METHOD FOR STAINING MITOCHONDRIA

Abstract

Methods for staining mitochondria are disclosed involving using a composition containing a cationic species of the formula: (I) wherein at least one of Y and Z is a substituted or unsubstituted azetidine group; X is selected from O, S, SO.sub.2, Se, NR.sub.12, P(O)R.sub.12, CR.sub.13R.sub.14, SiR.sub.13R.sub.14, Te, and GeR.sub.13R.sub.14. Also disclosed are methods for analysing mitochondria, involving staining a sample of mitochondria. illuminating the stained sample using light of an appropriate wavelength to fluoresce the compound, and observing or imaging a magnified image of the sample.

##STR00001##

Claims

1. A method for staining mitochondria, the method comprising: providing a sample containing mitochondria, and incubating the sample in a composition comprising a cationic species of formula (I): ##STR00017## or a solvate, or tautomer thereof; and a counter ion; wherein: Y is a substituted or unsubstituted azetidine ring and Z is selected from OR.sub.17 or a substituted or unsubstituted azetidine ring; X is selected from O, S, SO.sub.2, Se, NR.sub.12, P(O)R.sub.12, CR.sub.13R.sub.14, SiR.sub.13R.sub.14, Te, and GeR.sub.13R.sub.14; R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are each independently selected from H, C.sub.1 to C.sub.8 alkyl, OR.sub.15, C(O)OR.sub.16, NHCOR.sub.15, CONHR.sub.15 and halo; R.sub.v, R.sub.w, R.sub.x, R.sub.y, R.sub.6, R.sub.7 are each independently selected from H, C.sub.1 to C.sub.8 alkyl and halo; R.sub.12, R.sub.13, R.sub.14, and R.sub.15 are each independently selected from H, C.sub.1 to C.sub.8 alkyl, optionally substituted aryl or optionally substituted heteroaryl; R.sub.16 is selected from C.sub.1 to C.sub.8 alkyl, optionally substituted aryl or optionally substituted heteroaryl, and R.sub.17 is selected from H, C.sub.1 to C.sub.8 alkyl, optionally substituted aryl or optionally substituted heteroaryl.

2. A method as claimed in claim 1, wherein at least one of Y and Z is a substituted or unsubstituted azetidine group of formula: ##STR00018## wherein R.sub.A and R.sub.B are independently selected from H, halo, C.sub.1 to C.sub.8 alkyl, optionally substituted aryl or optionally substituted heteroaryl.

3. A method as claimed in claim 1, wherein the cationic species is of formula (II): ##STR00019## wherein R.sub.8 and R.sub.9 are independently selected from H, halo, C.sub.1 to C.sub.8 alkyl, optionally substituted aryl or optionally substituted heteroaryl.

4. A method as claimed in claim 1, wherein the counter ion is a biologically compatible counterion.

5. A method as claimed in claim 1, wherein the counter ion is selected from halide, carboxylate, oxalate, sulfate, alkanesulfonate, arylsulfonate, phosphate, perchlorate, trifluoroacetate, tetrafluoroborate, tetraphenylboride, hexafluorophosphate, nitrate and anions of aromatic or aliphatic carboxylic acids.

6. A method as claimed in claim 1, wherein incubating the sample is for a predetermined time, in the range 10 mins to 2hours and at a predetermined temperature, in the range 20? C. to 39? C.

7. A method as claimed in claim 1, wherein the cationic species is of formula (III): ##STR00020## wherein R.sub.10 and R.sub.11 are independently selected from H, halo, C.sub.1 to C.sub.8 alkyl, optionally substituted aryl or optionally substituted heteroaryl.

8. A method as claimed in claim 1, wherein the cationic species is of formula (IV): ##STR00021##

9. A method as claimed in claim 1, wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.10, and R.sub.11 are independently H, fluoro or chloro.

10. A method as claimed in claim 1, wherein R.sub.1 and/or R.sub.5 are C.sub.1 to C.sub.8 alkyl.

11. A method as claimed in claim 10, wherein R.sub.1 and/or R.sub.5 are methyl.

12. A method as claimed in claim 1, wherein the composition further comprises at least one organic solvent.

13. A method as claimed in claim 12, wherein the at least one organic solvent is selected from DMSO, acetone, dimethylformamide, acetonitrile, dioxane, and THF.

14. A method as claimed in claim 1, wherein the sample containing mitochondria comprises a tissue sample.

15. A method as claimed in claim 1, wherein the sample containing mitochondria is a plant, animal or fungal tissue sample, a sample of plant, animal or fungal cells or isolated plant, animal or fungal mitochondria.

16. A method as claimed in claim 1, wherein the sample containing mitochondria comprises a sample containing live mitochondria and/or a sample containing mitochondria in live cells.

17. A method as claimed in claim 1, wherein the sample containing mitochondria does not contain fixed cells.

18. A method as claimed in claim 1, wherein the concentration of the cationic species in the composition is in the range 10 nM to 1 ?M.

19. A method as claimed in claim 1, wherein the cationic species of formula (I) is selected from species of formulae: ##STR00022## ##STR00023## ##STR00024## or solvates, or tautomers thereof.

20. A method of analysing mitochondria, the method comprising: staining a sample of mitochondria using a method as claimed in claim 1, illuminating the stained sample using light of an appropriate wavelength to fluoresce the compound, and observing or imaging a magnified image of the sample.

21. A method as claimed in claim 20, wherein the appropriate wavelength is in the range 400 nm to 800 nm.

22. A method of detecting a mitochondrial condition, the method comprising staining a sample of mitochondria as claimed in claim 1.

23. A method as claimed in claim 22, wherein the sample of mitochondria is a plant, animal or fungal tissue sample, a sample of plant, animal or fungal cells or isolated plant, animal or fungal mitochondria.

24.-25. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Embodiments of the present invention will now be described further, with reference to the accompanying drawings, in which:

[0097] FIG. 1 shows chemical structures of compounds used in the invention.

[0098] FIG. 2 shows images of HeLa cells pre-treated with COMPOUND 1 or a comparator, and subsequently treated with compounds (oligomycin and CCCP) that hyper-and de-polarize the mitochondrial membrane, respectively.

[0099] FIG. 3 shows time-course images of HeLa cells incubated with either COMPOUND 1 or a comparator and a graph of normalized intensity with time for both COMPOUND 1 and a comparator.

[0100] FIG. 4 shows time-course images of HeLa cells incubated with COMPOUND 1,COMPOUND 2 or COMPOUND 3 and the corresponding graph of normalized intensity with time.

[0101] FIG. 5 shows time-course images of HeLa cells incubated with COMPOUND 3 or COMPOUND 4 and the corresponding graph of normalized intensity with time.

[0102] FIG. 6 shows images of Hela cells incubated with different concentrations of COMPOUND 1 or a comparator. FIG. 7 shows normalised intensity (a.u) against wavelength for emission and absorption of COMPOUND 1.

[0103] FIG. 8 shows normalised intensity (a.u) against wavelength for emission and absorption of COMPOUND 4.

DESCRIPTION OF THE EMBODIMENTS

[0104] FIG. 1 shows chemical structures of cationic species of COMPOUND 1, COMPOUND 2,COMPOUND 3 and COMPOUND 4 for use in the invention. Azetidine substituted rosamine (or Si rosamine) with/without an ortho-methyl substituent. The compounds shown in FIG. 1 have been synthesized and have undergone tests to demonstrate their utility in the context of the described invention.

[0105] The compounds outlined in FIG. 1 cover two core series that are primarily defined by distinct excitation/emission profiles. Further compounds with cationic species as in formula I may have different excitation/emission wavelengths.

[0106] COMPOUND 1 was extensively tested and shown to be a mitochondrial stain that localizes specifically to the mitochondria due to the charge potential across the mitochondrial membrane (the same mechanism as an existing commercially available comparator compound MitoTracker DeepRed?).

[0107] FIG. 2 shows HeLa cells incubated with the comparator compound (50 nM) or COMPOUND 1 (50 nM) for 60 mins, followed by treatment with Oligomycin (5 ?g/mL) or CCCP (10 ?M) for up to 60 minutes: the images are taken at specified time points. FIG. 2 demonstrates that COMPOUND 1 localizes to the mitochondria via the same mechanism as the comparator, since hyper-and de-polarizing the mitochondrial membrane with oligomycin and CCCP treatment (respectively) causes accumulation and dispersion (respectively) of both the comparator and COMPOUND 1.

[0108] FIG. 3 shows HeLa cells incubated with 50 nM of the comparator or COMPOUND 1 for 60 mins, followed by live imaging with images taken every 5 seconds for 240 frames, shown at specified time points. There is a clear increase in non-specific free dye in the comparator images that accumulates in the nucleus and cytoplasm over time (thus the signal: noise ratio for the mitochondria was reduced)-this results in an apparent increase in intensity over the first 5 minutes, but a significant reduction in overall intensity is seen for the comparator over the full imaging period (20 minutes @1 fr/5 sec). The graph shows time versus normalized intensity-NB: 100% is taken as the maximum intensity peak during movie-hence the comparator probe values start lower, then peak as apparent intensity increases due to non-specific accumulation of dye. Within this time frame, COMPOUND 1 intensity drops by ?25% during the imaging period compared to the comparator which drops by 65%. COMPOUND 1 remains clearly localized at the mitochondria, whereas the comparator is no longer located in the mitochondria and the cells have begun to contract, indicating (photo)-toxicity. FIG. 3 demonstrates the improved performance of this invention versus the comparator. COMPOUND 1 shows significantly improved photostability and localization within the mitochondria over time, with no apparent toxicity, while the comparator is less photostable, does not remain in the mitochondria over time and exhibits some (photo)-toxicity after prolonged imaging.

[0109] In some compounds, an ortho-methyl group may improve the quantum yield (i.e., brightness. The ortho-methyl substituent was present in COMPOUND 2 and COMPOUND 3and absent in the corresponding matched-pair compounds COMPOUND 1 and COMPOUND 4 (FIG. 1).

[0110] FIG. 4 shows HeLa cells incubated with 50 nM COMPOUND 1, COMPOUND 2 or COMPOUND 3 for 60 mins, followed by live imaging with images taken every 5 seconds for 240 frames, shown at specified time points. COMPOUND 1 and COMPOUND 2 perform very similarly in terms of signal over time; however COMPOUND 1 appears to mark the mitochondria more clearly than COMPOUND 2 over time. COMPOUND 3 showed retention of signal during the first 10 mins of imaging, but then the signal rapidly drops and higher non-specific cytoplasmic background (and lower mitochondrial labelling) was seen.

[0111] FIG. 5 shows HeLa cells incubated with 50 nM COMPOUND 3 (two samples, repeats) for 60 mins, followed by live imaging with images taken every 5 seconds for 240 frames, shown at specified time points. COMPOUND 3 show retention of signal during the first 10 mins of imaging, but then the signal rapidly drops and higher non-specific cytoplasmic background (and lower mitochondrial labelling) was seen. Data from two samples of COMPOUND 3,measured at different timepoints is overlaid in the graph (right). COMPOUND 4 shows higher signal retention over time than COMPOUND 3 and appears to remain faithfully localized to mitochondria over the entire imaging period with no observable phototoxicity effects.

[0112] FIG. 6 shows optimization of concentrations required for imaging. HeLa cells were incubated with the comparator or COMPOUND 1 for 60 mins at indicated doses and imaged at the same laser power/settings. NOTE: no washout step to remove unbound comparator was performed (manufacturer recommends this) to enable direct comparison with COMPOUND 1. Some toxicity is observed with 100 nM of comparator. Both COMPOUND 1 and the comparator give excellent results at 50 nM.

[0113] FIG. 7 shows normalised intensity (a.u) against wavelength for emission and absorption of COMPOUND 1:1-(7-(azetidin-1-yl)-5,5-dimethyl-10-phenyldibenzo[b,e]silin-3 (5H)-ylidene)azetidin-1-ium chloride

[0114] FIG. 8 shows normalised intensity (a.u) against wavelength for emission and absorption of COMPOUND 4:1-(6-(azetidin-1-yl)-9-phenyl-3H-xanthen-3-ylidene)azetidin-1-ium chloride

Confocal Microscopy

[0115] Mitochondrial stains were diluted to working concentrations from 10 mM DMSO stock solutions into DMEM containing 10% FCS and 25 mM HEPES. Solutions were incubated with HeLa cells at 37? C. in a humidified 5% CO.sub.2 incubator for the indicated period of time prior to imaging, typically without a washout step (though a washout step can be performed). A Nikon A1R TiE confocal laser scanning microscope equipped with environmental chamber (37? C.) was used for live-cell imaging, employing 561 nm and 640 nm diode laser lines, a Nikon A1R Plan APO VC 60x Oil lens (NA 1.4), pinhole at 1AU. The images were acquired using NIS Elements software and processed using ImageJ.

General Chemistry Methods

[0116] All reagents and solvents were purchased from commercial sources and used without further purification. Nuclear magnetic resonance spectra were recorded on a Bruker Avance III HD spectrometer operating at 400 MHz for .sup.1H NMR and 100 MHz for .sup.13C NMR. .sup.1H NMR and .sup.13C NMR chemical shifts (?) are reported in parts per million (ppm) and are referenced to residual protium in solvent and to the carbon resonances of the residual solvent peak respectively.

[0117] Purification by flash chromatography was performed using pre-packed silica gel columns and either a Buchi Reveleris, a Biotage Isolera or a Biotage Selekt system. Analytical thin layer chromatography was performed on glass plates pre-coated with silica gel (Analtech, UNIPLATE? 250 ?m/UV254), with visualization being achieved using UV light (254 nm) and/or by staining with alkaline potassium permanganate dip.

[0118] Reaction monitoring LC-MS analyses were conducted using Agilent InfinityLab LC/MSD systems. High resolution mass spectral (HRMS) data was collected using an Agilent 6545 LC/Q-TOF system.

[0119] Normalized absorption and fluorescence emission spectra were recorded in 10 mM PBS pH 7.3 at the concentration noted for each sample following dilution of a DMSO stock solution. Absorption spectra were recorded with an Agilent Cary 60 UV-Vis spectrophotometer using genuine precision quartz cells from Lovibond with a 1 cm path length. Fluorescence spectra were recorded on an Agilent Cary Eclipse Fluorescence Spectrophotometer using high precision Quartz Suprasil cells from Hellma Analytics and a 1 cm path length.

EXAMPLES

[0120] The invention is further illustrated by the following Examples.

Example 1-1-(7-(azetidin-1-yl)-5,5-dimethyl-10-phenyldibenzo [b,e]silin-3 (5H)-ylidene)azetidin-1-ium acetate

[0121] ##STR00012##

Synthesis of 1-(3-bromophenyl)azetidine

[0122] 3-Bromoiodobenzene (30 g, 106 mmol), azetidine (7.27 g, 127 mmol) and K.sub.3PO.sub.4 (67.5 g, 318 mmol) were combined with ethylene glycol (14.2 mL) and 1-butanol (150 mL) in a round bottom flask. The flask was sealed and evacuated/backfilled three times with nitrogen. CuI (2.02 g, 10.6 mmol) was subsequently added and the flask was again sealed and evacuated/backfilled with nitrogen three times. The mixture was then heated at 100? C. under an atmosphere of N.sub.2 for 4 h. After cooling to RT, saturated aqueous NH.sub.4Cl and EtOAc were added with stirring until there were no solids remaining. The layers were separated and the aqueous was extracted twice with EtOAc. The combined organic layers were washed with brine, then dried (MgSO.sub.4) and filtered and the solvent was removed in vacuo. The residue was further dried under high vacuum. The crude product was purified by flash chromatography (0 to 10% Et.sub.2O/PE) to give the title compound as a pale-yellow oil (18.2g, 81%).

[0123] .sup.1H NMR (CDCl.sub.3, 400 MHZ) ? 7.04 (1H, t), 6.85-6.80 (1H, m), 6.55 (1H, t), 6.36-6.31 (1H, m), 3.87 (4H, t), 2.37 (2H, p).

Synthesis of bis(3-(azetidin-1-yl) phenyl)dimethylsilane

[0124] A solution of 1-(3-bromophenyl) azetidine (12.6 g, 59.5 mmol) in THF (115 mL) was cooled to ?78? C. under nitrogen. A solution of n-butyllithium in hexane (2.5 M, 23.8 mL, 59.5 mmol) was slowly added so that the internal temperature was maintained below ?60? C. during the addition. The reaction mixture was subsequently stirred at ?78? C. for 30 min. A solution of dichlorodimethylsilane (3.20 g, 24.8 mmol) in THF (10 mL) was then added at a rate such that the internal temperature was kept below ?60? C. The cooling bath was removed, and the reaction was stirred at room temperature for 3 h. It was subsequently quenched with saturated aqueous NH.sub.4Cl (20 mL), diluted with water, and extracted twice with EtOAc. The combined organic extracts were washed with brine, dried over anhydrous MgSO.sub.4, filtered, and concentrated in vacuo. The resulting residue was co-evaporated twice with Et.sub.2O and purified by flash chromatography (0 to 30% Et.sub.2O/PE) to give the title compound as a colourless oil (8.00 g, 84%).

[0125] .sup.1H NMR (CDCl.sub.3, 400 MHZ) ? 7.20 (2H, t), 6.90 (2H, d), 6.61 (2H, d), 6.46 (2H, ddd), 3.86 (8H, t), 2.34 (4H, p), 0.51 (6H, s).

Synthesis of bis(5-(azetidin-1-yl)-2-bromophenyl)dimethylsilane

[0126] N-Bromosuccinimide (7.45 g, 41.9 mmol) was added in portions over 5 minutes to a solution of bis(3-(azetidin-1-yl)phenyl)dimethylsilane (6.75 g, 20.9 mmol) in DMF (120 mL). The resulting mixture was stirred for 5 days. Following removal of the solvent in vacuo, the resulting residue was diluted with water and extracted with EtOAc and then with DCM. The combined organic layers were washed with water and brine, then dried (MgSO.sub.4) and filtered and the solvent was removed in vacuo. The crude product was purified by recrystallisation from EtOAc to give the title compound as a white solid (5.69 g, 57%).

[0127] .sup.1H NMR (CDCl.sub.3, 400 MHZ) ? 7.31 (2H, d), 6.51 (2H, d), 6.31 (2H, dd), 3.81 (8H, t), 2.36 (4H, p), 0.71 (6H, s).

Synthesis of 1-(7-(azetidin-1-yl)-5,5-dimethyl-10-phenyldibenzo[b,e]silin-3 (5H)-ylidene)azetidin-1-ium acetate

[0128] A solution of t-BuLi in pentane (1.7 M, 1.96 mL) was added dropwise to a cooled (?78? C.) solution of bis(5-(azetidin-1-yl)-2-bromophenyl)dimethylsilane (0.40 g, 0.83 mmol) in THF (40 mL). After stirring for 20 minutes, the reaction mixture was warmed to ?20? C. and a solution of methyl benzoate (0.25 g, 1.83 mmol) in THF (7 mL) was added over 20 minutes, maintaining the internal temperature below ?20? C. over the course of the addition. The resulting mixture was allowed to warm to room temperature and was stirred overnight. The following day, saturated aqueous NH.sub.4Cl and water were added and the product was extracted twice with EtOAc The combined organic layers were washed with brine, then dried (MgSO.sub.4) and filtered and the solvent was removed in vacuo. The resulting residue was dissolved in MeOH (20 mL) and AcOH (0.2 mL) was added. After stirring for 15 minutes, the mixture was concentrated in vacuo. The crude product was purified by flash chromatography (1% MeOH in DCM (+1% AcOH) to 15% MeOH in DCM (+1% AcOH) to give the title compound as a blue solid (0.27 g, 69%).

[0129] .sup.1H NMR (MeOD, 400 MHZ) ? 7.60-7.52 (3H, m), 7.28-7.21 (2H, m), 7.08 (2H, d), 6.94 (2H, d), 6.34 (2H, dd), 4.36 (8H, t), 2.55 (4H, p), 1.96 (3H, s), 0.55 (6H, s).

[0130] LC/MS (ES+): m/z 409.3 (100%, M.sup.+).

Example 2-1-(7-(azetidin-1-yl)-5,5-dimethyl-10-phenyldibenzo[b,e]silin-3 (5H)-ylidene)azetidin-1-ium chloride

[0131] ##STR00013##

Synthesis of 3,7-di(azetidin-1-yl)-5,5-dimethyldibenzo[b,e]silin-10 (5H)-one

[0132] A solution of t-BuLi in pentane (1.7 M, 10.9 mL) was added dropwise to a cooled (?78? C.) solution of bis(5-(azetidin-1-yl)-2-bromophenyl)dimethylsilane (2.00 g, 4.16 mmol) in THF (160 mL). After stirring for 20 min, N,N-dimethylcarbamoyl chloride (0.49 g, 4.58 mmol) was added dropwise over 20 minutes. The reaction mixture was allowed to room temperature and was stirred overnight. The following day it was diluted with saturated aqueous NH.sub.4Cl and THF and the product was extracted twice with additional THF. The combined organic layers were washed with brine, then dried (MgSO.sub.4) and filtered and the solvent was removed in vacuo. The resulting residue was dissolved in DCM, dried (MgSO.sub.4) and filtered and the solvent was removed in vacuo. The crude product was purified by flash chromatography (0 to 15% MeOH in DCM) followed by trituration with hot EtOAc to give the title compound as a yellow solid (1.12 g, 77%).

[0133] .sup.1H NMR (CDCl.sub.3, 400 MHZ) ? 8.36 (2H, d), 6.53 (2H, dd), 6.48 (2H, d), 4.03 (8H, t), 2.44 (4H, p), 0.43 (6H, s).

[0134] HRMS (ESI) calcd. for C21H24N2OSi [M+H].sup.+, 349.1660, found 349.1733.

Synthesis of 1-(7-(azetidin-1-yl)-5,5-dimethyl-10-phenyldibenzo[b,e]silin-3 (5H)-ylidene)azetidin-1-ium chloride

[0135] A solution of phenylmagnesium chloride in THF (2M, 1.40 mL) was added to a stirred suspension of 3,7-di (azetidin-1-yl)-5,5-dimethyldibenzo[b,e]silin-10 (5H)-one (0.75 g, 2.15 mmol) in THF (25 mL). After stirring for 1 h, the reaction mixture was diluted with DCM and saturated aqueous NH.sub.4Cl. The layers were separated and the aqueous was further extracted three times with DCM. The combined organic layers were dried (MgSO.sub.4), filtered and the solvent was removed in vacuo. The resulting residue was dissolved in DCM and 1 drop of 2 M aqueous HCl was added. The mixture was subsequently concentrated in vacuo and then co-evaporated with firstly MeOH and then with DCM. The crude product was purified by flash chromatography (5 to 20% MeOH in DCM) followed by drying in the vacuum oven to give the title compound as a blue/green solid (0.15 g, 10%).

[0136] .sup.1H NMR (CDCl.sub.3, 400 MHZ) ? 7.52-7.49 (3H, m), 7.21-7.18 (2H, m), 7.05 (2H, d), 6.88 (2H, d), 6.25 (2H, dd), 4.49-4.38 (8H, m), 2.62 (4H, p), 0.60 (6H, s).

[0137] LC/MS (ES.sup.+): m/z 409.3 (100%, M.sup.+).

Example 3-1-(6-(azetidin-1-yl)-9-phenyl-3H-xanthen-3-ylidene)azetidin-1-ium chloride

[0138] ##STR00014##

Synthesis of 9-oxo-9H-xanthene-3,6-diyl bis(trifluoromethanesulfonate)

[0139] Pyridine was slowly added to a cooled (0? C.) suspension of 3,6-dihydroxyxanthen-9-one (3.65 g, 16.0 mmol) in DCM (60 mL). The reaction mixture was stirred for 5 minutes before trifluoromethanesulfonic anhydride (13.5 g, 47.8 mmol) was added dropwise keeping the internal temperature of the mixture below 15? C. The reaction mixture was allowed to warm to room temperature and was stirred overnight. The following day, water and DCM were added and the layers were separated. The organic layer was washed with water, 1 M aqueous HCl and brine then dried (MgSO.sub.4) and filtered and the solvent was removed in vacuo. The crude product was purified by precipitation of the product from a DCM solution by treatment with petroleum ether to give the title compound as a white solid (5.63 g, 72%).

[0140] .sup.1H NMR (CDCl.sub.3, 400 MHZ) ? 8.46 (2H, d), 7.50 (2H, d), 7.36 (2H, dd).

Synthesis of 3,6-di(azetidin-1-yl)-9H-xanthen-9-one

[0141] A mixture of 9-oxo-9H-xanthene-3,6-diyl bis (trifluoromethanesulfonate (5.63 g, 11.4 mmol), Pd.sub.2 (dba).sub.3 (1.05 g, 1.15 mmol), XPhos (1.49 g, 3.4 mmol) and Cs2CO3 (17.9 g, 54.9 mmol) in a round bottom flask was evacuated/backfilled with nitrogen (X3). To this mixture was added azetidine (1.44 g, 25.2 mmol) and 1,4-dioxane (65 ml) and the flask was again evacuated/backfilled three times with nitrogen. The flask was then inserted into a pre-heated metal heating block and stirred at 105? C. for 8 h. After cooling to room temperature, the reaction mixture was diluted with DCM/water and the layers were separated. The aqueous layer was extracted twice with additional DCM. The combined organic layers were dried (MgSO.sub.4), filtered and the solvent was removed in vacuo. The crude product was purified by flash chromatography (10 to 20% MeOH in DCM) followed by trituration with acetone to give the title compound as a yellow solid (0.46 g, 13%).

[0142] .sup.1H NMR (CDCl.sub.3, 400 MHZ) ? 8.21 (2H, d), 6.44 (2H, dd), 6.26 (2H, d), 4.10 (8H, t), 2.50 (4H, p).

Synthesis of 1-(6-(azetidin-1-yl)-9-phenyl-3H-xanthen-3-ylidene)azetidin-1-ium chloride

[0143] A solution of phenylmagnesium chloride in THF (2M, 0.55 mL) was added to a stirred suspension of 3,6-di(azetidin-1-yl)-9H-xanthen-9-one (0.28 g, 0.91 mmol) in THF (10 mL). After stirring for 1 h, the reaction mixture was diluted with DCM and saturated aqueous NH.sub.4Cl. The layers were separated and the aqueous was extracted three times with DCM. The combined organic layers were dried (MgSO.sub.4), filtered and the solvent was removed in vacuo. The resulting residue was dissolved in DCM and 1 drop of 2 M aqueous HCl was added. The mixture was subsequently concentrated in vacuo and then co-evaporated with firstly MeOH and then with DCM. The crude product was purified by flash chromatography (10 to 20% MeOH in DCM) followed by precipitation of the product from a solution in DCM with EtOAc to give the title compound as a red solid (0.024 g, 7%).

[0144] .sup.1H NMR (d6-DMSO, 400 MHZ) ? 7.70-7.66 (3H, m), 7.49-7.47 (2H, m), 7.19 (2H, d), 6.70 (2H, dd), 6.58 (2H, d), 4.28 (8H, t), 2.51 (4H, p).

[0145] HRMS (ESI) calcd for C25H23N2O [M].sup.+, 367.1810, found 367.1809.

Example 4-Synthesis of 1-(7-(azetidin-1-yl)-5,5-dimethyl-10-phenyldibenzo[b,e]silin-3 (5H)-ylidene)azetidin-1-ium trifluoroacetate

[0146] ##STR00015##

Synthesis of 1-(7-(azetidin-1-yl)-5,5-dimethyl-10-phenyldibenzo[b,e]silin-3 (5H)-ylidene)azetidin-1-ium trifluoroacetate

[0147] A solution of t-BuLi in pentane (1.7 M, 1.96 mL) was added dropwise to a cooled (?78? C.) solution of bis(5-(azetidin-1-yl)-2-bromophenyl)dimethylsilane (0.40 g, 0.83 mmol) in THF (40 mL). After stirring for 20 minutes, the reaction mixture was warmed to ?20? C. and a solution of methyl benzoate (0.25 g, 1.83 mmol) in THF (7 mL) was added over 20 minutes, maintaining the internal temperature below ?20? C. over the course of the addition. The resulting mixture was allowed to warm to room temperature and was stirred overnight. The following day, saturated aqueous NH.sub.4Cl and water were added and the product was extracted twice with EtOAc. The combined organic layers were washed with brine, then dried (MgSO.sub.4) and filtered and the solvent was removed in vacuo. The resulting residue was dissolved in MeOH (20 mL) and AcOH (0.2 mL) was added. After stirring for 15 minutes, the mixture was concentrated in vacuo. The crude product was initially purified by flash chromatography (1% MeOH in DCM (+1% AcOH) to 15% MeOH in DCM (+1% AcOH). Subsequently, half of the crude product was purified by prep-LC (10-100% ACN/H2O (+0.1% TFA) and concentrated by lyophilisation to furnish the desired product as a blue/red solid (0.011 g)

[0148] .sup.1H NMR (d6-DMSO, 400 MHZ) ? 7.61-7.52 (3H, m), 7.30-7.22 (2H, m), 7.05 (2H, d), 6.91 (2H, d), 6.42 (2H, dd), 4.40-4.22 (8H, m), 2.48-2.39 (4H, m), 0.54 (6H, s).

[0149] LC/MS (ES+): m/z 409.3 (100%, M.sup.+).

Example 5-1-(6-(azetidin-1-yl)-9-(o-tolyl)-3H-xanthen-3-ylidene)azetidin-1-ium

[0150] ##STR00016##

Synthesis of 1-(6-(azetidin-1-yl)-9-(o-tolyl)-3H-xanthen-3-ylidene) azetidin-1-ium

[0151] A solution of o-tolylmagnesium bromide in Et.sub.2O (2M, 1.22 mL) was added to a stirred, pre-heated (50? C.) solution of 3,6-di(azetidin-1-yl)-9H-xanthen-9-one (0.25 g, 0.81 mmol) in THF (12 mL). After stirring at 50? C. for 9 h, the reaction mixture was allowed to cool to room temperature and stirred overnight. The mixture was diluted with DCM and saturated aqueous NH4Cl (10 mL). The layers were separated and the aqueous was extracted three times with DCM. The combined organic layers were dried (MgSO.sub.4), filtered and the solvent was removed in vacuo. The resulting residue was dissolved in DCM and 1 drop of 2 M aqueous HCl was added. The mixture was subsequently concentrated in vacuo and then co-evaporated with firstly MeOH and then with DCM. The crude product was purified by flash chromatography (7 to 20% MeOH in DCM) followed by trituration with EtOAc to give the title compound as a red solid (0.065 g, 19%).

[0152] .sup.1H NMR (MeOD, 400 MHZ) ? 8 7.61-7.44 (3H, m), 7.25 (1H, dd), 7.16-7.12 (2H, m), 6.67 (2H, dd), 6.59 (2H, d), 4.36 (8H, t), 2.60 (4H, p), 2.07 (3H, s).

[0153] LC/MS (ES+): m/z 381.1 (100%, M.sup.+).

REFERENCES

[0154] 1) Grimm et al., General Synthetic Method for Si-Fluoresceins and Si-Rhodamines. ACS Cent Sci. 2017; 3 (9): 975-985. [0155] 2) Macho et al., Chloromethyl-X-rosamine is an aldehyde-fixable potential-sensitive fluorochrome for the detection of early apoptosis. Cytometry. 1996; 25 (4): 333-340. [0156] 3) Poot et al.; Analysis of mitochondrial morphology and function with novel fixable fluorescent stains. J Histochem Cytochem. 1996; 44 (12): 1363-72. [0157] 4) Grimm et al., A general method to improve fluorophores for live-cell and single-molecule microscopy; Nat Methods. 2015 12 (3): 244-50. [0158] 6) EP 3 126 451 [0159] 7) U.S. Pat. No. 5,686,261

[0160] All publications mentioned in the above specification are herein incorporated by reference. Although illustrative embodiments of the invention have been disclosed in detail herein, with reference to the accompanying drawings, it is understood that the invention is not limited to the precise embodiment and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the invention as defined by the appended claims and their equivalents.