Related Application

[001] This application claims convention priority to Australian Provisional Patent Application 2020903689, filed 12 October 2020. The content of AU’689 is incorporated herein by reference in its entirety.

Field of the Invention

[002] The present invention relates to a method of measuring mitochondrial membrane potential using a “turn-on” fluorescent probe. Another form of the invention relates to the “turn- on” fluorescent probe compounds and their novel precursors.

[003] More generally, the invention relates to an imaging tool that can be used to measure the mitochondrial membrane potential (MMP) of live cells. The MMP is a physiological parameter that can dictate the health of the mitochondria. The MMP of the cell is known to change under cellular stress, and tools to study these changes in live cells are necessary for medical advancement, diagnosis and drug development. The inventive platform allows the real-time live cell MMP imaging, with the ability to resolve individual mitochondria. The invention is more selective than the existing options for measuring MMP as it only provides a fluorescent output when the probe accumulates inside the mitochondria itself. It is an easy to use dye system, that requires no specialist training beyond standard cell culture and light microscopy techniques; it is compatible with most existing light microscopy devices. Potential applications of this system include biological and medical research, screening of diseased cells and screening of drug candidates.

[004] Although the present invention will be described hereinafter with reference to its preferred embodiment, it will be appreciated by those of skill in the art that the spirit and scope of the invention may be embodied in many other forms.

Background of the Invention

[005] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge ine the field.

[006] Mitochondria are cell organelles vital for cellular energy production and respiration. Mitochondria are related to a range of diseases and indications. Mitochondrial membrane potential (MMP) is a physiological parameter that can dictate or be an indication of mitochondrial health. The MMP of the cell changes under cellular stress, and tools to measure these changes in live cells are useful for advancement medical knowledge, diagnosis and drug development.

[007] The present invention is set against a background in which state-of-the-art fluorescent probes rely on the accumulation of a single fluorescent probe, which is always fluorescent within the cell, leading to potential off target readings if the probe accumulates in an unexpected location.

[008] Fluorescent probes are a mainstay of molecular imaging, providing previously inaccessible information about the complex chemistry of cells and organisms. ^[1-3] Recent advances in biorthogonal chemistry have allowed for the development of fluorogenic probes, whose fluorescence intensities dramatically increase upon a “click” reaction with a biorthogonal reaction partner. ^[4,5] These fluorogenic probes have been extensively used to image cellular substructures, revealing changes in the biochemistry of the cell during health and disease. ^[6] [009] Of the suite of biorthogonal reactions developed for studies of biological processes, the tetrazine- cycloalkene ligation strategy has been the most extensively utilised for fluorogenic probes. ^[7,8] The 1,2,4,5-tetrazines have attracted much attention due to their ability to quench fluorescence via both through-space Forster Resonance Energy Transfer (FRET) interactions, ^[9] and through- bond energy transfer (TBET) processes. ^[10-12] They have been shown to react rapidly (second- order rate constants up to 10 ⁶ M ^-1 s ^-1 ) ^[4] in an inverse electron demand Diels Alder (IEDDA) reaction with a range of strained dienophiles such as trans-cyclooctenes (TCO) ^[8] and bicyclononynes (BCN). ^[13]

Scheme 1. Fluorogenic click reaction between a quenched tetrazine fluorophore and a biorthogonal group (e.g., BCN)

[010] The reaction of the tetrazine moiety with a dienophile halts FRET and TBET quenching processes, restoring fluorescence to the fluorophore. The reaction of a tetrazine with strained cycloalkynes such as BCN generates a pyridazine product with only N ₂ as a by- product, which makes these reactions biocompatible and useful for live cell imaging (see, Scheme 1, above, which depicts a schematic of a fluorogenic click reaction between a quenched tetrazine- fluorophore and BCN, producing a fluorescent pyridazine product).

[Oil] Many tetrazine-containing fluorogenic probes have been synthesised with emission wavelengths spanning the visible and infrared spectrum, commonly employing coumarin, ^[14] fluorescein, ^[15] rhodamine, ^[16] cyanine, ^[17] B0DIPY ^[12] and phenoxazine ^[18] scaffolds. All of these have been utilised in confocal microscopy, including super resolution imaging. ^[19-22] To date, the majority of reports have used tetrazine-fluorophore conjugates to label cellular macromolecules. The few notable exceptions, where fluorogenic tetrazines have been used for organelle-targeted sensing or analyte sensing include; a Mg ²⁺ fluorescent sensor with a tetrazine for organelle- localised Mg ²⁺ detection, ^[23] Selby et al. exploited a tetrazine fluorogenic reaction to quantify endocytosis of antibody conjugates. ^[24] For all of the above studies the cells needed to be transfected with non-native proteins and/or treated antibodies containing dienophiles to allow for this staining. We ascribe the lack of development in this area due to the challenge of finding fluorescent moieties which have straightforward synthetic handles that can be readily decorated with sensing or targeting groups.

[012] The 1,8-naphthalimides are a class of fluorophores for which tetrazine conjugates for bioimaging applications have not been reported to date. These fluorophores have great potential for bioimaging applications due to their brightness, large Stokes shifts and good photostability. ^[25] In addition, they can be readily synthetically modified at the i) imide and ii) amine positions and iii) 3-, 5- or 6-positions ^[25] of the naphthalene core providing three orthogonal points of synthetic attachment. Shown below is a representative 4- aminonaphthalimide structure showing the usual points of synthetic modification and the 3-, 5- and 6- positions of the naphthalene core to which other groups can be coupled:

[013] There are some reports of fluorogenic naphthalimides for biological imaging and protein labelling, with the Anorogenic changes arising from click reactions involving functional groups including azides, ^[26] SNAP Tags, ^[27] sydnones ^[28] and oximes. ^[29] However, there are no reports of fluorogenic 4-amino-naphthalimides incorporating tetrazines for biological imaging. Of the examples of naphthalimide tetrazines in the literature, most used an unsubstituted 1,8- naphthalimide, which has significantly blue- shifted excitation and emission compared to 4- aminonaphthalimides, which is unsuitable for any biological application. ^[30-33]

[014] A representative survey of the available patent literature comprises: WO 2000/68686, to Tularik Inc; WO 2009/035574, to Merck & Co Inc.; WO 2010/119389, to Koninkl Philips Electronics NV; WO 2017/078623, to the National University of Singapore; CN 106590630, to Shandong University; CN 109574922, to the University of Jinan; and CN 111057085, to Nankai University.

[015] In particular, WO 2010/119389, to Koninkl Philips Electronics NV describes a pre- targeting probe (i.e., component one) having a primary targeting moiety (targeting ligand to bind to a target within a human or animal body such as mitochondria) and a reactive group such as a cyclooctene or cy cooctyne (dienophiles). The reactive group of the pre-targeting probe can react with an effector probe (i.e., component two) functionalised with a tetrazine and is linked to a detectable label for diagnostic, imaging and/or therapeutic effect. Detectable labels include fluorescent molecules.

[016] Reference [29] cited herein (Devaraj, et al., Angew. Chemie Int. Ed. 2010, 49, 2869- 2872) describes the use of tetrazine-conjugated quenched fluorescent probes which react rapidly in an inverse - electron - demand [4+2] cycloaddition with strained dienophiles such as trans - cyclooctene, to turn-on fluorescence. These turn - on probes have been applied for intracellular live - cell imaging of a microtubuli - binding trans - cyclooctene modified taxol.

[017] What the prior art fails to teach, however, is the use of naphthalimide-tetrazine quenched fluorophores which turn-on when reacting with a dienophile and that the naphthalimide-tetrazine quenched fluorophores have a targeting ligand. To the best of the Inventors’ knowledge, there have only been four reports where naphthalimides and tetrazines have been incorporated into the same molecule. The first report of any naphthalimide tetrazine compound was for electrochemical experiments, and was shown to give a fluorogenic response when stimulated by an electric potential. ^[33] Since then, there have been several reports using this dye and an analogue for the tuning of redox and electrochemical properties on nanoparticles. ^[30-32] None of these sensors were used for biological imaging and none of them further comprised a biorthogonal click reaction between a tetrazine and reactive group. With tetrazines known to be superior florescence quenchers, there is hence a large scope for the development of the first biorthogonally-activated fluorogenic naphthalimide tetrazines.

[018] It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

[019] It is an object of an especially preferred form of the invention to provide for the efficient synthesis and photophysical properties of a series of tetrazine-naphthalimides that combine FRET and TBET quenching. These naphthalimides are preferably suitable for live cell imaging.

[020] It is an object of another especially preferred form of the invention to provide for a “turn on” fluorescent probe which activates once present in the mitochondria and provides site- specific, selective and sensitive readings once the probe has entered the mitochondria, providing a more specific and sensitive reading.

[021] It is an object of another especially preferred form of the invention to provide a new imaging tool (probe) that can be used to measure the mitochondrial membrane potential (MMP) of live cells and real-time live cell imaging of this potential, with the ability to resolve individual mitochondria.

[022] It is an object of another especially preferred form of the invention to provide for a probe that is more selective than the existing options for measuring the MMP with live cell imaging as the probe provides a fluorescent output when the probe accumulates inside the mitochondria itself and is subsequently activated.

[023] It is an object of another especially preferred form of the invention to provide a probe particularly amenable to the biological, medical research and/or diagnostics fields (screening of diseased cells and drug candidates).

[024] It is an object of another especially preferred form of the invention to provide synthetic protocols for the synthesis of the novel naphthalimide-pyridazine “clicked” compounds and also their novel naphthalimide-tetrazine precursors.

[025] Although the invention will be described with reference to specific examples it will be appreciated by those skilled in the art that the invention may be embodied in many other forms.

Definitions

[026] In describing and defining the present invention, the following terminology will be used in accordance with the definitions set out below. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one having ordinary skill in the art to which the invention pertains.

[027] Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising” and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

[028] As used herein, the phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. When the phrase “consists of” (or variations thereof) appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole. As used herein, the phrase “consisting essentially of” limits the scope of a claim to the specified elements or method steps, plus those that do not materially affect the basis and novel characteristic(s) of the claimed subject matter.

[029] With respect to the terms “comprising”, “consisting of” and “consisting essentially of”, where one of these three terms are used herein, the presently disclosed and claimed subject matter may include the use of either of the other two terms. Thus, in some embodiments not otherwise explicitly recited, any instance of “comprising” may be replaced by “consisting of” or, alternatively, by “consisting essentially of”.

[030] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term “about”, having regard to normal tolerances in the art. The examples are not intended to limit the scope of the invention. In what follows, or where otherwise indicated, “%” will mean “weight %”, “ratio” will mean “weight ratio” and “parts” will mean “weight parts”.

[031] The term “substantially” as used herein shall mean comprising more than 50%, where relevant, unless otherwise indicated.

[032] The recitation of a numerical range using endpoints includes all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[033] The terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[034] It must also be noted that, as used in the specification and the appended claims, the singular forms “a”, “ an” and “the” include plural referents unless the context clearly dictates otherwise.

[035] The person skilled in the art would appreciate that the embodiments described herein are exemplary only and that the electrical characteristics of the present application may be configured in a variety of alternative arrangements without departing from the spirit or the scope of the invention.

[036] Although example embodiments of the disclosed technology are explained in detail herein, it is to be understood that other embodiments are contemplated. Accordingly, it is not intended that the disclosed technology be limited in its scope to the details of construction and arrangement of components set forth in the following description or illustrated in the drawings. The disclosed technology is capable of other embodiments and of being practised or carried out in various ways.

Nomenclature

[037] The compounds exemplified herein are denoted “NpxxTz”, wherein Np is a naphthalimide moiety, Tz is a tetrazine moiety, and xx represents the attachment of the tetrazine relative to the naphthalimide, e.g., 3p denoting substitution at the 3-position of the naphthalimide with the tetrazine located para- to the position of substitution, viz.

[038] Alternatively, the “clicked” pyridazine compounds of the invention are denoted “NpxxPz”. In like manner, Np is a naphthalimide moiety, Pz is the pyridazine moiety, and xx represents the attachment of the pyridazine relative to the naphthalimide, e.g., 6m denoting substitution at the 6-position of the naphthalimide with the pyridazine located meta- to the position of substitution, viz.

[039] Compounds containing a mitochondrial targeting group such as the triphenylphosphonium (TPP) group, are designated by the prefix “Mito”, but otherwise named by analogy with the naphthalimide-tetrazine and naphthalimide-pyridazine compounds referenced above, for example, MitoNp6mTz:

[040] Finally, the colloquial term “MitoMPP” (equiv. MitoNp6mPz) represents “Mitochondrial Membrane Probe Potential”. For the avoidance of any doubt, it is synonymous with “MitoClick” as used in the provisional application, AU 2020903689. “MitoMPP” is preferred herein due to potential confusion with literature sources naming “MitoClick” in contexts other than those presently described.

Summary of the Invention

[041] The invention relates to a novel system for the live cell and organism imaging of the mitochondrial membrane potential in real time. The system comprises a two-part probe, with each of the parts targeted to the mitochondria by the triphenylphosphonium (TPP) group. Each half of the probe also contains a reactive partner, which undergo a biorthogonal reaction when in close proximity (see, Figure 1). The tetrazine reactive partner doubles as a selective biorthogonal reactive group, as well as a fluorescence quencher. When the two halves of the probe localise inside the mitochondria, the reaction breaks the tetrazine, restoring fluorescence to the quenched fluorophore. The only by-product of this reaction is nitrogen and a stable, covalent linkage is made between the two halves of the probe. This provides an observable signal that can be captured with standard confocal light microscopy techniques.

Scheme 2. Probe “A” + Probe “B”→ “Clicked” probe (general)

[042] An exemplary design is summarised in Scheme 2, above, and a specific example is provided in Scheme 3, below.

Scheme 3. Probe “A” + Probe “B”→ “Clicked” probe (exemplary, using MitoNp6mTz as naphthalimide-tetrazine precursor and MitoBCN as the biorthogonal group)

[043] The membrane potential governs the accumulation of hydrophobic cationic groups such as the TPP group into the mitochondrial and since each half of the probe contains this group, the fluorescent probe is highly sensitive to the membrane potential. The tetrazine moiety could be applied to a range of fluorophores to provide a general scaffold to generate this system. In addition, the tetrazine can react with a range of well-known, exogenously applied biorthogonal partners within the cellular environment.

[044] According to a first aspect of the present invention there is provided a naphthalimide- tetrazine compound for use, when used for, or adapted for use as a fluorogenic probe in biological imaging.

[045] In an embodiment, the naphthalimide moiety of the naphthalimide-tetrazine compound is a 4-amino derivative thereof.

[046] In an embodiment, the naphthalimide moiety of the naphthalimide-tetrazine compound is of the general formula (I), and wherein the tetrazine moiety couples to the naphthalimide moiety through the 3-, 5- or 6-positions.

[047] In an embodiment, the tetrazine moiety of the naphthalimide-tetrazine compound is a phenyl-substituted derivative thereof.

[048] In an embodiment, the tetrazine moiety of the naphthalimide-tetrazine compound is of the general formula (II), and wherein the naphthalimide moiety couples to the tetrazine moiety through the ortho-, meta- or para-positions:

[049] In an embodiment, the compound is selected from the group consisting of: Np3oTz, Np3mTz, Np3pTz, Np5oTz, Np5mTz, Np5pTz, Np6oTz, Np6mTz, Np6pTz:

[050] In an embodiment, the compound is selected from the group consisting of: Np3mTz, Np3pTz, Np6mTz, Np6pTz:

[051] In an embodiment, the compound further comprises a mitochondria targeting group at the nitrogen of the naphthalimide moiety.

[052] In an embodiment, the mitochondria targeting group is a triphenylphosphonium salt.

[053] In an embodiment, the compound comprising a mitochondria targeting group at the nitrogen of the naphthalimide moiety is selected from the group consisting of: MitoNp3oTz, MitoNp3mTz, MitoNp3pTz, MitoNp5oTz, MitoNp5mTz, MitoNp5pTz, MitoNp6oTz,

MitoNp6mTz, MitoNp6pTz: [054] In an embodiment, the compound comprising a mitochondria targeting group at the nitrogen of the naphthalimide moiety is selected from the group consisting of: MitoNp3mTz,

MitoNp3pTz, MitoNp6mTz, MitoNp6pTz:

[055] In an embodiment, the compound comprising a mitochondria targeting group at the nitrogen of the naphthalimide moiety is MitoNp6mTz: [056] According to a second aspect of the present invention there is provided a naphthalimide- pyridazine compound formed from the reaction of a naphthalimide-tetrazine compound as defined according to the first aspect of the present invention with a biorthogonal group.

[057] In an embodiment, the biorthogonal group is selected from the group consisting of styrenes, norbomenes, cyclopropenes, bicyclononynes and trans-cyclooctenes.

[058] In an embodiment, the biorthogonal group reacting with the naphthalimide-tetrazine compound to form the naphthalimide-pyridazine compound is bicyclononyne (BCN).

[059] In an embodiment, the naphthalimide-pyridazine compound further comprises a mitochondria targeting group at the apical carbon of the cyclopropane moiety.

[060] In an embodiment, the mitochondria targeting group is a triphenylphosphonium salt.

[061] In an embodiment, the biorthogonal group having a triphenylphosphonium salt is MitoBCN:

[062] In an embodiment, the naphthalimide-pyridazine compound is selected from the group consisting of: Np3oPz, Np3mPz, Np3pPz, Np5oPz, Np5mPz, Np5pPz, Np6oPz, Np6mPz, Np6pPz:

[063] In an embodiment, the naphthalimide-pyridazine compound is selected from the group consisting of: Np3mPz, Np3pPz, Np6mPz, Np6pPz: [064] In an embodiment, both the nitrogen of the naphthalimide moiety and the apical carbon of the cyclopropane moiety comprise a mitochondria targeting group.

[065] In an embodiment, the compound is selected from the group consisting of: MitoNp3oPz,

MitoNp3mPz, MitoNp3pPz, MitoNp5oPz, MitoNp5mPz, MitoNp5pPz, MitoNp6oPz,

MitoNp6mPz, MitoNp6pPz: [066] In an embodiment, the compound is selected from the group consisting of: MitoNp3mPz,

MitoNp3pPz, MitoNp6mPz, MitoNp6pPz:

[067] In an embodiment, the compound is MitoNp6mPz:

[068] According to a third aspect of the present invention there is provided a method of biological imaging, said method comprising the steps of:

[069] a) introducing to the mitochondria of a cell selected for imaging a predetermined amount of a naphthalimide-tetrazine compound as defined according to the first aspect of the invention, the naphthalimide-tetrazine compound further incorporating a mitochondria-targeting group;

[070] b) simultaneously or sequentially introducing to said cell a predetermined amount of a biorthogonal group comprising a mitochondria targeting moiety;

[071] c) observing and measuring the fluorescence emitted from a click reaction between the naphthalimide-tetrazine and the biorthogonal group;

[072] d) determining the mitochondrial membrane potential (MMP) as a proxy of the health of the cell by correlating the measured fluorescence against a known standard.

[073] In an embodiment, the predetermined amount of the naphthalimide-tetrazine compound is between about 1 μM and about 10 μM.

[074] In an embodiment, the predetermined amount of the naphthalimide-tetrazine compound is about 1 μM.

[075] In an embodiment, the predetermined amount of the biorthogonal group is about 50 μM.

[076] In an embodiment, step b) is performed sequentially, following a time lag of between about 15 and about 90 minutes following completion of step a).

[077] In an embodiment, the naphthalimide-tetrazine compound is Np6mTz or Np6pTz, and wherein the compound is substituted with a mitochondria targeting group in the form of a triphenylphosphonium salt.

[078] In an embodiment, the biorthogonal group is bicyclononyne (BCN) substituted with a mitochondria targeting group in the form of a triphenylphosphonium salt. In an embodiment, the biorthogonal group is MitoBCN.

[079] In an embodiment, the cell is excited at about 488 nm and the fluorescence emission is collected between about 510 and 610 nm.

[080] According to a fourth aspect of the present invention there is provided a method for the synthesis of a naphthalimide-tetrazine compound as defined according to the first aspect of the present invention, the method comprising the steps of:

[081] a) obtaining bromophenyl-substituted tetrazines from a respective one of commercially- available o-, m- or p-bromobenzonitrile by catalysis by 3 -mercaptopropionic acid;

[082] b) subjecting the respective bromophenyl-substituted tetrazines to Miyaura borylation using bis(pinacolato)diboron to generate the respective tetrazine coupling partner;

[083] c) brominating a naphthalimide compound and its 3, 5- or 6-position to generate the respective naphthalimide coupling partner;

[084] d) subjecting a tetrazine coupling partner to Suzuki cross-coupling with a naphthalimide coupling partner, thereby to couple the tetrazine and naphthalimide moieties and form a respective isomer of the naphthalimide-tetrazine compound; and

[085] e) isolating the naphthalimide-tetrazine compound.

[086] According to a fifth aspect of the present invention there is provided a method for the synthesis of a naphthalimide-pyridazine compound as defined according to the second aspect of the invention, the method comprising the steps of:

[087] a) selecting a naphthalimide-tetrazine compound as defined according to the first aspect of the invention;

[088] b) reacting the naphthalimide-tetrazine compound with a biorthogonal group; and

[089] c) obtaining the naphthalimide-pyridazine compound.

[090] In an embodiment, the biorthogonal group reacting with the naphthalimide-tetrazine compound to form the naphthalimide-pyridazine compound is bicyclononyne (BCN).

[091] In an embodiment, the molar ratio of naphthalimide-tetrazine compound to biorthogonal group in step b) is about 1:5.

[092] According to a sixth aspect of the present invention there is provided a method of measuring the mitochondrial membrane potential (MMP) of a cell, the method comprising the steps of:

[093] providing a fluorophore quenched probe having a targeting ligand to bind with the mitochondria;

[094] within the mitochondria, contacting the fluorophore quenched probe with an activator to form a fluorescent adduct; and

[095] measuring the magnitude of the fluorescence, the magnitude of the fluorescence being proportional to the MMP.

[096] According to a seventh aspect of the present invention there is provided a kit for biological imaging, the kit comprising in sperate containers:

[097] a) a naphthalimide-tetrazine compound as defined according to the first aspect of the invention, the naphthalimide-tetrazine compound further incorporating a mitochondria-targeting group; and

[098] b) a biorthogonal group comprising a mitochondria targeting moiety.

[099] In an embodiment, the kit further comprises instructions for the simultaneous or sequential administration of the naphthalimide-tetrazine compound and the biorthogonal group. [0100] In an embodiment, the amount of the naphthalimide-tetrazine compound is about 1 μM; and wherein the amount of the biorthogonal group is about 50 μM.

[0101] Finally, according to an eighth aspect of the present invention there is provided a method for the synthesis of a naphthalimide-tetrazine compound comprising a mitochondria targeting group as defined according to the first aspect of the present invention, the method comprising a five-step synthesis substantially as shown in Scheme 9.

Brief Description of the Drawings

[0102] A preferred embodiment of the invention will now be described, by way of example only, with reference to the accompanying drawings in which:

[0103] Figure 1 is a schematic representation of a preferred embodiment of the invention in which the targeted quenched fluorophore (e.g., the naphthalimide tetrazine) and the targeted reactive biorthogonal group (e.g., BCN) react (or “click”) in the mitochondria. The MMP causes rapid accumulation of each half of the probe; when the two halves meet, the probe becomes strongly fluorescent; and the fluorescence is proportional to the MMP.

[0104] Figure 2 are the absorbance spectra of a) Np3mTz and Np3mPz, b) Np3pTz and Np3pPz, c) Np6mTz and Np6mPz and d) Np6pTz and Np6pPz, measured in absolute EtOH.

[0105] Figure 3 are the excitation and emission spectra of 5 μM solutions of a) Np3mTz, b) Np3pTz, c) Np6mTz and d) Np6pTz before (orange) and after (black) reaction with 5 equiv. BCN to form the pyridazine products. Spectra recorded in EtOH.

[0106] Figure 4 shows the excitation and emission spectra of a) Np3mPz, b) Np3pPz, c) Np6mPz and d) Np6pPz in CH ₂ CI ₂ , MeCN, EtOH or HEPES buffer.

[0107] Figure 5 shows fold turn-on of naphthalimide tetrazines after reaction with BCN (5 equiv.) in CH ₂ CI ₂ , MeCN, EtOH and HEPES (20 mM, pH = 7.4). Fold turn-on evaluated by dividing integrated fluorescence of the naphthalimide pyridazine products by the integrated fluorescence of the naphthalimide tetrazine product.

[0108] Figure 6 depicts cell viability of A549 cells after exposure to 1% DMSO, 250 μM BCN or 25 μM tetrazine naphthalimide or pyridazine naphthalimide after 3 h exposure.

[0109] Figure 7 comprises representative images of A549 cells dosed with A) 10 μM Np3mTz for 20 min, B) 10 μM Np3mTz for 90 min, C) 1 μM Np3mTz for 20 min with wash, D) 1 μM Np3mTz for 20 min without wash. Samples were excited at 405 nm and fluorescence was collected between 500-b00 nm. Scale bar represents 20 μm.

[0110] Figure 8 depicts representative images of A549 cells dosed with 1 μM naphthalimide- tetrazines or 1 μM naphthalimide pyridazines for 20 min as indicated. 3-position naphthalimides were excited at 405 nm and 6-position naphthalimides excited at 488 nm. Fluorescence emission was collected between 510-610 nm. Scale bar represents 20 μm.

[0111] Figure 9 shows representative images of A549 cells stained with Np3mTz, Np3pTz, Np6mTz or Np6pTz for 30 mins followed by 2 h incubation with 50 μM BCN. 3-position naphthalimides were excited at 405 nm and 6-position naphthalimides excited at 488 nm. Fluorescence emission spectra were collected between 510-610 nm. Scale bar represents 20 μm. [0112] Figure 10 shows images of A549 cells dosed with 2.5 μM Np6mTz for 30 min, followed by Fluorobrite media (Rows 1 and 2) or 50 μM BCN in Fluorobrite media (Rows 3 and 4) and imaged at the times indicated. Rows 1 and 3 are fluorescence images (collected 510-610 nm) and rows 2 and 4 are overlays with brightfield images. Samples were excited at 488 nm. Scale bar represents 20 μm.

[0113] Figure 11 displays images of A549 cells dosed with 50 μM BCN for 30 min, followed by 2.5 μM Np6mTz and imaged at the times indicated. Samples were excited at 488 nm. Scale bar represents 20 μm.

[0114] Figure 12 are the comparative absorbance profiles of “MitoNpTz” (properly, MitoNp6mTz by the nomenclature used throughout this application) and MitoMPP (equiv. MitoNp6mPz). Inset is a zoom of the 500-600 nm region, which shows a loss of the tetrazine peak.

[0115] Figure 13 shows A) the fluorescence enhancement/fold turn on after click reaction (in EtOH) for “MitoNpTz” (properly, MitoNp6mTz by the nomenclature used throughout this application) and MitoMPP (equiv. MitoNp6mPz) at the same concentrations; B) Fluorescence of MitoMPP in different solvents; and C) Fold turn-on based on these fluorescence spectra.

[0116] Figure 14 shows representative images of A549 cells treated with A) MitoNp6mTz (5 μM) for 30 min and B) MitoNp6mTz (5 μM) and MitoNp6mPz (5 μM) for 30 min. Samples were excited at 488 nm and emission collected between 510-610 nm. Scale bar represents 20 μm. [0117] Figure 15 shows representative images of MitoNp6mTz (5 μM) and MitoNp6mPz (5 μM) (green channel) and MitoTracker Deep Red (100 nM, red channel). The green channel was excited at 488 nm, and emission collected between 510-610 nm. The red channel was excited at 640 nm and emission collected between 650-750 nm. Pearson’s correlation coefficient = 0.87.

Scale bar represents 20 μm.

[0118] Figure 16 shows the cytotoxicity of MMP probes using the AlamarBlue assay. A549 cells were exposed to 1% DMSO (vehicle control) or 25 μM of the relevant probe for 3 h. [0119] Figure 17 shows the cytotoxicity of MMP probes using the MTT assay. A549 cells were exposed to 1% DMSO (vehicle control) or the indicated concentrations of the relevant probe for 3 h.

[0120] Figure 18 shows mean fluorescence intensity of A549 cells treated with MitoNpTz (5 μM) and MitoBCN (10 μM) and varying concentrations of FCCP. Fluorescence intensities are presented as the mean intensity of 40-60 individual cells from at least 7 different images for each condition. The error has been plotted as the 95% confidence interval of each data set. * = P < 0.01, **** = P < 0.0001 as determined by one-way ANOVA with Tukey’s multiple comparisons test.

[0121] Figure 19 is a comparison of MitoMPP with a commercial dye. A) Ratio of red to green fluorescence observed in A549 cells treated with JC-1 (2 μM) and 0, 0.1 or 1 μM FCCP for 1 h. B) Mean fluorescence intensities observed in A549 cells treated with MitoNpTz (5 μM) and MitoBCN (10 μM) and 0, 0.1 or 1 μM FCCP for 1 h. The error has been plotted as the 95% confidence interval of each data set. * = P < 0.05, **** = P < 0.0001 as determined by one-way ANOVA with Tukey’s multiple comparisons test.

[0122] Figure 20 depicts mean fluorescence intensity of A549 cells treated with 10 μM platinum complexes for 1 h and stained with MitoNpTz (5 μM) and MitoBCN (10 μM). Fluorescence intensities are presented as the mean intensity of at least 38 individual cells from at least 8 different images for each condition. The error has been plotted as the 95% confidence interval of each data set. * = P < 0.05, **** = P < 0.0001 as determined by one-way ANOVA with Tukey’s multiple comparisons test.

Detailed Description of a Preferred Embodiment

[0123] In accordance with the foregoing discussion, the probes embodied in the prior art are not as sensitive as the two-component probes of the invention and they cannot be used to quantify mitochondrial membrane potential due to non-specific uptake and binding or measure mitochondrial membrane potential on live cells in real time.

[0124] Due to the two-component nature of a preferred embodiment of the invention, the probes are envisaged to be more sensitive to dynamic changes in the mitochondrial membrane potential in a range of conditions. Existing commercial probes rely on the accumulation of a single fluorescent probe, which is always fluorescent within the cell, leading to potential off target readings if the probe accumulates in an unexpected location. The inventive probe will “turn-on” when it is in the mitochondria and therefore give a signal once the two-component probe has entered the mitochondria (which is more specific and sensitive).

[0125] In order to address one or more of the limitations of the prior art identified above, the present Inventors have proposed the efficient synthesis and photophysical properties of a series of tetrazine-naphthalimides that combine FRET and TBET quenching. Data confirm that naphthalimides are suitable for live cell imaging. Using the best available scaffold and the synthetic versatility and practicality of the inventive approach, the present Inventors have developed a lysosome-targeted variant as a novel fluorogenic tetrazine based organelle sensor that does not require genetic modification or antibody stains in live cells. In other embodiments, the fluorescent probes of the invention can be tailored to have specific targeting ligands to different parts of cells and/or organelles.

A) Material and methods i) General synthesis and analysis

[0126] All reactions were performed under a nitrogen atmosphere unless otherwise indicated. All reagents were obtained from Sigma-Aldrich, Merck or Combi-Blocks. Tetrahydrofuran (THE) and dichloromethane (CH ₂ CI ₂ ) were obtained from a Pure-Solv 400 Solvent Purification System. Anhydrous 1,4-dioxane was obtained from Sigma Aldrich. Peptide-grade N,N- dimethylformamide (DMF) was obtained from LabScan and used for synthesis. All other solvents were laboratory grade and used without further purification. Where indicated, solvents were degassed by nitrogen sparging unless otherwise specified. Reactions were monitored by silica gel thin-layer chromatography plates (Merck, TLC Silica gel 60 F ₂₅₄ ). All column chromatography was performed on silica gel 60 (Merck, 0.040-0.063 nm) using a Biotage Isolera One. Compounds 1, ^[37] 7, ^[38] 8 ^[39] and 9 were prepared according to literature methods.

[0127] All NMR spectra were obtained at 300 K on Bruker AVANCE III 400 or Bruker AVANCE III 500 spectrometers equipped with a 5 mm BBFO probe with z-gradients. Deuterated solvents (CDCl ₃ , DMSO-d ₆ ,) were obtained from Cambridge Isotope Laboratories. All chemical shifts are reported in ppm and all coupling constants are reported in Hz. ¹ H NMR spectra are calibrated to trace isotopic impurities of the solvent used (δ = 7.26 ppm for CDCl ₃ , δ = 2.50 ppm for DMSO-d ₆ . ¹ H NMR data are reported as: chemical shift, multiplicity, coupling constant(s) (J) and relative integral. The multiplicities are reported as one or more of the following: s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, m = multiplet, br = broad. ¹³ C{ ¹ H } NMR spectra are calibrated to trace isotopic impurities of the solvent used (δ = 77.16 ppm for CDCl ₃ , δ = 39.52 ppm for DMSO-d ₆ ). ¹³ C{ ¹ H } NMR data is reported as chemical shift.

[0128] Low resolution ESI and APCI mass spectrometry was performed on a Bruker AmaZon SL ion trap mass spectrometer. For low resolution ESI, samples were dissolved as indicated and injected via flow injection at 0.3 mL/min in methanol or acetonitrile into an Apollo II source with nitrogen drying gas at 180 °C. For low resolution APCI, samples were placed in a melting point tube and inserted into the Broker Apollo II APCI source with an atmospheric solid analysis probe attachment added with vaporisation temperature 400 °C and corona current 4 μA. High resolution ESI and APCI mass spectrometry was performed on a Bruker solarix 2XR Fourier Transform Ion Cyclotron Resonance Mass Spectrometer. For high resolution ESI, samples were injected using the supplied syringe pump at 180 μL/h with nebuliser flow 1 L/min and drying gas 4 L/min at 180 °C. For high resolution APCI, samples were placed in a melting point tube and inserted into the Bruker Apollo II APCI source with an atmospheric solid analysis probe attachment added with vaporisation temperature 400 °C and corona current 4 μA. ii) Photophysical studies

[0129] The solvents used in all photophysical studies were dichloromethane (HPLC grade, Sigma Aldrich), acetonitrile (Spectroscopy Grade, AJAX), absolute ethanol (200 proof, HPLC/spectrophotometric grade, Sigma Aldrich) or HEPES (20 mM, pH 7.4). All of the compounds were made up as stock solutions in DMSO (Spectroscopy grade, Sigma Aldrich) and the concentrated stock solution was diluted to the required concentration in the appropriate solvent. The DMSO concentration in all experiments was <0.5% v/v.

[0130] Absorption spectra were obtained for each compound in absolute ethanol on a Varian Cary 400 UV- Vis spectrophotometer using 10 mm pathlength quartz cuvettes. Fluorescence spectra were obtained for each compound in CH ₂ CI ₂ , MeCN, absolute EtOH or HEPES buffer on a Varian Cary Eclipse fluorometer using quartz cuvettes. iii) Extinction coefficients

[0131] Extinction coefficients were determined from the absorption spectra of three independent solutions (5, 7.5 and 10 μM) of each compound. The absorption value at λ _max above 400 nm was obtained for each concentration and a linear regression of these values was performed with Prism 8. iv) Fold turn-on

[0132] Fold turn-on values for each of the naphthalimide tetrazines were measured in black 96- well plates (655209, Greiner Bio-One) on a Perkin Elmer EnSpire Plate Reader. Stock solutions in DMSO of both the unreacted tetrazine as well its reacted form were prepared. These stocks were diluted into CH ₂ CI ₂ , MeCN, absolute EtOH or HEPES buffer to give 5 μM solutions of each compound and the corresponding reacted form in triplicate. 200 μL of each solution was added to each well. Excitation was provided at the maximal excitation wavelength for each compound in each solvent, and emission was collected at 1 nm intervals from 500-700 nm. Blank solutions, containing an equal amount of DMSO and the relevant solvent were also collected. Each set of intensity values were added together to give an integrated intensity, and the blank solvent sums were subtracted from these. The integrated intensities of the unreacted tetrazines were averaged, and each of the integrated intensities of the reacted forms were divided by this average to give the fold turn-on value. The values are presented as the mean ± SD of three independent solutions. v) Kinetics

[0133] Second order rate constants were derived using the fluorescence output of the reaction under pseudo- first order conditions. All fluorescence values were determined in black 96-well plates (655209, Greiner Bio-One) on a Perkin Elmer EnSpire Plate Reader. 10 mM stock solutions of the naphthalimide tetrazines in DMSO were diluted to 2 μM in 1:1 MeCN:H ₂ O solution. Stock solutions of BCN were prepared to concentrations of 200, 400, 600 and 800 μM in 1:1 MeCN:H ₂ O. For each compound, 100 μL of the tetrazine solution was added to five wells of the plate. 100 μL of 1:1

[0134] MeCN:H ₂ O was added to the control, and 100 μL of each of the BCN stock solutions were added to the well, giving solutions with 0, 100, 200, 300 and 400 equivalents of BCN. The solutions were excited at 450 nm and fluorescence intensity at 540 nm was recorded every 10 seconds for 20 minutes. The resultant curves were fitted to “one-phase association” models in GraphPad Prism 8 to determine the experimental first order rate constant, k _obs . A linear plot of BCN concentration against k _obs was used to determine the second order rate constants. vi) Absolute quantum yields

[0135] Absolute quantum yields were measured in absolute EtOH on a PTI QuantaMaster 400 fluorometer with an integrating sphere setup in 3.5 mL quartz cuvettes. Reported values are the average of three measurements of three independent optically dilute (A < 0.1, ~2-5 μM) solutions. The values were corrected for self-absorption as described previously, using estimates of the self-absorption parameter (a) from the area overlapping between the excitation and emission spectra. vii) LCMS reaction analysis

[0136] For the unreacted tetrazines, 2 μL of a 10 mM stock solution in DMSO was diluted to approximately 300 μL in 1:1 MeCN:H ₂ O. For the reacted products, 2 μL of a 10 mM stock solution of a tefrazine-naphthalimide in DMSO was mixed with 2 μL of a 50 mM stock solution of BCN in DMSO. This was similarly diluted to approximately 300 μL. Samples were syringe filtered before analysis.

[0137] Liquid-chromatography mass spectrometry (LCMS) was conducted on a Shimadzu LC- MS 2020 instrument consisting of a LC-M20A pump and a SPD-20A UV-VIS detector coupled to a Shimadzu 2020 mass spectrometer. Separations were performed on a Waters Sunfire C 18 column (5 μm, 2.1x150 mm), operating at a flow rate of 0.2 mL min ^-1 using a mobile phase of 0.1% formic acid in Milli-Q H2O and 0.1% formic acid in MeCN (HPLC grade, Sigma Aldrich). All LCMS traces are taken over a solvent gradient of 0% MeCN to 100% MeCN run for 30 min. viii) Cell culture

[0138] A549 cells were maintained at 37 °C in 5% carbon dioxide. Cells were cultured in Advanced Dulbecco’s Modified Eagle’s Medium (ADMEM, Thermo Fisher Scientific) supplemented with 2% foetal calf serum (FCS, Thermo Fisher Scientific) and 2.5 mM L- glutamine (Sigma- Aldrich). Fluorobrite DMEM (FDMEM, Thermo Fisher Scientific) was supplemented with 2% FCS and 2.5 mM L-glutamine, unless otherwise stated. Cells for imaging and toxicity experiments had a passage number lower than 20. Probe stock solutions were made in DMSO and were freshly dissolved and used within a week. For treatments, the DMSO stock was diluted into ADMEM or FDMEM at the appropriate concentrations and then added to cells. ix) Alamar blue cell viability assay

[0139] Cells were seeded in 96 well plates in 100 μL of ADMEM with a density of 25000 cells/well and allowed to adhere to 16 h. Solutions of probes were made up to 50 μM in ADMEM, containing 1% DMSO, whereas BCN was made up to a concentration of 500 μM in ADMEM, containing 1% DMSO. 100 μL of ADMEM (for controls), ADMEM containing 1% DMSO, 50 μM probe solution or 500 μM BCN solution were added to wells in triplicate. The cells were allowed to incubate for a further 3 hours, before the addition of Alamar Blue reagent. After a further 3 h incubation to allow the conversion of resorufin to reazurin, the fluorescence of each well was measured (λ _ex =570 nm, λ _em =590 nm) in a Perkin Enspire Plate Reader. Values were normalised to controls containing ADMEM only and the data was collated and graphed in GraphPad Prism 8. x) Confocal microscopy

[0140] For all imaging experiments, 100000 cells were seeded into 35 mm glass bottom dishes (MatTek) and allowed to adhere overnight. For the static images, the media was removed from the cells and replaced with 1 μM or 10 μM solutions of the probe in ADMEM (1.0 mL). Cells were incubated for 20 min. When complete, the media was removed, cells washed with PBS (3 x 0.5 mL) and resuspended in FDMEM (1.0 mL).

[0141] For images where BCN was dosed after the addition of probe, the cells were incubated with the relevant dose of probe for 30 mins. The media was removed, and cells were washed with PBS (3 x 0.5 mL). 50 μM BCN solution in FDMEM (1.0 mL) was added and the cells were incubated for a further 120 min before imaging.

[0142] Images were obtained at 37 °C on an Olympus FluoView FV3000 Confocal Laser Scanning Microscope, equipped with an Olympus 60 x water objective (UPLSAPO60XW) and 405, 488 and 640 nm lasers. Images were processed using FIJI software.

[0143] For the time course experiments, the cells were dosed with either Np6mTz (2.5 μM) or BCN (50 μM) for 30 mins. The cells were then quickly washed with PBS (3 x 0.5 mL) and the cells were suspended in BCN (50 μM) or Np6mTz (2.5 μM) solutions in FDMEM (1.0 mL) containing 10% FCS and 2.5 mM L-glutamine.

[0144] For the control, the cells were suspended in FDMEM (1.0 mL). The cells were immediately imaged every 2 mins with the first image obtained 2 minutes post-washing. The images were collected at 37 °C in 5% CO ₂ atmosphere, on an Olympus FluoView FV3000 Confocal Laser Scanning Microscope, equipped with an Olympus 40 x dry objective (UPLSAPO40X2) and 488 nm laser.

B) Synthetic protocols

[0145] The Inventors first set out to develop a series of tetrazine-naphthalimide candidates that explore how the position of the tetrazine relative to the naphthalimide affects the quenching and fluorescence emission of the naphthalimides themselves. The Inventors identified that tetrazine probes with the greatest fold turn-on after reaction with a biorthogonal partner exploited both FRET and TBET quenching. This requires the tetrazine to be in conjugation with the aromatic system of the naphthalimide. Thus, it was sought to install the tetrazine at the 3-, 5- or 6-position of the naphthalimide.

[0146] It was decided that a phenyl ring would be a suitable linker between the tetrazine and naphthalimides to ensure conjugation and twisted π bonds which allows for efficient TBET. ^[11] This means that the tetrazine could be oriented in an ortho-, meta- or para- configuration relative to the naphthalimide. In order to study the effect of these substitution points, the Inventors chose to install standard n-butylamine groups at the imide and 4-positions of the naphthalimide series. The Inventors designed a series of naphthalimides with tetrazines appended at the 3-, 5- and 6- position (Scheme 4):

Scheme 4. Exemplary naphthalimide tetrazines of the present invention

[0147] Initially, the synthetic design centred around a linear synthesis, with the tetrazine installed in the final step using a typical Zn-catalysed tetrazine formation. ^[34] However, the yields of assymetric tetrazines using this method is highly dependent on the appended groups, and some of the products could only be isolated in low yields. Hence, the Inventors changed focus to identify a convergent synthetic route to achieve the desired compounds.

[0148] A facile synthesis was sought using a Suzuki-Miyaura coupling in the final step, leading to generate naphthalimides with bromines at the 3- and 6-positions, and tetrazines functionalised with boronate esters. The synthesis of the naphthalimide with bromine at the 3-position (2) was performed in accordance with literature procedures (Scheme 5). For the 6-bromo-naphthalimide (4), there was only one report of its synthesis, with very low yields, ^[25] so a new synthetic route was required to convert this into a useful probe.

Scheme 5. Synthesis of brominated naphthalimide building blocks, having bromine at the 3- position (2) and 6-position (4)

[0149] The bromination of 4-nitronaphthalic anhydride gave approximately 90% conversion to 3

(Scheme 6). A simultaneous imide condensation and nucleophilic aromatic substitution at the 4- position with n-butylamine gave 4 in moderate yields, significantly higher than any previously reported yield.

[0150] In the next step, the Inventors exploited a recently reported thiol-catalysed tetrazine reaction ^[35] to afford the key building blocks in adequate amounts. The synthesis of both 3- bromophenyltetrazine (5) and 4-bromophenyltetrazine (7) proceeded in moderate yields on the gram scale when catalysed by 3 -mercaptopropionic acid. The Miyaura borylation on these intermediates to form 6 and 8 proceeded efficiently in good yields (see, Scheme 6).

Scheme 6. Synthesis of boronate tetrazines for Suzuki cross-coupling [0151] These substrates were then directly coupled to the naphthalimide precursors using standard Suzuki cross-coupling conditions, affording the four desired products in moderate yields (see, Table 7).

Table 1. Synthesis of naphthalimide tetrazines C) Photophysical properties

[0152] With the four candidates in hand, the Inventors first established that none of the unreacted tetrazine products was significantly fluorescent. Whilst all of the compounds showed very quenched fluorescence, weak fluorescent excitation and emission peaks could be observed in organic solvents. For all of the conjugated naphthalimides, a weak emission peak was observed around 530 nm in EtOH. This indicates that for biological application, these probes would not fluoresce in aqueous cellular media, and therefore could be applied for “no wash” cellular imaging. To quantify the lack of fluorescence, attempts were made to measure the quantum yield of the unreacted dye in absolute ethanol (see, Table 2).

Table 2. Fluorescent properties of tetrazine naphthalimides in absolute ethanol ^a only Np3pTz had a measurable quantum yield, and this is the only brightness that could be determined accurately

[0153] All of the dyes, with the exception of Np3pTz had a fluorescence quantum yield below the detection limit of the instrument. This indicates that Np3pTz would not be an ideal candidate for biological imaging, as the residual fluorescence from the unclicked product would interfere with the fluorescence readout. The estimated brightness for each of these was < 260 M ^-1 cm ^-1 , whereas dyes used for molecular imaging typically exhibit brightness in the range of 10 ³ -10 ⁶ M- 1cm ^-1 . ^[1] Satisfied that all of the tetrazine candidates had a quenched fluorescence, particularly in aqueous media, attempts were then made to evaluate the fluorogenicity of this series of compounds.

[0154] Due to its ready availability, a BCN derivative was chosen as the reaction partner to test the fluorogenic reaction of the new naphthalimide tetrazines. The relevant tetrazine was incubated with five equivalents of BCN in DMSO for at least 10 minutes (Scheme 7, general; Scheme 8, exemplary).

Scheme 7. Generic naphthalimide tetrazine and its generic reaction product with BCN

Scheme 8. Exemplary naphthalimide tetrazines (Np3pTz, Np6mTz and Np6pTz) and their respective reaction products with BCN (Np3pPz, Np6mPz and Np6pPz)

[0155] After reaction with BCN, a significant change in the colour of the solution was observed, with a corresponding shift in the absorption spectrum (see, Figure 2). This indicated the formation of the naphthalimide pyridazine products, NpxxPz (see, section headed “Nomenclature”). Reaction completion was verified, as was the formation of the desired products using LCMS and the masses of these products were confirmed with high resolution mass spectrometry (Table 3).

Table 3. Exact masses of unreacted naphthalimide tetrazines and exact masses after reaction with BCN

[0156] The second order rate constants of the reaction were determined experimentally using the resultant fluorescent increase after treatment with BCN. As depicted in Table 4, the rate constants were in the range of 4-8 M ^-1 s ^-1 in a 1:1 MeCN:H ₂ O mixture, which is comparable to other tetrazine-BCN reactions[36] and fast enough for live-cell labelling. ^[4]

[0157] The excitation and emission spectra of equal concentrations of the tetrazines and corresponding pyridazine products were compared, with strong fold increases in fluorescence observed in all cases (see, Figure 3). Table 4. Measured second-order rate constants of the click reaction between naphthalimide tetrazines and BCN in 1:1 MeCN:H ₂ O at 25 °C. Rate constants were determined from a series of pseudo first order reactions. The error value in each figure correlates to the error of the linear regression performed between experiments

Table 5. Fluorescent properties of tetrazine naphthalimides after reaction with BCN to form pyridazine products. All properties measured in absolute EtOH

[0158] The fluorescence properties of the pyridazine products in EtOH are presented in Table 5. The derivatives with tetrazines at the 6-position had slightly red-shifted excitation maxima compared to 3-position but similar emission maxima. The fluorescence spectra of each of the pyridazine products were then collected in DCM, MeCN, EtOH and HEPES buffer (see, Figure 4). As expected for ICT fluorophores such as naphthalimides, a strong solvatochromism was observed for all compounds, with fluorescence emission being most blue shifted in the non-polar solvent and most red-shifted in aqueous media.

[0159] The quantum yields of the reacted products were significantly greater than those of the unclicked tetrazines. All reacted products exhibited a brightness greater than 10 ³ M ^-1 cm ^-1 , ^[1] confirming that the reacted products were sufficiently bright for molecular imaging. Of these compounds, Np6mPz was the brightest, which suggests that this compound is most promising for the design of a naphthalimide-based fluorogenic probe. Np6mTz exhibited a 200-fold turn- on in ethanol, which was significantly higher than any of the other compounds.

[0160] Since the cellular environment is not homogenous in polarity, the fold turn on of these tetrazine-naphthalimides was investigated after reaction with BCN using integrated fluorescence emission intensities in the same solvents employed for solvatochromism studies (see, Figure 5). Np6mTz had the highest fold turn on in all solvents, as high as 250-fold in acetonitrile.

[0161] It is notable that despite the significant differences between the compounds across the range of solvents, all of the compounds had a similar 70-130-fold turn-on in HEPES buffer. For all tetrazines, except Np6mTz, this was the highest observed value in any of the solvents tested. This is ascribed to the extremely low fluorescence of all the naphthalimide tetrazines in aqueous solvents. These studies reinforce the suitability of the inventive naphthalimide tetrazines for no- wash cellular imaging. The combined photophysical data indicate that Np6mTz is the best candidate for a tetrazine-based fluorogenic probe.

D) Molecular imaging

[0162] Before carrying out imaging studies, it was first established that the tetrazine- naphthalimides, BCN and the clicked product were not significantly toxic to cells at doses relevant to imaging. A549 cells were dosed with tetrazine probes (25 μM), BCN (250 μM) or a combination of both for 3 h. No significant changes in viability were observed during this time (see, Figure 6), indicating that the probes and reaction partners are not toxic at doses and incubation times greater than those required for imaging.

[0163] Np3mTz was initially studied to gauge the conditions under which to test the series of naphthalimides (see, Figure 7). In order to determine whether any residual fluorescence could be observed in the images, the cells were dosed at both 1 and 10 μM for 20 min, and at 10 μM for 90 min. Some residual, punctate fluorescence signals could be observed in these images (see, Figure 7A-C), potentially corresponding to the lipid droplets, but no significant differences were observed between higher and lower concentrations. Another sample was prepared where the compound was not washed off the cells after dosing (see, Figure 7D), yielding very similar images to cells that were washed. This indicated that the probes would be useful for no-wash labelling and imaging.

[0164] Having established that low doses of 1 μM were sufficient, all the probes were screened at this concentration in both their tetrazine and pyridazine forms. All of the unclicked probes had a low brightness in cells compared to their reacted forms. Weak intracellular fluorescence was observed in the 3-position tetrazine images, but not for the 6-position analogues, potentially due to their slightly red shifted excitation profiles (see, Figure 8). This red shifted excitation profile suggests the use of a 488 nm wavelength excitation, and the excitation provided by 405 and 488 nm sources were compared for each of the unreacted and reacted compounds. Importantly, no punctate fluorescence was observed for the 6-position naphthalimides under 405 nm excitation, indicating that these compounds would be more favourable for biorthogonal labelling due to a lack of interference. The lipid interference was less pronounced for Np3mTz under these settings, but a significant amount of interference was observed for Np3pTz.

[0165] It was then tested whether the sequential dosing of the reaction partners would lead to an increase in fluorescence. The cells were dosed with 1 μM tetrazine naphthalimide for 30 mins, before the addition of BCN (50 μM) for 2 h (see, Figure 9). These images confirm that the naphthalimide tetrazine is stable in the cellular environment and does not degrade to fluorescent products within this time. The bright images obtained after a 2 h incubation with BCN confirm that the biorthogonal reaction happens within the cell and that the fluorescent product is sufficiently bright for confocal microscopy imaging with low power.

[0166] It was then evaluated how fast this reaction would occur within cells, and based on the photophysical data, Np6mTz was selected as the candidate for testing. The conditions of the previous experiment were replicated, except for the use of a higher dose of probe (2.5 μM) to ensure a bright signal and collected images every 2 min for 30 min (see, Figure 10). Maximum brightness was observed within 20 min, indicating that the BCN partner rapidly enters the cell. However, when the sequence of doses was reversed, only a very weak fluorescence was detected after 30 min (see, Figure 11). Since it is known that significant amounts of Np6mTz enters the cell within this time, this observation was ascribed to the BCN being metabolised by or removed from the cell during the 30 min dosing period.

E) Synthesis and characterisation of mitochondrial membrane potential probe

[0167] Exemplary mitochondrial membrane potential probe MitoNp6mTz was synthesised in five steps from brominated naphthalimide 3, as per Scheme 9, below. MitoNp6mTz was subsequently reacted with MitoBCN to form MitoNp6mPz, or “MitoMPP” (see, Scheme 3, above). Comparative photophysical data for MitoNp6mTz and MitoNp6mPz are provided below (see also, Figure 12 and Figure 13), and the fold turn-on data augur well for the use of the inventive compounds as probes for measuring MMP.

Scheme 9. Five- step synthesis of MMP probe MitoNp6mTz Table 5. Summary of comparative photophysical data for MitoNp6mTz and MitoNp6mPz

Table 6. Fold turn-on comparative data for MitoNp6mTz and MitoNp6mPz (“MitoMPP”)

F) Cellular toxicity data

[0168] Initial toxicity data for the probes was assessed using both AlamarBlue and MTT cytotoxicity assays using standard protocols. In an initial screen, both probe components and the clicked product MitoMPP were non-toxic to cells dosed at the high concentrations 25 μM for 3 h

(see, Figure 16). To verify that the probes did induce any cytotoxic effects at the concentrations at which they were used in cellular applications, a MTT assay was performed using the probes at the concentrations which were used for imaging (see, Figure 17).

G) MMP disruption with FCCP

[0169] Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) was used as a mitochondrial membrane disruptor. FCCP is a known protonophore that disrupts the proton gradient, raising the mitochondrial membrane potential towards zero (i.e., more positive), and therefore the accumulation of probe should be inhibited. A significant decrease in fluorescence intensity was observed in cells that had been exposed to 1 μM FCCP compared to those exposed to 0-0. IμM FCCP (see, Figure 18). This indicates that the two part probe system is sensitive to mitochondrial membrane potential.

H) JC-1 initial testing

[0170] The fluorescence of the new probe was compared to “JC-1”, a known mitochondrial membrane potential dye. JC-1 (CBIC2) is a fluorescent lipophilic carbocyanine dye used to measure mitochondrial membrane potential. JC-1 forms complexes known as J-aggregates at high ΔΨm. Aggregates of JC-1 emit an orange-red fluorescence (Ex/Em 585/590 nm). While in cells with low ΔΨm, JC-1 remains in the monomeric form. JC-1 monomers emit a green fluorescence (Ex/Em 510/527 nm).

[0171] The purpose of this test was to investigate whether the fluorescence response correlated to that of known probes. The fluorescence response of the new MMP probe under the same conditions as JC-1 showed a more dramatic difference between the control and treatments with

0.1 and 1 μM FCCP (see, Figure 19). This indicates that the probe is sensitive to changes in the MMP.

1) Platinum complexes used to induce mitochondrial stress

[0172] Mitochondrial stress was also induced using the clinically approved platinum-based chemotherapeutics cisplatin and oxaliplatin, as well as the novel complex, phenanthriplatin, which is more toxic than cisplatin. It was observed that within 1 h of treatment, that the two more toxic compounds (cisplatin and phenanthriplatin) induced decreases in the fluorescence of the probe, where oxaliplatin did not (see, Figure 20). This may indicate that the difference in their potency is dependent on inhibition of mitochondria, and invites further investigation.

Experimental Data

[0173] 3-Bromo-4-butylamino-1 ,8-naphthalimide (2)

[0174] 1 (500 mg, 1.5 mmol) was dissolved in DMF (7 mL) and cooled to 0 °C. A solution of freshly-recrystallised N-bromosuccinimide (0.37 g, 2.1 mmol) in DMF (10 mL) was added dropwise over 1 h. The reaction was warmed to rt and stirred for 3 h. The reaction mixture was diluted with 5% LiCl solution (100 mL) and extracted with EtOAc (50 mL). The organic extracts were washed with 5% LiCl solution (5 x 50 mL), dried over MgSO ₄ and concentrated in vacuo. The residue was purified with column chromatography (SiO ₂ , 6% EtOAc in hexanes). 2 was obtained as a bright yellow powder (0.49 g, 79%).

[0175] ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.62 (s, 1H), 8.57 (d, J = 7.3 Hz, 1H), 8.45 (d, J = 8.6 Hz, 1H), 7.64 (t, J = 7.6 Hz, 1H), 4.15 (t, J = 7.6 Hz, 2H), 3.67 (t, J = 6.9 Hz, 2H), 1.78-1.64 (m, 4H), 1.53-1.37 (m, 4H), 0.99-0.94 (m, 6H). ¹³ C NMR (101 MHz, CDCl ₃ ): 164.3, 163.2, 149.8, 135.6, 131.5, 130.7, 129.8, 125.3, 123.5, 123.4, 114.4, 110.0, 50.9, 40.3, 33.7, 30.6, 20.5, 20.1, 14.0, 13.9. LRMS (APCI) m/z. [M+H] ⁺ Calcd for C ₂₀ H ₂₃ BrN ₂ O ₂ ⁺ 403 and 405; Found 403 and 405. Characterisation data match those reported in the literature. ^[1] [0176] 3-Bromo-5-nitro-1,8-naphthalic anhydride (3)

[0177] 4-Nitro-l,8-naphthalic anhydride (2.50 g, 10.3 mmol) was dissolved in cone. H ₂ SO ₄ (40 mL). N-bromosuccinimide (1.52 g, 8.54 mmol) was added and the resultant mixture heated to 60 °C and stirred for 16 h. The reaction mixture was then cooled and poured over ice, then extracted with CH ₂ CI ₂ (3 x 200 mL). The combined organics were dried (Na ₂ SO ₄ ), filtered, and concentrated in vacuo to give the desired product as a pale yellow solid (2.85 g, 86%).

[0178] ¹ H NMR (500 MHz, CDCl ₃ ): δ 9.17 (1H, d, J = 1.8 Hz), 8.84 (1H, d, J = 1.8 Hz), 8.75 (1H, d, J = 8.0 Hz), 8.54 (1H, d, J = 8.0 Hz). ¹³ C NMR (125 MHz, CDCl ₃ ): δ 158.1, 158.0, 148.9, 137.6, 133.0, 132.1, 129.6, 125.6, 125.5, 125.0, 123.5, 120.6. HRMS (APPI) m/z: [M] ⁺ Calcd for C ₁₂ H ₄ BrNO ₅ ⁺ 320.9267; Found 320.9267.

[0179] 3-Bromo-5-butylamino-N-butyl- 1 ,8-naphthalimide (4)

[0180] 1 (0.20 g, 0.62 mmol) and Et ₃ N (0.18 mL, 1.3 mmol) were suspended in absolute EtOH (10 mL). n-Butylamine (0.13 mL, 1.3 mmol) was added dropwise and the reacted heated to reflux for 16 h. The reaction was diluted with 1 M HCl (50 mL) and extracted with CH ₂ CI ₂ (3 x 30 mL). The organic extracts were washed with 1 M HCl (2 x 100 mL) and brine (100 mL), dried over Na ₂ SO ₄ and concentrated in vacuo. The residue was purified by column chromatography (SiO ₂ , 0-7.5% EtOAc in hexanes) to afford 4 (0.13 g, 53%) as an orange powder.

[0181] ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.56 (d, J = 1.8 Hz, 1H), 8.40 (d, J = 8.4 Hz, 1H), 8.16 (d, J = 1.8 Hz, 1H), 6.69 (d, J = 8.4 Hz, 1H), 4.13 (t, J = 7.5 Hz, 2H), 3.38 (t, J = 7.2 Hz, 2H), 1.83- 1.76 (m, 2H), 1.71-1.64 (m, 2H), 1.58-1.49 (m, 2H), 1.46-1.37 (m, 2H), 1.02 (t, J = 7.4 Hz, 3H), 0.95 (t, J = 7.4 Hz, 3H). ¹³ C NMR (101 MHz, CDCl ₃ ): δ 163.8, 163.6, 148.6, 134.7, 133.8, 128.3, 124.9, 121.7, 118.7, 110.4, 105.2, 43.7, 40.3, 31.1, 30.4, 20.5, 20.5, 14.0, 14.0. LRMS (APCI) m/z: [M+H] ⁺ Calcd for C ₂₀ H ₂₃ BrN ₂ O ₂ ⁺ 403 and 405; Found 403 and 405.

[0182] 3 -( 3 -bromophenyl)-6-methyl- 1,2,4, 5 -tetrazine (7)

[0183] 3-Bromobenzonitrile (1.0 g, 5.5 mmol) was suspended in absolute EtOH (0.75 mL) and MeCN (2.3 mL, 44 mmol) and cooled to 0 °C. 3 -mercaptopropionic acid (0.48 mL, 5.5 mmol) and hydrazine hydrate (4.3 mL, 88 mmol) were sequentially added dropwise. The reaction was stirred for 30 min before being warmed to 40 °C for 16 h. The reaction was slowly poured into a solution of NaNO ₂ (5.7 g, 15 equiv.) in water, open to air. 1 M HCl was then added dropwise until the mixture was pH 3, with no effervescence and was vivid pink/red colour (SAFETY: During the addition of acid, small amounts of toxic NO _x gases are produced. Perform this step in a well-ventilated fume hood). The mixture was extracted with CH ₂ CI ₂ (3 x 50 mL) and the organic extracts were washed with brine (150 mL), dried over Na ₂ SO ₄ and concentrated to dryness in vacuo. Purification by column chromatography (SiO2, 0-35% CH ₂ CI ₂ in hexanes) afforded 7 (0.78 g, 56%) as pink crystals.

[0184] ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.75 (s, 1H), 8.53 (d, J = 7.8 Hz, 1H), 7.75 (d, J = 7.8 Hz, 1H), 7.47 (t, J = 7.9 Hz, lH), 3.12 (s, 3H). ¹³ C NMR (101 MHz, CDCl ₃ ): δ 167.8, 163.3, 135.6, 133.9, 131.0, 130.9, 126.5, 123.6, 21.4. LRMS (APCI) m/z: [M+H] ⁺ Calcd for C ₉ H ₈ BrN ₄ 251 and 253; Found 251 and 253.

[0185] 3-methyl-6-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)p henyl)-1,2,4,5-tetrazine (8) [0186] 7 (0.25 g, 1.0 mmol), B2pin2 (0.30 g, 1.2 mmol), KOAc (0.15 g, 1.5 mmol) and PdCl ₂ (dppf) (36 mg, 5 mol%) were put under N2 atmosphere. Degassed (3 x freeze-pump-thaw cycles) THF (7 mL) was added, and the reaction heated to 65 °C for 16 h. The reaction mixture was diluted with water (20 mL) and extracted with CH ₂ CI ₂ (3 x 15 mL). The organic extracts were washed with brine (50 mL), dried over Na ₂ SO ₄ and concentrated in vacuo. Purification by column chromatography (SiO ₂ , 2-10% EtOAc in hexanes) afforded 8 (pink solid; 0.22 g, 74%). [0187] ¹ H NMR (500 MHz, CDCl ₃ ): δ 9.03 (s, 1H), 8.68-8.64 (m, 1H), 8.05 (d, J = 7.2 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 3.10 (s, 3H), 1.38 (s, 12H). ¹³ C NMR (101 MHz, CDCl ₃ ): δ 167.4, 164.4, 138.9, 134.5, 131.4, 130.7, 128.8, 84.3, 25.1, 21.3. HRMS (ESI) m/z: [M+Na] ⁺ Calcd for C ₁₅ H ₁₉ BNaN ₄ O ₂ 321.1493; Found 321.1495.

[0188] General method for Suzuki coupling

[0189] The relevant brominated naphthalimide (1 equiv.), boronate ester (1.5-2 equiv.), K ₂ CO ₃ (2 equiv.) and Pd(PPh ₃ ) ₄ (5-10 mol% catalyst loading) were added to a flask under a N ₂ atmosphere. The flask was evacuated and refilled with N2 thrice. A degassed solvent mixture was added (4:1 THF:H ₂ O; final reaction concentration approximately 0.01-0.02 M relative to brominated naphthalimide), and the mixture was heated at the indicated temperature for the indicated time. Upon completion of the reaction (as determined by TLC analysis), the reaction was diluted with water (30 mL) and extracted thrice with CH ₂ CI ₂ (3x30 mL). The organic extracts were washed with brine (100 mL), dried over Na ₂ SO ₄ and concentrated in vacuo. The crude residue was purified by column chromatography to afford the desired products. [0190] Np3mTz

[0191] 2 (30 mg, 0.074 mmol), 6 (44 mg, 0.15 mmol), K2CO3 (21 mg, 0.15 mmol) and Pd(PPh3)4 (4 mg, 5 mol%) in 4: 1 THF:H ₂ O (7 mL) were subjected to the General Method for Suzuki Coupling at 65 °C for 5 h. Purification by column chromatography (SiO ₂ , 0-4% EtOAc in CH ₂ CI ₂ ) afforded Np3mTz as a red powder (22 mg, 60%).

[0192] ¹ H NMR (500 MHz, CDCl ₃ ): δ 8.72 (s, 1H), 8.66-8.65 (m, 1H), 8.61 (d, J = 7.2 Hz, 1H), 8.45 (s, 1H), 8.40 (d, J = 8.4 Hz, 1H), 7.76-7.72 (m, 2H), 7.69 (t, J = 7.7 Hz, 1H), 4.69 (br s, 1H), 4.18 (t, J = 7.5 Hz, 2H), 3.28 (t, J = 7.0 Hz, 2H), 3.12 (s, 3H), 1.72 (quint, J = 7.6 Hz, 2H), 1.51- 1.42 (m, 4H), 1.24-1.17 (m, 2H), 0.97 (t, J = 7.4 Hz, 3H), 0.78 (t, J =7.4 Hz, 3H). ¹³ C NMR (126 MHz, CDCl3): δ 167.7, 164.7, 164.2, 164.0, 149.7, 140.5, 135.3, 133.7, 132.8, 131.4, 130.2, 129.9, 129.5, 128.9, 127.4, 125.4, 125.3, 123.7, 123.4, 113.5, 50.2, 40.2, 33.2, 30.4, 21.4, 20.5, 20.0, 14.0, 13.8. HRMS (ESI) m/z: [M+Na] ⁺ Calcd for C ₂₉ H ₂₉ N ₆ NaO ₂ 517.2322; Found 517.2323.

[0193] Np3pTz

[0194] 2 (30 mg, 0.074 mmol), 8 (44 mg, 0.15 mmol), K2CO3 (21 mg, 0.15 mmol) and Pd(PPh3)4 (4 mg, 5 mol%) in 4: 1 THF:H ₂ O (7 mL) were subjected to the General Method for Suzuki Coupling at 65 °C for 4 h. Purification by column chromatography (SiO ₂ , 0-4% EtOAc in CH ₂ Cl ₂ ) afforded Np3pTz as a red powder (17 mg, 48%).

[0195] ¹ H NMR (500 MHz, CDCl ₃ ): δ 8.74-8.71 (m, 2H), 8.61 (dd, J = 7.3, 0.8 Hz, 1H), 8.44 (s, 1H), 8.39 (dd, J = 8.4, 0.8 Hz, 1H), 7.73-7.67 (m, 3H), 4.75 (br s, 1H), 4.16 (t, J = 7.5 Hz, 2H), 3.26 (t, J = 7.0 Hz, 2H), 3.13 (s, 3H), 1.75-1.69 (m, 2H), 1.52-1.41 (m, 4H), 1.27-1.19 (m, 2H), 0.96 (t, J = 7.5 Hz, 3H), 0.80 (t, J = 7.3 Hz, 3H). ¹³ C NMR (126 MHz, CDCl ₃ ): δ 167.5, 164.7, 164.1, 164.0, 149.6, 143.9, 135.2, 131.4, 131.4, 130.4, 129.9, 129.3, 128.7, 125.4, 125.1, 123.6, 123.4, 113.5, 50.1, 40.2, 33.3, 30.4, 21.4, 20.5, 20.0, 14.0, 13.8. HRMS (ESI) m/z: [M+Na] ⁺ Calcd for C ₂₉ H ₂₉ N ₆ NaO ₂ 517.2322; Found 517.2323.

[0196] Np6mTz

[0197] 4 (35 mg, 0.087 mmol), 6 (44 mg, 0.15 mmol), K2CO3 (24 mg, 0.17 mmol) and Pd(PPh ₃ ) ₄ (5 mg, 5 mol%) in 4: 1 THF:H ₂ O (8 mL) were subjected to the General Method for Suzuki Coupling at 60 °C for 3 h. Purification by column chromatography (SiO ₂ , 0-6% EtOAc in CH ₂ Cl ₂ ) afforded Np6mTz as a red powder (18 mg, 43%). [0198] ¹ H NMR (500 MHz, CDCl ₃ ): δ 8.84 (s, 1H), 8.80 (d, J = 1.2 Hz, 1H), 8.55 (d, J = 7.8 Hz, 1H), 8.41(d, J = 8.5 Hz, 1H), 8.31 (d, J = 1.2 Hz, 1H), 7.89 (d, J = 7.8 Hz, 1H) , 7.66 (t, J = 7.8 Hz, 1H), 6.73 (d, J = 8.5 Hz, 1H), 5.64 (br s, 1H), 4.18 (t, J = 7.6 Hz, 2H), 3.46 (t, J = 7.3 Hz, 2H), 3.12 (s, 3H), 1.89-1.81 (m, 2H), 1.76-1.69 (m, 2H), 1.59-1.41 (m, 4H), 1.03 (t, J = 7.4 Hz, 3H), 0.98 (t, J = 7.4 Hz, 3H). ¹³ C NMR (101 MHz, CDCl ₃ ): 167.7, 164.7, 164.1, 163.9, 149.9, 141.1, 136.8, 134.6, 132.6, 131.6, 130.3, 130.1, 129.2, 127.4, 126.5, 124.2, 123.9, 120.8, 110.1, 104.9, 43.8, 40.2, 31.2, 30.5, 21.4, 20.6, 20.6, 14.0, 14.0. HRMS (ESI) m/z: [M+Na] ⁺ Calcd for C ₂₉ H ₂₉ N ₆ NaO ₂ 517.2322; Found 517.2323.

[0199] Np6pTz

[0200] 4 (40 mg, 0.099 mmol), 8 (50 mg, 0.17 mmol), K ₂ CO ₃ (27 mg, 0.20 mmol) and Pd(PPh ₃ ) ₄ (6 mg, 5 mol%) in 4: 1 THF:H ₂ O (7 mL) were subjected to the General Method for Suzuki Coupling at 60 °C for 16 h. Purification by column chromatography (SiO ₂ , 0-4% EtOAc in CH ₂ Cl ₂ ) afforded Np6pTz as a red powder (25 mg, 69%).

[0201] ¹ H NMR (500 MHz, CDCl ₃ ): δ 8.78 (s, 1H), 8.61 (d, J = 7.6 Hz, 2H), 8.40 (d, J = 8.2 Hz, 1H), 8.27 (s, 1H), 7.84 (d, J = 7.6 Hz, 2H), 6.74 (d, J = 7.8 Hz, 1H), 4.16 (t, J = 7.4 Hz, 2H), 3.46 (t, J = 7.2 Hz, 2H), 3.10 (s, 3H), 1.90-1.81 (m, 2H), 1.75-1.67 (m, 2H), 1.61-1.52 (m, 2H), 1.49-1.39 (m, 2H), 1.04 (t, J = 7.2 Hz, 3H), 0.97 (t, J = 7.2 Hz, 3H). ¹³ C NMR (101 MHz, CDCl ₃ ): δ 167.4, 164.7, 164.1, 163.8, 149.9, 143.8, 136.5, 134.7, 131.3, 130.2, 129.3, 128.6, 128.1, 124.2, 123.9, 120.8, 110.3, 105.0, 43.8, 40.2, 31.2, 30.5, 21.3, 20.6, 14.0. HRMS (APPI) m/z: [M] ⁺ Calcd for C ₂₉ H ₂₉ N ₆ O ₂ 494.2425; Found 494.2424.

[0202] Tert-butyl (2-(5-bromo-7-nitro-1,3-dioxo-lH-benzo[de]isoauinolin-2(3H)- yl)ethyl)- carbamate (9)

[0203] 3 (0.50 g, 1.6 mmol) was dissolved in anhydrous 1,4-dioxane (20 mL). N-Boc- ethylenediamine (0.30 mL, 1.9 mmol) and Et ₃ N (0.24 mL, 1.7 mmol) were added and the reaction heated to 100 °C for 4 h. The reaction mixture was diluted with water (50 mL, pH 4, acidified with 1 M HCl) and extracted with EtOAc (50 mL). The organic extracts were washed with water (50 mL, pH 4) and brine (2 x 50 mL), dried over Na ₂ SO ₄ and concentrated in vacuo. Purification by column chromatography (SiO ₂ , 0-7% EtOAc in CH ₂ Cl ₂ ), affording 9 (0.38 g, 52%) as a beige powder.

[0204] ¹ H NMR (500 MHz, CDCl ₃ ): δ 9.07 (s, 1H), 8.79 (s, 1H). 8.69 (d, J = 7.8 Hz, 1H), 8.46 (d, J = 7.8 Hz, 1H), 4.83 (s, 1H), 4.35 (t, J = 5.2 Hz, 2H), 3.58-3.47 (m, 2H), 1.22 (s, 9H). ¹³ C NMR (126 MHz, CDCl ₃ ): δ 162.7, 162.5, 156.3, 148.3, 135.8, 131.6, 130.1, 127.9, 127.3, 125.4, 125.3, 124.9, 124.4, 79.5, 40.9, 39.2, 28.3. HRMS (ESI) m/z: [M+Na] ⁺ Calcd for C ₁₉ H ₁₈ BrN ₃ O ₆ Na 486.0271; Found 486.0270.

[0205] Tert-butyl (2-(5-bromo-7-(butylamino)-1,3-dioxo-1H-benzo[de]isoquinolin -2(3H)- yl)ethyl)carbamate (10)

[0206] 9 (0.18 g, 0.38 mmol) was suspended in MeCN (17 mL). n-Butylamine (0.12 mL, 1.13 mmol) was added dropwise, and the reaction heated to 70 °C for 16 h. The solvent was removed in vacuo and the residue purified by column chromatography (SiO ₂ , 2-15% EtOAc in CH ₂ CI ₂ ). This gave 10 (0.15 g, 80%) as an orange-yellow powder.

[0207] ¹ H NMR (500 MHz, DMSO-d ₆ ): δ 8.97 (s, 1H), 8.37-8.34 (m, 1H), 8.22 (t, J = 8.5 Hz, 1H), 7.75 (t J = 4.9 Hz, 1H), 6.82 (t, J = 6.0 Hz, 1H), 6.77 (d, J = 8.7 Hz, 1H), 4.07 (t, J = 6.0 Hz, 2H), 3.34 (q, J = 6.8 Hz, 2H), 3.20 (q, J = 5.9 Hz, 2H), 1.72-1.66 (m, 2H), 1.47-1.42 (m, 2H), 1.24 (s, 9H), 0.95 (t, J = 7.4 Hz, 3H). ¹³ C NMR (126 MHz, DMSO-d ₆ ): δ 162.8, 162.7, 155.6, 149.6, 134.4, 132.3, 130.4, 128.2, 124.1, 121.6, 117.0, 107.7, 104.5, 77.4, 42.7, 37.9,

29.8, 28.1, 19.8, 13.7. NCH ₂ not visible but confirmed under solvent at 39.5 ppm by HSQC.

HRMS (ESI) m/z: [M+Na] ⁺ Calcd for C ₂₃ H ₂₈ BrN ₃ O ₄ Na 512.1155; Found 512.1154.

[0208] Tert-butyl (2-(7-(butylamino)-5-(3-(6-methyl-1,2,4,5-tetrazin-3-yl)phen yl)-1,3-dioxo-1H- benzo[de]isoquinolin-2(3H)-yl)ethyl)carbamate (11)

[0209] 10 (70 mg, 0.14 mmol), 19 (64 mg, 0.21 mmol), CS ₂ CO ₃ (93 mg, 0.27 mmol) and Pd(PPh ₃ ) ₄ (19 mg, 10 mol%) in degassed (3 x freeze-pump-thaw cycles) 9:1 THF:H ₂ O (7 mL) were subjected to General Method for Suzuki Coupling at 65 °C for 4 h. Purification by silica column chromatography (SiO ₂ , 3-20% EtOAc in CH ₂ CI ₂ ) afforded 11 (49 mg, 59%) as a red powder.

[0210] ¹ H NMR (500 MHz, CDCl ₃ ): δ 8.89 (s, 1H), 8.85 (s, 1H), 8.62 (d, J = 7.7 Hz, 1H), 8.44 (d, J = 8.4 Hz, 1H), 8.33 (s, 1H), 7.95 (d, J = 7.7 Hz, 1H), 7.72 (t, J = 7.7 Hz, 1H), 6.75 (d, J = 8.4 Hz, 1H), 5.65-5.59 (m, 1H), 5.20 (br s, 1H), 4.38 (br s, 2H), 3.55 (br s, 2H), 3.47 (q, J = 6.7 Hz, 2H), 3.13 (s, 3H), 1.85 (quint, J = 7.3 Hz, 2H), 1.59-1.51 (m, 2H), 1.33 (s, 9H), 1.03 (t, J = 7.3 Hz, 3H). ¹³ C NMR (126 MHz, CDCl ₃ ): δ 167.7, 165.1, 164.5, 164.0, 156.3, 150.1, 141.0, 141.0, 136.9, 135.0, 132.7, 131.7, 130.6, 130.3, 129.4, 127.5, 126.6, 124.4, 123.7, 120.8, 109.8, 104.9, 79.2, 43.8, 40.4, 39.8, 31.2, 28.5, 21.4, 20.6, 14.0. HRMS (ESI) m/z: [M+H] ⁺ Calcd for C ₃₂ H ₃₆ N ₇ O ₄ 582.2823; Found 582.2820. [0211] 2-(7-(Butylamino)-5-(3-(6-methvl-1,2,4,5-tetrazin-3-yl)pheny l)-1,3-dioxo-1H- benzo[de]isoquinolin-2(3H)-yl]ethan-1-aminium 2,2,2-trifluoroacetate (12)

[0212] 11 (17 mg, 0.030 mmol) was dissolved in CH ₂ CI ₂ (2.5 mL). Trifluoroacetic acid (0.75 mL) was added and the reaction stirred for 30 min at rt. The solvent was removed under a stream of N ₂ . This afforded 12 in quantitative yield as a red residue that was used without further purification.

[0213] ¹ H NMR (500 MHz, MeOD): δ 8.80 (t, J = 1.7 Hz, 1H), 8.74 (d, J = 1.7 Hz, 1H), 8.66 (d, J = 1.7 Hz, 1H), 8.47 (dt, J = 7.8, 1.0 Hz, 1H), 8.23 (d, J = 8.7 Hz, 1H), 8.04-8.00 (m, 1H), 7.71 (t, J = 7.8 Hz, 1H), 6.71 (d, J = 8.7 Hz, 1H), 4.43(t, J = 5.9Hz, 2H), 3.44 (t, J = 7.5 Hz, 2H), 3.34 (t, J = 5.7 Hz, 2H), 3.08 (s, 3H), 1.82-1.75 (m, 2H), 1.56-1.48 (m, 2H), 1.03 (t, J = 7.5 Hz, 3H). ¹³ C NMR (126 MHz, MeOD): δ 169.0, 166.6, 165.7, 165.1, 153.2, 141.5, 137.1, 136.2, 134.2, 132.0, 131.1, 130.7, 128.2, 127.4, 127.0, 123.6, 122.2, 108.4, 105.5, 44.4, 40.3, 38.8, 31.6, 21.5, 21.2, 14.3. 1 x C not observed. HRMS (ESI) m/z. [M+H] ⁺ Calcd for C ₂₇ H ₂₈ N ₇ O ₂ 482.2299;

Found 482.2294.

[0214] MitoNp6mTz

[0215] 4-Carboxybutyltriphenylphosphonium bromide (11.6 mg, 29.8 μmol), HATU (21.3 mg, 56.0 μmol) and DIPEA (25 pL, 140 μmol) were dissolved in DMF (1.0 mL). A solution of 12 (17 mg, 30 μmol) in DMF (1.7 mL) was added dropwise and the reaction was protected from light and stirred at rt for 3 h. The mixture was diluted with 5% LiCl solution (20 mL). The mixture was extracted with CH ₂ CI ₂ (2 x 25 mL). The organic extracts were washed with 5% LiCl solution (2 x 50 mL) and brine (50 mL), dried over Na ₂ SO ₄ and concentrated in vacuo. The compound was purified by column chromatography (SiO ₂ , 0-5% MeOH in CH ₂ CI ₂ ). The pure fractions were triturated thrice into Et ₂ O with the solid isolated by centrifugation each time. This afforded MitoNp6mTz (10 mg, 44%) as a red solid.

[0216] ¹ H NMR (500 MHz, CDCl ₃ ): δ 8.49 (s, 1H), 8.46 (s, 1), 8.33 (d, J = 7.7 Hz, 1H) 8.23 (s, 1H), 8.07 (d, J = 8.5 Hz, 1H), 7.84 (d, J = 7.6 Hz, 1H), 7.75-7.69 (m, 3H), 7.64-7.55 (m, 13H), 6.78 (t, J = 4.9 Hz, 2H), 6.51 (d, J = 8.7 Hz, 1H), 6.06 (t, J = 4.9 Hz, 2H), 4.22 (t, J = 5.6 Hz, 2H), 3.58 (q, J = 5.2 Hz, 2H), 3.37 (q, J = 6.5 Hz, 2H), 3.19-3.13 (m, 2H), 3.06 (s, 3H), 2.26 (t, J = 6.7 Hz, 2H), 1.83 (quint, J = 6.7 Hz, 2H), 1.76 (quint, J = 13 Hz, 2H), 1.73- 1.63 (m, 2H), 1.50-1.44 (m, 2H), 0.97 (t, J = 13 Hz, 3H). ¹³ C NMR (126 MHz, CDCl ₃ ): δ 172.9, 167.5, 165.0, 164.1, 163.7, 150.6, 139.9, 135.8, 135.3 (d, J _C _ _P = 2.7 Hz), 134.6, 133.5 (d, J _C _ _P = 9.9 Hz), 132.1, 131.6, 130.6 (d, J _C _ _P = 12.5 Hz), 130.3, 129.6, 129.0, 127.2, 125.7, 124.7, 122.7, 120.6, 118.0

(d, J _C _ _P = 85.4 Hz), 108.3, 104.6, 43.7, 39.5, 39.0, 34.7, 30.8, 25.9 (d, J _C _ _P = 17 Hz), 21.8 (d, J _C _ _P = 56 Hz), 21.6, 21.3, 20.5, 14.0. ³¹ P NMR (202 MHz, CDCl ₃ ): δ 23.7. HRMS (ESI) m/z: [M] ²⁺ Calcd for C ₅₀ H ₄₉ N ₇ O ₃ P 826.3629; Found 826.3617.

Conclusions

[0217] The presently-described invention shows particular promise in several significant respects: As the first naphthalimide conjugated tetrazines in the literature, the inventive compounds show promise as biorthogonal labelling agents; and that the core naphthalimide tetrazine structure allows for easy synthetic modifications to be made to the scaffold [0218] The inventors have identified the following non-exhaustive list of practical and commercial advantages: real-time live cell imaging of mitochondrial membrane potential (MMP); ability to resolve individual mitochondria; ability to turn-on fluorescence of the probe; selective for measuring MMP; easy to use, requiring no specialist training beyond standard cell culture and light microscopy techniques; compatible with most existing light microscopy devices; and provides a fluorogenic response within the cellular environment.

Industrial Applicability

[0219] The invention allows for live cell and organism imaging of the mitochondrial membrane potential in real-time. In exemplary embodiments, the “turn-on” probe comprises two components, with each of the components having a targeting ligand (triphenylphosphonium group) which binds to the mitochondria. Each component of the probe also contains complimentary reactive groups, and the reactive groups of the two components undergo a biorthogonal reaction when in close proximity in the mitochondria to provide a covalent linkage. One reactive group is a tetrazine moiety (simultaneously quenches the fluorescent probe attached to the tetrazine component) which undergoes a selective biorthogonal reaction with the other complimentary reactive group being a strained dienophile such as a bicyclononyne or trans- cyclooctene. When the two complimentary reactive groups react, a pyridazine product is formed which restores fluorescence of the quenched fluorophore.

[0220] The only by-product of this reaction is nitrogen and the probe provides an observable signal that can be measured with standard confocal light microscopy techniques.

[0221] An increase in MMP increases the accumulation of hydrophobic cationic groups such as the targeting ligand of the probe into the mitochondria. Therefore, the inventive probe is expected to be highly sensitive to the membrane potential.

[0222] The invention is expectably compatible with most existing light microscopy devices.

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