GB2567124A

GB2567124A - Imaging agents and methods

Info

Publication number: GB2567124A
Application number: GB1707342.0A
Authority: GB
Inventors: Král Vladimir; Havlik Martin; Kaplánek Robert; Briza Tomás; Kejik Zdenek; Martásek Pavel; Krcová Lucie; Králová Jarmila; Ruml Tomás; Rimpelova Silvie
Original assignee: Charles Univ; Vysoka Akola Chemicko Tech V Praze
Current assignee: Charles Univ; Vysoka Akola Chemicko Tech V Praze
Priority date: 2017-05-08
Filing date: 2017-05-08
Publication date: 2019-04-10
Also published as: WO2018206126A1; GB201707342D0

Abstract

An imaging method comprising bringing a biological specimen into association with a compound of Formula I; irradiating the specimen with light; and observing two-photon excited fluorescence images emitted from the compound of Formula I; Wherein X and Y is as defined herein; B is an optionally substituted aromatic or heteroaromatic ring; M is 1-3; p is 1-3; Z is an anion having a negative charge of n; n is 1-3; q is 1-3; provided that m x p = n x q. B may be selected from phenyl, pyridyl, thienyl, furanyl, naphthyl, quinolyl and isoquinolyl, particularly 4-pyridyl. m, p, n and q are preferably 1. Z may be a halide anion, particularly iodide. Compounds of Formula 1 wherein B is phenyl substituted with 1-3 groups independently selected from fluoro and C1-C3, particularly trifluoromethyl are disclosed. The disclosed compounds are indicated to have greater imaging depth and photostability than known agents, whilst having lower toxicity. The methods are disclosed to have selectivity for mitochondria. The methods are disclosed to have utility in both in vitro and in vivo imaging, in particular, imaging of tumours.

Description

The invention relates, in one aspect to methods of imaging biological samples in vitro and in vivo, to methods of imaging and diagnosis, and of compounds useful in such methods.

Introduction

Fluorescence imaging is used as a non-destructive way of tracking or analysing biological molecules, both in vitro and in vivo. Certain small molecules or proteins present in cells are intrinsically fluorescent (such as NADH and tryptophan). Alternatively, biological molecules can be labelled with an extrinsic fluorophore. Examples of extrinsic fluorophores include fluorescent antibodies, proteins and small molecule probes. Several techniques exist to exploit additional properties of fluorophores, such as fluorescence resonance energy transfer, where the energy is passed non-radiatively to a particular neighbouring dye, allowing proximity or protein activation to be detected; another is the change in properties, such as intensity, of certain dyes depending on their environment allowing their use in structural studies.

Two-photon fluorescence microscopy is a technique that allows imaging of living tissue to a greater depth than conventional (single photon) fluorescence imaging. Being a type of multiphoton fluorescence, it employs red-shifted excitation light which is capable of exciting fluorescent dyes. Two-photon excitation is a phenomenon in which two photons are simultaneously absorbed by the same fluorophore by exposure to high photon density per unit volume and time by irradiation with a suitable light source. The use of infrared light minimizes scattering in the tissue. The background signal is strongly suppressed. Both effects lead to an increased penetration depth for these microscopes. Two-photon excitation can be a superior alternative to confocal microscopy due to its deeper tissue penetration, efficient light detection, and reduced phototoxicity.

Probes for use in two-photon microscopy are known. These include coumarin, rhodamine, xanthene, and cyanine dyes. Examples of rhodamine dyes include Alexa Fluor® 488 (J Biol Chem (2004) 279:37544-37550). Examples of cyanine dyes include Alexa Fluor® 647, available from Thermo Fisher Scientific of Waltham, Massachusetts, USA.

Mitochondria, an organelle found in almost all eukaryotic cells, play a vital role in the life and death of cells. The function of mitochondria is to produce ATP. The production of ATP involves a series of electron transport systems in the oxidative phosphorylation pathway, which is also found to be associated with the generation of reactive oxygen species (ROS). The production of ROS in mitochondria leads to the propagation of free radicals, damaging cells, and contributing to cell death, which is known as mitochondria-mediated apoptosis.

Fluorescent probes which selectively stain mitochondria are powerful tools for monitoring and studying cellular processes. Such probes must be photostable under the conditions of light exposure from fluorescent microscopes. Fluorescent dyes for mitochondrial staining have been developed, including those mentioned above. The photostability of known dyes is, however, rather poor. Moreover, known probes display significant systemic toxicity, rendering them less useful for in vivo imaging.

In view of the foregoing, there is an urgent need to develop two-photon fluorescent probes that can selectively stain mitochondria under in vivo and in vitro conditions. The ideal characteristics of such an agent would include one or more of the following: i) narrow excitation and fluorescence emission spectra; ii) minimal fluorescence in an aqueous environment, iii) maximal fluorescence after incorporation in the lipophilic environment; and iv) high binding selectivity for live cells and tissue; v) high binding photostability, and vi) low systemic toxicity.

Fluorescent probes displaying selectivity for cancer cells are extremely desirable. Improved visualization of tumours in vivo would aid diagnosis, facilitate surgical resection, investigate therapeutic efficacy, and improve prognosis. Fluorescence imaging has high specificity and sensitivity and has been utilized for medical imaging. The present invention addresses these, and other, problems.

Brief Description of the Figures

Figure 1 shows mitochondrial localization of compound 1 in opossum kidney cells in vitro.

Figure 2 shows mitochondrial localization of compound 2 in opossum kidney cells in vitro.

Figure 3 shows a time-lapse image performed by multiphoton fluorescence microscopy of podocytes in glomeruli of a living mouse labelled by compound 1.

Figure 4 shows a time-lapse image performed by multiphoton fluorescence microscopy of podocytes in glomeruli of a living mouse labelled by compound 2.

Figure 5 shows an excitation scan of compound 1 at a penetration depth of 25 pm.

Figure 6 shows an excitation scan of compound 2 at 25 pm penetration depth.

Figure 7 shows podocyte labelling in glomeruli in a living mouse by compound 1.

Figure 8 shows accumulation of compounds of the invention in mitochrondria of U-2 OS (human osteosarcoma) cells, compared with prior art compounds.

Figure 9 shows localization after intratumoral (top) and intravenous application (bottom) of Compound 7 of the invention in nu/nu mice.

Summary of the Invention

According to a first aspect, the invention provides compounds of Formula I,

wherein X is selected from the group consisting of

Y is selected from the group consisting of

A and A’ are independently selected substituents;

B is an optionally substituted aromatic or heteroaromatic group;

R1-R12 are independently selected from hydrogen and a substituent;

Q and Q’ independently selected from NH, N(Ci-C6 alkyl), oxygen, sulphur, selenium and di(Ci-Ce alkyl)-methylene, * indicates the point of attachment of a group to the remainder of the molecule; m is an integer 1, 2 or 3;

p is an integer 1,2 or 3;

Z is an anion having a negative charge of n, n is an integer 1,2 or 3;

q is an integer 1,2 or 3; provided that mxp=n*q for use as a two-photon probe for fluorescence imaging.

According to a second aspect, the invention provides an imaging method comprising bringing a biological specimen into association with a compound of formula I, irradiating the specimen with light, and observing two-photon excited fluorescence images emitted from the compound of formula I.

According to a third aspect, the invention provides a method for imaging live cells, the method comprising administering to an animal a compound of formula I, irradiating a portion of the animal with light, and observing two-photon excited fluorescence images emitted from the compound of formula I.

According to a fourth aspect, the invention provides a class of compounds of formula I as defined wherein B is phenyl substituted with from one to three groups independently selected from the group consisting of fluoro, and C1-C3 perfluoroalkyl, preferably trifluoromethyl.

Detailed Description and Preferred Embodiments

The invention relates to two-photon fluorescent dyes based of Formula I, which, in one embodiment, are employed as fluorescent probes for deep multiphoton imaging. The probes enable long-term real-time monitoring of in vivo samples and selective labelling of podocytes in glomerulus in living animals. The two-photon fluorescent dyes of the present invention can be employed for imaging of living samples to exceptionally large depth of up to 160 pm for periods of 90 minutes or longer.

Problems of the prior art are solved with polymethinium salts of Formula I, which show very strong fluorescence intensity, high target affinity, low systemic toxicity, high photostability and enable imaging to a significant penetration depth. The polymethinium salts of Formula I of the present invention can be used as two-photon probes for deep real-time imaging for podocytes in glomerulus, and cellular and subcellular visualization in general. These systems are based on the utilization of a structural motif of pentamethinium salts prepared from corresponding malondialdehydes.

The preparation of polymethinium salts of Formula I is based on condensation of a suitable aromatic malondialdehyde with a salt of the corresponding heteroaromatic compound. The syntheses has been described in Czech patent application 304094 (PV 2011-782); Chem. Commun. 2008,1901-1903; Bioconjug. Chem. 2013, 24,14451454; and Dyes Pigm. 2014, 107, 51-59, which are incorporated herein by reference.

Preferred compound for uses of the invention are those wherein X is a group wherein A, Q and Ri to R4 are as defined elsewhere herein.

Preferred compound for uses of the invention are those wherein Y is a group

wherein A’, Q’ and R7 to R10 are as defined elsewhere herein.

Preferably, B is a group selected from phenyl, pyridyl, thienyl, furanyl, naphthyl, quinolyl and isoquinolyl, optionally substituted as defined. More preferably, B is a pyridyl group. Still more preferably, B is a 4-pyridyl group. Most preferably, B is an unsubstituted 4-pyridyl group.

In a preferred class of compounds, B is phenyl substituted with from one to three groups independently selected from the group consisting of fluoro, and C1-C3 perfluoroalkyl. In one embodiment, B is phenyl substituted with from one to three fluoro groups. In an alternative, preferred embodiment, B is phenyl substituted with from one to three (preferably one) trifluoromethyl groups. Specifically preferred values of B are 4-fluorophenyl, 3,5-difluorophenyl, 4-trifluoromethyl and 3-trifluoromethyl. Such compounds offer unusually high photostability and duration of imaging.

Preferably, A is selected from a C1-C18 alkyl group, or a group having the formula

(CH2)_n1

wherein n1 is an integer from 1 to 6 (preferably 2), n2 is an integer from 1 to 8, and R13 is selected from the group consisting of C1-C18 alkyl, hydrogen, a sulphonic acid group SO3H or a lithium, sodium, potassium, cesium or rubidium salt thereof, benzyl, allyl, a cobalt bis(dicarbollide) preferably substituted in the 8-position or a cyclodextrin, preferably an α, β or y cyclodextrin.

More preferably, A is selected from a C1-C18 alkyl group. More preferably, A is a C1Ce alkyl group, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tertbutyl, pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 4methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2 dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3dimethylbutyl, and 2-ethylbutyl. Most preferably, A is n-butyl.

Preferably, A’ is selected from a Ci-Cis alkyl group, or a group having the formula

(CH2)_n1

wherein n1 is an integer from 1 to 6 (preferably 2), n2 is an integer from 1 to 8, and R™ is selected from the group consisting of Ci-Cis alkyl, hydrogen, a sulphonic acid group SO3H or a lithium, sodium, potassium, caesium or rubidium salt thereof, benzyl, allyl, a cobalt bis(dicarbollide) preferably substituted in the 8-position or a cyclodextrin, preferably an a, β or γ cyclodextrin.

More preferably, A’ is selected from a C1-C18 alkyl group. More preferably, A is a CiCe alkyl group, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tertbutyl, pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 4methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3dimethylbutyl, and 2-ethylbutyl. Most preferably, A’ is n-butyl.

Preferably groups R1-R12 (where present) are independently selected from hydrogen, halide, Ci-Ce alkyl, C3-C6 cycloalkyl, Ci-C_s alkoxy, Ci-Ce thioalkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-Ce haloalkyl, Ci-Ce trialkylsilyloxy, phenyl, NH2, NH(Ci-C6 alkyl), N(Ci-C6 alkyl)₂, N⁺(Ci-C6 alkyl)3, carboxylic acid, C1-C6 carboxyalkyl, hydroxy, azide, nitro, nitroso, nitrile, cyanate, isocyanate, thiocyanate, isothiocyanate, thiol, or a group having the formula

(CH2)_n1

wherein n1 is an integer from 1 to 6 (preferably 2), n2 is an integer from 1 to 8, and R13 is selected from the group consisting of C1-C18 alkyl, hydrogen, a sulphonic acid group SO3H or a lithium, sodium, potassium, cesium or rubidium salt thereof, benzyl, allyl, a cobalt bis(dicarbollide) preferably substituted in the 8-position or a cyclodextrin, preferably an α, β or γ cyclodextrin.

Preferably, R1-R12 (where present) are selected from fluorine and hydrogen. More preferably R1-R12 are hydrogen.

Preferably, Z is selected from the group consisting of chloride, bromide, iodide, sulfate, phosphate, methanesulfonate, nitrate, maleate, acetate, citrate, fumarate, tartrate, oxalate, succinate, benzoate and p-toluenesulfonate. More preferably, Z is selected from the group consisting of chloride, bromide and iodide. Most preferably, Z is iodide.

In certain embodiments, compounds of the formula I are partially fluorinated, that is at least some of the hydrogen atoms are replaced with fluorine. In other embodiments, the compounds are perfluorinated, i.e. all of the hydrogen atoms are replaced with fluorine. These compounds display enhanced photostability.

A preferred class of compounds is those having the formula

_Znwherein A, B, Q, Z and n are as defined elsewhere herein.

A very preferred compound for use in the invention is compound 1, (2Z)-3-propyl-2[(2Z,4E)-5-(3-propyl-1,3-benzothiazol-3-ium-2-yl)-3-(4-pyridyl)penta-2,4-dienylidene]-

1,3-benzothiazole, having the formula

Compound 1 wherein Z is defined above. Preferably, Z is chloride, bromide or iodide, most preferably iodide.

An alternative, very preferred compound for use in the invention is compound 2, 2[(1 E,3Z,5E)-5-(3,3-dimethyl-1-propyl-indolin-2-ylidene)-3-(4-pyridyl)penta-1,3-dienyl]-

3,3-dimethyl-1-propyl-indol-1-ium

wherein Z is defined above. Preferably, Z is chloride, bromide or iodide, most preferably iodide.

The compounds of the invention have utility in two-photon imaging methods. The methods of the invention are applicable to both in vitro and in vivo imaging.

In one aspect, the invention relates to a method for imaging living cells, the method comprising administering to an animal a compound of formula I, irradiating a portion of the animal with light, and observing two-photon excited fluorescence images emitted from the compound of formula I.

Preferably, the irradiating light has a wavelength of from 600 to 1300 nm. In some embodiments the irradiating light has a wavelength of between 600 and 700 nm, more preferably between 640 and 680 nm, such as about 640 nm, about 655 nm, or about 680nm. In alternative embodiments, the irradiating light has a wavelength of from 680 to 1300 nm, more preferably from 1100 to 1200 nm, such as about 1180 nm.

Compounds of formula I are suitably administered to a live animal (including human) subject in an aqueous vehicle. Compounds of formula I are preferably present at a concentration of between 10 μΜ and 1 mM, more preferably between 100 μΜ and 500 μΜ, still more preferably 200 μΜ and 300 μΜ, such as about 250 μΜ. In other embodiments, compounds of the invention are employed at working concentrations of from 1 to 100 nM, preferably from 10 to 50 nM. This compares very favourably with mitochondrial probes known in the art, such as MitoTrackers® (available from Thermo Fisher Scientific of Waltham, Massachusetts, USA), typically used at concentrations of 100-200 nM, and rhodamine dyes, typically used at concentrations of 500-1000 nM.

Compounds of formula I are suitably administered to a live animal via intravenous injection. However, other modes of administration are contemplated.

The methods of the invention can be used to image any organ, organelle or cellular structure in living tissue. Preferably, the methods are used for imaging the kidney, and in particular the glomerulus. The compounds of the invention show high affinity for the glomerulus, and in particular the podocyte of the glomerulus.

The methods of the invention are capable of providing images of living tissue to a substantial depth compared with known methods. For example, the methods of the invention are capable of providing images at a depth of at least 50 pm, more preferably at least 100 pm, still more preferably at least 150 pm.

A problem with known mitochondrial probes is that they exhibit an undesirable degree of nonspecific accumulation, and are frequently redistributed within the endoplasmic reticulum. The probes of the invention, namely the compounds of formula I, exhibit an extremely high degree of selectivity for mitochondria.

A further issue with known classes of imaging agent (coumarin, rhodamine, xanthene and cyanine dyes) is their toxicity, which limits their utility in diagnostic applications. The compounds of formula demonstrate low cytotoxicity, and may display lower systemic toxicity.

Compounds of formula I are particularly suitable for forming conjugates with a variety of species. In particular, the compounds may be attached (covalently, e.g. via a linker or otherwise) to various substrates such as biomolecules (including antibodies, oligonucleotides, enzymes, proteins, saccharides, etc.), aptamers, nanoparticles, drug molecules and polymers.

Conjugation of the compounds of formula I to the substrate may suitably be achieved via a chemical linker. Various linking strategies are known, and the skilled person will be able to select the appropriate one. A preferred class of linkers is the Nhydroxysuccinimide or succinimidyl ester. Alternative linkers are described e.g. in W02004018493, which is incorporated herein by reference.

The compounds of formula I of the present invention under appropriate conditions, are selectively sequestered in mitochondria.

Definitions

As used herein, the term “substituent” refers to a group other than hydrogen. Preferred substituents include alkyl (such as Ci-Ce), alkenyl (such as C2-C6), alkoxy (such as C1C6), aryl (such as C6-C12), aryloxy (such as C6-C12), heteroaryl, fluoroalkyl (such as C1Ce), perfluoroalkyl (such as C1-C6) and perfluoroaryl (such as C6-C12), unless otherwise stated.

As used herein, the term “aromatic group” group that contains any carbon-based aromatic group preferably having from 6 to 14 ring carbon atoms. Preferred aromatic groups are phenyl, naphthyl, biphenyl, and anthracenyl, with phenyl most preferred.

As used herein, the term “heteroaromatic group refers to a 5- or 6-membered monocyclic aromatic group wherein 1, 2, 3,4 ring heteroatoms independently selected from O, S and N; or to a 8- to 11 -membered bicyclic aromatic group wherein 1 , 2, 3, 4 or 5 ring heteroatoms independently selected from O, S and N.

Examples of 5- or 6-membered monocyclic heteroaromatic groups include pyrrolinyl, furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl, triazolyl, triazinyl, pyridazyl, pyrimidinyl, isothiazolyl, isoxazolyl, pyrazinyl, pyrazolyl and pyrimidinyl. Examples of 8- to 1 1 - membered bicyclic heteroaromatic groups include 6H-thieno[2,3-b]pyrrolyl, imidazo[2,1-b][1,3]thiazolyl, imidazo[5,1b][1,3]thiazolyl, indolyl, isoindolyl, indazolyl, benzimidazolyl, benzoxazolyl e.g. benzoxazol-2-yl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzothienyl, benzofuranyl, naphthridinyl, quinolyl, quinoxalinyl, quinazolinyl, cinnolinyl and isoquinolyl.

Where the aromatic or heteroaromatic groups are described as substituted, substituents are from one to three groups independently selected from CrCe alkyl, C3C₆ cycloalkyl, C1-C6 alkoxy, C1-C6 thioalkyl, C2-C6 alkenyl, C2-C6 alkynyl, CrCe haloalkyl, Ci-Ce trialkylsilyloxy, phenyl, NH2, NH(Ci-C6 alkyl), N(Ci-C6 alkyl)2, N⁺(Cr Ce alkyl)3, carboxylic acid, Ci-Ce carboxyalkyl, halide, hydroxy, azide, nitro, nitroso, nitrile and thiol.

As used herein, the term alkyl, unless otherwise specified, refers to a saturated straight or branched hydrocarbon chain of typically Ci to Ce, and specifically includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 3- methylpentyl, 2,2-dimethylbutyl, and

2,3-dimethylbutyl, and the like.

The term “alkoxy,” as used herein, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Examples include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, t-butoxy, pentoxy, isopentoxy, neopentoxy, hexyloxy, isohexyloxy, cyclohexyloxy, 2,2-dimethylbutoxy, and 2,3-dimethylbutoxy, and the like.

As used herein, the term haloalkyl refers to an alkyl as defined herein, which is substituted by one or more halo groups as defined herein. The haloalkyl can be monohaloalkyl, dihaloalkyl, trihaloalkyl, or polyhaloalkyl including perhaloalkyl. A monohaloalkyl can have one iodo, bromo, chloro or fluoro within the alkyl group. Chloro and fluoro are preferred. Dihaloalkyl and polyhaloalkyl groups can have two or more of the same halo atoms or a combination of different halo groups within the alkyl. Examples of haloalkyl include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl and dichloropropyl. A perhaloalkyl refers to an alkyl having all hydrogen atoms replaced with halo atoms, e.g, trifluoromethyl.

The term “haloalkoxy,” as used herein, refers to a haloalkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Examples include fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, trichloromethoxy, pentafluoroethoxy, heptafluoropropoxy, difluorochloromethoxy, dichlorofluoromethoxy, difluoroethoxy, difluoropropoxy, dichloroethoxy and dichloropropoxy and the like.

Preparation of a Labeling Solution

Compounds of formula I generally have low solubility in water. Typically a stock solution is prepared dissolving the reagent in an organic solvent (suitably DMSO, dimethylformamide, acetonitrile, dioxane, tetrahydrofuran etc). The labeling solution is prepared by diluting the stock solution into aqueous buffer to the desired labeling concentration.

The amount of dye in the labeling solution is the minimum amount required to yield detectable staining in the sample, without significant background fluorescence or undesired staining of other organelles. The amount of reagent required for staining eukaryotic mitochondria depends on the sensitivity required for staining of intracellular vs. cell-free mitochondria, the number of cells present, and the permeability of the cell membrane to the reagent.

Typically cells incubated with 10-50 nM labeling solution will require about 2 to 5 minutes to acheive fluorescent staining, with a fully stable signal that is reached in about 10 minutes. Higher concentrations achieve more rapid staining. The exact concentration of stain to be used is dependent upon the experimental conditions and the desired results. The skilled person is able to determine these using routine trial and error.

Before or after exposure to the inventive methods, the cells may optionally be treated with solvents to fix, and optionally permeabilize, the membranes. Various fixatives and fixation conditions are suitable for achieving this, for example formaldehyde, methanol, and ethanol. Suitably, fixation is accomplished by incubating in a 4% solution of formaldehyde for 15 minutes.

Co-Iocation with additional agents

Methods according to the present invention may be combined with the use of an additional visualization reagent. One or more additional visualization reagents may be used in conjunction with the compounds of Formula I. The additional visualization reagent may be used to stain the entire cell, or a cellular substructure by selection of an appropriate reagent with the desired degree of selectivity, such as a labeled antibody, labeled oligonucleotide, an enzymic activity, or other indicator for a specific cellular component or substructure such as the cytoplasm, nucleus, membrane, lysosome, or Golgi apparatus. The imaging methods of the present invention and the response of the additional visualization reagent may be observed simultaneously or sequentially.

One class of appropriate additional detection reagents is fluorescent nucleic acid stains. A wide variety of appropriate nucleic acid stains are known, such as thiazole orange, propidium iodide, ethidium homodimer, and DAPI.

In another embodiment of the invention, an appropriate additional detection reagent is any probe that selectively stains a cellular organelle such as the cell membrane, nucleus, Golgi apparatus, endoplasmic reticulum, lysosomes, or a second mitochondrial stain, such as MitoTracker, Alexa-647 or Alexa-488.

Examples

Synthesis Examples

Example 1 - Synthesis of (2Z)-2-[(2Z,4E)-3-(4-fluorophenyl)-5-(3-methyl-1,3benzothiazol-3-ium-2-yl)penta-2,4-dienylidene]-3-methyl-1,3-benzothiazole iodide (Compound 3)

Compound 3

The flask was charged with 2-(4-fluorophenyl) malondialdehyde (166 mg, 1.0 mmol), 2,3-dimethyl-benzothiazolium iodide (611 mg, 2.1 mmol) and dry n-butanol (20 mL). The mixture was stirred at 110 °C for 18 h. After cooling to laboratory temperature, the product was separated. The product was separated by filtration and obtained solid was washed with ethanol (3x5 mL) and dried in vacuum. The crude product was recrystallized from ethanol-dichlormethane. Compound 3 was isolated as green solid. Yield of product was 323 mg, 55%.

Ή NMR (500 MHz, DMSO) δ: 8.02 (d, J= 7.9 Hz, 2H), 7.95 (d, J= 13.5 Hz, 2H), 7.69 (d, J = 8.2 Hz, 2H), 7.54 (t, J = 7.7 Hz, 2H), 5.82 (m, 6H), 3.61 (s, 6H); HRMS: calculated for C27H₂2FN₂S2⁺ = 457.12029, found: 457.12075.

Example 2 - Synthesis of 2-[(1 E,3Z,5E)-3-(4-fluorophenyl)-5-(1,3,3-trimethylindolin2-ylidene)penta-1,3-dienyl]-1,3,3-trimethyl-indol-1-ium iodide (compound 4)

The flask was charged with 2-(4-fluorophenyl) malondialdehyde (166 mg, 1.0 mmol), 1-propyl-2,3,3-trimethylindol iodide (691 mg, 2.1 mmol) and dry n-butanol (20 mL). The mixture was stirred at 110 °C for 18 h. After cooling to laboratory temperature, the product was separated. The crude product was purified by column chromatography on silica (4x30 cm, eluent dichlormethane-methanol 10:1). Compound 4 was isolated as green solid. The yield of product was 345 mg, 57%.

Ή NMR (500 MHz, DMSO) δ: 8.45 (d, J = 14.2 Hz, 2H), 7.64 (d, J = 7.0 Hz, 2H), 7.36 (m, 8H), 7.26 (t, J= 7.2 Hz, 2H), 5.60 (m, J= 14.0 Hz, 2H), 3.33 (s, 6H), 1.74 (s, 12H); HRMS: calculated for C₃₃H₃4FN2⁺ = 477.27005, found: 477.27026.

Example 3 - Synthesis of 2-((1 E,3Z,5Z)-3-(4-nitrophenyl)-5-(3-propyl-1,3benzothiazol-2-ylidene)penta-1,3-dienyl]-3-propyl-1,3-benzothiazol-3-ium iodide (compound 5)

no₂

Compound 5

The flask was charged with 2-(4-nitrophenyl) malondialdehyde (193 mg, 1.0 mmol), 2-methyl-3-propylbenzothiazolium iodide (670 mg, 2.1 mmol) and dry n butanol (20 mL). The mixture was stirred at 110 °C for 18 h. After cooling to laboratory temperature, the product was separated. The product was separated by filtration and the obtained solid was washed with ethanol (3x5 mL) and dried in vacuum. The crude product was recrystalized from ethanol-dichlormethane. Compound 5 was isolated as a green solid. The yield of product was 377 mg, 62%.

Example 4 - Synthesis of (2E)-2-[(2Z,4E)-5-(3,3-dimethyl-1-propyl-indol-1-ium-2-yl)3-(4-nitrophenyl)penta-2,4-dienylidene]-3,3-dimethyl-1-propyl-indoline iodide (compound 6)

The flask was charged with 2-(4-nitrophenyl) malondialdehyde (193 mg, 1.0 mmol), 1,2,3,3-tetramethylindol iodide (632 mg, 2.1 mmol) and dry n-butanol (20 mL). The mixture was stirred at 110 °C for 18 h. After cooling to laboratory temperature, the product was separated. The product was purified by column chromatography on silica (4x30 cm, eluent dichlormethane-methanol 10:1). Compound 6 was isolated as a green solid. The yield of product was 387 mg, 61%.

Example 5

The following compounds were prepared in a similar manner to Examples 1 to 4.

Intracellular study

Gamma-substituted polymethinium salts 1 and 2 were tested for in vitro and in vivo 5 fluorescence microscopy applications in conventional wide-field, confocal and multiphoton fluorescence microscopy techniques. Both compounds showed selective localization in mitochondria of various cell lines, stated by the example of opossum kidney cells. In addition, these polymethinium salts are cell-permanent, with compound 1 being retained in mitochondria even after treatment with an uncoupling io reagent, e.g. carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), which enables media exchange, fixation or even permeabilization of cells without loss of the staining (see Example 1). Compound 2 is not retained after FCCP staining, nevertheless it offers a broad application in long-term live-cell imaging. Both compounds did not show any signs of cytotoxicity in in vitro imaging experiments.

The newly prepared, positively charged symmetric polymethinium salts have convenient spectroscopic properties: narrow excitation and fluorescence emission spectra, minimal fluorescence in water environment, maximal fluorescence after incorporation in the lipophilic environment of mitochondrial membranes. Moreover, these dyes show extraordinary photostability, the significantly outperform commercially available xanthylium dyes broadly used for mitochondrial labelling.

Additionally and more importantly, both dyes can serve for in vivo imaging. Compounds 1 and 2 are two-photon fluorescent probes, which were successfully employed for podocyte labelling in glomeruli in living mice of C57BI/6J-Rj (see Examples 7-11). Compound 1 enables in vivo imaging for 90 minutes or longer, compound 2 up to several hours and was very well tolerated for several hours, and also with reinjection. In terms of the depth penetration, compound 1 outperforms commercially available Alexa-647-albumin for in vivo imaging, compound 1 enables imaging up to 160 pm (in contrast to Alexa-647 conjugate to 120 pm)

Animals injected with compound 1 died after about 1 to 1 ¹Λ hours. This indicates toxicity but no proof was found for direct toxicity in the kidney (n=2).

Compound 2 also demonstrated excitation at around 800 nm

Compound 1 is suitable for deep tissue imaging and was superior to Alexa 647Albumin.

Compound 2 was able to demonstrate long term imaging for several hours.

In vitro mitochondrial localization of compound 1: Independence on the mitochondrial membrane potential

Opossum kidney cell (OK) line was used for in vitro evaluation of the localization and fluorescence microscopy performance of compound 1. Compound 1 was incubated at 50 nM concentration with the cells for 30 minutes at physiological conditions and washed with phosphate buffered saline, then the cells were imaged using a Leica SP8 confocal fluorescence microscope equipped with a white light laser. The excitation wavelength employed for compound 1 was 655 nm. HyD-Detectors were used for image acquisition. Uncoupling was performed by 30 second treatment with carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, 100 nM - 10 μΜ) disrupting ATP synthesis.

Figure 1 shows mitochondrial localization of compound 1 in Opossum kidney cells in vitro, specifically:

Figure 1A) Mitochondria labelled with 50 nM compound 1 (before uncoupler treatment)

Figure 1B) co-treatment with a mitochondrial uncoupler FCCP

Figure 1C) tetramethylrhodamine methyl ester (TMRM) localization in opossum cells, Figure 1D) TMRM co-treated with FCCP uncoupler.

Compound 1 did not show any obvious signs of cytotoxicity.

Based on the localization of the dyes, it is obvious that, in contrast to a commercial mitochondria-specific dye TMRM (significantly decreased fluorescence intensity), localization of compound 1 based on a gamma-substituted polymethinium salt is quite independent of the mitochondrial membrane potential, since the mitochondrial staining is retained even after the FCCP treatment.

It is possible to combine mitochondrial labelling with compound 1 with staining using, for example, fluorescent proteins, labeled antibodies, nanobodies, quantum dots, other small organelles probes.

Example 6

In vitro mitochondrial localization of compound 2: mitochondrial membrane potential dependence

Opossum kidney cell (OK) line was used for in vitro evaluation of the localization and fluorescence microscopy performance of compound 2. Compound 2 was incubated at 50 nM concentration with the cells for 30 minutes at physiological conditions and washed with phosphate buffered saline, then the cells were imaged by Leica SP8 confocal fluorescence microscope equipped with a white light laser. The excitation wavelength used for compound 2 was 640 nm. HyD-Detectors were used for image acquisition. Uncoupling was performed by a 30 second treatment with carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, 100 nM - 10 μΜ) disrupting ATP synthesis.

Figure 2 shows mitochondrial localization of compound 2 in opossum kidney cells in vitro, specifically:

Figure 2 A) Mitochondria labelled with 50 nM compound 2 (before uncoupler treatmnent)

Figure 2 B) 30 second co-treatment with a mitochondrial uncoupler FCCP, according to the decreased fluorescence signal (the same manner as for commercial TMRM mitochondrial dye).

Compound 2 did not show any obvious signs of cytotoxicity. Retention of compound 2 in mitochondria depends on the mitochondrial membrane potential.

It is possible to combine mitochondrial labelling with compound 2 with staining using, for example, fluorescent proteins, labeled antibodies, nanobodies, quantum dots, other small organelles probes.

Example 7

Compound 1 imaging from basolateral site: Multiphoton fluorescence microscopy in C57BI/6J-Ri mouse

Model mouse strain C57BI/6J-Rj was used for this experiment. The mice were catheterized with a jugular-vein catheter for dye application. Compound 1 was used at 250 μΜ concentration in 0.9% NaCI (volume 120 pl). The imaging was performed immediately after dye application using a custom-built multiphoton fluorescence microscope setup equipped with a Spectra-Physics Insight Deep Sea laser (680 to 1300 nm) and the emission-BP-filter at 700/75 nm and a GaAsP-detector were used. The excitation wavelength used was 1180 nm and the frame rate was 1 fps.

Figure 3 shows a time-lapse image performed by multiphoton fluorescence microscopy of podocytes in glomeruli of a living mouse labelled by compound 1 (excitation at 1180 nm, 1fps), specifically:

Figure 3A) Tissue image prior to dye loading,

Figure 3B) Tissue image 20 seconds after dye application, and

Figure 3C) Tissue image 6 minutes after dye application.

The target structure was stained 6 minutes after application of compound 1.

Example 8

Compound 2 imaging from basolateral site: Multiphoton fluorescence microscopy in C57BI/6J-Rj mouse

Model mouse strain C57BI/6J-Rj was used for this experiment. The mice were catheterized with a jugular-vein catheter for dye application. Compound 2 was used at

250 μΜ concentration in 0.9% NaCI (volume 120 μΙ). The imaging was performed immediately after dye application using a custom-built Multiphoton fluorescence microscope setup equipped with a Spectra-Physics Insight Deep Sea laser (680 to 1300 nm) and the emission-BP-filter at 700/75 nm and a GaAsP-detector were used. The excitation wavelength used was 1180 nm and the frame rate was 1 fps.

Figure 4 shows a time-lapse image performed by multiphoton fluorescence microscopy of podocytes in glomeruli of a living mouse labelled by compound 2 (excitation at 1180 nm, 1fps), specifically:

Figure 4A) Tissue image prior to dye loading,

Figure 4B) Tissue image 90 seconds after dye application, and

Figure 4C) Tissue image 23 minutes after dye application.

Example 9

Excitation wavelength scan: Multiphoton fluorescence microscopy in C57BI/6J-Ri mouse by Compound 1

Model mouse strain C57BI/6J-Rj was used for this experiment. The mice were catheterized with a jugular-vein catheter for dye application. Compound 2 was used at 250 μΜ concentration in 0.9% NaCI (volume 120 μΙ). The imaging was performed using a custom-built Multiphoton fluorescence microscope setup equipped with a Spectra-Physics Insight Deep Sea laser (680 to 1300 nm) and the emission-BP-filter at 700/75 nm and a GaAsP-detector were used. The excitation wavelength scan was performed at 25 pm depth (px size 228 nm).

Figure 5 shows an excitation scan of compound 1 at a penetration depth of 25 pm. Selected wavelengths (maximal fluorescence intensities): 820 (max 4000), 1100 (max 2500), 1150 (max 5000), 1200 (max 4000), 1300 (max 5000). Podocyte labelling in the glomerulus in a living mouse was achieved by the methods of the invention. (Key: PT = proximal tubule, CD = collecting duct).

Example 10

Excitation wavelength scan: Multiphoton fluorescence microscopy in C57BI/6J-Ri mouse by Compound 2

Mouse model strain C57BI/6J-Rj was used for this experiment. The mice were catheterized with a jugular-vein catheter for dye application. Compound 2 was used at 250 μΜ concentration in 0.9% NaCI (volume 120 μΙ). The imaging was performed using a custom-built Multiphoton fluorescence microscope setup equipped with a Spectra-Physics Insight Deep Sea laser (680 to 1300 nm) and the emission-BP-filter at 700/75 nm and a GaAsP-detector were used. The excitation wavelength scan was performed at 25 pm depth (px size 381 nm).

Figure 6 shows an excitation scan of compound 2 at 25 pm penetration depth. Selected wavelengths (maximal fluorescence intensities): 820 (max 4000), 1100 (max 2500), 1150 (max 5000), 1200 (max 4000), 1300 (max 5000). Podocyte labelling in the glomerulus in a living mouse was achieved by the methods of the invention. (Key: PT = proximal tubule, CD = collecting duct).

Example 11

Multiphoton fluorescence in vivo microscopy in C57BI/6J-Ri mouse by Compound 1 Mouse model strain C57BI/6J-Rj was used for this experiment. The mice were catheterized using a jugular-vein catheter for dye application. Compound 1 was used at 250 pM concentration in 0.9% NaCI (volume 120 pl). The imaging was performed using a custom-built Multiphoton fluorescence microscope setup equipped with a Spectra-Physics Insight Deep Sea laser (680 to 1300 nm) and the emission-BP-filter at 700/75 nm and a GaAsP-detector were used.

Figure 7 shows podocyte labelling in glomeruli in a living mouse by polymethinium salt 1 at A) 133 pm and B) 160 pm penetration depth. The penetration depth exceeds the best-performing Alexa-647-Albumin, in which case only 120 pm penetration depth can be achieved.

Example 12 - UV-visible and fluorescence experiments

For UV-visible analysis, the absorption spectra in 6.25 pM of all studied compounds in DMSO were recorded over wavelengths of 300 - 800 nm using a GBC Cintra 404 spectrometer. The absorbance of these solutions was determined in a conventional 1 cm PMMA cell. The results are shown in Table 1.

Fluorescence spectroscopy studies were carried out using a SCINCO FluoroMate FS2 spectrometer. The compounds were dissolved in DMSO and the solution (39 nM) was placed in a 1 cm PMMA cell. The results are shown in Table. 1.

	Amax (nm)	Aemmax (nm)	Aexmax (nm)
Compound 3	645	668.6	643.7
Compound 7	642	657.1	641.7
Compound 8	652	674.7	650.9
Compound 9	643	658.3	642.6

TABLE 1

Amax - maximum absorbance wavelength Aemmax - maximum emission wavelength Aexmax - maximum excitation wavelength

Example 13 - Co-localization experiment

A mitochondria-specific probe, MitoTracker Green, was used to confirm the selective accumulation in mitochrondria of U-2 OS (human osteosarcoma) cells. Cells were incubated with the compounds for 30 minutes in the presence of 100 nM MitoTracker Green, washed with phosphate buffer saline, and then imaged. The images were acquired using an inverse fluorescent microscope (Olympus IX-81).

Results are shown in Figure 8.

Example 14 - Effect of Compound 7 on localization in vivo

Nu/nu mice bearing subcutaneously growing human pancreatic carcinoma (MIA PaCa2) received Compound 7 intravenously. Compound 7 was also injected intratumorally in mice bearing human glioblastoma (U-87). Mice were anaesthetised and subjected to fluorescence imaging. Imaging was performed immediately after dye application, and then after 1 and 72 hours using Bruker In-Vivo Xtreme II.

Figure 9 shows localization after intratumoral (top) and intravenous application (bottom) of Compound 7 in nu/nu mice.

Claims

1. An imaging method comprising:

bringing a biological specimen into association with a compound of formula I; irradiating the specimen with light; and observing two-photon excited fluorescence images emitted from the compound of formula I m+ [Zn']q

P

Formula I wherein X is selected from the group consisting of

A and A’ are independently selected substituents;

B is an optionally substituted aromatic or heteroaromatic ring

R1-R12 are independently selected from hydrogen and a substituent;

Q and Q’ independently selected from NH, N(Ci-Ce alkyl), oxygen, sulphur, selenium and di(Ci-C6)-methylene, * indicates the point of attachment of a group to the remainder of the molecule;

m is an integer 1, 2 or 3;

p is an integer 1,2 or 3;

Z is an anion having a negative charge of n, n is an integer 1,2 or 3;

q is an integer 1,2 or 3;

provided that mxp=nxq.

2. A method according to claim 1 wherein X is a group

Ft wherein A, Q and Ri to R4 are as defined in claim 1.

3. A method according to claim 1 or 2 wherein Y is a group wherein A’, Q’ and R7 to R10 are as defined in claim 1.

4. A method according to any preceding claim, wherein B is a group selected from phenyl, pyridyl, thienyl, furanyl, naphthyl, quinolyl and isoquinolyl.

5. A method according to claim 4 wherein B is a pyridyl group.

6. A method according to claim 5 wherein B is a 4-pyridyl group.

7. A method according to any preceding claim wherein A is selected from a Cr Cis alkyl group.

8. A method according to any preceding claim wherein A’ is selected from a Cr Cis alkyl group.

9. A method according to any preceding claim wherein R1-R12 (where present) are H.

10. A method according to any preceding claim wherein m is 1, p is 1, n is 1 and q is 1.

11. A method according to any preceding claim wherein Z is a halide anion, preferably iodide.

12. A compound of formula I as defined in any one of claims 1 to 3 or 7 to 11 wherein B is phenyl substituted with from one to three groups independently selected from the group consisting of fluoro, and C1-C3 perfluoroalkyl, preferably trifluoromethyl.

13. A compound according to claim 12 wherein B is selected from 4-fluorophenyl, 3,5-difluorophenyl, 4-trifluoromethyl and 3-trifluoromethyl.

14. A compound according to claim 12 which is one of:

J ?

and

15. A compound of formula I as defined in any one of claims 1 to 14 for use in diagnostic methods practised on the human or animal body.

16. A method of imaging live cells, the method comprising administering to an animal a compound as defined in any one of claims 1 to 15, irradiating a portion of the animal with light, and observing two-photon excited fluorescence images emitted from the compound of formula I.

17. The method of any one of claims 1 to 11 or 16 wherein the irradiating light has a wavelength of between 600 to 1300 nm.

18. The method of any one of claims 1 to 11, 16 or 17, wherein the cells to be imaged are at a depth of at least 100 pm.

19. The method of claim 18, wherein the cells to be imaged are at a depth of at least 150 pm.

20. The method of any one of claims 16 to 19 wherein the cells are podocytes.

21. The method according to any one of claims 16 to 20 wherein the imaging method is for detecting tumour cells.

22. A method for visualizing cellular organelles in tissue or in cell suspension, said method comprising the steps of:

(a) obtaining a tissue sample or cell sample, said sample containing a plurality of cells;

(b) contacting said tissue sample or cell sample with a compound of Formula I as defined in any of claims 1 to 14;

(c) irradiating the specimen with light; and (d) observing two-photon excited fluorescence images.

23. The method according to claim 22 wherein the organelle is a mitochondrion.