AZETIDINE SUBSTITUTED ROSAMINES USEFUL FOR STAINING MITOCHONDRIA

Abstract

Azetidine substitute rosamines useful for staining mitochondria of formula (I) are disclosed, 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, and there is at least one Q group on the pendant phenyl, the Q group comprising a group selected from halo, maleimidyl, OSO.sub.2R.sub.13 and epoxide. Also disclosed are methods for staining mitochondria involving incubating a sample in a composition comprising the compound, and analysing mitochondria, involving staining a sample of mitochondria, optionally fixing the cells, 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 compound comprising a cationic species of formula (I): ##STR00024## 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 selected from H, C.sub.1 to C.sub.8 alkyl, OR.sub.15, C(O)OR.sub.16, NHC(O)R.sub.15, C(O)NHR.sub.15, halo, and a group of formula Q, wherein at least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 is a group of formula Q; 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.16 is selected from C.sub.1 to C.sub.8 alkyl, optionally substituted aryl or optionally substituted heteroaryl; R.sub.17 is selected from H, C.sub.1 to C.sub.8 alkyl, optionally substituted aryl or optionally substituted heteroaryl; and Q is a group comprising L-M.sub.A, wherein M.sub.A is selected from CR.sub.18R.sub.19M.sub.B, NHC(O)CR.sub.18R.sub.19M.sub.B, C(O)NHCR.sub.18R.sub.19M.sub.B, NHC(O)-L-CR.sub.18R.sub.19M.sub.B, C(O)NH-L-CR.sub.18R.sub.19M.sub.B, OCR.sub.18R.sub.19M.sub.B and O-L-CR.sub.18R.sub.19M.sub.B; each M.sub.B is independently selected from halo, maleimidyl, OSO.sub.2R.sub.13, and ##STR00025## each L is an independently selected divalent linker group, substituted or unsubstituted phenylene or is absent, and R.sub.18 and R.sub.19; are each independently selected from H and CH.sub.3; and 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.

2. A compound as claimed in claim 1, wherein at least one of Y and Z is a substituted or unsubstituted azetidine group of formula: ##STR00026## wherein R.sub.A and R.sub.B are independently selected from H, C.sub.1 to C.sub.8 alkyl, OR.sub.20, C(O)OR.sub.20, NHC(O)R.sub.20, C(O)NHR.sub.20, halo, NR.sub.20R.sub.21, CN, NC, optionally substituted aryl or optionally substituted heteroaryl; wherein R.sub.20 and R.sub.21 are independently selected from H, and C.sub.1 to C.sub.8 alkyl.

3. A compound as claimed in claim 1, wherein the cationic species is of formula (II): ##STR00027## wherein R.sub.8 and R.sub.9 are independently selected from H, C.sub.1 to C.sub.8 alkyl, OR.sub.20, C(O)OR.sub.20, NHC(O)R.sub.20, C(O)NHR.sub.20, halo, NR.sub.20R.sub.21, CN, NC, optionally substituted aryl or optionally substituted heteroaryl.

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

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

6. A method as claimed in claim 1, wherein the cationic species is of formula (III): ##STR00028## wherein R.sub.10 and R.sub.11 are independently selected from H, C.sub.1 to C.sub.8 alkyl, OR.sub.20, C(O)OR.sub.20, NHC(O)R.sub.20, C(O)NHR.sub.20, halo, NR.sub.20R.sub.21, CN, NC, optionally substituted aryl or optionally substituted heteroaryl.

7. A compound as claimed in claim 1, wherein the cationic species is of formula (IV): ##STR00029##

8. A compound as claimed in claim 1, wherein at least one of R.sub.1 and R.sub.5 is Q.

9. A compound as claimed in claim 1, wherein R.sub.3 is Q.

10. A compound as claimed in claim 1, wherein at least one of R.sub.2 and R.sub.4 is Q.

11. A compound as claimed in claim 1, wherein L comprises an alkylene chain (CH.sub.2).sub.m, wherein m is 1 to 6.

12. A compound as claimed in claim 1, wherein M.sub.B is halo.

13. A compound as claimed in claim 10, wherein M.sub.B is chloro.

14. A compound as claimed in claim 1, wherein the compound comprises a cationic species selected from species of formulae: ##STR00030## ##STR00031## ##STR00032## ##STR00033## ##STR00034## ##STR00035##

15.-18. (canceled)

19. A method for staining mitochondria, the method comprising: providing a sample containing mitochondria, and incubating the sample in a composition comprising a compound as claimed in claim 1.

20. A method as claimed in claim 19, wherein incubating the sample is for a predetermined time in the range 10 mins to 2 hours and at a predetermined temperature in the range 20 C. to 39 C.

21. A method as claimed in claim 19, wherein the sample containing mitochondria comprises a tissue sample.

22. A method as claimed in claim 19, 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.

23. A method as claimed in claim 19, wherein the sample containing mitochondria comprises a sample containing fixed mitochondria and/or a sample containing mitochondria in fixed cells.

24. A method as claimed in claim 19, wherein the sample containing mitochondria contains substantially no live cells.

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

26. A method as claimed in claim 25, wherein the appropriate wavelength is in the range 400 nm to 800 nm.

27. A method of detecting a mitochondrial condition, the method comprising staining a sample of mitochondria as claimed in claim 18.

28. A method as claimed in claim 27, 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.

29. A compound as claimed in claim 1, wherein each L is independently selected from C.sub.1 to C.sub.8 alkylene.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Embodiments of the present invention will now be described further, with reference to the accompanying drawings, in which:

[0106] FIG. 1 shows chemical structures of compounds according to the invention.

[0107] FIG. 2 shows images of antibody NBP2-23489 incubated on HeLa cells co-stained with COMPOUND 5 (left, far red mitochondrial marker) and antibody (middle) and COMPOUND 5 (right) separately stained.

[0108] FIG. 3 shows in A, a schematic representation of mitochondrial membrane de-polarisation experiments, and in B, images of the results.

[0109] FIG. 4 shows images of fixed COS-7 cells stained with COMPOUND 5 compared to a Comparator (MitoTracker Deep Red FM) at varying concentrations.

[0110] FIG. 5 shows images of fixed HeLa cells stained with COMPOUND 5 compared to a Comparator (MitoTracker Deep Red FM) at a single concentration.

[0111] FIG. 6 shows images of fixed HeLa cells stained with either COMPOUND 8 or COMPOUND 9 (both added before fixation). In each experiment, an antibody for the mitochondrial marker TOM20 has also been added. Co-localization of TOM20 and mitochondrial probes is directly compared in the merged data.

[0112] FIG. 7 shows images of fixed HeLa cells stained with COMPOUND 5 (added after fixation) or a Comparator (added before fixation). COMPOUND 5 and the Comparator have distinct absorption and emission maxima. Intensity profiles (lower panel) taken across a set frame are shown for both COMPOUND 5 and Comparator.

[0113] FIG. 8 shows normalized absorption and fluorescence emission spectra for COMPOUND 5 1-(7-(azetidin-1-yl)-10-(2-(chloromethyl)phenyl)-5,5-dimethyldibenzo[b,e]silin-3(5H)-ylidene)azetidin-1-ium chloride.

[0114] FIG. 9 shows normalized absorption and fluorescence emission spectra for COMPOUND 6 1-(7-(azetidin-1-yl)-10-(4-(chloromethyl)phenyl)-5,5-dimethyldibenzo[b,e]silin-3(5H)-ylidene)azetidin-1-ium chloride.

[0115] FIG. 10 shows normalized absorption and fluorescence emission spectra for COMPOUND 8 1-(6-(azetidin-1-yl)-9-(2-(chloromethyl)phenyl)-3H-xanthen-3-ylidene)azetidin-1-ium chloride.

[0116] FIG. 11 shows normalized absorption and fluorescence emission spectra for COMPOUND 9 1-(9-(2-(chloromethyl)phenyl)-6-(3-methoxyazetidin-1-yl)-3H-xanthen-3-ylidene)-3-methoxyazetidin-1-ium chloride.

DESCRIPTION OF THE EMBODIMENTS

[0117] FIG. 1 shows the structures of compounds 5, 6, 7, 8 and 9 according to the invention. The compounds in FIG. 1 are azetidine-substituted rosamines and analogues, with ortho- or para-chloromethyl substituents. Compounds according to the invention are excellent for fixed-cell (and live-cell) fluorescent imaging of mitochondria.

[0118] 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.

[0119] COMPOUND 5 is an excellent mitochondrial stain that localizes specifically to the mitochondria as demonstrated in FIG. 2. FIG. 2 shows images for a series of experiments where NBP2-23489 DRP1 Antibody (Novus Biologicals; Catalog #NBP2-23489) was co-stained with COMPOUND 5 (far red mitochondrial marker). NBP2-23489 was incubated on fixed, permeabilized HeLa cells at 8 g/mL for 2 hours at room temperature. Cells were then incubated with an NL557 secondary antibody (R&D Systems; Catalog #NL007) for 1 hour at room temperature protected from light. Lastly, cells were incubated with COMPOUND 5 at 200 nM for 15 minutes at room temperature protected from light. Cells were cover-slipped with a DAPI-containing mounting media and imaged. Antibody staining was pseudo-coloured green, and mitochondrial marker staining was pseudo-coloured red. The images clearly show that antibody staining is localized to the mitochondria and cytoplasm and overlaps well with COMPOUND 5.

[0120] FIG. 3 demonstrates that COMPOUND 5 localizes to the mitochondria via the same mechanism as MitoTracker DeepRed (the Comparator), because de-polarizing the mitochondrial membrane with CCCP treatment prior to addition of the mitotrackers eliminates staining. Advantageously and surprisingly, COMPOUND 5 can stain the mitochondria more rapidly than the Comparator (MitoTracker deep red), since subsequent treatment with CCCP (CCCP chase) does not eliminate staining by COMPOUND 5.

[0121] In FIG. 3 (scale bar=10 m), A) shows a schematic representation of the experimental setup. COS-7 cells were pre-treated with media or 20 M CCCP for 30 minutes followed by a staining step with 100 nM COMPOUND 5 or 100 nM Comparator (MitoTracker Deep Red FM) for 30 minutes in the presence/absence of 20 M CCCP. After the staining, excessive dye was washed out with media, with or without 20 M CCCP for 30 minutes. B) shows depolarizing the mitochondrial membrane potential with CCCP reduces the mitochondrial accumulation of COMPOUND 5 or the Comparator (MitoTracker Deep Red FM). However, depolarizing the mitochondrial membrane potential for 30 minutes with CCCP after staining still results in a strong mitochondrial signal of COMPOUND 5, but not of the Comparator (MitoTracker Deep Red FM), see CCCP Chase. This suggests that COMPOUND 5 is still retained in mitochondria even after the loss of the mitochondrial membrane potential, whereas the mitochondrial accumulation of the Comparator (MitoTracker Deep Red FM) is reversible. In conclusion, mitochondrial accumulation of COMPOUND 5 and the Comparator (MitoTracker Deep Red FM) is membrane potential-dependent, but only reversible for the Comparator upon loss of the mitochondrial membrane potential.

[0122] FIG. 4 demonstrates the improved performance of compounds of the invention compared to an existing commercial product (MitoTracker Deep Red). COMPOUND 5 shows significantly improved brightness compared to the Comparator, particularly at lower concentrations (25 nM and 50 nM). FIG. 4 shows optimization of concentrations required for imaging. Both COMPOUND 5 and the Comparator show some toxicity at 200 nM. COMPOUND 5 gives nice staining at 25-100 nM.

[0123] In FIG. 4 (scale bar=10 m), images are shown after COS-7 cells were stained for 30 minutes with indicated concentrations followed by immediate fixation with 4% PFA for 20 minutes at 37 C. Images were acquired using a spinning disk confocal microscope with identical exposure time and laser power and were processed with identical settings to allow a direct comparison. Low concentrations of COMPOUND 5 (25-100 nM, 30 min) resulted in a good mitochondrial signal with little background and no obvious toxicity. Staining intensities for the Comparator decreased at low concentration. High concentration (above 200 nM) of COMPOUND 5 or the Comparator resulted in mitochondrial alterations (fragmentation and peri-nuclear clustering suggesting toxic effects). Moreover, both dyes stained additionally other cellular structures like the ER and the nuclear envelope at high concentrations.

[0124] FIG. 5 (scale bar=10 m), demonstrates that COMPOUND 5 retains clearer staining of mitochondria following fixation, compared to a Comparator. In FIG. 5 (scale bar=10 m), images are shown after HeLa cells were stained with 100 nM probe for 40 minutes, followed by fixation with 4% PFA for 10 minutes. Laser power and gain have been optimized for each individual probe in this figure.

[0125] FIG. 6 (scale bar=10 m), demonstrates that both COMPOUND 8 and COMPOUND 9 are selective mitochondrial stains that retain staining fidelity after fixation, since they both co-localize with the mitochondrial marker TOM20.

[0126] FIG. 7 shows images of fixed HeLa cells. Live HeLa cells were stained with a Comparator (250 nM, 45 min) and were then fixed (4% PFA, 20 min). After fixation, COMPOUND 5 (75 nM) was added and the cells were imaged. COMPOUND 5 and Comparator have distinct absorption and emission profiles and spill over between channels was not observed. Intensity profiles for both mitochondrial stains were taken across a set frame (lower panel) and compared. The data shows very high similarity in staining profile between the Comparator and COMPOUND 5, suggesting that COMPOUND 5 can be applied post-fixation.

[0127] FIG. 8 shows normalised intensity (a.u) against wavelength for emission and absorption of COMPOUND 5 1-(7-(azetidin-1-yl)-10-(2-(chloromethyl)phenyl)-5,5-dimethyldibenzo[b,e]silin-3(5H)-ylidene)azetidin-1-ium chloride.

[0128] FIG. 9 shows normalised intensity (a.u) against wavelength for emission and absorption of COMPOUND 6 1-(7-(azetidin-1-yl)-10-(4-(chloromethyl)phenyl)-5,5-dimethyldibenzo[b,e]silin-3(5H)-ylidene)azetidin-1-ium chloride.

[0129] FIG. 10 shows normalised intensity (a.u) against wavelength for emission and absorption of COMPOUND 8 1-(6-(azetidin-1-yl)-9-(2-(chloromethyl)phenyl)-3H-xanthen-3-ylidene)azetidin-1-ium chloride.

[0130] FIG. 11 shows normalised intensity (a.u) against wavelength for emission and absorption of COMPOUND 9 1-(9-(2-(chloromethyl)phenyl)-6-(3-methoxyazetidin-1-yl)-3H-xanthen-3-ylidene)-3-methoxyazetidin-1-ium chloride.

General Chemistry Methods

[0131] 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 (5) 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.

[0132] 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.

[0133] 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.

[0134] 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

[0135] The invention is further illustrated by the following Examples.

Example 11-(7-(azetidin-1-yl)-10-(2-(chloromethyl)phenyl)-5,5-dimethyldibenzo[b,e]silin-3(5H)-ylidene)azetidin-1-ium chloride

[0136] ##STR00020##

Synthesis of 1-(3-bromophenyl)azetidine

[0137] 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 with nitrogen three times. 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 room temperature, a saturated aqueous solution of 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 (18.2 g, 81%).

[0138] .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

[0139] 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 (o to 30% Et.sub.2O/PE) to give the title compound as a colourless oil (8.00 g, 84%).

[0140] .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

[0141] 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%).

[0142] .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 1,1-(5,5-dimethyl-3H,5H-spiro[dibenzo[b,e]siline-10,1-isobenzofuran]-3,7-diyl)bis(azetidine)

[0143] A solution of t-BuLi in pentane (1.7 M, 10.3 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 (180 mL). After stirring for 20 minutes, the reaction mixture was warmed to 10 C. and a solution of MgBr.sub.2.Math.OEt.sub.2 (2.37 g, 9.18 mmol) in THF (40 mL) was slowly added, keeping the internal temperature below 5 C. The reaction mixture was stirred at 10 C. for 30 minutes and then a solution of phthalide (1.23 g, 9.17 mmol) in THF (25 mL) was added dropwise over 30 minutes. The reaction mixture was allowed to warm to room temperature and was stirred for 60 h. Saturated aqueous NH.sub.4Cl and EtOAc were added and the layers were separated, the aqueous was further extracted twice with EtOAc. The combined organic layers were washed with saturated aqueous NaHCO.sub.3 and brine, dried over anhydrous MgSO.sub.4, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography (PE to 50% EtOAc/PE to a flush with 50% DCM/EtOAc) to give the title compound as an off-white solid (0.25 g, 14%).

[0144] .sup.1H NMR (CDCl.sub.3, 400 MHz) 7.31-7.21 (3H, m), 7.06 (1H, d), 6.95 (2H, d), 6.67 (2H, d), 6.31 (2H, dd), 5.21 (2H, s), 3.87 (8H, t), 2.34 (4H, p), 0.59 (3H, s), 0.52 (3H, s).

Synthesis of 1-(7-(azetidin-1-yl)-10-(2-(chloromethyl)phenyl)-5,5-dimethyldibenzo[b,e]silin-3(5H)-ylidene)azetidin-1-ium chloride

[0145] Thionyl chloride (0.06 g, 0.50 mmol) was added dropwise to a solution of 1,1-(5,5-dimethyl-3H,5H-spiro[dibenzo[b,e]siline-10,1-isobenzofuran]-3,7-diyl)bis(azetidine) (0.20 g, 0.46 mmol) in DCM (8 mL). After 20 minutes, the reaction mixture was diluted with water and DCM and the layers were separated. The aqueous layer was extracted twice with DCM and the combined organic layers were dried over anhydrous MgSO.sub.4, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography (5 to 20% MeOH/DCM) followed by precipitation from DCM/EtOAc to give the title compound as a blue/red solid (0.135 g, 60%).

[0146] .sup.1H NMR (CDCl.sub.3, 400 MHz) 7.63-7.61 (1H, m), 7.57-7.52 (1H, m), 7.49-7.45 (1H, m), 7.13 (1H, d), 6.93 (2H, d), 6.89 (2H, d), 6.26 (2H, dd), 4.51-4.36 (8H, m), 4.29 (2H, s), 2.60 (4H, p), 0.63 (3H, s), 0.60 (3H, s).

[0147] HRMS (ESI) calcd for C.sub.28H.sub.30ClN.sub.2Si [M].sup.+, 457.1867, found 457.1864.

Example 21-(7-(azetidin-1-yl)-10-(4-(chloromethyl)phenyl)-5,5-dimethyldibenzo[b,e]silin-3(5H)-ylidene)azetidin-1-ium chloride

[0148] ##STR00021##

Synthesis of 1-(7-(azetidin-1-yl)-10-(4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)-5,5-dimethyldibenzo[b,e]silin-3(5H)-ylidene)azetidin-1-ium chloride

[0149] A solution of t-BuLi in pentane (1.7 M, 2.49 mL) was added dropwise to a cooled (78 C.) solution of (4-bromophenyl)methoxy-tert-butyldimethylsilane (0.60 g, 2.00 mmol) in THF (8 mL). After stirring for 10 minutes, a portion of this solution (5 mL) was slowly added to a suspension of 3,7-bis(azetidin-1-yl)-5,5-dimethyl-benzo[b][1]benzosilin-10-one (0.58 g, 1.66 mmol) in THF (12.5 mL). The resulting mixture was stirred overnight and was then diluted with both DCM and a saturated aqueous solution of NH.sub.4Cl. The layers were separated and the aqueous was extracted three times with additional DCM. The combined organic layers were dried over anhydrous MgSO.sub.4, filtered, and concentrated in vacuo. The resulting residue was dissolved in DCM and 5 drops of 2M aqueous HCl were 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) to give the title compound as a blue/green solid (0.14 g, 12%).

[0150] .sup.1H NMR (CDCl.sub.3, 400 MHz) 7.47-7.437.46 (2H, dm), 7.18-7.13 (2H, m), 7.07 (2H, d), 6.87 (2H, d), 6.23 (2H, dd), 4.86 (2H, s) 4.55-4.32 (8H, m), 2.61 (4H, p), 0.99 (9H, s), 0.60 (6H, s), 0.18 (6H, s).

[0151] LC/MS (ES+): m/z 553.4 (100%, M.sup.+).

Synthesis of 1-(7-(azetidin-1-yl)-10-(4-(hydroxymethyl)phenyl)-5,5-dimethyldibenzo[b,e]silin-3(5H)-ylidene)azetidin-1-ium chloride

[0152] A 1M aqueous solution of HCl (0.4 mL) was added to a solution of 1-(7-(azetidin-1-yl)-10-(4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)-5,5-dimethyldibenzo[b,e]silin-3(5H)-ylidene)azetidin-1-ium chloride (0.056 g, 0.095 mmol) in MeOH (8 mL). After stirring for 30 minutes, the reaction mixture was diluted with DCM and water and the layers were separated. The aqueous was further extracted twice with DCM. The combined organic layers were dried over anhydrous MgSO.sub.4, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography (5 to 25% MeOH in DCM) to give the title compound as a blue/green solid (0.031 g, 71%).

[0153] .sup.1H NMR (CDCl.sub.3, 400 MHz) 7.59-7.55 (2H, m), 7.18-7.14 (2H, m), 7.12 (2H, d), 6.80 (2H, d), 6.26 (2H, dd), 4.87 (2H, s) 4.50-4.33 (8H, m), 3.03 (1H, s), 2.61 (4H, p), 0.58 (6H, s).

[0154] LC/MS (ES+): m/z 439.2 (100%, M.sup.+).

Synthesis of 1-(7-(azetidin-1-yl)-10-(4-(chloromethyl)phenyl)-5,5-dimethyldibenzo[b,e]silin-3(5H)-ylidene)azetidin-1-ium chloride

[0155] A solution of triphosgene (0.008 g, 0.027 mmol) in DCM (0.5 mL) was added to a solution of 1-(7-(azetidin-1-yl)-10-(4-(hydroxymethyl)phenyl)-5,5-dimethyldibenzo[b,e]silin-3(5H)-ylidene)azetidin-1-ium chloride (0.021 g, 0.044 mmol) in DCM (4 mL). The reaction was monitored by LC/MS and after 30 minutes additional triphosgene/pyridine were added to push the reaction to completion. The mixture was diluted with DCM and washed with a 1M aqueous solution of HCl. The combined organic layers were dried over anhydrous MgSO.sub.4, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography (5 to 30% MeOH in DCM) followed by trituration with EtOAc to give the title compound as a blue/green solid (0.08 g, 37%).

[0156] .sup.1H NMR (CDCl.sub.3, 400 MHz) 7.57-7.51 (2H, m), 7.24-7.18 (2H, m), 7.02 (2H, d), 6.88 (2H, d), 6.28 (2H, dd), 4.70 (2H, s) 4.52-4.34 (8H, m), 2.62 (4H, p), 0.60 (6H, s).

[0157] LC/MS (ES+): m/z 457.3 (100%, M.sup.+).

Example 31-(6-(azetidin-1-yl)-9-(2-(chloromethyl)phenyl)-3H-xanthen-3-ylidene)azetidin-1-ium chloride

[0158] ##STR00022##

Synthesis of 2-(3,6-dibromo-9H-xanthen-9-yl)benzoic acid

[0159] A mixture of phthalic anhydride (3.80 g, 25.7 mmol), 3-bromophenol (8.66 g, 50.0 mmol) and methanesulfonic acid (12.5 mL) was heated at 130 C. for 72 h. After cooling to room temperature, the reaction mixture was poured onto H.sub.2O (700 mL) resulting in the formation of a precipitate. The precipitate was collected by filtration, washing with H.sub.2O. The solid was subsequently dissolved in DCM (700 mL) with gentle heating and the solution was dried (MgSO.sub.4), filtered and solvent was removed in vacuo. The crude product was heated with EtOAc (100 mL) allowed to cool and the solid was collected by filtration, washing with EtOAc to afford the title compound as a pale pink solid (4.20 g, 36%).

[0160] .sup.1H NMR (CDCl.sub.3, 400 MHz) 8.06-8.02 (1H, m), 7.71-7.62 (2H, m), 7.50 (2H, d), 7.19 (2H, dd), 7.14-7.10 (1H, m), 6.70 (2H, d).

[0161] LC/MS (ES+): m/z 458.9 (100%, [M+H].sup.+).

Synthesis of (2-(3,6-dibromo-9H-xanthen-9-yl)phenyl)methanol

[0162] A solution of LiBH.sub.4 in THF (2M, 36.6 mL) was added dropwise to a cooled (o C.) suspension of 2-(3,6-dibromo-9H-xanthen-9-yl)benzoic acid (2.68 g, 5.85 mmol) in a mixture of THF (50 mL) and isopropanol (250 mL). The reaction mixture was allowed to warm to room temperature and stirred for 48 h. Subsequently, the reaction mixture was quenched by the cautious addition of saturated aqueous NH.sub.4Cl (100 mL) and the product was extracted three times with DCM. The combined organic layers were dried (MgSO.sub.4) and filtered and the solvent was removed in vacuo. The crude product was purified by flash chromatography (5 to 10% MeOH in DCM) to give the title compound (2.36 g, 90%) as a white solid.

[0163] .sup.1H NMR (d6-DMSO, 400 MHz) 8.20 (1H, dd), 7.53-7.46 (3H, m), 7.44-7.32 (2H, m), 7.25 (2H, dd), 6.86 (2H, d), 6.77 (1H, s), 4.86 (1H, t), 3.46 (2H, d).

Synthesis of 3,6-di(azetidin-1-yl)-3H-spiro[isobenzofuran-1,9-xanthene]

[0164] A mixture of (2-(3,6-dibromo-9H-xanthen-9-yl)phenyl)methanol (2.36 g, 5.29 mmol), Pd.sub.2(dba).sub.3 (0.484 g, 0.529 mmol), XPhos (0.69 g, 1.59 mmol) and Cs.sub.2CO.sub.3 (8.27 g, 25.4 mmol) in a round bottom flask was evacuated/backfilled with nitrogen (X3). To this mixture was added azetidine (0.664 g, 11.6 mmol) and 1,4-dioxane (40 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. overnight. 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 (2 to 15% MeOH in DCM) followed by trituration with EtOAc to give the title compound as a pale pink solid (0.62 g, 30%).

[0165] .sup.1H NMR (d6-DMSO, 400 MHz) 7.46-7.39 (1H, m), 7.34 (1H, t), 7.23 (1H, t), 6.73 (1H, d), 6.65 (2H, d), 6.15-6.10 (4H, m), 5.16 (2H, s), 3.80 (8H, t), 2.29 (4H, p).

[0166] LC/MS (ES+): m/z 397.2 (100%, [M+H].sup.+).

Synthesis of 1-(6-(azetidin-1-yl)-9-(2-(chloromethyl)phenyl)-3H-xanthen-3-ylidene)azetidin-1-ium chloride

[0167] A stock solution of thionyl chloride (300 mg) in DCM (5 mL) was prepared. A portion (0.5 mL) of this stock solution was added dropwise to a solution of 3,6-di(azetidin-1-yl)-3H-spiro[isobenzofuran-1,9-xanthene] (0.10 g, 0.252 mol) in DCM (10 mL). After 30 minutes, the reaction mixture was diluted with water and DCM and the layers were separated. The aqueous layer was extracted twice with DCM and the combined organic layers were dried over anhydrous MgSO.sub.4, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography (6 to 15% MeOH/DCM) followed by trituration with Et.sub.2O to give the title compound as a dark red solid (0.114 g, 26%).

[0168] .sup.1H NMR (CDCl.sub.3, 400 MHz) 7.70-7.65 (1H, m), 7.62 (1H, t), 7.54 (1H, t), 7.19 (1H, d), 7.06-6.99 (2H, m), 6.58 (2H, dd), 6.49 (2H, d), 4.37 (8H, t), 4.26 (2H, s), 2.60 (4H, p).

[0169] HRMS (ESI) calcd for C.sub.26H.sub.24ClN.sub.2O [M].sup.+, 415.1577, found 415.1575.

Example 41-(9-(2-(Chloromethyl)phenyl)-6-(3-methoxyazetidin-1-yl)-3H-xanthen-3-ylidene)-3-methoxyazetidin-1-ium chloride

[0170] ##STR00023##

Synthesis of 3,6-bis(3-methoxyazetidin-1-yl)-3H-spiro[isobenzofuran-1,9-xanthene]

[0171] A mixture of (2-(3,6-dibromo-9H-xanthen-9-yl)phenyl)methanol (1.10 g, 2.47 mmol), Pd.sub.2(dba).sub.3 (0.226 g, 0.247 mmol), XPhos (0.321 g, 0.74 mmol) and Cs.sub.2CO.sub.3 (3.86 g, 11.8 mmol) in a round bottom flask was evacuated/backfilled with nitrogen (X3). To this mixture was added 3-methoxyazetidine hydrochloride (0.67 g, 5.42 mmol) and 1,4-dioxane (20 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. overnight. 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 (4 to 15% MeOH in DCM) to give the title compound as a grey solid (0.32 g, 28%).

[0172] .sup.1H NMR (CDCl.sub.3, 400 MHz) 7.41-7.34 (2H, m), 7.32-7.24 (2H, m), 6.94 (1H, d), 6.77 (2H, d), 6.25 (1H, d), 6.17 (2H, dd), 5.26 (2H, s), 4.39-4.31 (2H, m), 4.11 (4H, t), 3.79-3.71 (4H, m), 3.35 (6H, s).

[0173] LC/MS (ES+): m/z 457.2 (100%, [M+H].sup.+).

Synthesis of 1-(9-(2-(chloromethyl)phenyl)-6-(3-methoxyazetidin-1-yl)-3H-xanthen-3-ylidene)-3-methoxyazetidin-1-ium chloride

[0174] A stock solution of thionyl chloride (235 mg) in DCM (10 mL) was prepared. A portion (1.0 mL) of this stock solution was added dropwise to a solution of 3,6-bis(3-methoxyazetidin-1-yl)-3H-spiro[isobenzofuran-1,9-xanthene] (0.10 g, 0.219 mol) in DCM (10 mL). After 20 minutes, the reaction mixture was diluted with water and DCM and the layers were separated. The aqueous layer was extracted twice with DCM and the combined organic layers were dried over anhydrous MgSO.sub.4, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography (7 to 15% MeOH/DCM) followed by trituration with Et.sub.2O to give the title compound as a dark purple solid (0.086 g, 77%).

[0175] .sup.1H NMR (CDCl.sub.3, 400 MHz) 7.70-7.59 (2H, m), 7.58-7.51 (1H, m), 7.19 (1H, d), 7.09-7.03 (2H, m), 6.64-6.55 (4H, m), 4.67-4.58 (4H, m), 4.56-4.49 (2H, m), 4.25 (2H, s), 4.17-4.11 (4H, m), 3.38 (6H, s).

[0176] HRMS (ESI) calcd for C.sub.28H.sub.28ClN.sub.2O.sub.3 [M].sup.+, 475.1788, found 475.1791.

REFERENCES

[0177] 1) Grimm et al., General Synthetic Method for Si-Fluoresceins and Si-Rhodamines. ACS Cent Sci. 2017; 3(9):975-985. [0178] 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. [0179] 3) Poot et al.; Analysis of mitochondrial morphology and function with novel fixable fluorescent stains. J Histochem Cytochem. 1996; 4 4 (12): 1363-72. [0180] 4) Grimm et al., A general method to improve fluorophores for live-cell and single-molecule microscopy; Nat Methods. 2015 12(3): 244-50. [0181] 6) EP3 126451 [0182] 7) U.S. Pat. No. 5,686,261

[0183] 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.