WO2020120636A1

WO2020120636A1 - Fluorescence dye

Info

Publication number: WO2020120636A1
Application number: PCT/EP2019/084763
Authority: WO
Inventors: Srinivas Banala; Fabian Kiessling; Magnus RUEPING; Jean Michel MERKES
Original assignee: Rheinisch-Westfälische Technische Hochschule (Rwth) Aachen
Priority date: 2018-12-14
Filing date: 2019-12-11
Publication date: 2020-06-18
Also published as: EP3894482A1

Abstract

The present invention relates to compound according to formulas (1), (15), (16) or (17), the use of the compounds as a fluorescent dye, and a fluorescent dye comprising the compounds. The invention also relates to a method for fluorescence-based identification, detection, or imaging, comprising the step of using the compounds.

Description

Fluorescence dye

The present invention relates to fluorescence dyes, and particularly relates to fluorescent compounds based on the BODIPY scaffold.

Fluorescent molecules are widely used in bioimaging and medicinal applications for several decades due to their high sensitivity and easy visibility. 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dyes are a class of organic fluorescence dyes noted for their high photostability, sharp absorption and emission bands, excitation and emission wavelengths in the visible/near infrared region, high fluorescence quantum yield, high photostability and resistance to chemical degradation. BODIPY compounds fulfil the rigorous requirements for bio-imaging, yet, the inherently small Stokes shift, mostly below 20 nm, limits the applications of BODIPY compounds especially in super -resolution imaging.

Due to the outstanding fluorescent properties, the BODIPY scaffold is widely used for the development of fluorescent derivatives, particularly by modifications of the substituents of the BODIPY core. Julian Knight et al. for example described in Org. Biomol. Chem., 2015, 13, 3819- 3829 the synthesis of 3 -amino BODIPY dyes and the absorption and fluorescence properties of the synthesized compounds. The emission yields of the compounds however were low, and some are described as being essentially non-fluorescent. WO 2014/104975 Al further discloses 3-amino- triazolyl-BODIPY compounds comprising a triazole group at the 5-position and a secondary amine group at the 3 -position. These substituents together provide for the described mega stokes shift. Yet, the compounds only have nominal fluorescence quantum yields of 2-4%. There is a need for new fluorescent compounds based on the BODIPY scaffold. Therefore, it is an object of the present invention to provide fluorescent compounds based on the BODIPY-based scaffold with improved fluorescence quantum yields.

This object is achieved by a compound according to formulas (1), (15), (16) or (17) as given as follows and pharmaceutically acceptable salts thereof:

wherein:

Ri is selected from the group of hydrogen, OH, Ci-CYalkyl, Ci-Cs-alkoxy, NH₂, NHR", NR"₂, -(CH₂)_m-COOR'; or

Ri together with two carbon atoms of the aryl ring to which it is bound forms a saturated or unsaturated 5- or 6-membered ring fused to the aryl, wherein the ring optionally comprises 1 to 3 nitrogen atoms;

R₂ is selected from the group of hydrogen, Ci-CYalkyl, -(CH₂)_m-COOR', G.-Cio-aryl, (C₂- C4-alkyl)aryl, (C₂-C4-alkyl)heteroaryl, (C₂-C4-alkenyl)aryl and (C₂-C4- alkenyl)heteroaryl, wherein aryl and heteroaryl optionally is substituted with OH, Ci- Ce-alkyl, Ci-Cs-alkoxy, NH₂, NHR", NR"₂ or -(CH₂)_m-COOR’; or R2 together with the carbon atom at position 6 for a 5- or 6-membered aromatic heterocycle or aromatic or non-aromatic carbocycle which optionally is fused to pyrrole or aryl which optionally is substituted with OH, CVCValkyl, Ci-Cs-alkoxy, NH₂, NHR", NR"₂ or -(CH₂)_m-COOR’;

R3 is selected from the group of hydrogen, CVCValkyl, -(CH₂)_m-COOR', Ce-Cio-aryl, (C₂- C4-alkyl)aryl, (C₂-C4-alkyl)heteroaryl, (C₂-C4-alkenyl)aryl and (C₂-C4- alkenyl)heteroaryl, wherein aryl and heteroaryl optionally is substituted with OH, Ci- C₆-alkyl, Ci-Cs-alkoxy, NH₂, NHR", NR"₂ or -(CH₂)_m-COOR’;

R₄, R₅ are independently selected from the group of hydrogen, CVCValkyl, -C(=0)-CH₃ and -(CH₂)_m-COOR, or R₄ and R₅ together with nitrogen atom, to which R₄ and R₅ bind, form a cyclic 3- to 6-membered, saturated or unsaturated amine;

R' is selected from the group of hydrogen, Ci-C3-alkyl and phenyl;

R" is selected from the group of Ci-C3-alkyl; and

m is 1, 2, 3, 4, 5 or 6.

Surprisingly it has been found that the compounds according to formulas (1), (15) and (17) provide bright and stable fluorophores. The compound (16) is non-fluorescent, but thermally convertible towards fluorophore (15). The compounds absorb light efficiently and thus exhibit high extinction coefficients, and the compounds emit good number of photons per excitation and thus provide large fluorescence yield. Further, the compounds showed improved Stokes shifts. Large Stokes shifts improve the quality, i.e. the signal to noise ratios, of fluorescent images, as large Stokes shifted compounds reduce the cross-talk between excitation source and emission light, and allow capturing of most of the emission light. Advantageously, the compounds thus provide a large fluorescence yield combined with large Stokes shifts, which enables their applicability in super-resolution imaging and multi-colour /multi-spectral imaging applications. Further, the compounds showed a good

photostability of over 200 seconds in 2-photon laser irradiation assays, and up to 85 pulses of 660 nm STED depletion laser and for over 5 months on a cover slip that was used for fixation of cells. The compounds moreover were found to be non-toxic on cell growth up to 24 pmol/mL. This shows that the compounds are biocompatible and can be used in biomedical imaging and microscopy.

The term“alkyl” according to the invention is to be understood as meaning straight-chain or branched alkyl groups. The term CVCValkyl as used herein refers to alkyl groups having 1 to 6 carbon atoms. Ci-Ce- Alkyl groups may be selected from the group comprising methyl, ethyl, propyl, butyl, pentyl and hexyl. Preferred are Ci-C3-alkyl groups, particularly methyl and ethyl.

The term“alkenyl” according to the invention is to be understood as meaning straight-chain or branched alkyl groups having at least one double bond between carbon atoms.

The term“Ci-Cs-alkoxy” according to the invention is to be understood as meaning a C i-G.-alkyl bonded to oxygen. Preferred are Ci-C3-alkoxy groups, particularly methoxy and ethoxy.

The term“aryl” or CVCm-aryl” according to the invention is to be understood as meaning a 6- to 10- membered aromatic ring, such as phenyl and naphthyl. The term“heteroaryl” or“Cs-Cio-heteroaryl” according to the invention is to be understood as meaning a 5- to 10-membered, preferably a 5- or 6- membered, aromatic ring comprising at least one hetero atom selected from O, S and N. In embodiments, the aryl group is phenyl. In other embodiments, the heteroaryl group is selected from pyrrole, thiophene or furan.

The terms“(C2-C4-alkyl)aryl”,“(C2-C4-alkyl)heteroaryl”,“(C2-C4-alkenyl)aryl” and“(C2-C4- alkenyl)heteroaryl” according to the invention are to be understood as binding via the alkyl or alkenyl group to the compound. In embodiments, the C2-C4-alkyl group is -(0¾)₂-. In embodiments, the C2- C4-alkenyl group is a C2-alkenyl group. In embodiments, the aryl group is phenyl. In embodiments, the heteroaryl group is pyrrole.

In preferred embodiments, the compound is a compound according to formula (1). The compounds of formula (1) may be denoted 2-aryl-3-amino substituted 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) compounds. Particularly the 2-aryl-3-amino BODIPY compounds provide bright and photostable dyes. Advantageously, the 2-aryl position on the 3-amino BODIPY scaffold massively increased the fluorescence quantum yields. Further, the 2-aryl substitution provided a redshift in the absorption and increased the Stokes shift of fluorescence emission. Without being bound to a specific theory, it is assumed that the 2-arylation on the 3-amino-BODIPY provides the exceptional properties of the compounds such as redshifted absorption, large Stokes shift, high fluorescent quantum yields and photostability. The substituent Ri may be selected from the group of hydrogen, OH, CVCYalkyl, Ci-Cs-alkoxy, N¾, NHR", NR"₂ and -(CH₂)_m-COOR' wherein m represents 1, 2 or 3. The substituent Ri may be selected from hydroxyl and Ci-C₃-alkyl. In embodiments, the substituent Ri is selected from the group of hydrogen, methyl and hydroxyl. In preferred embodiments, the substituent Ri is selected from methyl and hydroxyl. 2-Aryl-3-amino BODIPY compounds wherein the aryl group is substituted with methyl or hydroxyl showed high fluorescence emission quantum yield. The substituent Ri alternatively may be a carboxylic group -(CH₂)_m-COOR', preferably selected from -(CH₂)₂-COOH, -(CH₂)₂-COOCH₃ and -(CH₂)₂-COOEt. A carboxylic group allows to attach the compounds to targeting moieties, such as tumor-markers or docetaxel.

The substituent Ri in further embodiments together with two carbon atoms of the aryl ring to which it is bound may form a saturated or unsaturated 5- or 6-membered ring fused to the aryl, wherein the ring optionally comprises 1 to 3 nitrogen atoms. The ring system preferably comprises one nitrogen atom. In preferred embodiments, the substituent Ri together with the aryl ring may form indole.

The substituents R₂ and R₃ may be the same or independent from each other selected from the group of hydrogen, CVCValkyl, -(CH₂)_m-COOR', CVC ₁₀-ary 1, (C₂-C₄-alkyl)aryl, (C₂-C₄-alkyl)heteroaryl, (C₂-C₄-alkenyl)aryl or (C₂-C₄-alkenyl)heteroaryl which aryl or heteroaryl groups optionally are substituted with OH, CVCValkyl, Ci-Cs-alkoxy, N¾, NHR", NR' ₂ or -(CH₂)_m-COOR'. In preferred embodiments, R₂ and R₃ are selected independently from each other and are different groups.

The substituent R₂ may be selected from methyl and phenyl, wherein phenyl preferably is substituted with hydroxyl, methyl, methoxy or N(CH₃)₂- The substituent R₃ may be selected from hydrogen and phenyl, wherein phenyl may be substituted with methyl, methoxy, N(CH V or -(CH₂)_m-COOR'. In embodiments, at least one of R₂ and R₃ may be a (C₂-C₄-alkenyl)aryl or (C₂-C₄-alkenyl)heteroaryl group, preferably a (C₂-alkenyl)aryl or (C₂-alkenyl)heteroaryl group. The aryl group may be phenyl, preferably substituted with N(CH₃)₂- The heteroaryl group may be pyrrolyl, wherein pyrrolyl may be unsubstituted or substituted with one, two or three methyl groups. Electron-rich (methyl)_npyrroles conjugated to the BODIPY compounds yields conjugated dyes and advantageously the absorption maximum can be redshifted by increasing the number of methyl substituents at the pyrrole. In other embodiments, R₂ together with the carbon atom at position 6 of the BODIPY scaffold forms a 5- or 6-membered aromatic heterocycle or aromatic or non-aromatic carbocycle which optionally is condensed to pyrrolyl or aryl. This substitution allows for a larger conjugated system of the compound. The aryl or pyrrolyl group optionally is substituted with hydroxyl, methyl, methoxy or N(0¾)₂. The compounds of formulas (10), (11) and (12) show embodiments where R₂ together with the carbon atom at position 6 of the BODIPY scaffold forms a 6-membered non-aromatic carbocycle that is condensed to aryl, which is substituted with methoxy. In other embodiments, the aromatic heterocycle is thiophene and R₂ together with the carbon atom at position 6 of the BODIPY scaffold forms a 5,6-thiophene-fused BODIPYs structure.

In embodiments, R₂ and R₃ are the same or independent from each other selected from the group of hydrogen, methyl and phenyl, wherein phenyl optionally is substituted with hydroxyl, methyl, methoxy or N(CH₃)₂- Preferably, at least one of R₂ and R₃ is a phenyl group, which phenyl group may be substituted with hydroxyl, methyl, methoxy, N(CH3)2 or -(Ctbl_m-COOR'.

The substituents R₄ and R₅ may be independently selected from the group of hydrogen, Ci-CYalkyl, - C(=0)-CH₃ and -(Ctbl_m-COOR'. In preferred embodiments, R4 and R5 are hydrogen and the compound comprises a 3-NH₂ group. The compounds of formula (1) preferably are 2-aryl- and 3- amino-substituted BODIPY compounds. In other embodiments, R5 is hydrogen and R4 is -C(=0)-CH₃, and the compound comprises a 3-acetamide substituent. In other embodiments, R₅ is hydrogen and R₄ is -CH2-COOR' or -(CH₂)₅-C00R', wherein R' preferably is selected from hydrogen, methyl and phenyl. In other embodiments, R₄ and R₅ form together with the nitrogen atom to which they bind a 3-, 4-, 5- or 6-membered, saturated or unsaturated cyclic amine. Preferably, R₄ and R₅ form an unsaturated cyclic amine selected from pyrrole, aziridinyl, azetdinyl, pyrrolinyl and pyrrolidinyl.

In embodiments, the compound is selected from the group of compounds according to formulas (2) to (14) as given as follows:

Referring to the compounds according to formulas (2) to (14), Et denotes ethyl and Me denotes methyl. In preferred embodiments, the compound is selected from the group of compounds according to formulas (2), (3), (5), (10), (11), (13) and (14). It was found that these compounds exhibited exceptional advantageous optical properties. For the compound of formula (2), over 30 nm redshift in absorption compared to BODIPY dyes without the 2-aryl-3-amino substitution was observed, and a 39 nm Stokes shift along with high fluorescence quantum yields of <I>_t = 66%. Furthermore, the compound of formula (2) was applied in two-photon imaging and STED super-resolution imaging, which showed its potential suitability in super resolution imaging. For the compound of formula (5), an even larger Stokes shift of 75 nm with a quantum yield of 66%, as for the compound of formula (2), was observed. Also the compound of formula (11) showed a Stokes shift of 44 nm. These compounds further provide particularly bright fluorophores.

The compounds according to formulas (1) to (14), particularly the compounds according to formulas (2), (3), (5), (10), (11), (13) and (14), provide bright and photostable BODIPY dyes with large Stokes shifts, which are usable as fluorescent dyes in fluorescence-based imaging, and particularly are usable for super-resolution based bio-imaging modalities.

The compounds according to formulas (1) to (14) can be modified at the amine position, as in the compound (13), with a fatty chain carboxylic acid without affecting optical brightness and stability properties. Thus this position also is suitable for conjugating to targeting moieties such as small molecules like docetaxel, biotin, and phalloidine or to antibodys and other biomolecules.

A further aspect relates to a fluorescent dye comprising the compounds according to formulas (1) to (17), particularly the compounds according to formulas (2), (3), (5), (10), (11), (13) and (14). For the description of the compounds reference is made to the description above.

Another aspect relates to the use of a compound according to formulas (1) to (17), particularly a compound according to formulas (2), (3), (5), (10), (11), (13) and (14), as a fluorescent dye. For the description of the compounds reference is made to the description above.

The compounds advantageously were found to be non-toxic on cell growth. Biocompatibility allows for a use of the compounds in biomedical imaging and microscopy of living cells. Advantageously, the BODIPY compounds are usable in bio-imaging. The compounds may be conjugated to targeting moieties, such as tumor-markers or antibodies. The compounds may be attached to targeting moieties via polyethylene glycol (PEG) or fatty chain carboxylic acids or via‘click’ chemistry with biotin and others. The compounds also may be incorporated into nanoformulations, such as liposomes, nanocrystals, and microbubbles, for bio-imaging applications. The compounds may be used in form of their bio-hybrid conjugate as dyes for tumor targeting.

The compounds are usable in bio-imaging as a labeling reagent for labeling a target such as proteins, nucleic acids and other biomolecules. The compounds particularly are usable as reactive fluorescent dye or a fluorescent dye conjugate. For such uses the compounds may be formulated as succinimidyl (NFIS) esters for coupling to amino groups or as maleimides for coupling to thiol groups or may be functionalised with iodoacetamide or amine groups. Further, the compounds may be conjugated to phalloidin, streptavidin or biotin. The compounds also may be functionalised as an azide or alkyne for applications via‘click’ chemistry. The compounds also may be attached to tetrazine, via a modified benzylamine. Such groups undergo an inverse-electron demand cycloaddition reaction with labelled alkenes. The compounds can be conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection.

In embodiments, the fluorescent dye comprises a compound according to the invention comprising a succinimidyl ester, a phosphine group, a tetrazine-benzylamine group, a O⁶- (4-aminomethyl) benzylguanine group or a chloroalkane linker. Such compounds may be denoted an activated or reactive fluorescent dye.

NHS esters can be used to label the compound to primary amines (R-Nth) of peptides, proteins, amine-modified oligonucleotides, and other amine-containing molecules. Activated fluorescent dyes are usable to tag proteins, nucleic acids, and other biomolecules for use in life science applications including fluorescence microscopy, flow cytometry, fluorescence in situ hybridization (FISH), fluorescence resonance energy transfer (FRET) techniques, receptor binding assays, and enzyme assays.

A chloroalkane linker may comprise an alkyl chloride and one or more such as two three or four ethylene glycol units connected to the compound. A chloroalkane linker attached to the compound allows the fluorescent dye to be used with HaloTag® applications, and the compound can be used with the HaloTag® protein tagging system also in living cells. The protein tag (HaloTag®) is a modified haloalkane dehalogenase designed to covalently bind to such chloroalkane linkers. An O⁶- aminobenzylguanine group attached to the compound allows the fluorescent dye to be used in SNAPTag® applications, wherein cells express SNAPTag® fusion protein in living cells.

Phosphine-activated fluorescent dyes are uasable for specific labeling and detection of azide-tagged molecules, which enables use of the fluorescence dyes in metabolic labeling strategies. When used in combination with azide labeling, phosphine-activated compounds enable selective fluorescent labeling for detection of protein interactions and post-translational modifications using fluorescence imaging technologies. Further, the phosphine-tethered dyes are suitable for a labelling of the mitochondria in cells. In embodiments, the fluorescent dye comprises a compound according to the invention conjugated to phalloidin, streptavidin or biotin. Such compounds may be denoted a fluorescent conjugate. Phalloidin conjugates comprising a compound according to the invention conjugated to the mushroom toxin phalloidin may be used to label F-actin.

A further aspect relates to a method for fluorescence-based identification, detection, or imaging, particularly of cells, the method comprising the step of using a compound according to formulas (1) to (17), particularly a compound according to formulas (2), (3), (5), (10), (11), (13) and (14), and/or a fluorescent dye according to the invention as fluorescence dye. In embodiments, the method may be selected from dynamic fluorescence imaging, confocal microscopy imaging, two-photon microscopy, stochastic optical reconstruction microscopy (STORM), stimulated emission depletion (STED) microscopy, and single or multi-photon fluorescence microendoscopy. The compounds, particularly the compounds according to formulas formulas (2), (3), (5), (10), (11), (13) and (14), provide bright and stable fluorophores and thus advantageously are usable in high resolution methods such as confocal microscopy, two-photon excitation, and super-resolution techniques like stimulated emission depletion (STED) microscopy. The bright and photostable BODIPY compounds with large Stokes shifts are advantageously usable for bio-imaging.

Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The examples that follow serve to illustrate the invention in more detail but do not constitute a limitation thereof.

The figures show:

Figure 1 The extinction coefficient and the emission of the 2,7-ditolyl 3-amino BODIPY

compound of formula (2) in ethanol.

Figure 2 The absorption and the emission of the compound of formula (5) in ethanol.

Figure 3 The extinction coefficient and the emission of the compound of formula (11) in

ethanol. Figure 4 The stability of the compound of formula (2) under 2 -photon-laser irradiation using 10% power.

Figure 5 The quantification of cell labeling intensity using the compound of formula (2). Figure 6 The cell viability after incubation with the compound of formula (2).

Figure 7 2-Photon Images of A549 cells labelled with the compound of formula (2), using 0,2 mM in Figure 7a) and 0,6 mM in Figure 7b). Each image includes four emission channels, corresponding to, orange (540-580 nm) and red (580-650 nm), blue, (419- 465 nm), green (495-540 nm).

Figure 8 2-Photon Images using the dye of formula (10), using an excitation wavelength of 750 nm, 800 nm and 850 nm Figures 8a) b) and c), respectively. Each image includes four emission channels, corresponding to, orange (540-580 nm) and red (580-650 nm), blue, (419-465 nm), green (495-540 nm).

Figure 9 2-Photon Images using the dye of formula (11), using an excitation wavelength of 750 nm, 800 nm and 850 nm Figures 9a) b) and c), respectively. Each image includes four emission channels, corresponding to, orange (540-580 nm) and red (580-650 nm), blue, (419-465 nm), green (495-540 nm).

Figure 10 2-Photon Images using the dye of formula (15), using an excitation wavelength of 750 nm, 800 nm, 850 nm, 900 nm and 950 nm Figures 10a) b), c), d) and e), respectively. Each image includes four emission channels, corresponding to, orange (540-580 nm) and red (580-650 nm), blue, (419-465 nm), green (495-540 nm).

Figure 11 An 3D STED image of A549 cells labeled with the dye of formula (3) (bottom) and confocal image (top).

Figure 12 An 3D STED image of PC3 cells labeled with the dye of formula (3) (bottom) and confocal image (top).

Figure 13 Confocal images in Figures 13a) and c) and STED images in Figures 13b) and d) of

A549 cells labeled with 5 nmol/200 pL of the compound of formula (13).

Figure 14 A stack of 85 slices of STED images in Figures 14b) and confocal images Figures

14a) which were taken in the xz plane, and in Figure 14c) a confocal image in the xy plane with exposed cell centre.

Figure 15 Confocal (top) and 3D STED image (bottom) of A549 cells labeled with 5

nmol/200pL of the compound of formula (14). Figure 16 Confocal (bottom) and 3D STED (top) image of PC3 cells labeled with 5 nmol/200pL of the dye of formula (14).

Figure 17 Changes in UV-vis absorption spectra of compound (16) in DMSO over 9 days, towards the fluorescent isomer of formula (15).

Figure 18 Comparison of UV-vis absorption spectra of the compound of formulas (16) and (15) in DMSO over 12 days.

Figure 19 UV-vis absorption spectra for the thermal conversion of compound (16) to (15) in

DMSO, by heating for 15 min at 50 °C and 75 °C.

Example 1

Synthesis of the compounds according to formulas (2) to (4)

The compound according to formula (2) was prepared according to the following scheme 1 :

Scheme 1

In a round bottom flask 4-aryl-5-nitropentan-2-one was given in n-butanol. The mixture was degassed by argon ventilation and 35 equivalents of ammonium acetate were added. The argon flow was kept running for 10 minutes. The reaction mixture was than heated to 100 °C for 12-48 h under inert atmosphere. After cooling to room temperature the reaction mixture was diluted with dichloromethane (DCM) and washed with saturated sodium bicarbonate solution. The water phase was extracted with DCM three times. The combined organic solvent was dried over sodium sulfate and concentrated in vacuum. The crude mixture was than purified by column chromatography twice (1. silica gel, hexane: ethyl acetate; 2. silica gel, dichloromethane: methanol).

The obtained dipyrromethene was than dissolved in 0.1 M DCM under argon atmosphere. 5 equivalents triethylamine were added and the reaction was stirred at room temperature for 10 minutes. 7 equivalents Boron trifluoride diethyl etherate were given to the solution and the mixture was stirred for further 30 minutes. The reaction was quenched by addition of saturated sodium bicarbonate solution and extracted with DCM three times. The combined organic solvents were dried over sodium sulfate and concentrated in vacuum. The crude product was purified by column chromatography twice (1. silica gel, hexane: ethyl acetate; 2. silica gel, dichloromethane: methanol).

Also the compounds according to formulas (3) and (4) were prepared as described above using the starting materials listed in Table 1. Table 1: Structures of the compounds, their systematic names, and starting materials used in the synthesis

Example 2

Synthesis of the compound according to formula (5) to (14)

The compound according to formula (5) was prepared according to the following scheme 2: Scheme 2

In a round bottom flask were given 4-aryl-5-nitropentan-2-one in a suitable alcohol (0.3 M). The mixture was degassed by Argon. 1.5 equivalents of pyrrole and 35 equivalents of ammonium acetate were added to the reaction mixture. The reaction was than heated to 50-100 °C for 12-48 h under inert atmosphere. After cooling to room temperature the reaction mixture was diluted with DCM and washed with saturated sodium bicarbonate solution. The water phase was extracted with DCM three times. The combined organic solvent was dried over sodium sulfate and concentrated in vacuum. The crude mixture was than purified by column chromatography (silica gel, hexane: ethyl acetate) the obtained product was used without further purification in the BF2-complexation.

The obtained dipyrromethene was dissolved in DCM (0.1 M) under argon atmosphere. 5 equivalent triethylamine were added and the reaction was stirred at room temperature for 10 minutes. 7 equivalent Boron trifluoride diethyl etherate were given to the solution and the mixture was stirred for further 30 minutes. The reaction was quenched by addition of saturated sodium bicarbonate solution and extracted with DCM three times. The combined organic solvents were dried over sodium sulfate and concentrated in vacuum. The crude product was purified by column chromatography twice (1. silica gel, hexane: ethyl acetate; 2. silica gel, dichlorome thane).

Also the compounds according to formulas (6) to (8) and (10) to (14) were prepared as described above using the starting materials listed in Table 2.

Table 2: Structures of the compounds (5) to (8) and (10) to (14), their systematic names, and starting materials used in the synthesis:

Example 3

Synthesis of the compounds according to formula (9)

The compound according to formula (9) was prepared by Knoevenagel-Condensation starting from the compound of formula (3) according to the following scheme 3:

R= H, Me

Scheme 3 The compound of formula (3) was dissolved in dry toluene (0.1 M). 2.0 equivalents of 4- dimethylamino benzaldehyde, 0.25 mL piperidine and 3 drop of methyl sulfonic acid were added to the reaction mixture. The reaction was then heated above 120 °C using a distillation bridge or Dean- Stark apparatus until no solvent remains in the flask. The process was repeated until full conversion of the compound. The crude was taken up with dichloromethane and washed with saturated sodium bicarbonate. The water phase was extracted with DCM three times. The combined organic solvents were dried over sodium sulfate and concentrated in vacuum. The crude product was purified by column chromatography (silica gel, hexane: ethyl acetate). Table 3: Structures of the compounds, their systematic names, and starting materials used in the synthesis

Example 4

Synthesis of the compounds according to formula (15), (16)

The compounds according to formulas (15) and (16) was prepared according to the following scheme 4:

Scheme 4

2.2 equivalent of pyrrole were dissolved in degassed acetic acid anhydride (0.6 M). The mixture was continuously ventilated by argon flow. Two drops of trifluoro acetic acid and 1 equivalent of sodium nitrite in water (2.75 M) were added to the mixture and the reaction was warmed to 50 °C under inert atmosphere for 8 h. After cooling to room temperature the reaction mixture was quenched by addition into a saturated sodium bicarbonate solution and extracted with dichloromethane three times. The combined organic solvents were dried over sodium sulfate and concentrated in vacuum. The crude product was purified by column chromatography (silica gel, hexane: ethyl acetate or ethylacetate: methanol) to obtain the product as a one to one adduct of dipyrromethene and acetic acid. The obtained dipyrromethene adduct was dissolved in dry dichloromethane under inert atmosphere. 5 equivalents of triethylamine are added to the reaction mixture and the reaction was stirred for 10 minutes. The mixture was than cooled to 0 °C and 7 equivalents of boron trifluoride diethyl etherate were added. The reaction was stirred for 10 minutes, before it was quenched by addition of saturated sodium bicarbonate solution and extracted with DCM three times. The combined organic solvents were dried over sodium sulfate and concentrated in vacuum. The crude product was purified by column chromatography twice (1. silica gel, hexane: ethyl acetate; 2. silica gel, dichloromethane).

Example 5

Synthesis of the compound according to formula (17)

The compound according to formula (17) was prepared according to the following scheme 5:

2.2 equivalent of pyrrole were dissolved in a degassed acetic acid anhydride and acetic acid mixture (0.6 M). The mixture was continuously ventilated by argon flow and 1 equivalent of sodium nitrite in water (2.75 M) were added to the mixture and the reaction was warmed to 100 °C under inert atmosphere for 2 h. After cooling to room temperature the reaction mixture was quenched by addition into a saturated sodium bicarbonate solution and extracted with dichloromethane three times. The combined organic solvents were dried over sodium sulfate and concentrated in vacuum. The crude product was purified by column chromatography (silica gel, hexane: ethyl acetate or ethylacetate: methanol) to obtain the product as a one to one adduct of dipyrromethene and acetic acid. The obtained dipyrromethene adduct was dissolved in dry dichloromethane under inert atmosphere. 5 equivalents of triethylamine are added to the reaction mixture and the reaction was stirred for 10 minutes. The mixture at room temperature, 7 equivalents of boron trifluoride diethyl etherate were added. The reaction was stirred for 60 minutes, before it was quenched by addition of saturated sodium bicarbonate solution and extracted with DCM three times. The combined organic solvents were dried over sodium sulfate and concentrated in vacuum. The crude product was purified by column chromatography twice (1. silica gel, hexane: ethyl acetate; 2. silica gel, dichloromethane).

Example 6

Determination of optical characteristics

The absorption and the emission of the compounds of formulas (2), (5) and (11) was determined in solution. UV/Vis spectra were obtained on a SHIMADZU UV-2600 and a TECAN (infinite 200) and fluorescence spectra were obtained on a PerkinElmer LS45 and a TECAN (infinite 200) at room temperature. Fluorescence quantum yields were obtained on a PicoQuant FT300 device using integrated sphere.

The Figure 1 shows the extinction coefficient and the emission of the compound of formula (2) in ethanol. As can be taken from the figure 1, the 2,7-ditolyl 3-amino BODIPY compound of formula (2) showed a red shifted absorption maximum (k_abS = 538 nm) with a 39 nm Stokes shift in fluorescence emission (k_Em = 577 nm). Furthermore, the molar extinction coefficient (e) was 50,000 M 'em and fluorescence quantum yield (Fp) was 66%. This shows that the compound (2) provides a highly bright (brightness factor, e * F_A = 33,000) fluorophore.

The Figure 2 shows the absorption and the emission of the compound of formula (5) in ethanol. As can be taken from the figure 2, the 5-aryl substituted 3-amino BODIPY compound (5) exhibited an even larger Stokes shift of 75 nm, with absorption as in simple BODIPYs, k_abS = 515 nm, with emission maximum at k_Em = 590 nm, and fluorescence quantum yield (Fp) of 66%.

The Figure 3 shows the extinction coefficient and the emission of the compound (11) in ethanol. As can be taken from the figure 3, the 3 -amino BODIPY compound (11) showed a large Stokes shift of 44 nm, with absorption,

= 586 nm, and fluorescence emission of l_Eh, = 590 nm. The molar extinction coefficient (e) was 78,000 M 'em

This shows that the 2-aryl 3-amino BODIPY compounds provide exceptional optical properties such as redshifted absorption, large Stokes shift, and high fluorescent quantum yields. It is assumed that the 2-arylation is necessary on the 3-amino-BODIPY to gain these optical properties and photostability.

Example 7

Determination of photostability

For the determination of the photostability, the stability of the 2,7-ditolyl 3-amino BODIPY compound of formula (2) was determined under one and two-photon excitation in phantom studies in three different mediums: in DMSO solution (on a concave glass), in 5% gelatine and PVA (Tissue-Tek) 100 mM thick slices.

Measurements were conducted on a LaVision Biotec Trimscope and a Olympus Fluoview 1000 MPE. The tests were made using dye of formula (2). Measurements in DMSO were done on a concave object slide. The well was filled with a DMSO dye solution in three different concentrations and the signal intensity was measured in the two photon setup over 15 min. Analog the signal intensity in immobilised dye formulation in a 5% gelantine/water phantom and a commercial PVA (Tissue-Tek) phantom was investigated. Here for the obtained solids were cut into 100 mM thick slices and placed on a object slide. The imaging of the slices was done in a single position using the same system as for the DMSO.

The phantoms containing the compound of formula (2) were excited with a two-photon laser

(EaVision Biotec Trimscope) at 750 nm, at 10% of the maximum irradiation intensity, as usually required for bio imaging, for 30 sec to test its photostability. The figure 4 shows the results of the stability tests of the compound of formula (2) under 2-photon-laser irradiation in 5% gelatine, DMSO and PVA. As can be seen in figure 4, the 2 -photon-laser irradiation resulted in over 40% of remaining dye which would still be sufficient for two-photon microscopy. This shows that the compounds are usable in two-photon microscopy. Example 8

Determination of cell labeling intensity and Dynamic Fluorescence Imaging

Dynamic Fluorescence imaging was done using a Yokogawa Cell Voyager CV 8000. The determination of cellular uptake of the 2,7-ditolyl 3-amino BODIPY compound of formula (2) was carried out by dynamic fluorescence microscopy for up to 15 h using HT29 cells. A blank control was used for control. Fiving HT29 cells (6000 cells per well) were stained using 100 pF RPMI medium with a loading of 1 mM, 2,5 pM and 5 pM dye. Images were taken by between 1 and 30 minutes after beginning of the incubation. The dye was excited at 561 nm and emission was recorded at 600 nm to 637 nm. The excitation time was 50 ms.

The Figure 5 shows the images of cells incubated with 5 pM, 2.5 pM or 1 pM compound of formula (2) for 1 min and 30 min, respectively, in the Figures 5a) to g). As can be seen in the figure 5, the compound of formula (2) was taken up by cells within one minute and showed normal cell growth. The compound of formula (2) was visible even after continuous exposure for up to 15 h in biological medium without any adverse effect on cell growth. Further, fluorescence images indicated that the compound crossed the cell membrane, and showed accumulation in lysosomal compartments of cells (cytosol).

The Figure 5h) shows the quantification of cell uptake. The cell uptake of dye was observed in living HT29 cells. It could be observed that the dye was taken up very fast into the cell membran. After 1 minute incubation a strong signal at all concentrations could already be observed. Fow concentrations of lpM proofed hereby to be most efficient seeing that already for 2,5 pM concentration self quenching could be observed after 15 minutes. This effect was even more enhanced at 5 pM concentration, which cells never showed a signal stronger than cells incubated with 2,5 pM dye. As uch the dye is well suited for living cell images when used in concentration of 1 pM and below (depending on the planned time line of the experiments). In all cases no signal due to rest dye in the medium could be observed. Making this a well suited dye for cell imaging and general microscopy applications.

Example 9

Determination of cell viability The biocompatibility of the 2,7-ditolyl 3-amino BODIPY compound of formula (2) was tested using a colorimetric cell proliferation (XTT) assay. The cells were incubated with dye in medium over 24 h, before the supernatant solution was removed. 100 pL XTT activator (N-methyl dibenzopyrazine methyl sulfate) was added, followed by 50 pL freshly prepared XTT- solution (5 mL) (by XTT test kit, from Gibco) to each well of the above cells. After 2-4 h the absorption was measured in TECAN-reader at 475 nm wavelength using 660 nm as reference.

Figure 6 shows the percentage of living A549 and PC3 cells incubated with 0.8, 0.64, 0.4, 0.24 or 0.08 pmol/ml (=mM) of the compound of formula (2) and control cells with 5% DMSO (CTRL), respectively. As can be seen in the figure 6, the compound of formula (2) was non-toxic and cell growth was unaffected in up to 0.24 pmol/mL.

This shows that the compounds are non-toxic, will not compromise cell growth and thus are usable for bio-imaging of cells.

Example 10

2-Photon microscopic images of cells labelled with the compounds of formula (2), (10), (11) and (15)

All images were taken using a LaVision Biotec Trimscope and a Olympus Fluoview 1000 MPE.

50000 cells were incubated on the object slides using commonly used growthmedium, DMEM for A549 and RPMI for PC3, for 24-48 hours to guarantee adherence to the slides. The cells were than incubated with the corresponding amount of dye for 2h, before they were washed with PBS thrice. The cells were than fixated with 4 % Formalin solution for 15 min. The slides were than fixed on a object slide using DABCO containing Muviol.

10.1 - 2-Photon Images using the compound of formula (2):

A549 cells were incubated in medium containing 0,2 pM or 0,6 pM dye of formula (2) as described above. 2-Photon axcitation was conducted at 600 nm and the emission was recorded between 580 and 650 nm, corresponding to the red channel in most commercial systems. Figure 7 shows the 2-Photon Images using the dye of formula (2), for Figure 7a) using 0,2 m M and in Figure 7b) 0,6 m M of the compound. As can be taken from Figure 7, the compound (2) showed significant uptake in A549 cells by incubation at concentrations 0,2 mM and above.

10.2 - 2-Photon Images using the compound of formula (10):

PC3 cells were incubated in RPMI medium containing 5 mM dye of formula (10) as described above. 2-Photon images were taken at different excitation wavelength between 750 and 850 nm at 3 % laser energy setting. The images were split into 4 channels, correspondingly named blue (emission 419-465 nm), green (emission 495-540 nm), orange (emission 540-580 nm) and red (emission 580-650 nm).

Figure 8 shows the 2-Photon Images using the dye of formula (10), using an excitation wavelength of 750, 800 and 850 nm Figures 8a) b) and c) respectively. As can be taken from Figure 8, the dye of formula (10) showed a strong signal in the red channel at 750 and 800 nm excitation. Above 850 nm excitation no signal could be detected. At 750, 800 and 850 nm excitation a weak signal in the green channel could be observed. The dye was excited at the low energy 3 % laser power, but still showed a strong signal. This shows that the dye can be used together with DAPI at 750 and 850 nm excitation.

10.3 - 2-Photon Images using the compound of formula (11):

PC3 cells were incubated in RPMI medium containing 5 mM dye of formula (11) as described above. 2-Photon images were taken at different excitation wavelength between 750 and 850 nm at 3 % laser energy setting. The images were split into 4 channels, correspondingly named blue (emission 419-465 nm), green (emission 495-540 nm), orange (emission 540-580 nm) and red (emission 580-650 nm).

Figure 9 shows the 2-Photon Images using the dye of formula (11), using an excitation wavelength of 750, 800 and 850 nm Figures 9a) b) and c) respectively. As can be taken from Figure 9, the dye of formula (11) showed a strong signal in the red channel at 750 and 800 nm excitation. Above 850 nm excitation no signal could be detected. At 750, 800 and 850 nm excitation a weak signal in the green channel could be observed. The dye was excited at the low energy 3 % laser power, but still showed a strong signal. The dye can be used together with DAPI at 750 and 850 nm excitation.

10.4 - 2-Photon Images using the compound of formula (15): PC3 cells were incubated in RPMI medium containing 5 mM dye of formula (15) as described above. 2-Photon images were taken at different excitation wavelength between 750 and 950 nm at 8 % laser energy setting. The images were split into 4 channels, correspondingly named blue (emission 419-465 nm), green (emission 495-540 nm), orange (emission 540-580 nm) and red (emission 580-650 nm).

Figure 10 shows the 2-Photon Images using the dye of formula (15), using an excitation wavelength of 750, 800, 850, 900 and 950 nm Figures 10a) b), c), d) and e) respectively. As can be taken from Figure 10, the images show the strongest fluorescence emission at around 850 nm excitation, but were overall good visible in the green channel at excitation between 750 and 900 nm. For excitation 750 and 800 nm the dye can be used together with DAPI as costain, where DAPI gives a good signal in the blue channel. At all excitation wavelength slight bleeding into the orange channel could be observed.

Example 11

STED Imaging of cells labelled with the compound of formulas (3), (13) and (14)

Ah STED images were obtained using gated STED microscopy on a Leica TCS SP8 STED 3X.

11.1 - STED imaging of labeled cells with the compound of of formula (3):

Each object slide was prepared with 5000 cells per slide adhered over 24-48 hours. The Cells were than labelled using corresponding growth medium, DMEM for A549, RPMI for PC3, the dye of formula (3) was incubated at 5 nmol/200 pL and 1 nmol/200pL concentrations for 2 h. Afterwards the cells were washed with PBS and fixated with 4 % Formalin for 15 minutes. The finished slides were fixed to a carrier by using a Muviol formulation with DAB SO as additive. The images were taken using a laser of 510 nm at 50 % power to excite the dye. The depletion laser was used at a power setting of 50 % (660 nm depletion). Emission was measured between 551-651 nm with a time gate starting at Ins and ending at 6 ns.

Figure 11 shows confocal and 3D STED image of A549 cells labeled with the dye of formula (3), wherein the top shows the confocal image and the bottom shows the STED image. Figure 12 shows confocal and 3D STED image of PC3 cells labeled with the dye of formula (3), wherein the top shows the confocal image and the bottom shows the STED image. As can be taken from the Figures 11 and 12, the gain in resolution by using the dye of formula (3) for STED by 660 nm depletion was significant. The membranes could be observed in perfect clarity. Furthermore no anti-Stokes emission could be observed. The stability of the compound of formula (3) is well suited for imaging purposes.

11.2 - STED imaging of labeled cells with the compound of formula (13):

Each object slide was prepared with 5000 cells per slide adhered over 24-48 hours. The Cells were than labelled using corresponding growth medium, DMEM for A549, RPMI for PC3, the dye of formula (13) was incubated at 5 nmol/200 pL and 1 nmol/200pL concentrations for 2 h. Afterwards the cells were washed with PBS and fixated with 4 % Formalin for 15 minutes. The finished slides were fixed to a carrier by using a Muviol formulation with DABCO as additive. The images were taken using a laser of 561 nm for excitation at 15 % power. The depletion laser was used at 50 % power (660 nm). The detection gate was chosen from 1.2 ns to 6 ns, with an emission area of 565-650 nm.

Figure 13 shows confocal microscopy in Figures 13a) and c) and STED images in Figures 13b) and d) of A549 cell labeled with 5 nmol/200 pL of the compound of formula (13). Depletion Wavelength of 660 nm by 561 nm excitation. Figure 14 shows a stack of 85 slices of STED images in Figures 14b) and confocal images Figures 14a) which were taken in the xz plane. Figure 14c) shows a confocal image in the xy plane afterwards with exposed cell centre. For the slices, 5 pM dye incubated A549 cells were used. As can be taken from the Figures 13 and 14, the compound of formula (13) showed in all imaging tests a very high stability. Simple STED images in the xy plane could be stacked up to 5 times before bleaching began. Even stacks of up to 85 images could be taken, using cells labeled with 1 nmol dye, in the xz plane (one image every 50 nm in direction of y-axis), while still retaining enough signal to again see signal in confocal imaging. The gain in resolution at 660 nm depletion was exceptional allowed to see and differentiate between inner membranes of cells. No anti-Stokes emission could be detected.

11.3 - STED imaging of labeled cells with the compound of formula (14):

Each object slide was prepared with 5000 cells per slide adhered over 24-48 hours. The Cells were than labeled using corresponding growth medium, DMEM for A549, RPMI for PC3, the dye of formula (14) was incubated at 5 nmol/200 pL and 1 nmol/200pL concentrations for 2 h. Afterwards the cells were washed with PBS and fixated with 4 % Formalin for 15 minutes. The finished slides were fixed to a carrier by using a muviol formulation with DABSO as additive. The images were taken using a laser of 561 nm for excitation at 15 % power. The depletion laser was used at 40 % power (660 nm). The detection gate was chosen from 1 ns to 6 ns, with an emission area of 565-650 nm.

Figure 15 shows confocal and 3D STED image of A549 cells labeled with 5 nmol/200pL of the compound of formula (14), wherein the top shows the confocal image and the bottom shows the STED image. Figure 16 shows confocal and 3D STED image of PC3 cells labeled with 5 nmol/200pL of the dye of formula (14), wherein the top shows the STED image and the bottom shows the confocal image. As can be taken from the Figures 15 and 16, the gain in resolution by using the dye formula (14) for STED by 660 nm depletion was significant. The membranes could be observed in perfect clarity. Furthermore no anti-Stokes emission could be observed. The stability of the dye is well suited for imaging purposes.

Example 12

Thermal conversion of the compound of formula (16) to (15) and determination of the fluorescence quantum yield of the compound according to formula (15)

The thermal conversion of the compound according to formula (16) to the compound according to formula (15) in solution was determined in DMSO at room temperature and by heating for 15 min at 50 °C and 75 °C. The Figure 17 shows the changes in UV-vis absorption spectra of compound (16) in DMSO, at room temperature over 9 days, towards the fluorescent isomer of formula (15). The Figure 18 shows the comparison of UV-vis absorption spectra of the compound of formula (16) in DMSO at room temperature over 12 days, along with the compound of formula (15). The Figure 19 shows the comparison of UV-vis absorption spectra of the compound of formula (16) in DMSO for accelerated conversion towards formula (15) at high temperatures.

Further, the fluorescence quantum yield of the compound of formula (15) was obtained in ethanol on a PicoQuant FT300 device using integrated sphere. The fluorescence quantum yield (Fίΐ) was determined to 81%.

Example 13

Preparation of a phalloidin conjugate with compound (13) 600 mg (0.625 mihoΐ, 3.0 eq) of phalloidin amine tosylate and 102 mg (0.208 mhioΐ, 1.0 eq) of the compound (13) were dissolved under argon atmosphere in water-free dimethylsulfoxide (DMSO) and 94.1 pg (0.312 mihoΐ, 1.5 eq) O-(N-succinimidyl)- 1 , 1 ,3,3-tctramcthyluronium tetrafluoroborate (TSTU) were added thereto together with 126 pg (1.25 pmol, 6.0 eq) triethanolamine (TEA). The reaction was run at room temperature for 4 days. The reaction mixture was purifed on reverse phase HPLC (water:MeCN 95:5 to5:95 in 15 min + 5 min water:MeCN 5:95) to obtain the product as an orange solid.

Example 14

Preparation of the compound according to formula (13) provided with a chloroalkane linker

The compound according to formula (13) provided with a chloroalkane linker was prepared according to the following scheme 6:

Step 14.1: Preparation of tosylated 6-chloro hexanol

6.67 mL (6.83 g, 50 mmol, 1.0 eq) 6-chlorohexanol was dissolved in 250 mL water-free

dichloromethane (DCM) and cooled to 0 °C. To this, 7.62 mL (5.57 g, 55 mmol, 1.1 eq) of TEA and 1.22 g (10 mmol, 0,2 eq) 4-dimethylaminopyridine (DMAP) were added. To the cooled solution, 10.5 g (55 mmol, 1.1 eq) of tosylchloride in 50 mL DCM was added and the reaction was allowed to warm to room temperature. The reaction was stirred over night and after full conversion the mixture was diluted with 1 M HC1, and extracted with DCM. The organic solvent was dried over sodium sulfate and concentrated in vacuum. The crude was purified by column chromatography (silica, hexane: ethyl acetate 4: 1) to obtain the product as white solid.

Step 14.2: Preparation of hoc protected aminoethanol

4.97 mL (5.26 g, 50 mmol, 1.0 eq) aminoethoxy ethanol was dissolved in 15 mL water free DCM under argon atmosphere and cooled to 0 °C. To this, 13.1 g (60 mmol, 1.2 eq) tert-butyloxycarbonyl anhydride (Boc-anhydride) was added and the reaction was stirred at room temperature, until full conversion, for 2 hours. The reaction mixture was diluted with DCM and washed with saturated sodium bicarbonate. The organic solvent was dried over sodium sulfate and concentrated in vacuum. The product was purified by column chromatography (silica, hexane: ethyl acetate 2: 1) and obtained as a colorless oil.

Step 14.3

500 mg (2.44 mmol, 1.0 eq) of the hoc protected aminoethanol from step 14.2 and 779 mg (2.68 mmol, 1.1 eq) of the tosylated 6-chloro hexanol from step 14.1 were dissolved in 10 mL water free DMF under argon atmosphere and 410 mg (3.65 mmol, 1.5 eq) of potassium tert.-butoxide was added. The solution was stirred at room temperature for 8 hours, diluted with water and extracted with diethyl ether. The organic solvent was dried over sodium sulfate and concentrated in vacuum. The crude product was purified by column chromatography (silica, hexane: ethyl acetate 2: 1) and obtained as a colorless oil.

Step 14.4

700 mg (2.16 mmol, 1.0 eq) product of step 14.3 was dissolved in 2.5 mL 4 M HC1 in dioxane (9.73 mmol, 4.55 eq) and was stirred at room temperature for 2 hours. The solvent was removed in vacuum and the product was obtained as white solid, which was used without further purification.

Step 14.5

2 mg (4.1 pmol, l.O eq) compound according to formula (13) and 1.4 mg (5.33 pmol, 1.3 eq) of the linker of step 14.4 were dissolved in 100 pL dry DMF under argon atmosphere and cooled to 0 °C. To this, 10.7 qL (0.5 M in DMF, 5.33 mmol, 1.3 eq) of l-hydroxy-7-azabenzotriazol was added, followed by 1.71 qL (1.25 qg, 12.3 qmol, 3.0 eq) TEA and 1.02 mg (5.33 qmol, 1.3 eq) 1 -Ethyl-3 -(3'- dimethylaminopropyl)carbodiimide hydrochloride (EDC HC1). The mixture was stirred over night at room temperature. After full conversion the reaction was dried, crude directly purified by column chromatography (silica fine grain, hexane: ethyl acetate 2: 1 to 0: 1). The product was obtained as a red solid.

Example 15

Preparation of the compound according to formula (13) provided with a phosphine group

The compound according to formula (13) provided with a phosphine group was prepared according to the following scheme 7 :

Scheme 7

5 mg (13.4 qmol, 1.0 eq) of the compound according to formula (13) was dissolved in 1,6- dibromohexane followed by addition of 5.6 mg (40.2 qmol, 3.0 eq) potassium carbonate. The reaction was stirred over nigth at 50 °C. Reaction progress was monitored by thin-layer chromatography (TLC) and UV/Vis. The reaction was purified by filtration over silica (hex:ea 10:1). The prodcut was obtained as red solid in quantitative yields.

The product was than diluted in 0.5 mL dry MeCN and 6.9 mg (26.1 qmol, 2.0 eq) triphenyl phosphine were added and the reaction mixture was refluxed for 5 days. The crude was purified on reverse phase HPLC (water: MeCN 95:5 to 5:95 in 12 minutes (water: MeCN 95:5 to 5:95 in 12 minutes)) to obtain the product as red solid. Example 16

Preparation of the compound according to formula (13) provided with a NHS ester group

The compound according to formula (13) provided with a NHS ester group was prepared according to the following scheme 8:

The compound according to formula (13) was dissolved in water-free DMF under argon atmosphere and TSTU (1.5 eq) and TEA (3.0 eq) are added. The mixture is stirred for 2 hours and the resulting NHS ester is purified on column (silica, hexane: ethyl acetate 4:1 to 1 :2) to obtain the product as red solid. In summary, the results show that the compounds of the invention provide bright and stable fluorophores. Particularly the 2-aryl 3-amino BODIPY compounds provide exceptional optical properties such as redshifted absorption, large Stokes shift, and high fluorescent quantum yields.

Claims

C l a i m s

1. A compound according to formulas (1), (15), (16) or (17) as given as follows and

pharmaceutically acceptable salts thereof:

wherein:

Ri is selected from the group of hydrogen, OH, Ci-CValkyl, Ci-Cs-alkoxy, NH₂, NHR", NR"₂, -(CH₂)_m-COOR'; or

R₂ is selected from the group of hydrogen, Ci-CValkyl, -(CH₂)_m-COOR', CVCio-aryk (C₂- C4-alkyl)aryl, (C₂-C4-alkyl)heteroaryl, (C₂-C4-alkenyl)aryl and (C₂-C4- alkenyl)heteroaryl, wherein aryl and heteroaryl optionally is substituted with OH, Ci- Ce-alkyl, Ci-Cs-alkoxy, NH₂, NHR", NR"₂ or -(CH₂)_m-COOR’; or

R₂ together with the carbon atom at position 6 forms a 5- or 6-membered aromatic

heterocycle or aromatic or non-aromatic carbocycle which optionally is fused to pyrrole or aryl which optionally is substituted with OH, CVCValkyl, Ci-Cs-alkoxy, NH₂, NHR", NR"₂ or -(CH₂)_m-COOR’; R3 is selected from the group of hydrogen, Ci-CValkyl, -(CH2)_m-COOR', Ce-Cio-aryl, (C2- C4-alkyl)aryl, (C2-C4-alkyl)heteroaryl, (C2-C4-alkenyl)aryl and (C2-C4- alkenyl)heteroaryl, wherein aryl and heteroaryl optionally is substituted with OH, Ci- Ce-alkyl, Ci-C₅-alkoxy, NH₂, NHR", NR"₂ or -(CH₂)_m-COOR’;

R₄, R₅ are independently selected from the group of hydrogen, CVCValkyl, -C(=0)-CH₃ and -(CH₂)_m-COOR, or R4 and R5 together with nitrogen atom, to which R4 and R5 bind, form a cyclic 3- to 6-membered, saturated or unsaturated amine;

R' is selected from the group of hydrogen, Ci-C3-alkyl and phenyl;

R" is selected from the group of Ci-C3-alkyl; and

m is 1, 2, 3, 4, 5 or 6.

2. The compound according to claim 1, wherein the compound is a compound according to

formula (1).

3. The compound according to claims 1 or 2, wherein Ri is selected from the group of hydrogen, methyl and hydroxyl.

4. The compound according to any one of the foregoing claims, wherein R2 and R3 are the same or independent from each other selected from the group of methyl and phenyl, wherein phenyl optionally is substituted with hydroxyl, methyl, methoxy or N(CH3)2-

5. The compound according to any one of the foregoing claims, wherein R4 and R5 are hydrogen.

6. The compound according to any one of the foregoing claims, wherein the compound is

selected from the group of compounds according to formulas (2) to (14) as given as follows:

7. The compound according to any one of the foregoing claims, wherein the compound is selected from the group of compounds according to formulas (2), (3), (5), (10), (11), (13) and (14).

8. A fluorescent dye comprising a compound according to any one of claims claim 1 to 7.

9. The fluorescent dye according to claim 8, wherein the fluorescent dye comprises a compound according to any one of claims claim 1 to 7 comprising a succinimidyl ester, a phosphine group, a tetrazine-benzylamine group, a O⁶- (4-aminomethyl) benzylguanine group or a chloroalkane linker.

10. The fluorescent dye according to claim 8, wherein the fluorescent dye comprises a compound according to any one of claims claim 1 to 7 conjugated to phalloidin, streptavidin or biotin.

11. Use of a compound of any one of claims claim 1 to 7 as a fluorescent dye.

12. A method for fluorescence-based identification, detection, or imaging, the method comprising the step of using a compound according to any one of claims claim 1 to 7 and/or a fluorescent dye according to claims 8 to 10 as fluorescence dye.

13. The method according to claim 12, wherein the method is selected from dynamic fluorescence imaging, confocal microscopy imaging, two-photon microscopy, stochastic optical reconstruction microscopy (STORM), stimulated emission depletion (STED) microscopy, and two-photon fluorescence microendoscopy.