfa Aesar silica gel, particle size 230-400 mesh. Melting points were determined using a MELTEMP melting point apparatus and were uncorrected. ^XH and ¹³C NMR spectra were measured with a Varian UNITY INOVA instrument at 400 MHz and 100 MHz, respectively. The chemical shifts (δ) were reported in reference to solvent peaks (residue CHCI₃ at δ 7.24 ppm for ^XH and CDCI₃ at δ 77.00 ppm for ¹³C). High-resolution mass spectra (HR-MS) were obtained on a JEOL JMS HX 11 OA mass spectrometer. UV-vis spectra were recorded on a Perkin Elmer Lambda 35 UV/Vis Spectrometer equipped with PTP 1+1 Peltier Temperature Programmer instrument at 37°C. Fluorescence spectra were obtained with a Horiba Jobin Yvon Fluoromax-4 spectrofluorometer. Fluroscence imaging was performed on a Zeiss Axiovert 200M with Apotome.

[00117] tert-butyl 4-(prop-2-ynyl)piperazine-l-carboxylate (2). To a solution of tert- butyl 1-piperazinecarboxylate 1 (2.8 g, 15 mmol) and diisopropylethylamine (2.8 mL, 15.75 mmol) in anhydrous dicholoroform (DCM) (10 mL) was added propargyl bromide (1.7 mL, 15 mmol) at 0°C. The reaction mixture was stirred at room temperature overnight until TLC indicated that the starting material disappeared. The resulting mixture was diluted with dichloromethane and then washed with saturated aHC0₃ and brine. The combined organic layers were dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The crude residue was purified by flash column chromatography on silica gel (Benzene:Hexane, 1 : 1) to yield compound 2 as yellow solid (2.5 g, 74% yield). ^XH NMR (400 MHz, CDCI₃) δ 1.44 (s, 9H), 2.24 (t, J=2.4, 1H), 2.49 (t, J=4.8, 4H), 3.29 (d, J=2.4, 2H), 3.45 (t, J=4.8, 4H). ¹³C NMR (100 MHz, CDC1₃) δ 28.35, 46.93, 46.94, 51.56, 73.36, 78.37, 79.63, 154.62.

[00118] l-(prop-2-ynyl)piperazine (3). To a solution of compound 2 in DCM (10 mL) trifluoroacetic acid (TFA, 10 mL) was added slowly at 0°C. The reacting mixture was stirred overnight at room temperature until TLC indicated that the starting material disappeared. The reaction mixture was evaporated under reduced pressure. The resulting crude material was co-evaporated with toluene. The product was precipitated using hexanes and then filtered, dried under vacuum to obtain compound 3 as colorless solid (1.19 g, 86 % yield). ^XH NMR (400 MHz, CDC1₃) δ 2.26 (t, J=2.4, 1H), 2.72 (t, J=7.2, 4H), 3.12 (t, J=4.8, 4H), 3.31 (d, J=2.4, 2H). ¹³C NMR (100 MHz, CDC1₃) δ 45.79, 47.32, 52.93, 73.13, 78.65.

[00119] Rhodamine B 4-(propargyl)piperazine amide (4). To a stirring solution of Rhodamine B (2.50 g, 5.22 mmol) in DCM (20 mL) was sequentially added compound 3 (0.83g, 6.68 mmol), 0-benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate (HBTU) (2.2 g, 5.80 mmol), and triethylamine (0.87 niL, 5.80 mmol). The resulting mixture was stirred overnight at room temperature until TLC indicated that the starting material disappeared. The reaction mixture was diluted with DCM (50 mL) and then washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. This crude residue was purified by flash column chromatography on silica gel (DCM:MeOH, 20: 1) to give compound 4 as purple solid (2.27 g, 79.1 % yield). ^XH NMR (400 MHz, CDC1₃) δ 1.30 (t, J=7.2, 12H), 2.20 (t, J=2.4, 1H), 2.35 (br s, 4H), 3.23 (d, J=2.4, 2H), 3.32 (br s, 2H), 3.42 (br s, 2H), 3.59 (q, J=7.2, 8H), 6.75-6.76 (m, 2H), 6.89-6.92 (m, 2H), 7.22-7.24 (m, 2H), 7.30-7.32 (m, 1H), 7.51-7.53 (m, 1H), 7.63-7.66 (m, 2H). ¹³C NMR (100 MHz, CDC1₃) δ 12.50, 41.31, 46.00, 46.44, 47.17, 50.86, 51.45, 74.12, 96.28, 1 13.72, 1 14.05, 127.61, 129.88, 130.16, 130.44, 132.10, 135.35, 155.63, 157.74, 167.50.

[00120] MitoProbe I. To a stirring solution of compound 4 (250mg, 0.397 mmol), 4- azido-TEMPO (94 mg, 0.476 mmol), Et₃N (0.5 mL) in anhydrous MeCN was added Cul (75 mg, 0.397 mmol). The reaction mixture was stirred under nitrogen for 6h at room temperature. The reaction mixture was washed with saturated aq NaHC0₃ and then extracted with EtOAc (30 mL x 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. This crude residue was purified by flash column chromatography on silica gel (DCM:MeOH, 1 :20) to give MitoProbe I (33% yield). Following the general procedure for reduction of TEMPO derivatives, the hydroxyl form of MitoProbe I was obtained. ¾ NMR (400 MHz, CDC1₃) δ ppm 7.72 (bs, 1H), 7.63 (bs, 2H), 7.53 (bs, 1H), 7.26 (m, 3H), 7.14-6.82 (m, 2H), 6.73 (s, 2H), 5.07-4.60 (m, 1H), 3.62- 3.55 (m, 10H), 3.52-4.40 (m, 4H), 2.38 (bs, 4H), 2.08-2.01 (m, 4H), 1-35-1.15 (m, 24H).

Table 1: Absorbance and Emission Data of the Hydroxyl Form of MitoProbe I

[00121] Synthesis Scheme. The synthesis scheme for MitoProbe II is depicted in FIG. 26.

[00122] Reagents and Conditions. Reagents and solvents available from commercial sources were used as received unless otherwise noted. Thin layer chromatography (TLC) was performed using Sigma-Aldrich TLC plates, silica gel 60F-254 over glass support, 0.25 μηι thickness. Flash column chromatography was performed using Alfa Aesar silica gel, particle size 230-400 mesh. Melting points were determined using a MELTEMP melting point apparatus and were uncorrected. ^lH and ¹³C NMR spectra were measured with a Varian UNITY INOVA instrument at 400 MHz and 100 MHz, respectively. The chemical shifts (δ) were reported in reference to solvent peaks (residue CHCI3 at δ 7.24 ppm for ^XH and CDCI₃ at δ 77.00 ppm for ¹³C). High-resolution mass spectra (HR-MS) were obtained on a JEOL JMS HX 1 1 OA mass spectrometer. UV-vis spectra were recorded on a Perkin Elmer Lambda 35 UV/Vis Spectrometer equipped with PTP 1+1 Peltier Temperature Programmer instrument at 37°C. Fluorescence spectra were obtained with a Horiba Jobin Yvon Fluoromax-4 spectrofluorometer. Fluroscence imaging was performed on a Zeiss Axiovert 200M with Apotome.

[00123] teri-butyl 4-(undec-10-ynoyl)piperazine-l-carboxylate (6). To a stirring solution of 10-undecynoic acid (1.18 g, 6.48 mmol), HBTU (2.45 g, 6.48 mmol) and Et₃N (0.9 mL, 6.48 mmol) in DCM (10 mL) was added 1-Boc-piperazine (1.0 g, 5.4 mmol). The reaction mixture was stirred overnight at room temperature. The resulting mixture was diluted with dichloromethane and then washed with saturated aHC0₃ and brine. The combined organic layers were dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The crude residue was purified by flash column chromatography on silica gel (Hexanes/EtOAc, 3: 1) to afford compound 6 as a yellow syrup (1.6 g, 84.6% yield). ^XH NMR (400 MHz, CDC1₃) δ 1.15-1.30 (m, 8H), 1.34 (s, 9H), 1.36-1.54 (m, 4H), 1.82 (td, J= 0.8, 2.4, 1H), 2.04 (td, J=2.4, 7.2, 2H), 2.20 (t, J=7.2, 2H), 3.27 (t, J=5.2, 2H), 3.32 (s, 4H), 3.46 (t, J=5.2, 2H). ¹³C NMR (100 MHz, CDC1₃) δ 18.06, 24.95, 28.08, 28.12, 28.35, 28.62, 28.96, 29.06, 33.01, 41.03, 45.12, 67.94, 79.91, 84.33, 154.26, 171.53.

[00124] l-(piperazin-l-yl)undec-10-yn-l-one (7). To a stirring solution of compound 6 (1.0 g, 2.85 mmol) in DCM (10 mL) was added TFA (10 mL, 130 mmol) at 0°C. The reaction mixture was stirred overnight at room temperature. The solvent was evaporated under reduced pressure and co-evaporated with toluene to remove excess TFA, and the resultant yellow residue was dissolved in DCM (50 mL) and washed with aqueous NaHC0₃ solution. The organic layer was separated, and the aqueous layer was extracted with DCM (3 x 30 mL). The combined organic layers were washed with brine (3 x 30 mL), dried over Na₂S0₄, and concentrated under reduced pressure to afford 7 as a yellow syrup (0.43 g, 60% yield). ¾ NMR (400 MHz, CDC1₃) δ 1.28-1.40 (m, 8H), 1.44-1.61 (m, 4H), 1.91 (t, J=2.4, 1H), 2.14 (dt, J=2.4, 7.2, 2H), 2.34 (t, J=7.2, 2H), 3.23 (br s, 4H), 3.78 (br s, 2H), 3.87 (br s, 2H), 9.2 (br s, 1H). ¹³C NMR (100 MHz, CDC1₃) δ 18.26, 25.00, 28.31, 28.50, 28.78, 29.03, 29.08, 32.78, 38.35, 42.35, 68.09, 84.62, 173.08.

[00125] Rhodamine B 4-(undec-10-ynoyl) piperazine amide (8). To a stirring solution of compound 7 (1.35 g, 5.42 mmol) in DCM (20 mL) was sequentially added Rhodamine B (2.59 g, 5.42 mmol), HBTU (2.05 g, 5.42 mmol), and Et₃N at 0°C. The reaction mixture was stirred overnight at room temperature. After evaporation, the residue was dissolved in DCM (100 mL), and washed with aqueous NaHCC^ solution (3 x 30 mL) and brine (3 x 30 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The crude residue was purified by flash column chromatography on silica gel (DCM:MeOH, 40: 1) to afford the compound 8 as a purple solid (2.74 g, 74.8 % yield). ^XH NMR (400 MHz, CDC1₃) δ 1.18-1.37 (m, 8H), 1.26 (t, J=7.2, 12H), 1.39-1.54 (m, 4H), 1.86 (t, J=2.8, 1H), 2.09 (dt, J=2.8, 7.2, 2H), 2.18-2.34 (m, 2H), 3.19-3.44 (m, 8H), 3.47-3.64 (m, 8H), 6.64-6.85 (m, 3H), 6.97-6.99 (m, 1H), 7.13-7.19 (m, 2H), 7.23-7.28 (bs, 1H), 7.43- 7.48 (m, 1H), 7.58 (bs, 2H). ¹³C NMR (100 MHz, CDC1₃) δ 12.38, 18.20, 25.01, 28.29, 28.50, 28.73, 29.00, 29.13, 32.83, 40.75, 45.35, 45.90, 67.91, 84.70, 95.85, 113.67, 114.31, 127.33, 129.94, 130.84, 132.01, 134.97, 155.52, 157.57, 167.62, 172.50. [00126] MitoProbe II. To a stirring solution of compound 8 (260 mg, 0.351 mmol), 4- azido-tempo (83 mg, 0.422 mmol), Et₃N (0.5ml) in anhydrous MeCN was added Cul (75 mg, 0.397 mmol). The reaction mixture was stirred at room temperature for 6h under nitrogen. The reaction mixture was washed with saturated aq aHC0₃ and then extracted with EtOAc (30 mL x 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. This crude residue was purified by flash column chromatography on silica gel (DCM:MeOH, 100:4) to give MitoProbe II (31% yield). Following the general procedure for reduction of TEMPO derivatives, the hydroxyl form of MitoProbe II was obtained. 1H NMR (400 MHz, CDC1₃) δ 1.12-1.22 (m, 32H), 1.47-1.54 (m, 4H), 2.12-1.89 (m, 4H), 2.28-2.32 (m, 2H), 2.65 (bs, 2H), 3.32-3.37 (m, 8H), 3.56-3.59 (m, 8H), 4.80 (m, 1H), 6.68-6.88 (m, 3H), 7.04 (bs, 1H), 7.27-7.40 (bs, 4H), 7.50 (bs, 2H), 7.65 (bs, 1H).

Table 2: Absorbance and Emission Data of the Hydroxyl Form of MitoProbe II

Example 3. Synthesis of MitoProbes III and IV.

(MitoProbe IV)

[00127] Synthesis Scheme. The synthesis scheme for MitoProbes III and IV is depicted in FIG. 27. [00128] Reagents and Conditions, (i): N₃CH₂CH₂-(OCH₂CH₂)_n-COOH, Et₃N, HBTU, CH₂C1₂, 2 h; (ii): Tripropargylamine, Cul, ACN, 3 h; (iii) 4-azido TEMPO, Cul, ACN, 3 h.

[00129] Rhodamine B 3-(2-(2-azidoethoxy)ethoxy)-l-(piperazin-l-yl)propan-l-one

(2) . To a stirring solution of Rhodamine B piperazine amide (1, 538 mg, 1.08 mmol), N₃- PEG2-COOH (200 mg, 0.99 mmol) and Et₃N (327 mg, 3.24 mmol) in anhydrous CH₂C1₂ (30 mL), was added HBTU (409 g, 1.08 mmol). The mixture was stirred for 3 h at room temperature and diluted in CH₂C1₂ (100 mL). The CH₂C1₂ layer was washed with brine (30 mL x 2). The organic phase was dried on anhydrous Na₂S0₄, filtered and concentrated. Flash column chromatography of the residue on silica gel (CH₂Cl₂:MeOH, 20: 1) afforded the compound (2) as a purple foam (410 mg, 60.6%). ^XH NMR (400 MHz, CDC1₃) δ ppm 7.62- 7.56 (m, 2H), 7.48 (s, 1H), 7.25 (s, 1H), 7.16 (d, J=9.5 Hz, 2H), 6.86-6.71 (m, 2H), 6.68 (s, 2H), 3.69 (t, J=6.2 Hz, 2H), 3.63-3.45 (m, 12H), 3.36-3.27 (m, 6H), 3.27-3.15 (m, 6H), 2.59- 2.52 (m, 2H), 1.25 (t, J=7.1 Hz, 12H). ¹³C NMR (100 MHz, CDC1₃) δ ppm 170.46, 167.92, 157.88, 155.85, 135.22, 132.25, 130.29, 127.74, 114.53, 113.96, 96.27, 70.57, 70.35, 69.99, 67.22, 50.87, 46.24, 41.93, 33.37, 12.73.

[00130] Rhodamine B l-azido-15-(piperazin-l-yl)-3,6,9,12-tetraoxapentadecan-15-one

(3) . 3 was synthesized from 1 (370 mg, 0.74 mmol) as described for 2. The product (3) was obtained as a purple foam (310 mg, 59.1%). ^XH NMR (400 MHz, CDC1₃) δ ppm 7.64-7.52 (m, 2H), 7.58-7.49 (m, 1H), 7.35-7.23 (m, 1H), 7.29-7.19 (m, 1H), 7.04-6.79 (m, 2H), 6.84- 6.72 (m, 2H), 3.71 (d, J=10.6 Hz, 2H), 3.67-3.47 (m, 20H), 3.46-3.22 (m, 10H), 2.68-2.49 (m, 4H), 1.28 (t, J=7.1 Hz, 12H). ¹³C NMR (100 MHz, CDC1₃) δ ppm 178.45, 167.93, 157.89, 157.85, 155.86, 135.21, 132.30, 130.35, 128.51, 114.00, 112.52, 96.23, 87.97, 73.05, 70.49, 70.34, 70.02, 67.04, 53.85, 50.79, 48.32, 46.26, 45.79, 41.91, 41.23, 34.15, 33.19, 13.87, 12.75.

[00131] Rhodamine B 3-(2-(2-(4-((di(prop-2-yn-l-yl)amino)methyl)-lH-l,2,3-triazol- l-yl)ethoxy)ethoxy)-l-(piperazin-l-yl)propan-l-one (4). Rhodamine B 3-(2-(2- azidoethoxy)ethoxy)-l-(piperazin-l-yl)propan-l-one (2, 400 mg, 0.59 mmol) and tripropargylamine (153 mg, 1.17 mmol) in anhydrous acetonitrile (20 mL), was added copper iodide (20 mg, 0.11 mmol). The mixture was stirred for 3 h at room temperature, concentrated and diluted in CH₂C1₂ (50 mL). The CH₂C1₂ layer was washed with brine (10 mL x 2). The organic phase was dried on anhydrous Na₂S0₄, filtered and concentrated. Flash column chromatography of the residue on silica gel (CH₂Cl₂:MeOH, 20: 1) afforded the compound (4) as a purple foam (150 mg, 30.5%). ¾ NMR (400 MHz, CDCI3) δ ppm 7.68 (s, 1H), 7.66-7.55 (m, 2H), 7.49-7.45 (m, 1H), 7.29-7.21 (m, 1H), 7.17 (d, J=9.3 Hz, 2H), 7.00-6.76 (m, 2H), 6.75-6.67 (m, 2H), 4.48-4.42 (m, 2H), 3.83-3.72 (m, 4H), 3.72-3.61 (m, 2H), 3.63-3.43 (m, 12H), 3.42-3.36 (m, 8H), 3.34-3.17 (m, 4H), 2.62-2.46 (m, 2H), 2.21 (t, J=2.4 Hz, 2H), 1.25 (t, J=7.1 Hz, 12H). ¹³C NMR (100 MHz, CDC1₃) δ ppm 167.92, 157.87, 155.85, 144.12, 135.24, 132.29, 130.28, 127.69, 124.45, 114.61, 113.98, 96.21, 78.85, 73.67, 70.55, 70.23, 69.52, 67.14, 50.39, 48.10, 46.24, 42.04, 33.29, 12.74.

[00132] Rhodamine B l-(4-((di(prop-2-yn-l-yl)amino)methyl)-lH-l,2,3-triazol-l-yl)- 15-(piperazin-l-yl)-3,6,9,12-tetraoxapentadecan-15-one (5). 5 was synthesized from 3 (300 mg, 0.39 mmol) as described for 4. The product (5) was obtained as a purple foam (160 mg, 46.2%). ¾ NMR (400 MHz, CDC1₃) δ ppm 7.67 (s, 1H), 7.71-7.59 (m, 2H), 7.60-7.47 (m, 1H), 7.35-7.24 (m, 1H), 7.15 (d, J=9.5 Hz, 2H), 6.97-6.78 (m, 2H), 6.77-6.66 (m, 2H), 4.47 (t, J=7.4 Hz, 2H), 3.80 (t, J=4.8 Hz, 2H), 3.75 (s, 2H), 3.74-3.65 (m, 2H), 3.61-3.44 (m, 20H), 3.42-3.37 (m, 8H), 3.34-3.20 (m, 4H), 2.62-2.45 (m, 2H), 2.21 (t, J=2.4 Hz, 2H), 1.23 (t, J=7.1 Hz, 12H). ¹³C NMR (100 MHz, CDCB) δ ppm 170.58, 167.85, 157.86, 155.83, 144.18, 135.23, 132.20, 130.24, 124.46, 114.54, 113.93, 96.25, 78.83, 73.70, 70.35, 69.64, 67.03, 50.26, 48.12, 46.23, 42.04, 33.24, 12.73.

[00133] MitoProbe III (6). To a stirring solution of Rhodamine B 3-(2-(2-(4-((di(prop-2- yn- 1 -yl)amino)methyl)- 1 H- 1 ,2,3 -triazol- 1 -yl)ethoxy)ethoxy)- 1 -(piperazin- 1 -yl)propan- 1 -one (4, 150 mg, 0.18 mmol) and 4-azido TEMPO (109 mg, 0.55 mmol) in anhydrous acetonitrile (10 mL), was added copper iodide (11 mg, 0.06 mmol). The mixture was stirred for 3 h at room temperature, concentrated and diluted in CH₂CI₂ (50 mL). The CH₂CI₂ layer was washed with brine (10 mL x 2). The organic phase was dried on anhydrous Na₂S0₄, filtered and concentrated. Flash column chromatography of the residue on silica gel (CH₂Cl₂:MeOH, 20: 1) afforded the compound (6) as a purple foam (80 mg, 36.8%). ¾ NMR (400 MHz, CDCI₃) δ ppm 8.01 (br.s), 7.67 (br.s), 7.38 (br.s), 6.87 (br.s), 4.70 (br.s), 3.75 (br.s), 2.76 (br.s), 1.49 (br.s). ¹³C NMR (100 MHz, CDCI3) No peaks.

[00134] MitoProbe IV (7). 7 was synthesized from 5 (160 mg, 0.18 mmol) as described for 6. The product (7) was obtained as a purple foam (70 mg, 30.0%). ^XH NMR (400 MHz, CDCI₃) δ ppm 8.15 (br.s), 7.75 (br.s), 7.36 (br.s), 7.23 (br.s), 6.84 (br.s), 4.68 (br.s), 3.71 (br.s), 2.72 (br.s), 1.46 (br.s). ¹³C NMR (100 MHz, CDC1₃) No peaks.

[00135] Synthesis Scheme. The synthesis scheme for MitoProbe V is depicted in FIG. 28.

[00136] Reagents and Conditions, (i): Boc-piperazine, Et₃N, HBTU, CH₂CI₂, 2 h; (ii): TFA:CH₂C1₂ 1 :5, 1 h; (iii): 3-azidopropanoic acid, Et₃N, HBTU, CH₂C1₂, 2 h; (iv): Tripropargylamine, Cul, ACN, 3 h; (v): 4-azido TEMPO, Cul, ACN, 3 h.

[00137] Rhodamine B Boc-piperazine Amide (2). To a stirring solution of Rhodamine B (1, 2.00 g, 4.18 mmol), Boc-piperizine (0.78 g, 4.18 mmol) and Et₃N (1.02 g, 10.04 mmol) in CH₂CI₂ (50 mL), was added HBTU (1.90 g, 5.02 mmol). The mixture was stirred for 2 h at room temperature and diluted in CH₂CI₂ (50 mL). The solution was washed with brine (50 mL x 2), dried on anhydrous Na₂S0₄, filtered and concentrated. Flash column chromatography of the residue on silica gel (CLLC MeOH, 20: 1) afforded the compound (2) as a purple foam (2.28 g, 91.2%). ^XH NMR (400 MHz, CDC1₃) δ ppm 7.62-7.57 (m, 2H), 7.48-7.44 (m, 1H), 7.25 (m, 1H), 7.16-7.1 1 (m, 2H), 6.84 (d, J=9.0 Hz, 2H), 6.69 (d, J=2.4 Hz, 2H), 3.54 (h, J=7.8 Hz, 8H), 3.29 (s, 4H), 3.19 (s, 4H), 1.33 (s, 9H), 1.22 (t, J=7.8 Hz, 12H). ¹³C NMR (100 MHz, CDC1₃) δ ppm 167.84, 157.87, 155.95, 155.78, 154.63, 135.23, 132.13, 130.92, 130.47, 130.26, 127.75, 114.23, 1 13.84, 96.44, 80.54, 46.22, 28.43, 12.70.

[00138] Rhodamine B Piperazine Amide (3). To a stirring solution of Rhodamine B Boc-piperazine amide (2, 11.01 g, 18.41 mmol) in CH₂C1₂ (150 mL), was added TFA (30 mL) dropwise under ice bath. The mixture was stirred for 1 h at room temperature and adjusted to pH 8 with saturated NaHCC>3 solution. CH₂CI₂ layer was separated and water phase was extracted with CH₂CI₂ (150 mL x 2). The combined CH₂CI₂ layers were dried on anhydrous a₂S0₄, filtered and concentrated. Flash column chromatography of the residue on silica gel (CH₂Cl₂:MeOH, 10: 1) afforded the compound (3) as a purple foam (6.09 g, 66.3%). ¾ NMR (400 MHz, CDC1₃) δ ppm 7.66-7.59 (m, 2H), 7.55-7.49 (m, 1H), 7.33-7.26 (m, 1H), 7.10 (d, J=9.5 Hz, 2H), 6.86 (dd, J=9.6, 2.3 Hz, 2H), 6.69 (d, J=2.4 Hz, 2H), 3.71 (s, 4H), 3.63-3.57 (m, 4H), 3.52-3.48 (m, 4H), 3.10 (s, 2H), 3.02 (s, 2H), 1.26 (t, J=7.1 Hz, 12H). ¹³C NMR (100 MHz, CDC1₃) δ ppm 167.35, 157.88, 155.87, 155.82, 134.63, 131.67, 131.19, 130.60, 130.40, 130.32, 127.80, 118.56, 115.63, 114.42, 113.76, 96.51, 46.24, 44.44, 42.76, 38.57, 12.65.

[00139] Rhodamine B 3-azido-l-(piperazin-l-yl)propan-l-one (4). To a stirring solution of Rhodamine B piperazine amide (3, 2.23 g, 4.48 mmol), 3-azidopropanoic acid (0.50 g, 4.93 mmol) and Et₃N (0.91 g, 8.96 mmol) in anhydrous CH₂C1₂ (50 mL), was added HBTU (2.04 g, 5.38 mmol). The mixture was stirred for 3 h at room temperature and diluted in CH₂CI₂ (100 mL). The CH₂CI₂ layer was washed with brine (30 mL x 2). The organic phase was dried on anhydrous a2S0₄, filtered and concentrated. Flash column chromatography of the residue on silica gel (CH₂Ci₂:MeOH, 20: 1) afforded the compound (4) as a purple foam (0.74 g, 28.3%). ^XH NMR (400 MHz, CDC1₃) δ ppm 7.69-7.56 (m, 2H), 7.47 (d, J=6.3 Hz, 1H), 7.32-7.22 (m, 1H), 7.15 (d, J=9.5 Hz, 2H), 6.96 (d, J=9.0 Hz, 1H), 6.89-6.74 (m, 1H), 6.74-6.61 (m, 2H), 3.66-3.45 (m, 10H), 3.44-3.36 (m, 4H), 3.35-3.17 (m, 4H), 2.63-2.45 (m, 2H), 1.25 (t, J=7.1 Hz, 12H). ¹³C NMR (100 MHz, CDC1₃) δ ppm 169.70, 167.98, 157.87, 155.86, 135.23, 132.20, 130.27, 127.66, 114.18, 113.94, 96.24, 94.85, 47.24, 46.23, 41.84, 41.36, 32.30, 12.72.

[00140] Rhodamine B 3-(4-((di(prop-2-yn-l-yl)amino)methyl)-lH-l,2,3-triazol-l-yl)- l-(piperazin-l-yl)propan-l-one (5). To a stirring solution of Rhodamine B 3-azido-l- (piperazin-l-yl)propan-l-one (4, 0.33 g, 0.57 mmol) and tripropargylamine (0.11 g, 0.85 mmol) in anhydrous acetonitrile (20 mL), was added copper iodide (17 mg, 0.09 mmol). The mixture was stirred for 3 h at room temperature, concentrated and diluted in CH₂CI₂ (50 mL). The CH₂CI₂ layer was washed with brine (10 mL x 2). The organic phase was dried on anhydrous Na₂S0₄, filtered and concentrated. Flash column chromatography of the residue on silica gel (CH₂Cl₂:MeOH, 20:1) afforded the compound (5) as a purple foam (0.24 g, 59.6%). ¾ NMR (400 MHz, CDC1₃) δ ppm 7.71 (s, 1H), 7.62 (d, J=3.9 Hz, 2H), 7.48 (s, 1H), 7.34-7.24 (m, 1H), 7.20-7.12 (m, 2H), 7.03-6.77 (m, 2H), 6.68 (s, 2H), 4.59 (t, J=6.4 Hz, 2H), 3.76 (s, 2H), 3.70-3.46 (m, 10H), 3.45-3.35 (m, 6H), 3.27-3.20 (m, 4H), 3.01-2.84 (m, 2H), 2.24 (s, 2H), 1.26 (t, J=7.1 Hz, 12H). ¹³C NMR (100 MHz, CDC1₃) δ ppm 168.86, 167.93, 157.88, 155.88, 135.25, 132.17, 130.25, 127.69, 124.59, 114.68, 113.95, 96.22, 79.01, 73.66, 48.02, 47.89, 46.26, 45.42, 42.15, 41.83, 41.24 33.15, 12.76. [00141] MitoProbe V (6). To a stirring solution of Rhodamine B 3-(4-((di(prop-2-yn-l- yl)amino)methyl)-lH-l,2,3-triazol-l-yl)-l-(piperazin-l-yl)propan-l-one (5, 120 mg, 0.17 mmol) and 4-azido TEMPO (83 mg, 0.42 mmol) in anhydrous acetonitrile (10 mL), was added copper iodide (8 mg, 0.04 mmol). The mixture was stirred for 3 h at room temperature, concentrated and diluted in (¾(¾ (50 mL). The (¾(¾ layer was washed with brine (10 mL x 2). The organic phase was dried on anhydrous Na₂S0₄, filtered and concentrated. Flash column chromatography of the residue on silica gel

20: 1) afforded the compound (6) as a purple foam (0.08 g, 51.8%). ^XH NMR (400 MHz, CDC1₃) δ ppm 7.72 (br.s), 7.24 (br.s), 6.66 (br.s), 4.68 (br.s), 3.59 (br.s), 1.38 (br.s).

[00142] Synthetic Scheme. The synthesis scheme for MitoProbe VI is depicted in FIG. 29.

[00143] tert-butyl 4-(undec-10-ynoyl)piperazine-l-carboxylate. To a stirring solution of 10-undecynoic acid (1.18 g, 6.48 mmol), HBTU (2.45 g, 6.48 mmol) and Et₃N (0.9 mL, 6.48 mmol) in DCM (10 mL) was added 1-Boc-piperazine (1.0 g, 5.4 mmol). The reaction mixture was stirred overnight at room temperature. The resulting mixture was diluted with dichloromethane and then washed with saturated aHC03 and brine. The combined organic layers were dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The crude residue was purified by flash column chromatography on silica gel (Hexanes/EtOAc, 3: 1) to afford the compound tert-butyl 4-(undec-10-ynoyl)piperazine-l- carboxylate as a yellow syrup (1.6 g, 84.6% yield). ^XH NMR (400 MHz, CDC1₃) δ 1.15-1.30 (m, 8H), 1.34 (s, 9H), 1.36-1.54 (m, 4H), 1.82 (td, J=0.8, 2.4, 1H), 2.04 (td, J=2.4, 7.2, 2H), 2.20 (t, J=7.2, 2H), 3.27 (t, J=5.2, 2H), 3.32 (s, 4H), 3.46 (t, J=5.2, 2H). ¹³C NMR (100 MHz, CDCI₃) δ 18.06, 24.95, 28.08, 28.12, 28.35, 28.62, 28.96, 29.06, 33.01, 41.03, 45.12, 67.94, 79.91, 84.33, 154.26, 171.53.

[00144] l-(piperazin-l-yl)undec-10-yn-l-one. To a stirring solution of tert-butyl 4- (undec-lO-ynoyl)piperazine-l-carboxylate (1.0 g, 2.85 mmol) in DCM (10 mL) was added TFA (10 niL, 130 mmol) at 0°C. The reaction mixture was stirred overnight at room temperature. The solvent was evaporated under reduced pressure and co-evaporated with toluene to remove excess TFA, and the resultant yellow residue was dissolved in DCM (50 mL) and washed with aqueous aHC03 solution. The organic layer was separated, and the aqueous layer was extracted with DCM (3 x 30 mL). The combined organic layers were washed with brine (3 x 30 mL), dried over Na₂S0₄, and concentrated under reduced pressure to afford the compound l-(piperazin-l-yl)undec-10-yn-l-one as a yellow syrup (0.43 g, 60% yield). ¾ NMR (400 MHz, CDC1₃) δ 1.28-1.40 (m, 8H), 1.44-1.61 (m, 4H), 1.91 (t, J=2.4, 1H), 2.14 (dt, J=2.4, 7.2, 2H), 2.34 (t, J=7.2, 2H), 3.23 (br s, 4H), 3.78 (br s, 2H), 3.87 (br s, 2H), 9.2 (br s, 1H). ¹³C NMR (100 MHz, CDC1₃) δ 18.26, 25.00, 28.31, 28.50, 28.78, 29.03, 29.08, 32.78, 38.35, 42.35, 68.09, 84.62, 173.08.

[00145] Rhodamine 101 4-(undec-10-ynoyl) piperazine amide (compound 2). To a stirring solution of l-(piperazin-l-yl)undec-10-yn-l-one (1.35 g, 5.42 mmol) in DCM (20 mL) was sequentially added Rhodamine 101 (5.40 mmol), HBTU (2.05 g, 5.42 mmol), and Ets at 0° C. The reaction mixture was stirred overnight at room temperature. After evaporation, the residue was dissolved in DCM (100 mL), and washed with aqueous aHC03 solution (3 x 30 mL) and brine (3 x 30 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The crude residue was purified by flash column chromatography on silica gel (DCM:MeOH, 40: 1) to afford the compound 2 as a purple solid [M+H]+: 724.4.

[00146] MitoProbe VI. To a stirring solution of compound 2 (0.35 mmol), 4-azido-tempo (83 mg, 0.422 mmol), Et₃N (0.5ml) in anhydrous MeCN was added Cul (75 mg, 0.397 mmol). The reaction mixture was stirred at room temperature for 6h under nitrogen. The reaction mixture was washed with saturated aq aHC03 and then extracted with EtOAc (30 mL x 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. This crude residue was purified by flash column chromatography on silica gel (DCM:MeOH, 100:4) to give MitoProbe VI. [M+H]: 921.56.

[00147] Synthetic Scheme. The synthesis for the MitoProbe VII is depicted in FIG. 30.

[00148] Reagents and conditions, (i): Boc-piperazine, Et₃N, HBTU, CH₂C1₂, 2 h; (ii): TFA:CH₂C1₂ 1 :5, 1 h; (iii): 3-azidopropanoic acid, Et₃N, HBTU, CH₂C1₂, 2 h; (iv): Tripropargylamine, Cul, ACN, 3 h; (v): 4-azido TEMPO, Cul, ACN, 3 h.

[00149] Rhodamine 101 Boc-piperazine Amide (3). To a stirring solution of Rhodamine 101 (1, 4.20 mmol), Boc-piperizine (0.78 g, 4.18 mmol) and Et₃N (1.02 g, 10.04 mmol) in CH₂C1₂ (50 mL), was added HBTU (1.90 g, 5.02 mmol). The mixture was stirred for 2 h at room temperature and diluted in CH₂C1₂ (50 mL). The solution was washed with brine (50 mL x 2), dried on anhydrous Na₂S0₄, filtered and concentrated. Flash column chromatography of the residue on silica gel (CH₂Cl₂:MeOH, 20: 1) afforded the compound 3 as a purple foam. HRMS (FAB+) m/z 659.9568, calculated for C₄iH₄₇N₄0₄ 659.8357.

[00150] Compound 4. To a stirring solution of Rhodamine 101 Boc-piperazine amide (3, 18.4 mmol) in CH₂C1₂ (150 mL), was added TFA (30 mL) dropwise under ice bath. The mixture was stirred for 1 h at room temperature and adjusted to pH 8 with saturated NaHCC solution. CH₂C1₂ layer was separated and water phase was extracted with CH₂C1₂ (150 mL x 2). The combined CH₂C1₂ layers were dried on anhydrous Na₂S0₄, filtered and concentrated. Flash column chromatography of the residue on silica gel (CH₂Cl₂:MeOH, 10: 1) afforded the compound (4) as a purple foam.

[00151] Rhodamine 101 3-azido-l-(piperazin-l-yl) propan-l-one (5). To a stirring solution of Rhodamine 101 piperazine amide (4, 4.50 mmol), 3-azidopropanoic acid (0.50 g, 4.93 mmol) and Et₃N (0.91 g, 8.96 mmol) in anhydrous CH₂C1₂ (50 mL), was added HBTU (2.04 g, 5.38 mmol). The mixture was stirred for 3 h at room temperature and diluted in CH₂C1₂ (100 mL). The CH₂C1₂ layer was washed with brine (30 mL x 2). The organic phase was dried on anhydrous a2S0₄, filtered and concentrated. Flash column chromatography of the residue on silica gel (CH₂Cl₂:MeOH, 20: 1) afforded the compound 5 as a purple foam. HR/MS (FAB+) m/z 656.8044, calculated for C₃5H_{42 7}03 656.7953.

[00152] Rhodamine 101 3-(4-((di(prop-2-yn-l-yl)amino)methyl)-lH-l,2,3-triazol-l- yl)-l-(piperazin-l-yl)propan-l-one (6). To a stirring solution of Rhodamine B 3-azido-l- (piperazin-l-yl)propan-l-one (5, 0.60 mmol) and tripropargylamine (0.1 1 g, 0.85 mmol) in anhydrous acetonitrile (20 mL), was added copper iodide (17 mg, 0.09 mmol). The mixture was stirred for 3 h at room temperature, concentrated and diluted in CH2CI2 (50 mL). The CH2CI2 layer was washed with brine (10 mL x 2). The organic phase was dried on anhydrous a2S0₄, filtered and concentrated. Flash column chromatography of the residue on silica gel (CH₂Cl₂:MeOH, 20: 1) afforded the compound 6 as a purple foam. HRMS (FAB+) m/z 787.4095, calculated for QsHsi sOs 787.4049.

[00153] MitoProbe VII. To a stirring solution of Rhodamine 101 3-(4-((di(prop-2-yn-l- yl)amino)methyl)-lH-l,2,3-triazol-l-yl)-l-(piperazin-l-yl) propan-l-one (6, 0.18 mmol) and 4-azido TEMPO (83 mg, 0.42 mmol) in anhydrous acetonitrile (10 mL), was added copper iodide (8 mg, 0.04 mmol). The mixture was stirred for 3 h at room temperature, concentrated and diluted in CH2CI2 (50 mL). The CH2CI2 layer was washed with brine (10 mL x 2). The organic phase was dried on anhydrous Na₂S0₄, filtered and concentrated. Flash column chromatography of the residue on silica gel (CH₂Cl₂:MeOH, 20: 1) afforded the compound MitoProbe VII as a purple foam. MS (FAB+) m/z 1 181.59, calculated for C₆₆H₈5N1₆O5 1 181.70.

Ex

(MitoProbe IX)

[00154] Synthetic Scheme. The synthesis scheme for MitoProbe VIII and MitoProbe IX is depicted in FIG. 31.

[00155] Reagents and conditions, (i) N₃CH₂CH₂-(OCH₂CH₂)_n-COOH, Et₃N, HBTU, CH₂C1₂, 2 h; (ii) Tripropargylamine, Cul, ACN, 3 h; (iii) 4-azido TEMPO, Cul, ACN, 3 h.

[00156] Compound 7. To a stirring solution of compound 4 (560mg, 1.00 mmol), N₃- PEG2-COOH (200 mg, 0.99 mmol) and Et₃N (327 mg, 3.24 mmol) in anhydrous CH₂C1₂ (30 mL), was added HBTU (409 g, 1.08 mmol). The mixture was stirred for 3 h at room temperature and diluted in CH₂C1₂ (100 mL). The CH₂C1₂ layer was washed with brine (30 mL x 2). The organic phase was dried on anhydrous Na₂S0₄, filtered and concentrated. Flash column chromatography of the residue on silica gel (CH₂Cl₂:MeOH, 20: 1) afforded the compound 7 as a purple foam [M+H]+ MS: 701.8. [00157] Compound 8. To a stirring solution of compound 4 (560mg, 1.00 mmol), N₃- PEG4-COOH (200 mg, 0.99 mmol) and Et₃N (327 mg, 3.24 mmol) in anhydrous CH₂C1₂ (30 mL), was added HBTU (409 g, 1.08 mmol). The mixture was stirred for 3 h at room temperature and diluted in CH2CI2 (100 mL). The CH2CI2 layer was washed with brine (30 mL x 2). The organic phase was dried on anhydrous Na₂S0₄, filtered and concentrated. Flash column chromatography of the residue on silica gel (CH₂Cl₂:MeOH, 20: 1) afforded the compound 8 as a purple foam [M+H]+ MS: 745.9.

[00158] Compound 9. Compound 7 (0.60 mmol) and tripropargylamine (153 mg, 1.17 mmol) in anhydrous acetonitrile (20 mL), was added copper iodide (20 mg, 0.1 1 mmol). The mixture was stirred for 3 h at room temperature, concentrated and diluted in CH2CI2 (50 mL). The CH2CI2 layer was washed with brine (10 mL x 2). The organic phase was dried on anhydrous a2S0₄, filtered and concentrated. Flash column chromatography of the residue on silica gel (CH₂Cl₂:MeOH, 20: 1) afforded the compound 9 as a purple foam (150 mg, 30.5%) [M+H]+ MS: 877.1.

[00159] Compound 10. Compound 8 (0.60 mmol) and tripropargylamine (153 mg, 1.17 mmol) in anhydrous acetonitrile (20 mL), was added copper iodide (20 mg, 0.1 1 mmol). The mixture was stirred for 3 h at room temperature, concentrated and diluted in CH2CI2 (50 mL). The CH2CI2 layer was washed with brine (10 mL x 2). The organic phase was dried on anhydrous a2S0₄, filtered and concentrated. Flash column chromatography of the residue on silica gel (CH₂Cl₂:MeOH, 20: 1) afforded the compound 10 as a purple foam. [M+H]+ MS: 965.2.

[00160] MitoProbe VIII. To a stirring solution of Rhodamine 101 3-(2-(2-(4-((di(prop-2- yn- 1 -yl)amino)methyl)- 1 H- 1 ,2,3 -triazol- 1 -yl)ethoxy)ethoxy)- 1 -(piperazin- 1 -yl)propan- 1 -one (compound 9, 0.18 mmol) and 4-azido TEMPO (109 mg, 0.55 mmol) in anhydrous acetonitrile (10 mL), was added copper iodide (11 mg, 0.06 mmol). The mixture was stirred for 3 h at room temperature, concentrated and diluted in CH2CI2 (50 mL). The CH2CI2 layer was washed with brine (10 mL x 2). The organic phase was dried on anhydrous Na₂S0₄, filtered and concentrated. Flash column chromatography of the residue on silica gel (CH₂Cl₂:MeOH, 20: 1) afforded the compound (MitoProbe VIII) as a purple foam (80 mg, 36.8%). MS (FAB+) m/z 1272.8, calculated for C₇oH₉₃ i₆0₇ 1272.6

[00161] MitoProbe IX. To a stirring solution of compound 10 (0.18 mmol) and 4-azido TEMPO (109 mg, 0.55 mmol) in anhydrous acetonitrile (10 mL), was added copper iodide (11 mg, 0.06 mmol). The mixture was stirred for 3 h at room temperature, concentrated and diluted in CH2CI2 (50 niL). The CH2CI2 layer was washed with brine (10 mL x 2). The organic phase was dried on anhydrous a₂S04, filtered and concentrated. Flash column chromatography of the residue on silica gel (CH₂Ci2:MeOH, 20: 1) afforded the compound (MitoProbe IX) as a purple foam MS (FAB+) m/z 1360.9, calculated for C74H10I 16O9 1360.7.

(MitoProbe 11)

[00162] Synthetic Scheme. The synthesis scheme for MitoProbes 10 and 11 is depicted in FIG. 32.

[00163] 3,6-Dihydroxy-xanthen-9-one (S2). A stirred suspension of 2,2',4,4'- tetrahydroxybenzophenone (SI, 4.00 g, 16.3 mmol) in distilled water (24 mL) was heated in an autoclave at 200°C for 6 h. The mixture was cooled to 20°C, and 3,6-dihydroxy-9H- xanthen-9-one was obtained as a cluster of needles. It was filtered off, washed with hot distilled water (3 x 10 mL), and dried under reduced pressure to give a pure title product. Yield: 3.55 g (96%). Light orange needles. ^XH NMR (400 MHz, DMSO-d6): δ (ppm) 6.82 (d, 2H, J=2.0 Hz), 6.86 (dd, 2H, Jl=8.7 Hz, J2=2.1 Hz), 7.98 (d, 2H, J=8.7 Hz), 10.82 (s, 2H, -OH).

[00164] 3,6-Ditrifyl-xanthone (S3). 3,6-Dihydroxy-xanthen-9-one (S2, 6.85 g, 30 mmol) was dissolved in 150 mL DCM and pyridine (24.5 mL, 300 mmol) was added slowly over 5 min at 0°C. The mixture was stirred at 0°C for 10 min then Tf₂0 (15 mL, 90 mmol) was added dropwise over 10 min. The reaction mixture was warmed to room temperature slowly and stirred for 24 h. The reaction was quenched with water and the organic layer was washed with water (1 x 30 mL), IN HC1 (3 x 30 mL), brine (1 x 30 mL) and dried over Na₂S0₄. The solvents were removed under reduced pressure and the residue was recrystallized from CH₂Cl₂/hexanes to afford the pure product as a white crystal. ^XH NMR (500 MHz, CDC1₃): δ (ppm) 8.43 (d, 2H, J=8.9 Hz), 7.47 (d, 2H, J=2.3 Hz), 7.33 (dd, 2H, J=8.9, 2.3 Hz).

[00165] 3,6-Dipiperidin-l-yl-xanthen-9-one (S4). 3,6-Ditriflate-xanthone 6 (S3, 4.91 g, 10 mmol) was dissolved in DMSO (50 mL) and piperidine (8.5 g, 100 mmo) was added. The reaction mixture was heated to 90°C and stirred for 12 h. After cooling to room temperature, the reaction was quenched with water. The precipitate was collected, washed with saturated a₂C0₃ (aq.) and water to give the crude product, which was recrystallized from EtOAc/hexanes to afford the pure product as a yellow solid. ¾ NMR (300 MHz, d6- DMSO): δ (ppm) 7.86 (d, 2H, J=9.0 Hz), 6.98 (dd, 2H, J=9.0, 2.3 Hz), 6.74 (d, 2H, J=2.3 Hz), 3.41 (br, 8H), 1.60 (br, 12H).

[00166] Rosamine 1. 2,6-dimethoxyphenyl lithium (prepared from 1,3-dimethoxybenzene with nBuLi, 0.144 g, 1 mmol) was added dropwise over 1 min to the solution of 3,6- Dipiperidin-l-yl-xanthen-9-one (S4, 96 mg, 0.2 mmol) in 5 mL THF at 0°C. After stirring for 12 h at room temperature, the reaction mixture was quenched by 2 mL 2N HC1 (aq.) and stirred for 10 min then diluted with 20 mL DCM. The organic layer was washed with water, brine, dried over Na₂S0₄ and concentrated under reduced pressure. The residue was purified by flash chromatography (5% to 10% MeOH/DCM) to give the pure product as a green solid. ^XH NMR (500 MHz, CDC1₃): δ (ppm) 7.49 (t, 1H, J=8.5 Hz), 7.18 (d, 2H, J=9.5 Hz), 7.01 (dd, 2H, J=9.5, 2.5 Hz), 6.88 (d, 2H, J=2.5 Hz), 6.71 (d, 2H, J=8.5 Hz), 3.69-3.67 (m, 8H), 3.62 (s, 6H), 1.73 (br, 12H); ¹³C NMR (125 MHz, CDC1₃): δ (ppm) 158.3, 157.4, 156.5, 153.7, 132.3, 131.7, 1 14.6, 114.5, 108.4, 104.0, 97.0, 55.9, 48.9, 25.8, 24.1.

[00167] Rosamine 2. BBr3 (0.19 mL, 2.0 mmol) was added dropwise over 1 min to the solution of Rosamine 1 (124 mg, 0.2 mmol) in 4 mL dry DCM at -78°C. The solution was warmed to room temperature slowly and stirred for 12 h. The reaction was quenched with ice- water and the mixture was extracted with 1 : 1 iPrOH/DCM (3 x 15 mL). The organic layer was washed with water (1 x 20 mL), brine (1 x 20 mL), dried over Na₂S0₄ and concentrated under reduced pressure. The residue was dissolved in 10 mL MeOH and 0.5 g Amberlite IRA-400 (CI) ion exchange resin was added. The mixture was stirred at room temperature for lh and filtered through celite. The solvent was removed under reduced pressure. The ionexchange process was repeated twice. The crude product was purified by flash chromotagraphy (5 % to 10 % MeOH/DCM) to afford the product 2 as a green solid. ^XH NMR (500 MHz, CDCI₃/CD₃OD 1 : 1) : δ (ppm) 7.44 (d, 2H, J=9.6 Hz), 7.23 (t, 1H, J=8.3 Hz), 7.07 (dd, 2H, J=9.6, 2.6 Hz), 6.94 (d, 2H, J=2.6 Hz), 6.54 (d, 2H, J=8.3 Hz), 3.71-3.69 (m, 8H), 1.79-1.74 (m, 12H); ¹³C NMR (125 MHz, CDCI₃/CD₃OD 1 : 1): δ (ppm) 159.3, 157.4, 156.4, 156.1, 133.2, 132.5, 115.5, 114.9, 107.4, 107.3, 97.5, 49.4, 26.5, 24.8.

[00168] Rosamine 3. Rosamine 2 (137 mg, 0.28 mmol), Cs₂C0₃ (456 mg, 1.4 mmol), Bu₄NI (310 mg, 0.84 mmol) were dissolved in 5 mL DMF and tert-butyl bromoacetate (0.41 mL, 2.8 mmol) was added. The reaction mixture was stirred at 25°C for 12 h then diluted with 30 mL DCM, washed with H₂0 (3 x 20 mL), brine (1 x 20 mL) and dried over Na₂S0₄. The solvents were removed under reduced pressure and the residue was passed through a short pad of silica gel eluting with 5 % MeOH/DCM to give the crude product which was used in the next step without further purification. The crude material was dissolved in 10 mL TFA/DCM (1 :1) and stirred at 25°C for 1 h. The solvents were removed with a N₂ stream. The residue was dissolved in 30 mL DCM, washed with H₂0 (2 x 20 mL), brine (1 x 20 mL) and dried over Na₂S0₄. The solvents were removed under reduced pressure. The residue was dissolved in 20 mL MeOH/DCM (1: 1) and 1.0 g Amberlite IRA-400 (CI) ion exchange resin was added. The mixture was stirred at room temperature for 1 h and filtered through celite. The solvent was removed under reduced pressure. The ion-exchange process was repeated twice. The crude product was purified by reverse phase MPLC (H₂0 - 60 % CH₃CN/H₂0) to afford the pure product 3 as a green solid (128 mg, 75 %). ^XH NMR (500 MHz, CDCI₃/CD₃OD 1: 1): δ (ppm) 7.49 (t, 1H, J=8.5 Hz), 7.46 (d, 2H, J=9.6 Hz), 7.05 (dd, 2H, J=9.6, 2.5Hz), 6.93 (d, 2H, J=2.5 Hz), 6.69 (d, 2H, J=8.5 Hz), 4.52 (s, 4H), 3.72-3.70 (m, 8H), 1.80-1.75 (m, 12H); ¹³C NMR (125 MHz, CDCI₃/CD₃OD 1 :1): δ (ppm) 171.0, 159.2, 157.4, 156.9, 154.0, 133.3, 132.7, 115.4, 114.9, 110.1, 105.7, 97.4, 65.5, 49.4, 26.5, 24.8.

[00169] Rosamine 4. To a stirred solution of rosamine 3 (0.4 mmol) in DCM (10 mL) was sequentially added l-(prop-2-yn-l-yl)piperazine trifluoroacetate (130 mg, 0.55 mmol), HBTU (416mg, 1.1 mmol) and Et₃N (0.5 mL). The reaction mixture was stirred for 6 h room temperature. The solvent was then removed by distillation under reduced pressure, and the crude residue was purified via flash chromatograph to obtain the pure rosamine 4.

[00170] MitoProbe 10. To a solution of 0.75 mmol rosamine 4 and 177 mg (0.90 mmol) 4-azido-TEMPO in 3 ml acetonitrile was added 15.2 mg (0.08 mmol) copper (I) iodide at room temperature. The reaction mixture was stirred at room temperature for 6 h. The reaction mixture was washed with saturated aqueous NaHC03 solution (3 x 10 mL) and then extracted with ethyl acetate (3 x30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated. The crude residue was purified by flash column chromatography on silica gel to yield the target mitoprobe 10.

[00171] MitoProbe 11. To a stirred solution of rosamine 3 (0.4 mmol) in DCM (10 mL) was sequentially added l-(piperazin-l-yl)undec-10-yn-l-one trifluoroacetate (prepared from 10-undecynoic acid with tert-butyl piperazine-l-carboxylate, 190 mg, 0.55 mmol), HBTU (416mg, 1.1 mmol) and Ets (0.5 mL). The reaction mixture was stirred for 6 h room temperature. The solvent was then removed by distillation under reduced pressure, and the crude residue was purified via flash chromatograph to obtain the. To a solution of the intermediate (0.75 mmol) and 177 mg (0.90 mmol) 4-azido-TEMPO in 3 ml acetonitrile was added 15.2 mg (0.08 mmol) copper (I) iodide at room temperature. The reaction mixture was stirred at room temperature for 6 h. The reaction mixture was washed with saturated aqueous aHCOs solution (3 x 10 mL) and then extracted with ethyl acetate (3 x30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated. The crude residue was purified by flash column chromatography on silica gel to yield the target mitoprobe 11.

Example 9. S nthesis of MitoProbes 12 and 13.

(MitoProbe 13)

[00172] Synthetic Scheme. The synthesis scheme for MitoProbes 12 and 13 is depicted in FIG. 33. [00173] 7-Diethylamino-4-hydroxycoumarin (2). 3-Diethylaminophenol 1 (12.8 g, 77.5 mmol) and diphenyl malonate 8 (18.8 g, 73.4 mmol) were added into anhydrous toluene (50 mL). The mixture was heated to reflux for 4 h, and 7-Diethylamino-4-hydroxycoumarin was obtained by filtration as gray solid (5.2 g, 30%): ¾ NMR (400 MHz, DMSO): δ (ppm) 1 1.98 (s, 1H), 7.55-7.53 (d, 1H, J=9.0 Hz), 6.67-6.64 (dd, 1H, Jl=2.2 Hz, J2=9.0 Hz), 6.45-6.44 (d, 1H, J=2.2 Hz), 5.23 (s, 1H), 3.43-3.38 (q, 4H, J=7.0 Hz), 1.13-1.09 (t, 6H, J=7.0 Hz); ¹³C NMR (100 MHz, DMSO): δ (ppm) 166.7, 162.8, 156.1, 150.8, 124.1, 108.1, 103.6, 96.4, 86.1, 44.0, 12.3.

[00174] Compound 3. A solution of 3-diethylamino phenol (4.11 g, 30.0 mmol) and phthalic anhydride (4.66 g, 31.5 mmol) in toluene (30 mL) was refluxed under 2 for 3 h, and cooled to 50-60°C. Then 30mL of 35% aqueous NaOH (w/w) was added and heated at 90°C for 6 h. The resulting mixture was poured into H₂O(300mL), acidified with HC1 (10.0 M), and allowed to stand at room temperature for 2 h. The suspension was then filtered. The solid was recrystallized from a mixture of water and methanol, and dried to afford the 2-(4- diethylamino-2-hydroxy)-benzoylbenzoic acid. ¾ NMR (500MHz, CD₃0D:CDC1₃ = 5: 1): δ (ppm) 1.15 (t, J=7.0 Hz, 6H), 3.40 (q, J=7.0 Hz, 4H), 6.09 (d, J=2.5 Hz, 1H), 6.15 (d, J=9.0 Hz, 1H), 6.84 (d, J=9.5 Hz, 1H), 7.35 (dd, J=7.5, 1.0 Hz, 1H), 7.58 (t, J=7.5 Hz, 1H), 7.66 (t, J=7.5 Hz, 1H), 8.03 (d, J = 8.0 Hz, 1H).

[00175] Compound 2 (440 mg, 1.9 mmol) and 2-(4-diethylamino-2-hydroxy)- benzoylbenzoic acid (629 mg, 2.0 mmol) were heated to reflux in 15 mL of 1,1,2,2- tetrachloroethane until the substances have dissolved completely. A total of 2.8 g phosphorus pentoxide was then added in portions, and the reaction mixture was heated to reflux for 4 h. After cooling, the mixture was dissolved using water (25 mL) and chloroform (25 mL), and the water phase was extracted with chloroform (total 100 mL for three times). The combined organic phases were concentrated under vacuum and purified by column chromatography using v(CH₂Cl₂)/v(CH₃0H) = 100: 1 as the eluent. The dye fractions were concentrated under vacuum, and the residue was dissolved in 2 mL ethanol. After addition of 3 mL perchloric acid (note: the operation should be executed very carefully due to the instability of perchlorate salts), the solution was precipitated by dropwise addition of water (100 mL). Compound 3 was obtained by filtration as a dark green solid (682 mg, 59%): mp 287-289°C; ^XH NMR (400 MHz, DMSO): δ (ppm) 8.02-7.94 (m, 2H), 7.71-7.60 (m, 2H), 7.31-7.29 (d, 1H), 6.90-6.87 (m, 2H), 6.68 (s, 1H), 6.60-6.55 (m, 2H), 3.52-3.44 (m, 8H), 1.16-1.1 1 (m, 12H); ¹³C NMR (100 MHz, CD₃0D/CD₂C1₂): δ (ppm)166.9, 163.6, 163.4, 158.7, 158.4, 158.0, 157.9, 156.6, 136.8, 133.7, 132.9, 131.4, 130.4, 129.6, 128.8, 127.8, 1 16.9, 1 16.7, 1 12.7, 104.6, 101.3, 98.0, 97.9, 52.9, 47.2, 46.4, 12.7.

[00176] MitoProbe 12. To a stirred solution of compound 3 (0.4 mmol) in DCM (10 mL) was sequentially added l-(prop-2-yn-l-yl)piperazine trifluoroacetate (130 mg, 0.55 mmol), HBTU (416mg, 1.1 mmol) and Et₃N (0.5 mL). The reaction mixture was stirred for 6 h room temperature. The solvent was then removed by distillation under reduced pressure, and the crude residue was purified via flash chromatograph to obtain the pure the intermediate.

[00177] To a solution of intermediate (0.75 mmol) and 177 mg (0.90 mmol) 4-azido- TEMPO in 3 ml acetonitrile was added 15.2 mg (0.08 mmol) copper (I) iodide at room temperature. The reaction mixture was stirred at room temperature for 6 h. The reaction mixture was washed with saturated aqueous NaHCC solution (3 x 10 mL) and then extracted with ethyl acetate (3 x30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated. The crude residue was purified by flash column chromatography on silica gel to yield the target mitoprobe 12.

[00178] MitoProbe 13. To a stirred solution of compound 3 (0.4 mmol) in DCM (10 mL) was sequentially added l-(piperazin-l-yl)undec-10-yn-l-one trifluoroacetate (prepared from 10-undecynoic acid with tert-butyl piperazine-l-carboxylate, 190 mg, 0.55 mmol), HBTU (416mg, 1.1 mmol) and Ets (0.5 mL). The reaction mixture was stirred for 6 h room temperature. The solvent was then removed by distillation under reduced pressure, and the crude residue was purified via flash chromatograph to obtain the intermediate.

[00179] To a solution of the intermediate (0.75 mmol) and 177 mg (0.90 mmol) 4-azido- TEMPO in 3 ml acetonitrile was added 15.2 mg (0.08 mmol) copper (I) iodide at room temperature. The reaction mixture was stirred at room temperature for 6 h. The reaction mixture was washed with saturated aqueous aHC0₃ solution (3 x 10 mL) and then extracted with ethyl acetate (3 x30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated. The crude residue was purified by flash column chromatography on silica gel to yield the target mitoprobe 13. Example 10.

(MitoProbe 15)

[00180] Synthetic Scheme. The synthesis scheme for MitoProbes 14 and 15 is depicted in FIG. 34.

[00181] 7-Hydroxy-l-methyl-l,2,3,4-tetrahydroquinolin (2). 1 -methyl- 1,2,3,4- tetrahydroquinolin-7-amine (3.2 g, 1.6 mmol) was added into 85% phosphoric acid (15 mL) and heated to reflux for 24 h. After being cooled to room temperature, the reaction solution was neutralized with 200 mL of 40% NaOH aqueous solution. A large amount of a3P0₄ was precipitated and filtrated. The filtrate was extracted with CH2CI2 (total 60 mL for three times). The organic phase was concentrated under vacuum and purified by column chromatography to afford Compound 2 as white solid using v(CH₃COOC2H5)/v(petroleum ether) = 1 :3 as the eluent (1.3g, 48%): mp 103-104°C; 'H NMR (400 MHz, DMSO): δ (ppm) 8.74 (s, 1H), 6.64-6.62 (d, 1H, J=7.9 Hz), 5.98-5.97 (d, 1H, J=2.1 Hz), 5.95-5.93 (dd, 1H, Jl=2.2 Hz, J2=7.9 Hz), 3.13-3.10 (t, 2H, J=5.6 Hz), 2.75 (s, 3H), 2.57-2.54 (t, 2H, J=6.4 Hz), 1.86-1.80 (m, 2H); ¹³C NMR (100 MHz, DMSO): δ (ppm) 156.4, 147.3, 128.9, 1 12.9, 102.9, 98.2, 50.5, 38.7, 26.5, 22.3.

[00182] 4-Hydroxy-9-methyl-6,7,8,9-tetrahydropyrano-[3,2-g]quinolin-2-one (3).

Compound 2 (1.0 g, 3.9 mmol) and diphenyl malonate (0.64 g, 3.9 mmol) were added into anhydrous toluene (8 mL). The mixture was heated to reflux for 6 h, and Compound 3 was obtained by filtration as gray solid (0.8 g, 88%): mp 255-257°C; ¾ NMR (400 MHz, DMSO): δ (ppm) 1 1.83 (s, 1H), 7.26 (s, 1H), 6.35 (s, 1H), 5.23 (s, 1H), 3.33-3.31 (t, 2H, J=5.6 Hz), 2.92 (s, 3H), 2.73-2.70 (t, 2H, J=6.1 Hz), 1.87-1.84 (t, 2H); ¹³C NMR (100 MHz, DMSO): δ (ppm) 166.4, 162.7, 154.7, 149.7, 121.7, 118.8, 103.2, 95.5, 86.0, 50.0, 38.5, 26.6, 21.2.

[00183] Compound 4. A solution of 3-diethylamino phenol (4.11 g, 30.0 mmol) and phthalic anhydride (4.66 g, 31.5 mmol) in toluene (30 mL) was refluxed under 2 for 3 h, and cooled to 50-60°C. Then 30mL of 35% aqueous NaOH (w/w) was added and heated at 90°C for 6 h. The resulting mixture was poured into H₂0 (300mL), acidified with HC1 (10.0 M), and allowed to stand at room temperature for 2 h. The suspension was then filtered. The solid was recrystallized from a mixture of water and methanol, and dried to afford the 2-(4- diethylamino-2-hydroxy)-benzoylbenzoic acid. ¾ NMR (500MHz, CD₃0D:CDC1₃ = 5: 1): δ (ppm) 1.15 (t, J=7.0 Hz, 6H), 3.40 (q, J=7.0 Hz, 4H), 6.09 (d, J=2.5 Hz, 1H), 6.15 (d, J=9.0 Hz, 1H), 6.84 (d, J=9.5 Hz, 1H), 7.35 (dd, J=7.5, 1.0 Hz, 1H), 7.58 (t, J=7.5 Hz, 1H), 7.66 (t, J=7.5 Hz, 1H), 8.03 (d, J = 8.0 Hz, 1H).

[00184] Compound 3 (659 mg, 2.9 mmol) and 2-(4-diethylamino-2- hydroxy)benzoylbenzoic acid (1.1 g, 3.5 mmol) were heated to reflux in 20 mL of 1, 1,2,2- tetrachloroethane until the substances have dissolved completely. A total of 4.0 g of phosphorus pentoxide was then added in portions, and the reaction mixture was heated to reflux for 4 h. After cooling, the mixture was dissolved using water (25 mL) and chloroform (25 mL), and the water phase was extracted with chloroform (total 100 mL for three times). The combined organic phases were concentrated under vacuum and purified by column chromatography using v(CH₂Cl₂)/v(CH₃OH) = 100: 1 as the eluent. The dye fractions were concentrated under vacuum, and the residue was dissolved in 3 mL ethanol. After addition of 5 mL perchloric acid (note: the operation should be executed very carefully due to the instability of perchlorate salts), the solution was precipitated by dropwise addition of water (100 mL). Compound 4 was obtained by filtration as dark green solid (720 mg, 42%): mp 289-290°C; ^XH NMR (400 MHz, DMSO): δ (ppm) 7.97-7.96 (m, 1H), 7.74-7.60 (m, 3H), 7.29-7.27 (m, 1H), 6.87 (s, 1H), 6.69 (s, 1H), 6.57 (s, 1H), 6.49 (s, 1H), 3.44 (s, 6H), 3.01 (s, 3H), 2.84-2.81 (t, 2H, J=5.8 Hz), 1.92 (s, 2H), 1.15-1.12 (t, 6H, J=6.8 Hz); ¹³C NMR (100 MHz, CD₃0D/CD₂C1₂): δ (ppm) 168.2, 162.9, 158.2, 157.9, 157.5, 155.7, 137.5, 133.5, 132.8, 131.7, 130.4, 130.3, 128.5, 124.3, 124.2, 1 16.4, 104.4, 101.2, 98.0, 96.7, 52.4, 47.2, 40.0, 27.8, 21.8, 12.8. [00185] MitoProbe 14. To a stirred solution of compound 4 (0.4 mmol) in DCM (10 mL) was sequentially added l-(prop-2-yn-l-yl)piperazine trifluoroacetate (130 mg, 0.55 mmol), HBTU (416mg, 1.1 mmol) and Ets (0.5 mL). The reaction mixture was stirred for 6 h room temperature. The solvent was then removed by distillation under reduced pressure, and the crude residue was purified via flash chromatograph to obtain the pure the intermediate.

[00186] To a solution of intermediate (0.75 mmol) and 177 mg (0.90 mmol) 4-azido- TEMPO in 3 ml acetonitrile was added 15.2 mg (0.08 mmol) copper (I) iodide at room temperature. The reaction mixture was stirred at room temperature for 6 h. The reaction mixture was washed with saturated aqueous NaHCC solution (3 x 10 mL) and then extracted with ethyl acetate (3 x30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated. The crude residue was purified by flash column chromatography on silica gel to yield the target mitoprobe 14.

[00187] MitoProbe 15. To a stirred solution of compound 4 (0.4 mmol) in DCM (10 mL) was sequentially added l-(piperazin-l-yl)undec-10-yn-l-one trifluoroacetate (prepared from 10-undecynoic acid with tert-butyl piperazine-l-carboxylate, 190 mg, 0.55 mmol), HBTU (416mg, 1.1 mmol) and Ets (0.5 mL). The reaction mixture was stirred for 6 h room temperature. The solvent was then removed by distillation under reduced pressure, and the crude residue was purified via flash chromatograph to obtain the intermediate.

[00188] To a solution of the intermediate (0.75 mmol) and 177 mg (0.90 mmol) 4-azido- TEMPO in 3 ml acetonitrile was added 15.2 mg (0.08 mmol) copper (I) iodide at room temperature. The reaction mixture was stirred at room temperature for 6 h. The reaction mixture was washed with saturated aqueous aHC0₃ solution (3 x 10 mL) and then extracted with ethyl acetate (3 x30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated. The crude residue was purified by flash column chromatography on silica gel to yield the target mitoprobe 15.

Example 11. Synthesis of MitoProbes 16 and 17.

(MitoProbe 16)

(MitoProbe 17)

[00189] Synthetic Scheme. The synthesis scheme for MitoProbes 16 and 17 is depicted in FIG. 35.

[00190] 4-Hydroxypyrano[3,2-g]julolidine-2-one (2). Hydroxyjulolidine (3.7 g, 19.3 mmol) and diphenyl malonate (4.1 g, 15.9 mmol) were added into anhydrous toluene (10 mL). The mixture was heated to reflux for 4 h, and compound 2 was obtained by filtration as a straw yellow solid (3.6 g, 88%): mp 268-270°C; ¾ NMR (400 MHz, DMSO): δ (ppm) 1 1.73 (s, 1H), 7.15 (s, 1H), 5.21 (s, 1H), 3.24-3.20 (m, 4H), 2.70 (s, 4H), 1.91-1.83 (m, 4H); ¹³C NMR (100 MHz, DMSO): δ (ppm) 166.5, 162.8, 151.0, 146.0, 119.8, 117.3, 105.3, 103.1, 85.8, 49.2, 48.7, 26.9, 21.0, 20.1, 20.0.

[00191] Compound 3. A solution of 3-diethylamino phenol (4.11 g, 30.0 mmol) and phthalic anhydride (4.66 g, 31.5 mmol) in toluene (30 mL) was refluxed under 2 for 3 h, and cooled to 50-60 °C. Then 30mL of 35% aqueous NaOH (w/w) was added and heated at 90°C for 6 h. The resulting mixture was poured into H₂0 (300mL), acidified with HC1 (10.0 M), and allowed to stand at room temperature for 2 h. The suspension was then filtered. The solid was recrystallized from a mixture of water and methanol, and dried to afford the 2-(4- diethylamino-2-hydroxy)-benzoylbenzoic acid. ¾ NMR (500MHz, CD₃0D:CDC1₃ = 5: 1): δ (ppm) 1.15 (t, J=7.0 Hz, 6H), 3.40 (q, J=7.0 Hz, 4H), 6.09 (d, J=2.5 Hz, 1H), 6.15 (d, J=9.0 Hz, 1H), 6.84 (d, J=9.5 Hz, 1H), 7.35 (dd, J=7.5, 1.0 Hz, 1H), 7.58 (t, J=7.5 Hz, 1H), 7.66 (t, J=7.5 Hz, 1H), 8.03 (d, J=8.0 Hz, 1H).

[00192] Compound 2 (1.2 g, 4.7 mmol) and 2-(4-diethylamino-2-hydroxy)benzoylbenzoic acid (1.8 g, 5.7 mmol) were heated to reflux in 50 mL of 1, 1,2,2-tetrachloroethane until the substances have dissolved completely. A total of 7.8 g of phosphorus pentoxide was then added in portions, and the reaction mixture was heated to reflux for 4 h. After cooling, the mixture was dissolved using water (25 mL) and chloroform (25 mL), and the water phase was extracted with chloroform (total 100 mL for three times). The combined organic phases were concentrated under vacuum and purified by column chromatography using v(CH₂Cl2)/v(CH₃OH) = 100: 1 as the eluent. The dye fractions were concentrated under vacuum, and the residue was dissolved in 5 mL of ethanol. After addition of 10 mL of perchloric acid (note: the operation should be executed very carefully due to the instability of perchlorate salts), the solution was precipitated by dropwise addition of water (150 mL). Compound 3 was obtained by filtration as dark green solid (982 mg, 33%): mp 290-292°C; ¾ NMR (400 MHz, DMSO): δ (ppm) 8.04-8.03 (m, 1H), 7.74-7.62 (m, 3H), 7.28-7.26 (m, 1H), 6.92-6.64 (m, 3H), 3.49-3.36 (m, 8H), 2.81-2.78 (m, 2H), 2.65-2.64 (m, 2H), 1.94-1.83 (m, 4H), 1.16-1.13 (t, 6H, J=6.9 Hz); ¹³C NMR (100 MHz, CD30D/CD2C12) δ (ppm) 167.1, 161.8, 157.4, 157.3, 156.5, 152.3, 152.0, 136.3, 132.5, 131.8, 130.9, 129.4, 129.3, 127.6, 122.3, 122.1, 115.2, 1 15.1, 106.3, 103.5, 100.0, 96.9, 50.8, 50.3, 46.2, 27.2, 20.6, 19.6, 19.5, 11.9.

[00193] MitoProbe 16. To a stirred solution of compound 3 (0.4 mmol) in DCM (10 mL) was sequentially added l-(prop-2-yn-l-yl)piperazine trifluoroacetate (130 mg, 0.55 mmol), HBTU (416mg, 1.1 mmol) and Ets (0.5 mL). The reaction mixture was stirred for 6 h room temperature. The solvent was then removed by distillation under reduced pressure, and the crude residue was purified via flash chromatograph to obtain the pure the intermediate.

[00194] To a solution of intermediate (0.75 mmol) and 177 mg (0.90 mmol) 4-azido- TEMPO in 3 ml acetonitrile was added 15.2 mg (0.08 mmol) copper (I) iodide at room temperature. The reaction mixture was stirred at room temperature for 6 h. The reaction mixture was washed with saturated aqueous NaHC0₃ solution (3 x 10 mL) and then extracted with ethyl acetate (3 x30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated. The crude residue was purified by flash column chromatography on silica gel to yield the target mitoprobe 16.

[00195] MitoProbe 17. To a stirred solution of compound 3 (0.4 mmol) in DCM (10 mL) was sequentially added l-(piperazin-l-yl)undec-10-yn-l-one trifluoroacetate (prepared from 10-undecynoic acid with tert-butyl piperazine-l-carboxylate, 190 mg, 0.55 mmol), HBTU (416mg, 1.1 mmol) and Et₃N (0.5 mL). The reaction mixture was stirred for 6 h room temperature. The solvent was then removed by distillation under reduced pressure, and the crude residue was purified via flash chromatograph to obtain the intermediate.

[00196] To a solution of the intermediate (0.75 mmol) and 177 mg (0.90 mmol) 4-azido- TEMPO in 3 ml acetonitrile was added 15.2 mg (0.08 mmol) copper (I) iodide at room temperature. The reaction mixture was stirred at room temperature for 6 h. The reaction mixture was washed with saturated aqueous NaHCC solution (3 x 10 mL) and then extracted with ethyl acetate (3 x30 niL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated. The crude residue was purified by flash column chromatography on silica gel to yield the target mitoprobe 17.

Example 12. Synthesis of MitoProbes 18 and 19.

(MitoProbe 19)

Synthetic Scheme. The synthesis scheme for MitoProbes 18 and 19 is depicted [00198] N-((lE)-3-(phenylimino)prop-l-enyl)benzenamine (3). A solution of distilled water (200 mL), HC1 (40 mL), and aniline (30 g, 0.32 mol) was added dropwise to a solution of distilled water (200 mL), HC1 (35 mL), and 1, 1,3,3-tetramethoxypropane (40 g, 0.24 mol) with stirring at 50°C. The precipitate was isolated by filtration and washed again with ether to give 3 (26 g, 49 %) as a brown-red powder. ¾ NMR (400 MHz, DMSO-d₆) δ: 6.45 (t, J=1 1.2 Hz, 1H), 7.21 (m, 2H), 7.37-7.46 (m, 8H), 8.83 (t, J=12.4 Hz, 1H).

[00199] 2,3,3-trimethyl-3H-indole (5). To a mixture of phenylhydrazine (32.4 g, 0.3 mol), glacial acetic acid (250 mL) was added 3-methylbutanone (50 mL, 0.36 mol). The mixture was heated at reflux for 5 h and then was cooled to room temperature. After removed the volatile components under vacuum, the residue was partitioned between ether (350 mL) and water (200 mL). The aqueous phase was washed again with ether, and then the combined organic solutions were dried (Na₂S0₄), filtered, and evaporated under vacuum to afford 5 (39 g, 81%). The material was used without further purification. ^XH NMR (400 MHz, CDC1₃) δ: 1.25 (s, 6H), 2.23 (s, 3H), 7.14 (m, 1H), 7.22-7.27 (m, 2H), 7.49 (d, J=8.0 Hz, 1H);

[00200] l-ethyl-2,3,3-trimethyl-3H-indolium Iodide (6). To a solution of 5 (16 g, 100 mmol) in acetonitrile (40 mL) was added ethyl iodide (20 mL). The mixture was heated at reflux under argon for 48 h, and then most of the volatile components were removed by distillation. The residue was triturated with ether (100 mL) to give a powder. The solid was filtered off, washed with ether, and dried to afford 6 as an orange solid (16 g, 85%). ¾ NMR (400 MHz, CDCI₃) δ: 1.54-1.58 (m, 9H), 3.06 (s, 3H), 4.66 (m, 2H), 7.51 (m, 3H), 7.54 (m, 1H).

[00201] l-(4-pentynyl)-2,3,3-trimethyl-3H-indolium iodide (7). Potassium iodide (11.50 g, 69.08 mmol, 2.2 eq) was suspended in acetonitrile (50 mL), then 5-chloro-l- pentyne (5.0 mL, 31.40 mmol, 1 eq) was added and the yellow suspension was stirred at 50°C. After 10 min compound 6 (5.05 mL, 31.40 mmol, 1 eq) was added dropwise and the reaction mixture was heated at reflux overnight then cooled to room temperature. The inorganic salt was filtered off, washed with DCM, and the filtrate was evaporated to dryness. The residue was purified by column chromatography (0-10 % MeOH in DCM) and the pure product 7 was obtained as a purple solid in 58 % yield (6.41 g, 18.15 mmol). ^XH NMR (400 MHz, CD₃OD) δ: 7.96 - 7.90, 7.82 - 7.77 (1 H, m), 7.70 - 7.63 (2 H, m), 4.66 (2 H, dd, J=8.3, 7.0 Hz), 2.52 - 2.45 (3 H, m), 2.26 - 2.16 (2 H, m), 1.64 (6 H, s); ¹³C NMR (100 MHz, CD₃OD) δ: 198.6, 143.5, 142.7, 131.4, 130.6, 124.9, 116.6, 83.3, 72.1, 56.2, 49.2, 27.7, 23.0, 16.7.

[00202] 2-[5-(2,3-Dihydro-l-(4-pentynyl)-3,3-dimethyl-lH-indol-2-ylidene)-l,3- pentadienyl]-l,3,3-trimethyl-3H-indolium Iodide (9). l-(4-Pentynyl)-2,3,3-trimethyl-3H- indolium iodide 7 (1.76 g, 5.0 mmol, 1 eq) and compound 3 (1.68 g, 6.50 mmol, 1.3 eq) were suspended in acetic anhydride (17 mL) and heated at reflux under argon. After 30 min the reaction mixture was cooled to room temperature and the solution of compound 6 (3.11 g, 10.0 mmol, 2 eq) in anhydrous pyridine (21 mL) was added and the reaction mixture stirred under argon at ambient temperature for 15 h. After precipitation with cold diethyl ether, the green solution was decanted off and the precipitate was purified by column chromatography (0-5 % MeOH in EtOAc) to give the product 9 as a green-gold foam.

[00203] Compound 10. To a stirred solution of compound 9 (2.5 mmol) in THF: H₂0: t- BuOH (30 mL, 3 : 1 : 1) was sequentially added 2-azidoethyl 4-methylbenzenesulfonate (0.72 g, 3 mmol), Na L-ascorbate (1 g, 0.5 mmol) and Q1SO₄. The reaction was allowed to stir for 6h at room temperature. After evaporation, the residue was dissolved in EtOAc and washed with water. The combined organic layers were dried over a₂S0₄ and filtered. The filtrate was concentrated to dryness under reduced pressure and the residue was purified via flash chromatograph to give the desired compound 10.

[00204] Compound 11. To a stirred solution of compound 10 (1.11 mmol) in DMF (20 mL) was added NaN₃ (130 mg, 2.0 mmol). The reaction was heated at 1 10°C overnight. After cooling to room temperature, the reaction mixture was poured to ice-water and stirred for 0.5 hr, and then diluted with EtOAc, and washed with water and aq aHC0₃. The organic layer was dried over a₂S0₄, and the filtrate was concentrate and puritied by silica column chromatography to give the desired compound 11.

[00205] Compound 12. To a stirred solution of compound 11 (0.93 mmol) in THF: H₂0: ?-BuOH (20 mL, 3 : 1 : 1) was sequentially added tripropargylamine (288 mg, 2.2 mmol), Na L- ascorbate (600 mg, 3 mmol) and CuS0₄. The reaction was allowed to stir overnight at room temperature. After evaporation,the residue was dissolved in EtOAc and washed with water. The combined organic layers were dried over a₂S0₄ and filtered. The filtrate was concentrated to dryness under reduced pressure and the residue was purified via flash chromatograph to give the desired compound 12.

[00206] NIR-based MitoProbe 18. To a stirring suspension of NaH (600 mg, 15 mmol, 60 % dispersion in mineral oil) in dry DMF (30 mL) was added 4-hydroxytempo (2 g, 1 1.6 mmol) in 30 ml DMF at 0°C followed by stirring at room temperature for 30 min. Propargyl bromide (1.4 mL) was added dropwise at 0°C. The resulting mixture was stirred for 3 h at room temperature. The reaction mixture was washed with brine and extracted with EtOAc (30 mL x 3). The combined organic layers were dried over anhydrous Na₂S04, filtered and concentrated. The crude residue was purified by flash column chromatography on silica gel (EtOAc:Hexane, 1 :5) to yield propargyl ether TEMPO as an orange solid (1.63 g, 69 %). ^XH NMR (400 MHz, CDC1₃) δ: 1.15 (s, 6H), 1.20 (s, 6H), 1.43 (t, J=11.7 Hz, 2H), 1.91 (dd, J=0.6 Hz, J=3.8 Hz, 2H), 2.42 (s, 1H), 3.83 (tt, J=1.2 Hz, J=3.8 Hz, 1H), 4.16 (s, 2H). ¹³C NMR (100 MHz, CDC1₃)5: 20.7, 32.6, 44.7, 55.5, 59.3, 70.0, 74.5, 80.4.

[00207] To a stirring solution of propargyl ether TEMPO (1 g, 1.73 mmol) and compound 11 (2.30 mmol) in anhydrous acetonitrile was added Cul (33 mg, 10 mmol %). The reaction mixture was stirred for 6 h at room temperature until TLC indicated that the starting material was consumed. The reaction was washed with saturated aq aHC0₃ and then extracted with EtOAc (30 mL x 3). The combined organic layers were dried over anhydrous Na₂S0₄, filtered and concentrated. The crude residue was purified by flash column chromatography on silica gel to yield the desired MitoProbe 18.

[00208] NIR-based MitoProbe 19. To a solution of the compound 12 (0.75 mmol) and 177 mg (0.90 mmol) 4-azido-TEMPO in 3 ml acetonitrile was added 15.2 mg (0.08 mmol) copper (I) iodide at room temperature. The reaction mixture was stirred at room temperature for 6 h. The reaction mixture was washed with saturated aqueous NaHC0₃ solution (3 x 10 mL) and then extracted with ethyl acetate (3 x30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated. The crude residue was purified by flash column chromatography on silica gel to yield the target mitoprobe 19.

Example 13. Materials and Methods for Examples 14-25.

[00209] Hind Limb Ischemia-Reperfusion Injury Model. Animals were housed in cages in a temperature and humidity-controlled room, and they were fed rat chow and water ad libitum. Male Wistar rats were randomly allocated into 3 groups: (1) Sham group: rats subjected to the procedures described below, except for ischemia/reperfusion (LR) (n=6); (2) I/R group: rats subjected to the procedures described below underwent limb ischemia for 3h followed by reperfusion for 4 hours (n=8). All animals were maintained under anesthesia for the duration of the experiment.

[00210] Male Wistar rats weighing 250-300g were anesthetized with an intraperitoneal injection of sodium pentobarbital (80mg/kg). Throughout the experiments body temperature was maintained at 37°C with the aid of a heating pad. Unilateral rubber bands were applied above the greater trochanter to interrupt the arterial blood supply to the hind limbs. After 3 h of hind limb ischemia, the rubber bands were removed, thereby initiating hind limb reperfusion for 4 h. At the end of all experiments, rats were sacrificed by a sodium pentobarbital overdose.

[00211] Tissues were taken immediately and then subjected to snap-frozen using liquid nitrogen. Subsequently, the tissue sections were prepared using a cryostat microtome (Leica CM1850 UV clinical cryostat) at -30°C for tissue staining and analyzed by confocal fluorescent microscope.

[00212] SOD-l^G93A Mice. Transgenic mice over-expressing mutant SOD-l^G93A and B6SJLF1 hybrids were purchased from Jackson Laboratory (Bar Harbor, ME). The animals were housed in a room with 12 h-dark/12 h-light cycle and provided with free access to water and diet (standard diet purchased from Beijing Vital River Experimental Animal Co. Ltd, Beijing, China). Transgenic SOD-l^G93A mice and their non-trans genie littermates were generated by breeding male hemizygous carriers (B6SJL-Tg (SOD-l^G93A) IGur/J) to female B6SJLF1 hybrids. PCR-based genotyping of tail DNA was used to identify the transgenic mice. This transgenic mouse line expressed high copy number of mutant SOD-l^G93A and showed a rapid disease onset and progression.

[00213] To monitor disease progression, the animals were subjected to a tail suspension test daily and weighed twice a week from 12 weeks of age. When the earliest gait abnormalities were presented or two successive weight losses were recorded or when an animal could not remain on the rotating cylinder (d = 30 mm) of a Rota-Rod apparatus at a constant speed of 30 rpm (YLS-4C Rota-Rod, Shandong Academy of Medical Sciences) for 90 s, it was concluded that disease onset was reached. The lumbar spinal cord from clinical onset of SOD-l^G93A and age-matched non-trans genie littermates were used for comparative study. The animals were considered as end stage and sacrificed when they were unable to right themselves within 20 s after being placed on either side or their back.

[00214] EPR Spectroscopy. 1 mM stock solution of MitoProbe I/II was prepared in DMSO, and then 100 μΜ final concentration was prepared by diluting with Ι¾0. The EPR signal was then measured. After addition of 1 mM final concentration of sodium L-ascobate, EPR signal was measured. In presence of lower concentrations of sodium L-ascorbate, signal recovery was observed. [00215] Cell Culture and Growth Media. ARPE-19 and HeLa cell lines were obtained from American Type Culture Collection (ATCC). Cells were grown in Eagle's Minimal Essential Medium (EMEM) and 10% FBS (Sigma-Aldrich, heat inactivated). The retinal pigment epithelial cell line, ARPE-19, was grown in DMEM/Ham's F12 1 : 1 (Hyclone, Fisher Sci.) containing 10% FBS (Sigma-Aldrich, heat inactivated). All cells were maintained in a 5% CO₂ humidified atmosphere at 37°C.

[00216] Tissue Preparation. Twenty-four female animals (12 SOD-l^G93A mice and 12 littermates, 4 per group) were anesthetized with 10% chloral hydrate (0.2 mL/mouse) and perfused transcardially with 4% paraformaldehyde and 0.05% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. The spinal cords were carefully dissected. The lumbar enlargements (L3-5) were fixed in 4% paraformaldehyde, or incubated in 4% glutaraldehyde for 24 h and then in 1% osmium tetroxide for 1 h. Frozen human breast and colon cancer tissues were purchased from OriGene Technologies Inc.

[00217] Live Cell Imaging. Cells were grown in a 35 mm glass bottom dish (Mattek Glass Bottom Dishes) in media for 24 h. The media was removed and cells were washed three times with DPBS without Ca²⁺ or Mg²⁺ (Hyclone, Fisher Scientific). MitoProbes were dissolved in DMSO and diluted with PBS buffer to get stock solution. After incubation with fluorescent dyes, cells were washed with DPBS buffer. Then, cells were imaged in non- phenol red and non-FBS media.

[00218] Molecular Dynamics Simulation. Molecular dynamics evaluations were performed based on an MM2 force field (CambridgeSoft Chem & Bio 3D 12.0). Some parameters employed included the following: Step Interval: 2.0 fs; Frame Interval: 10 fs; Terminate After: 10000 steps; Heating/Cooling Rate: 1.000 Kcal/atom/ps; Target Temperature: 300°K.

Example 14. Analysis of MitoProbes I and II.

[00219] Thus, the synthesized fluorescent probes (MitoProbe I/II) that contained both cationic and hydrophobic residues provided an electrostatic driving force for uptake through mitochondrial membranes (FIG. 1). MitoProbes were analyzed by EPR spectroscopy to further confirm the presence of the intact nitroxide label (FIG. 2).

Example 15. Intracellular Localization of MitoProbe I/II.

[00220] Compared to non-neoplastic cells, oxidative stress is significantly enhanced in tumor cells. Multiple human tumor cell lines produce large amounts of ROS in vitro. ROS generation by tumor cells may help them mutate or display other malignant properties such as tissue invasion. To analyze the exact intracellular localization of MitoProbe I/II inside the cells, counter staining with a mitochondria-specific fluorescent marker MitoTracker (Life Technologies) was performed in living HeLa (human cervical cancer) cells. HeLa cells were treated with MitoProbe I/II for 45 min, and followed by washing with DPBS buffer. HeLa cells were then stained with MitoTracker FM, a well-established mitochondrial dye, and subjected to confocal fluorescence microscopy. A high proportion of overlay between MitoProbe I/II and MitoTracker was clearly visible, indicating a good degree of co- localization between the two stains (FIG. 3). This result was a good indication that MitoProbe I/II was indeed localizing in mitochondria after 45 min. incubation in the cell. MitoProbe I/II was punctately localized in the mitochondria, which was due to the presence of the membrane-permeable and cationic rhodamine moiety, because of the highly inside- negative membrane potential across the inner mitochondrial membrane.

Example 16. Detection of Mitochondrial Oxidative Stress in Living Human Retinal Pigment Epithelial (ARPE) Cells under Stress Condition.

[00221] Oxidative stress is believed to contribute to the pathogenesis of many diseases, including age-related macular degeneration (AMD). The retina is particularly susceptible to oxidative stress because of its high consumption of oxygen, its high proportion of polyunsaturated fatty acids, and its exposure to visible light. Although the vision loss of AMD results from photoreceptor damage in the central retina, the initial pathogenesis involves degeneration of retinal pigment epithelial cells. Herein, ARPE- 19 cells (human retinal pigment epithelial cells) were used to verify the ability of the MitoProbes to detect mitochondrial oxidative stress in a biological environment. First, the effect of phorbol 12- myristate 13 -acetate (PMA) stimulation upon MitoProbe I/II in ARPE- 19 cells was investigaged. Phorbol esters are involved with redox-sensitive promoter regions. Upon simulation with PMA, the cytosolic phox proteins translocated and associated with the membrane components leading to the activation of NADPH oxidase. Superoxide (0₂ ^") is first produced by activated NADPH oxidase. Subsequently, the initially formed ( is converted into other reactive oxygen species (ROS), such as ¾(¾, ^'OH, ^lC>2 and hypochlorous acid (HOC1). Treatment with 0.19 μΜ PMA resulted in a 7-8 fold elevation of ROS levels (FIG. 4). This was similar to the ROS increase that occurred in transient ischemia-related disease such as central retinal artery occlusion, angle-closure glaucoma, and carotid artery disease. Furthermore, bright-filed transmission measurements after MitoProbe I/II incubation and PMA treatment confirmed that the cells are viable throughout the experiments. These results demonstrated that MitoProbe I/II can passively enter live cells and monitor free radicals generation in the mitochondria.

Example 17. Mitochondria undergo Distinct Morphological Changes under Oxidative Stress.

[00222] Mitochondrial morphology changes to adapt an oxidative environment are critical for the survival of retinal and retinal pigment epithelial (RPE) cells, because of their high oxygen demand and up-regulated metabolism. Little is known about the consequences of a short-term ROS boost on the cellular and especially on the mitochondrial level. Next, the effect of oxidative stress on mitochondrial morphology in human retina pigment epithelial (ARPE) cells was investigated. To determine how the mitochondria of ARPE cells were affected by oxidative stress, the mitochondrial morphology was visualized with MitoProbes I/II and monitored by confocal fluorescent microscope. Mitochondria underwent distinct morphological changes under oxidative stress. After PMA treatment, the mitochondria morphology of the ARPE- 19 cells was visualized by MitoProbes and was classified into three categories: tubular (close to normal), intermediate (tubular with swollen regions) and fragmented (small and globular) (FIGS. 5-7). The cellular viability was verified with DIC images (FIGS. 8 and 9).

[00223] Without PMA treatment, ARPE-19 cells had tubular mitochondria and exhibited weak rhodamine fluorescence from MitoProbes (FIGS. 6 and 7, row 1). After treatment with low concentration of PMA (0.5 μΜ), a proportion of the cells appeared to have enhanced mitochondrial fluorescence, with MitoProbe VII being restricted to a dispersed network of tubular structure around the nucleus (FIGS. 6 and 7, row 2). The identification of intracellular localizations of MitoProbe I/II in ARPE- 19 cells as mitochondria was further confirmed by double labeling with MitoTracker. In contrast to the tubular staining pattern observed in the cells treated with low concentration of PMA, a more dispersed and irregular staining pattern with strong mitochondrial fluorescence was detected in the ARPE- 19 cells treated with higher concentration of PMA (FIGS. 6 and 7, row 3-6). Large amounts of reactive oxygen species (ROS) were produced by activated NADPH oxidase stimulated with PMA. ROS level correlated positively with the fluorescence intensity of MitoProbe I/II. The correlation between the concentration of PMA and the severity of mitochondrial fragmentation in ARPE-19 cell lines was also investigated. A significant positive correlation between the concentration of PMA and the percentage of ARPE-19 cells with fragmented mitochondria was observed. Overall, ARPE- 19 cells treated with a higher concentration of PMA had more mitochondria with fragmented and punctiform morphology than the cells treated with lower concentration of PMA.

[00224] Non-treated ARPE cells contained mostly long tubular mitochondria, distributed evenly throughout the whole cell. With increasing the PMA concentration, mitochondria displayed intermediate/fragmented structure, indicating that oxidative stress induced mitochondrial fragmentation. I ntermediate mitochondria and the fragmented mitochondria mainly accumulated in perinuclear region in ARPE-19 cells. With increasing concentration of PMA, the amount of ARPE-19 cells with intermediate and fragmented mitochondria was significantly increased.

Example 18. Mitochondria Recover Partially after Treatment with Nonlethal

Concentration of PMA.

[00225] The mitochondrial change of ARPE cells in response to PMA stimulation was further examined. A protocol in which ARPE cells were activated with PMA and then rested in culture without PMA was employed. A 2h PMA treatment, followed by resting in culture without PMA, induced significant mitochondria morphology alterations: transition from the "spaghetti like" pattern of the ARPE cells to vacuolar structures to tubular structures again (FIGS. 10 and 1 1). Interestingly, the mitochondrial morphology started to recover after 24h resting in culture without PMA. The fragmented mitochondria were maintained longer than the intermediate mitochondria, indicating that intermediate mitochondria were less damaged and recovered earlier.

[00226] After 24h resting in culture without PMA, ARPE-19 cells exhibited tubular mitochondria again, implying that a low concentration of PMA treatment transiently altered the mitochondrial morphology. Recovery of the mitochondrial morphology was caused by fusion of tubular mitochondria, synthesis of new mitochondria or both. However, when ARPE-19 cells were treated with a higher concentration of PMA, ARPE-19 cells contained significantly damaged mitochondria and exhibited slower recovery than the cells treated with low-concentration of PMA. This result indicated that oxidative damage induced by a continuous higher concentration of PMA had been too strong for the mitochondrial repair system to recover.

[00227] Changes in mitochondrial morphology have been linked to apoptotic cell death. In response to oxidative stress, cells undergo mitochondrial fission, generating fragmented mitochondria. However, the presence of mitochondrial fragmentation does not necessarily indicate that the cell is undergoing apoptosis. Accordingly, the cellular viability was verified with DIC images (FIGS. 12 and 13).

[00228] Mitochondrial morphology depends on the balance between mitochondrial fission and fusion, and is controlled by multiple proteins that mediate remodeling of the outer and inner mitochondrial membrane. In this assay, mitochondrial morphology recovery partially after 24h resting in culture without PMA was observed. This data indicated that oxidative stress following exposure of cells to PMA in a nonlethal concentration rapidly induced time- and dose- dependent morphology change of mitochondria (FIGS. 6, 7, 10, and 11). Unbalanced fusion led to mitochondrial elongation, and unbalanced fission led to excessive mitochondrial fragmentation, both of which impair mitochondrial function. However, these mitochondrial morphology alterations were all reversible as long as cells were exposed to nonlethal amounts of PMA. The mitochondrial fission and fusion acted as a rescue mechanism for the recovery of damaged mitochondria.

Example 19. Imaging Mitochondrial Oxidative Stress in Ischemia/Reperfusion Injury.

[00229] Mitochondria are dynamic organelles that undergo continual fusion and fission to maintain their structure and functions. Imbalanced fission-fusion of mitochondria is involved in many pathological processes, including neuronal injury, muscle atrophy and ischemia-reperfusion injury. Mitochondrial morphological abnormality has frequently been observed in disease conditions. The probes previously developed are inadequate for imaging of oxidative stress, because of the low sensitivity response to oxidative stress and/or use of short wavelengths of fluorescent probes that potentially renders living cells and tissues vulnerable.

[00230] Accordingly, the MitoProbes were examined for use in imaging mitochondrial oxidative stress induced by ischemia/reperfusion (I/R) injury. In this assay, a rubber band tourniquet model was utilized to induce limb I/R damage in rats, an animal model widely employed to mimic the clinical setting of acute I/R damage. This model has clinical relevance because tourniquet application was broadly employed in a variety of surgical protocols in order to ensure a bloodless surgical field. The severity of mitochondrial oxidative stress was evaluated using MitoProbe I/II. As shown in FIGS. 14-16, the fluorescence intensities of the mitochondria were significantly different among the tissues from sham and I/R injury group rats.

[00231] In skeletal muscle from I/R group rats, the mitochondria with strong fluorescence appeared to be contained within membranous compartments along the myofibers (FIG. 14). There was a massive swollen mitochondria with irregular distribution that was observed in the skeletal muscle taken from I/R groups rats, which was indicative of widespread mitochondrial damage. To extend the studies beyond the mitochondria damage that occurred in local tissues induced by limb I R, mitochondrial morphological changes in remote organs were also examined.

[00232] In other tissues, mitochondrial sizes varied greatly among the different cell types. In liver from ischemia/reperfusion group rats, the enlarged mitochondria presented with a less clear envelope and strong fluorescence, and were mainly located in more perinuclear region (FIG. 15). In kidney from I/R group rats, a large number of mitochondria with strong fluorescence were observed in the interdigitations of the tubules because the mitochondria in this region are more sensitive to ischemia/reperfusion injury.

[00233] In addition, the mitochondrial morphology was very different between the damaged convoluted tubules and glomus. Larger mitochondria were observed in cells of convoluted tubules and smaller mitochondria were observed in cells of the glomus (FIG. 16). Tissues from the sham group rats exhibited weak mitochondrial fluorescence using the same image settings. These findings indicated that limb I/R injury also led to secondary mitochondrial oxidative damage to other remote organs. Large amount of ROS are produced in the ischemic tissue, and upon tourniquet release, these ROS exacerbated endothelial injury, increased microactivated permeability, and tissue edema. Thus, beyond the regional injury, limb I R injury also resulted in multiple remote organ failure, which maybe mediated via free radical accumulation and inflammatory cytokines.

Example 20. Imaging Mitochondrial Oxidative Stress in Amyotrophic Lateral Sclerosis (ALS).

[00234] Mitochondrial oxidative stress also plays an important role in the pathogenesis of various neurodegenerative diseases. ALS is an age-related neurodegenerative disease that mainly affects motor neurons in cortex, brain stem, and spinal cord. Little information is available regarding the morphological changes in the mitochondria in amyotrophic lateral sclerosis (ALS). S0D1^G93A mice developing a phenotype similar to that of ALS patients were used in the present study. SOD1 transgenic mice are the most widely used animal model of amyotrophic lateral sclerosis.

[00235] Through the use of MitoProbe LII, the oxidative stress and morphology changes of mitochondria in amyotrophic lateral sclerosis was assessed. Confocal laser-scanning fluorescent images of semi-thin tissue sections revealed the overall redox status and morphology changes of mitochondria in SOD 1 mice. As shown in FIG. 17, mitochondria with strong fluorescence were observed in a dotted pattern within motor neurons of the spinal anterior horns of onset stage S0D 1^G93A mice and were comprised of a mix of both tubular and punctate morphologies. In addition, the rounded swollen mitochondria with irregular distribution mitochondria and strong fluorescence were also observed in a few small interneurons of onset stage S0D1^G93A. The nuclei of all the cells were always euchromatic and no noticeable morphology changes were observed. Using MitoProbes, the changes in the mitochondrial morphology and vacuolization during the progression of the disease were observed.

[00236] In fact, mitochondrial swelling and vacuolization were common at the clinical onset stage in S0D1^G93A mice. The mitochondria in motor neurons were becoming less tubular and increasingly round (FIG. 17). The mitochondrial degeneration occurred much earlier than the death of motor neurons. Massive vacuolization of cytoplasm and swollen mitochondria were commonly observed in the motor neuron of transgenic S0D1^G93A mice, suggesting that mitochondrial pathology was an early preclinical feature of motor neuron damage in these ALS mice.

[00237] ALS is a disease mainly involved in motor neurons degeneration, which leads to muscle atrophy and paralysis. It is generally thought that muscle alterations in ALS patients are due to motor neuron loss in the periphery. In the present assay, histological examination of the skeletal muscle from the end stage S0D1^G93A mice showed the characteristic features of neurogenic atrophy including small or larger groups of elongated atrophic muscle fibers. In addition, the typical morphological hallmarks of mitochondrial myopathies such as "ragged red fibers" or SDH hyper-reactive fibers were also observed at a low frequency.

[00238] With visualization of the skeletal muscle samples using MitoProbe I/II, it was observed that the mitochondrial defects in muscle tissues taken from the end-stage SOD1G93A mice were distributed heterogeneous ly (FIG. 18). Interestingly, mitochondrial dysfunction seemed detectable not only in the spinal cord mitochondria but also in the skeletal muscle, indicating that mitochondrial functional abnormalities in S0D1^G93A mice at the endstage might be systemic. Muscle mitochondrial dysfunction generally occurred late in the pathogenesis of ALS. The occurrence of mitochondrial morphological abnormalities in the muscle might simply be the late consequence of muscle denervation and not the pathological stimulator activating it. Example 21. Mitochondrial Redox Imaging of Cancer Metastatic Potential.

[00239] Intracellular free radical species are known to attack mitochondrial proteins and DNA and to inhibit the activities of specific mitochondria enzymes. This may cause the inhibition of the mitochondrial electron transport chain and the mitochondrial morphology changes. ROS are involved in tumor initiation and progression. Aberrant ROS generation can result in accumulated DNA damage, which increases susceptibility towards the onset of cancer. Cancer cells, myofibroblasts, macrophages and neutrophils are thought to be the largest producers of reactive oxygen species. Mitochondria play an important role in tumor formation and cancer pathogenesis, thus establishing a direct link between mitochondrial oxidative stress and tumor pathogenesis.

[00240] Next, confocal laser-scanning fluorescence microscopy of fresh tumor slices following incubation with MitoProbe I/II for 45 min (FIG. 19) was used. The fluorescence intensity of MitoProbe I/II was more pronounced in selected tumor areas, although present in all tumor regions to varying extents. In some regions, concentrated localization of MitoProbes appeared to be giant mitochondria. In some cells, the sizes of mitochondria were observed to be much smaller in comparison with neighboring cells. It seemed that mitochondria were broken down into smaller fragments during apoptosis. However, it was not clear whether mitochonrial fragmentation has a significant impact on the rate of cell death, or merely as a phenomenon accompanies with apoptosis. This result may be explained by increased mitochondrial oxidative damage in the more aggressive (metastatic) tumor areas as compared to those areas that are less aggressively expanding.

[00241] Mitochondrial oxidative stress and functional abnormalities have been associated with tumorgenesis and progression to metastasis. Mitochondrial redox state was used as potential indicator for cancer metastatic potential, and thus MitoProbes can be used to differentiate the tumors with different metastatic potentials by imaging the in vivo mitochondrial redox states of tumor tissues. Although further characterization is needed, it could be concluded that MitoProbe I II was useful for diagnosis of tumors in their early stages, as well as assisting in differentiation of more aggressive lesions through evaluation of the mitochondrial morphology and mitochondrial redox states of tumor tissues.

Example 22. MitoProbe I/II Staining of Live Cells and Fixed, Permeabilized Cells.

[00242] The fluorescence of rhodamine 123 almost completely disappears upon fixation of stained cells. Accordingly, the effect of cell fixation and acetone permeabilization on fluorescence intensity of HeLa cells stained with MitoProbe VII was investigated. The fluorescence intensity of MitoProbe LII was well-retained after HeLa cell fixation with formaldehyde and acetone treatment (FIG. 20).

Example 23. Molecular Dynamic Simulation.

[00243] When fluorescent and nitroxide probes are combined into one molecule, this yielded a non- or weakly fluorescent compound because of the quenching of the excited singlet state of aromatic fluorescent compounds by the nitroxide moiety. MitoProbe I contained a relatively rigid linker which was capable to ensure electron exchange between the nitroxide and the fluorophore "through bond" (FIG. 21).

[00244] MitoProbe II contained a relatively flexible linker that provided the interaction between nitroxide and fluophore upon "through space" formation of the collision complex. In a biological environment, nitroxides underwent one electron reduction and oxidation, which provided the feature of their free radical scavenging capability. The free radical scavenging effect of nitroxide derivatives was correlated with selected molecular and biochemical parameters such as the highest occupied molecular orbital (HOMO) energy, the net charge, and the difference in heat of formation between hydroxylamine and its radical.

[00245] Along these lines, the ionization energy of the HOMO was employed as a measure of a free radical scavenger's capacity to participate in radical scavenging reaction. For example, the HOMO energy of melatonin (a natural antioxidant) was -10.425 eV. In comparison, the HOMO energy of MitoProbes was as follows: E_HOMO (MitoProbe I) = -4.983 eV and EHOMO (MitoProbe II) = -5.224 eV. The higher the HOMO energy, the more active the compound was as a free radical scavenger. MitoProbes possessed much higher radical trapping potential than melatonin. MitoProbe I was a slightly more active free radical scavenger than MitoProbe II. However, there were no statistically significant differences between these two fluorescent probes in the studies described above.

Example 24. MitoProbe VI Staining of Cells.

[00246] As discussed above in Example 15, MitoProbes Vll were localized to in the mitochondria. To determine if MitoProbe VI could localize in the mitochondria, HeLa (human cervical cancer) cells were incubated with MitoProbe VI. In particular, to analyze the exact intracellular localization of MitoProbe VI inside the cells, counter staining with a mitochondria-specific fluorescent marker MitoTracker (Life Technologies) was performed in living HeLa cells. HeLa cells were treated with MitoProbe VI for 45 min, and followed by washing with DPBS buffer. HeLa cells were then stained with MitoTracker FM, a well- established mitochondrial dye, and subjected to confocal fluorescence microscopy. A high proportion of overlay between MitoProbe VI and MitoTracker was visible, indicating a good degree of co-localization between the two stains (FIG. 23). This result was a good indication that MitoProbe VI was localizing in mitochondria after 45 min. incubation in the cell.

Example 25. MitoProbe VII Staining of Cells.

[00247] As discussed above in Examples 15 and 24, MitoProbes I/II and VI were localized to in the mitochondria. To determine if MitoProbe VII could localize in the mitochondria, HeLa (human cervical cancer) cells were incubated with MitoProbe VII. In particular, to analyze the exact intracellular localization of MitoProbe VII inside the cells, counter staining with a mitochondria-specific fluorescent marker MitoTracker (Life Technologies) was performed in living HeLa cells. HeLa cells were treated with MitoProbe VII for 45 min, and followed by washing with DPBS buffer. HeLa cells were then stained with MitoTracker FM, a well-established mitochondrial dye, and subjected to confocal fluorescence microscopy. A high proportion of overlay between MitoProbe VII and MitoTracker was visible, indicating a good degree of co-localization between the two stains (FIG. 24). This result was a good indication that MitoProbe VII was localizing in mitochondria after 45 min. incubation in the cell.

[00248] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the invention, which is defined solely by the appended claims and their equivalents.

[00249] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the invention, may be made without departing from the spirit and scope thereof.

Claims

1. A compound according to Formula (I):

(I)

wherein

A is a fluorophore;

W is a direct bond, -C(0)NH-, or

;

L is a linker;

n 1 or 2.

2. A compound according to claim 1, wherein the linker comprises at least one triazole.

3. A compound according to any one of claims 1 or 2, wherein at least one triazole is a 1,2,3 triazole.

4. A compound according to any of the preceding claims, wherein the linker comprises a moiety selected from the group consisting of:

5. A compound according to any one of claims 1-3, wherein the linker comprises a

group consisting of N~N and

6. A compound according to any one of the preceding claims wherein the linker further comprises a polyethylene glycol moiety.

7. A compound according to any one of the preceding claims, wherein the linker further comprises an alkylene moiety.

8. A compound according to any one of the preceding claims, wherein the fluorophore is selected from the group consisting of rhodamines, fluoresceins, coumarins, cyanines, and boron-dipyrromethenes.

9. A compound according to any one of the preceding claims wherein W is

10. A compound according to any one of the preceding claims selected from the group consisting of:

72

73

1 1. A compound according to any one of claims 1 -9, wherein the compound is selected from the group consisting of:

PCT/US2013/065649

12. A method of detecting, determining or identifying oxidative stress in cells comprising: a. contacting a sample containing cells with an effective amount of a compound according to any one of claims 1-1 1;

b. washing away excess compound to generate a washed sample;

c. exposing the washed sample to a wavelength of light to generate fluorescence d. detecting the fluorescence of the washed sample; and

e. comparing the fluorescence level of the washed sample to the fluorescence level of a control sample, wherein the fluorescence level of the washed sample that is higher than the fluorescence level of the control sample indicates that that the cells have oxidative stress.

13. A method of detecting a disease or disorder related to oxidative stress in a subject comprising:

a. contacting a biological sample from a subject with an effective amount of a compound according to any one of claims 1-11 ; b. washing away excess compound to generate a washed sample;

c. exposing the washed sample to a wavelength of light to generate fluorescence d. detecting the fluorescence level of the washed sample; and

e. comparing the fluorescence level of the washed sample to the fluorescence level of a control sample, wherein the fluorescence level of the washed sample that is higher than the fluorescence level of the control sample indicates that that the subject has a disease or disorder related to oxidative stress.

14. A method of screening for an anti-oxidative stress agent comprising:

a. administering an anti-oxidative stress candidate agent to a sample containing cells under oxidative stress conditions to generate a treated sample;

b. contacting the treated sample with an effective amount of a compound according to any one of claims 1-1 1;

c. contacting a control sample with an effective amount of a compound according to any one of claims 1-11 ;

d. washing away excess compound to generate a washed sample and washed control sample;

e. exposing the washed sample and washed control sample to a wavelength of light to generate fluorescence;

f. detecting the fluorescence level of the washed sample and the washed control sample; and

g. comparing the fluorescence level of the washed sample to the fluorescence level of a washed control sample, wherein the fluorescence level of the washed sample that is lower than the fluorescence level of the washed control sample indicates that the anti-oxidative stress candidate agent is an anti-oxidative stress agent.

15. A method of in vivo imaging of oxidative stress comprising:

a. administering an effective amount of a compound according to any one of claims 1-11 to a subject; and

b. detecting fluorescence, wherein the fluorescence indicates the presence of oxidative stress.

16. The method of claim 16, wherein the subject has a disease or disorder related to oxidative stress.

17. The method of claim 13 or 16, wherein the disease or disorder is selected from the group consisting of cancer, diabetes, arteriosclerosis, obesity, hepatitis, AIDS, neurological diseases, such Alzheimer's disease, Parkinson's disease, Huntington's disease, motor neuron diseases, including Lou Gehrig's disease (Amyotrophic Lateral Sclerosis (ALS)), apoptosis, inflammatory diseases, shock, ischemia/reperfusion injury, asthma, eczema, high bone mass syndrome, osteopetrosis, osteoporosis-pseudoglioma syndrome, digestive diseases such as gastric ulcer, irritable bowel syndrome, and ulcerative colitis, hypertension, angina pectoris, myocardial infarction, cardiomyopathy, chronic rheumatoid arthritis, Friedreich's Ataxia, musculoskeletal diseases such as migraine and tension headache, respiratory diseases such as bronchial asthma and hyperventilation syndrome, various diabetes complications, cranial nerve disease, Leber's hereditary optic neuropathy (LHON), optic neuritis, multiple sclerosis, systemic lupus erythematosis, stroke, traumatic hemorrhage, spinal cord trauma, Crohn's disease, autoimmune diseases, cataract formation, age-related macular degeneration, uveitis, emphysema, oxygen toxicity, neoplasia, and radiation sickness.

18. The method of any one of claims 15-17, wherein the imaging is during a surgical procedure.

19. The method of claim 18, wherein the presence of fluorescence indicates the presence of diseased tissue.

20. A kit comprising a compound according to any one of claims 1-1 1.