STYRYL QUINOLINIUM, PROCESS FOR THEIR PREPARATION AND USE THEREOF AS FLUORESCENT PROBES FOR IMAGING

Abstract

The present invention is directed to styryl quinolinium compounds, a process for their synthesis and their use for selective nucleoli staining in cells, preferably in living cells and for imaging rRNA.

Description

TECHNOLOGICAL FIELD

The present invention concerns styryl quinolinium compounds, their preparation and use as fluorescent probes for imaging, by rapid and selective stain of nucleoli in both fixed and living cells.

BACKGROUND ART

References considered to be relevant as background to the presently disclosed subject matter are listed below:

• • Seiad, L. L.; Villemin, D.; Bar, N.; Hachemi, M., Solvent-Free condensation of methyl pyridinium and quinolinium salts with aldehydes catalyzed by DBU. Synth. Commun. 2012, 42 (5), 650-657.
  • Ramani, A.; Chanda, B.; Velu, S.; Sivasanker, S., One-pot synthesis of coumarins. Catalysis by the solid base, calcined Mg—Al hydrotalcite. Green Chem 1999, 1 (3), 163-165.
  • Li, Y.; Xu, D.; Ho, S.-L.; Li, H.-W.; Yang, R.; Wong, M. S., A theranostic agent for in vivo near-infrared imaging of β-amyloid species and inhibition of β-amyloid aggregation. Biomaterials 2016, 94, 84-92.
  • Teshome, A.; Bhuiyan, M. D. H.; Gainsford, G. J.; Ashraf, M.; Asselberghs, I.; Williams, G. V.; Kay, A. J.; Clays, K., Synthesis, linear and quadratic nonlinear optical properties of ionic indoline and N, N-dimethylaniline based chromophores. Opt. Mater 2011, 33 (3), 336-345.
  • Ashwell, G. J.; Mohib, A., Improved molecular rectification from self-assembled monolayers of a sterically hindered dye. J. Am. Chem. Soc 2005, 127 (46), 16238-16244.
  • Dondoni, A.; Massi, A.; Minghini, E.; Bertolasi, V., Dihydropyridine C-Glycoconjugates by Hantzsch Cyclocondensation. Synthesis of a C (6)-Glycosylated Nifedipine Analogue. Helv. Chim. Acta 2002, 85 (10), 3331-3348.
  • Saunders, J., Top Drugs: Top Synthetic Routes. Oxford Chemistry Primers 2002, 90 (1).
  • Taylor Jr, R.; Markley, E.; Ellenbogen, L., The inhibition of monoamine oxidase by styrylquinoliniums. Biochem. Pharmacol. 1967, 16 (1), 79-86.
  • Kumar, R.; Lingstram, B.; Darreh-Shori, T., Novel ligands of Choline Acetyltransferase designed by in silico molecular docking, hologram QSAR and lead optimization. Sci Rep 2016, 6, 31247.
  • Fortuna, C. G.; Barresi, V.; Musso, N.; Musumarra, G., Synthesis and applications of new trans 1-indolyl-2-(1-methyl pyridinium and quinolinium-2-yl) ethylenes. Arkivoc 2009, 8, 222-229.
  • Jones G. The knoevengel Condensation. Org React 2004; 15; 204-599.

Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.

BACKGROUND

Dyes emitting at long wavelengths, green to red regions have are used in various applications, including visualization of biomolecules in living systems, real-time tracking of cellular events, and fluorescent dye guidance during surgeries. These applications are based on the minimal auto-fluorescence of cells at wavelengths above 600 nm, the penetration depths of NIR-dyes, as well as their sensitivity and minimal photo-damage on cells and tissues. Styryl quinolinium- and merocyanine-dye classes constitute scaffolds that are applied for the development of NIR dyes for the above applications of visualization of biomolecules, tracking cellular events and guidance during surgery. Other uses of styryl-quinolinium derivatives are used in therapeutic treatment of various conditions. Specific 1-methyl-4-(substituted) styryl-quinolinium analogs are used for treatment of tuberculosis, other styryl quinolinium derivatives are used as choline acetyltransferase inhibitors or in antitumor activity. Specifically, a styryl quinolinium dye contains two non-identical aryl groups usually joined by an ethylene moiety. One of the aryl groups is electron deficient (e.g., pyridinium moiety) while the other one is electron rich, thus resulting in a push-pull system. Unlike the positively charged styryl quinolinium dyes, merocyanine dyes are zwitterions and hence are essentially neutral. Merocyanine push-pull dyes contain two ethylene-bridged aryl groups, one of which is positively charged (e.g., pyridinium moiety) and the other one is negatively charged (e.g., phenolate moiety). In particular, merocyanine dyes are used for detection of DNA or RNA since they often exhibit enhanced fluorescence intensity by binding to nucleic acids.

Various dyes have been reported for selective nucleoli staining, however, there remain a demand for new fluorescent dyes that are photochemically stable easily synthesized and purified and stain the nucleoli rapidly.

General Description

The present invention is based on the findings of a new family of styryl quinolinium compounds, an improved process for their facile and high yield synthesis and their use for selective nucleoli staining in cells, preferably in living cells and for imaging rRNA.

In a first aspect, the present invention is directed to a compound of formula (I):

wherein:R₁, R₂, R₃, R₄, R₅ are independently selected from H, F, Cl, Br, I, OR, NHR, NR₂, CN,R′SO₃, (R′)₄N, (R′)₃NH, NO₂.R is independently selected from H or straight or branched C_1-6alkyl;R′ is straight or branched C_1-6 alkyl; and

X⁻ is Cl, Br, I;

provided that:in case R₃ is Cl, then one of R₁, R₂, R₄, R₅ is not H;in case R₃ is N(CH₃)₂, then one of R₁, R₂, R₄, R₅ is not H;in case R₂ and R₃ are both OCH₃, then one of R₁, R₄, R₅ is not H.

More particularly, R₁ is H, F, Cl, or OR, R₂ is H, F, Cl, OR, NO₂ or CN, R₃ is H, OR, NHR or NR₂, R₄ is H, F, Cl, CN, NO₂, R₅ is H, F, Cl; R being H, or straight or branched C_1-6alkyl and X is I.

In a further aspect the present invention is directed to a process for the synthesis of a compound of formula (I) by reacting compound of formula (II) with a compound of formula (III) in the presence of a base in an appropriate solvent under heating:

In particular, the base is a pyridine or piperidine, the alcohol is ethanol and heating is done by microwave irradiation at a temperature of 70-80° C.

Yet in a further aspect the present invention is directed to the use of the styryl quinolinium compounds of the present invention for staining cells, particularly living cells and fluorescent probes for imaging, particularly rRNA.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

FIG. 1 provides evidence for variance of color of the solution solubilizing a compound of the present invention (I-e) depending on the solvent and its polarity. The solvents are (left to right): acetic acid, water, methanol, ethanol, isopropanol, DMSO, DMF, acetone and dichloromethane.

FIG. 2 demonstrates the photostability of dyes I-a-I-l measured by the loss of fluorescence intensity (2 μM dye, in PBS buffer).

FIG. 3 provides emission spectra of compound I-e with RNA in water. Compound I-e alone (black), RNA solution+compound I-e (blue), compound I-e+RNA solution+RNASE, control, (green), water+RNASE, control, (red).

FIGS. 4A-D: provides spectrum scan showing peak of excitation/emission.

FIG. 4A; compound I-e was added to HeLa cells and these were scanned as described in the Methods section. The strongest excitation, at the emission range of 647-667 nm was found to be 570 nm. Bar=10 μm.

FIG. 4B; images were analyzed as described in the Methods and the image emission intensities were plotted. The excitation that resulted in the strongest emission was found to be at 570 nm.

FIG. 4C; 2D heat map exported from acquisition software showing the excitation vs. the emission profiles.

FIG. 4D; compound I-e localizes to nucleoli in treated cells. Nucleoli are identified by staining with an antibody to the nucleolar marker fibrillarin (green). Hoechst DNA stain is in cyan. No 12e signal is detected in untreated cells. Bar=10 μm.

FIGS. 5A-C: Compound I-e rapidly stains living HeLa cells and does not affect viability.

FIG. 5A; Cells were imaged every 5 sec for 5 min. The compound entered the cells within 30 sec and stained nucleoli within 150 sec.

FIG. 5B; Cells were then imaged every 15 min for 26 hours and the compound intensity remained strong and specific to the nucleoli. The cells remained viable. Bar=20 μm.

FIG. 5C; Signal-to-noise ratio pixel intensity graph of nucleoli/nucleus and nucleoli/outside of the cell derived from ImageJ software.

FIGS. 6A-B: Compound I-e binds to RNA in cells.

FIG. 6A; Wide-field fluorescence images of untreated or DNase treated cells stained with the compound I-e. Hoechst DNA stain is in cyan.

FIG. 6B; Untreated cells or cells treated with RNase or with the actinomycin D transcriptional inhibitor. DIC images are in grey. Bar=10 μm.

FIGS. 7A-B: demonstrates the binding of compound I-e to rRNA in cells.

FIG. 7A; transcriptional inhibition of RNA polymerase I by CX-5461 and BMH-21 diminishes the fluorescence signal of compound I-e in cells.

FIG. 7B; stress granules formed by arsenite treatment and labeled with anti-G3BP1 (green) do not contain compound I-e (white). Hoechst DNA stain is in cyan. DIC is in grey. Bar=10 μm.

FIG. 8. Provides evidence that compound I-e binds to secondary structures in rRNA. The binding of compound I-e to rRNA and mRNA in solution at room temperature and at 90° C. In the presence of structured rRNA there is an increase in the fluorescent signal; however, when the samples were heated and the secondary structures of the rRNA are dismantled, then no increase in fluorescence was observed.

FIG. 9. Provides fluorescence spectra evidencing the binding of compound I-e to DNA without histones

DETAILED DESCRIPTION OF EMBODIMENTS

The present invention is directed to styryl quinolinium compounds of formula (I), their synthesis and use. The unique process of synthesis by employing microwave irradiation provides a much higher yield than refluxing the solvent and a shorter duration of the reaction.

Straight or branched alkyl groups according to the present application are straight or branched —CH₃, —C₂H₅, C₃H₇, C₄H₉, C₅H₁₁, C₆H₁₃ groups.

Generally, the synthesis of the styryl quinolinium compounds of formula (I), was conducted by the following path (Knoevenagel condensation):

Where heating varied from 5-40 minutes in a microwave at a temperature of 65° C.-85° C. or for 5-20 hours under reflux. The former process is superior by its yield and duration. The base may be selected from pyridine, piperidine, the C_1-4alcohol is selected from methanol, ethanol, propanol, isopropanol, butanol, sec-butyl alcohol, tert-butyl alcohol.

More particularly, the reaction was carried in the presence of piperidine in ethanol in a microwave as follows:

In particular, specific compounds of formula (I) are:

• (E)-2-(4-hydroxystyryl)-1,4-dimethylpyridinium iodide (I-a);
• (E)-2-(4-Methoxystyryl)-1,4-dimethylpyridinium iodide (I-b);
• (E)-2-(3-Flouro-4-hydroxystyryl)-1,4-dimethylpyridinium iodide (I-c);
• (E)-2-(3-Flouro-4-methoxystyryl)-1,4-dimethylpyridinium iodide (I-d);
• (E)-2-(3, 5-Difluoro-4-hydroxystyryl)-1,4-dimethylpyridinium iodide (I-e);
• (E)-2-(3, 5-Difluoro-4-methoxystyryl)-1,4-dimethylpyridinium iodide (I-f);
• (E)-2-(2,3,5,6-Tetrafluoro-4-hydroxystyryl)-1,4-dimethylpyridinium iodide (I-g);
• (E)-2-(2,3,5,6-Tetrafluoro-4-methoxystyryl)-1,4-dimethylpyridinium iodide (I-h);
• (E)-2-(4-Dimethylaminostyryl)-1,4-dimethylpyridinium iodide (I-i);
• (E)-2-(3,4-Dihydroxystyryl)-1,4-dimethylpyridinium iodide (I-j);
• (E)-2-(2-Hydroxy-3-methoxy-5-nitrostyryl)-1,4-dimethylpyridinium iodide (I-k); and
• (E)-2-(4-Hydroxy-3-nitrostyryl)-1,4-dimethylpyridinium iodide (I-l).

The compounds, depending on their substituents vary in their properties such as their solubility, absorbance wavelength, quantum yield and photostability. These properties vary depending on the nature of the solvent as well. The styryl quinolinium compounds of formula (I), were found to be nontoxic to leaving cells and therefore may be used for staining cells, particularly living cells and fluorescent probes for imaging, particularly rRNA. The compounds of the present invention display unique and specific properties when stain and dye living cells. The compounds clearly show preference to binding to secondary structures in the rRNA, wherein under heating when the secondary structure disappears, binding is lost.

The invention will now be described with reference to the following non-limiting Examples and drawings.

Example 1: Chemical Synthesis

A mixture of compounds (II) and (III) was irradiated at a temperature of about 65° C.-85° C. To a 10 mL microwave reaction vessel equipped with a magnetic stirring bar were added 1,4-dimethyl quinolinium iodide ((II); 0.5 mmol), absolute ethanol (5 mL), piperidine (cat. amount), and a substituted benzaldehyde ((III); 0.5 mmol). Thus 3,5-Difluoro-4-hydroxybenzaldehyde, or 2,3,5,6-Tetrafluoro-4-hydroxybenzaldehyde (whose ¹H NMR, ¹³C NMR, ¹⁹F NMR and High Resolution Mass Spectrum (HRMS) are given below are exemplified. The vessel was sealed, and the mixture was irradiated in a microwave oven (CEM Focused Microwave type Discover) for 5 to 30 min at 70-80° C. (as indicated in Table 1). Then the reaction mixture was cooled to RT, and diethyl ether (10 mL) was added to precipitate the products as black crystals. The latter were vacuum-filtered and washed 3×3 mL with diethyl ether to yield pure products. In a similar manner all compounds I-a-I-l were isolated in a high yield, >95% purity e as determined by ¹H NMR, ¹³C NMR, ¹⁹F NMR, DEPT, UV and HRMS.

Following are the ¹H NMR, ¹³C NMR and/or ¹⁹F NMR and HRMS of compounds of two compounds of formula (III) and of the I-a-I-l styryl quinolinium compounds.

3,5-Difluoro-4-hydroxybenzaldehyde

¹H-NMR (400 MHz, CDCl₃): 9.82 (t, J=1.8 Hz, 1H), 7.49 (d, J=6.4 Hz, 2H). ¹³C NMR (ppm): 190.93, 152.32, 137.15, 132.74, 111.53. ¹⁹F NMR (376 MHz, CDCl₃) δ −135.14 ppm. HRMS Calcd for C₇H₄F₂O₂ m/z 158.03035, Found 158.03024.

2,3,5,6-Tetrafluoro-4-hydroxybenzaldehyde

¹H NMR (DMSO, 400 MHz): δ 10.22 (s, 1H), 3.36 (bs, 1H). ¹³C NMR (DMSO 75.45 MHz): δ 91.5, 98.7, 136.1, 139.5, 143.2, 146.5, 182.3. Calcd for C₇H₂F₄O₂ m/z 194.09183, Found 193.09189.

(E)-2-(4-hydroxystyryl)-1,4-dimethylpyridinium Iodide (I-a)

¹H-NMR (400 MHz, DMSO (d6)): 8.97 (d, 5.89 Hz, 1H, Ar), 8.68 (d, 8.5 Hz, 1H, Ar), 7.93 (d, 16.1 Hz, 1H, CH═CH), 7.91 (d, 16.1 Hz, 1H, CH═CH), 7.8 (d, 7.8 Hz, 1H, Ar), 7.66 (dd, 8.5, 7.8 Hz, 1H, Ar), 7.57 (d, 5.8 Hz, 1H, Ar), 7.31 (d, 8.72 Hz, 2H, Ar), 7.16 (dd, 8.5, 7.8 Hz, 1H, Ar), 6.95 (d, 8.72 Hz, 2H, Ar), 4.31 (s, 3H, +NCH₃). ¹³C NMR (ppm): 157.71, 157.32, 142.85, 132.54, 130.83, 130.52, 129.98, 129.54, 128.14, 126.91, 126.71, 125.02, 123.82, 119.87, 116.16, 115.95, 46.92. HRMS Calcd for C₁₈H₁₆NOI m/z 389.23025, Found 389.23024.

(E)-2-(4-Methoxystyryl)-1,4-dimethylpyridinium Iodide (I-b)

¹H-NMR (400 MHz, DMSO (d6)): 8.96 (d, 5.89 Hz, 1H, Ar), 8.68 (d, 8.5 Hz, 1H, Ar), 7.93 (d, 16.1 Hz, 1H, CH═CH), 7.91 (d, 16.1 Hz, 1H, CH═CH), 7.8 (d, 7.8 Hz, 1H, Ar), 7.66 (dd, 8.5, 7.8 Hz, 1H, Ar), 7.57 (d, 5.8 Hz, 1H, Ar), 7.31 (d, 8.72 Hz, 2H, Ar), 7.16 (dd, 8.5, 7.8 Hz, 1H, Ar), 6.95 (d, 8.72 Hz, 2H, Ar), 4.31 (s, 3H, +NCH₃), 3.92 (s, 3H, OCH₃). ¹³C NMR (ppm): 157.71, 157.32, 142.85, 132.54, 130.83, 130.52, 129.98, 129.54, 128.14, 126.91, 126.71, 125.02, 123.82, 119.87, 116.16, 115.95, 55.52, 46.92. HRMS Calcd for C₁₉H₁₈NOI m/z 403.48018, Found 403.48026.

(E)-2-(3-Flouro-4-hydroxystyryl)-1,4-dimethylpyridinium Iodide (I-c)

¹H-NMR (400 MHz, DMSO (d6)): 9.06 (d, 5.89 Hz, 1H, Ar), 8.68 (d, 8.5 Hz, 1H, Ar), 8.43 (d, 16.1 Hz, 1H, CH═CH), 7.91 (d, 16.1 Hz, 1H, CH═CH), 7.8 (d, 7.8 Hz, 1H, Ar), 7.66 (dd, 8.5, 7.8 Hz, 1H, Ar), 7.57 (d, 5.8 Hz, 1H, Ar), 7.31 (d, 8.72 Hz, 2H, Ar), 7.16 (dd, 8.5, 7.8 Hz, 1H, Ar), 6.95 (d, 8.72 Hz, 1H, Ar), 4.31 (s, 3H, +NCH₃). ¹³C NMR (ppm): 157.81, 153.33, 147.45, 146.94, 139.83, 136.62, 133.44, 129.98, 126.14, 125.14, 124.91, 123.76, 123.09, 122.12, 118.67, 117.46, 112.65, 45.32. ¹⁹F NMR −130.94 ppm. HRMS Calcd for C₁₈H₁₅FNOI m/z 407.22431, Found 407.21909.

(E)-2-(3-Flouro-4-methoxystyryl)-1,4-dimethylpyridinium Iodide (I-d)

¹H-NMR (400 MHz, DMSO (d6)): 9.06 (d, 5.89 Hz, 1H, Ar), 8.68 (d, 8.5 Hz, 1H, Ar), 8.43 (d, 16.1 Hz, 1H, CH═CH), 7.91 (d, 16.1 Hz, 1H, CH═CH), 7.8 (d, 7.8 Hz, 1H, Ar), 7.66 (dd, 8.5, 7.8 Hz, 1H, Ar), 7.57 (d, 5.8 Hz, 1H, Ar), 7.31 (d, 8.72 Hz, 2H, Ar), 7.16 (dd, 8.5, 7.8 Hz, 1H, Ar), 6.95 (d, 8.72 Hz, 1H, Ar), 4.31 (s, 3H, +NCH₃), 3.82 (s, 3H, OCH₃). ¹³C NMR (ppm): 157.81, 153.33, 147.45, 146.94, 139.83, 136.62, 133.44, 129.98, 126.14, 125.14, 124.91, 123.76, 123.09, 122.12, 118.67, 117.46, 112.65, 55.23, 45.32. ¹⁹F NMR −129.83 ppm. HRMS Calcd for C₁₉H₁₇FNOI m/z 421.49363, Found 421.51019.

(E)-2-(3, 5-Difluoro-4-hydroxystyryl)-1,4-dimethylpyridinium Iodide (I-e)

¹H-NMR (400 MHz, DMSO (d6)): 9.02 (d, 5.8 Hz, 1H, Ar), 8.68 (d, 8.5 Hz, 1H, Ar), 7.93 (d, 16.1 Hz, 1H, CH═CH), 7.91 (d, 16.1 Hz, 1H, CH═CH), 7.8 (d, 7.8 Hz, 1H, Ar), 7.66 (dd, 8.5, 7.8 Hz, 1H, Ar), 7.57 (d, 5.8 Hz, 1H, Ar), 7.31 (d, 8.72 Hz, 2H, Ar), 7.16 (dd, 8.5, 7.8 Hz, 1H, Ar), 4.31 (s, 3H, +NCH₃). ¹³C NMR (ppm): 157.81, 153.33, 147.45, 146.94, 139.83, 136.62, 133.44, 129.98, 126.14, 125.14, 124.91, 123.76, 123.09, 122.12, 118.67, 117.46, 112.65, 45.32. ¹⁹F NMR −133.13 ppm. HRMS Calcd for C₁₈H₁₄F₂NOI m/z 425.22031, Found 425.21901.

(E)-2-(3, 5-Difluoro-4-methoxystyryl)-1,4-dimethylpyridinium Iodide (I-f)

¹H-NMR (400 MHz, DMSO (d6)): 9.02 (d, 5.8 Hz, 1H, Ar), 8.68 (d, 8.5 Hz, 1H, Ar), 7.93 (d, 16.1 Hz, 1H, CH═CH), 7.91 (d, 16.1 Hz, 1H, CH═CH), 7.8 (d, 7.8 Hz, 1H, Ar), 7.66 (dd, 8.5, 7.8 Hz, 1H, Ar), 7.57 (d, 5.8 Hz, 1H, Ar), 7.31 (d, 8.72 Hz, 2H, Ar), 7.16 (dd, 8.5, 7.8 Hz, 1H, Ar), 4.31 (s, 3H, ⁺NCH₃), 3.83 (s, 3H, OCH₃). ¹³C NMR (ppm): 157.81, 153.33, 147.45, 146.94, 139.83, 136.62, 133.44, 129.98, 126.14, 125.14, 124.91, 123.76, 123.09, 122.12, 118.67, 117.46, 112.65, 62.35, 45.32. ¹⁹F NMR −133.13 ppm. HRMS Calcd for C₁₉H₁₆F₂NOI m/z 439.14021, Found 439.15001.

(E)-2-(2,3,5,6-Tetrafluoro-4-hydroxystyryl)-1,4-dimethylpyridinium Iodide (12g)

¹H-NMR (400 MHz, DMSO (d6)): 9.02 (d, 5.8 Hz, 1H, Ar), 8.63 (d, 7.8 Hz, 1H, Ar), 8.41 (d, 5.4 Hz, 1H, Ar), 8.23 (dd, 7.8, 5.4 Hz, 1H, Ar), 8.11 (dd, 8.5, 7.8 Hz, 1H, Ar), 7.84 (d, 5.8 Hz, 1H, Ar), 7.22 (d, 16.1 Hz, 1H, CH═CH), 6.78 (d, 16.1 Hz, 1H, CH═CH), 4.31 (s, 3H, ⁺NCH₃). ¹³C NMR (ppm): 157.81, 153.33, 147.45, 146.94, 139.83, 136.62, 133.44, 129.98, 126.14, 125.14, 124.91, 123.76, 123.09, 122.12, 118.67, 117.46, 112.65, 45.32. ¹⁹F NMR −156.27, −133.13 ppm. HRMS Calcd for C₁₈H₁₂F₄NOI m/z 461.77062, Found 461.77059.

(E)-2-(2,3,5,6-Tetrafluoro-4-methoxystyryl)-1,4-dimethylpyridinium Iodide (12-h)

The dye was not sufficiently soluble in any solvent in order to perform NMR tests. HRMS Calcd for C₁₉H₁₄F₄NOI m/z 475.26041, Found 475.26039.

(E)-2-(4-Dimethylaminostyryl)-1,4-dimethylpyridinium Iodide (I-i)

¹H-NMR (400 MHz, DMSO (d6)): 9.11 (d, 5.89 Hz, 1H, Ar), 8.68 (d, 8.5 Hz, 1H, Ar), 7.93 (d, 16.1 Hz, 1H, CH═CH), 7.91 (d, 16.1 Hz, 1H, CH═CH), 7.8 (d, 7.8 Hz, 1H, Ar), 7.66 (dd, 8.5, 7.8 Hz, 1H, Ar), 7.57 (d, 5.8 Hz, 1H, Ar), 7.31 (d, 8.72 Hz, 2H, Ar), 7.16 (dd, 8.5, 7.8 Hz, 1H, Ar), 6.95 (d, 8.72 Hz, 2H, Ar), 4.42 (s, 3H, +NCH₃), 3.01 (s, 6H, N(CH₃)₂). 157.71, 157.32, 142.85, 132.54, 130.83, 130.52, 129.98, 129.54, 128.14, 126.91, 126.71, 125.02, 123.82, 119.87, 116.16, 115.95, 46.92, 41.39. HRMS Calcd for C₂₀H₂₁N₂I m/z 416.71421, Found 416.71409.

(E)-2-(3,4-Dihydroxystyryl)-1,4-dimethylpyridinium Iodide (I-j)

¹H-NMR (400 MHz, DMSO (d6)): 9.02 (d, 5.89 Hz, 1H, Ar), 8.68 (d, 8.5 Hz, 1H, Ar), 7.93 (d, 16.1 Hz, 1H, CH═CH), 7.91 (d, 16.1 Hz, 1H, CH═CH), 7.8 (d, 7.8 Hz, 1H, Ar), 7.57 (d, 5.8 Hz, 1H, Ar), 7.29 (s, 1H, Ar), 7.17 (s, 1H, Ar), 7.16 (dd, 8.5, 7.8 Hz, 1H, Ar), 6.95 (d, 8.72 Hz, 2H, Ar), 6.64 (s, 1H, Ar), 4.31 (s, 3H, +NCH₃). ¹³C NMR (ppm): 160.6, 157.42, 146.32, 140.05, 139.51, 136.83, 133.44, 126.08, 125.84, 125.61, 124.91, 124.03, 123.99, 123.82, 122.87, 119.35, 118.95, 111.77, 46.02. HRMS Calcd for C₁₈H₁₅BrNOI m/z 405.13662, Found 405.13648.

(E)-2-(2-Hydroxy-3-methoxy-5-nitrostyryl)-1,4-dimethylpyridinium Iodide (I-k)

The dye was not sufficiently soluble in any solvent in order to perform NMR tests. HRMS Calcd for C₁₉H₁₇N₂O₄I m/z 464.26553, Found 464.25919.

(E)-2-(4-Hydroxy-3-nitrostyryl)-1,4-dimethylpyridinium Iodide (I-l)

¹H-NMR (400 MHz, DMSO (d6)): 9.06 (d, 5.89 Hz, 1H, Ar), 8.68 (d, 8.5 Hz, 1H, Ar), 8.43 (d, 16.1 Hz, 1H, CH═CH), 7.91 (d, 16.1 Hz, 1H, CH═CH), 7.8 (d, 7.8 Hz, 1H, Ar), 7.66 (dd, 8.5, 7.8 Hz, 1H, Ar), 7.57 (d, 5.8 Hz, 1H, Ar), 7.31 (d, 8.72 Hz, 2H, Ar), 7.16 (dd, 8.5, 7.8 Hz, 1H, Ar), 6.95 (d, 8.72 Hz, 1H, Ar), 4.31 (s, 3H, +NCH₃). ¹³C NMR (ppm): 157.81, 153.33, 147.45, 146.94, 139.83, 136.62, 133.44, 129.98, 126.14, 125.14, 124.91, 123.76, 123.09, 122.12, 118.67, 117.46, 112.65, 45.32. HRMS Calcd for C₁₈H₁₅N₂O₃I m/z 434.02911, Found 434.028922.

The process of preparation under microwave irradiation according to the present invention is superior to carrying the reaction of compounds (II) and (III) in the presence of a base and refluxing in a suitable alcohol (prior art) as apparent in Table 1. Yield is higher (90-98% compared to 27-85%) and reaction time shortened significantly to 5-30 minutes compared to 9-20 hours.

TABLE 1
Methos of synthesis-present invention (microwave) and
conventional reflux of solvent
		Microwave			Normal
		condition			condition
		in EtOH			in EtOH
		Reaction	Temper-		Reaction	Temper-
	Yield	time	ature	Yield	time	ature
Product	(%)	(min)	(° C.)	(%)	(h)	(° C.)
I-a	94	15	70	65	9	Reflux
I-b	96	25	80	53	6	Reflux
I-c	91	10	75	27	12	Reflux
I-d	95	15	75	55	12	Reflux
I-e	99	5	75	85	9	Reflux
I-f	90	10	75	73	14	Reflux
I-g	99	5	75	45	10	Reflux
I-h	95	15	80	37	16	Reflux
I-i	98	5	80	81	8	Reflux
I-j	94	20	80	64	12	Reflux
I-k	91	30	80	60	18	Reflux
I-l	90	30	80	59	20	Reflux

Physical Properties

Example 2: Solubility in Various Polar Solvents

The compounds (I-a-I-l) are soluble in nonpolar organic solvents only to a rather limited extent, while they are readily soluble in polar organic solvents. Some are readily soluble in water. In polar solvent such as water, the zwitterion form of the compounds dominates, while in nonpolar solvents such as chloroform, the neutral form prevails. More particularly, styryl quinolinium compounds having a hydroxyl group at the para position of the styryl moiety, are soluble in a wide range of solvents giving colored solutions, the color of which is solvent-dependent. Turning to FIG. 1, color change of the solution depending on the polarity of the solvent dissolving compound I-e is presented where compound I-e is dissolved in (from left to right): acetic acid, water, methanol, ethanol, isopropanol, DMSO, DMF, acetone and dichloromethane.

Example 3: Absorption Wavelength

The styryl quinolinium compounds of formula (I) are characterized by λ_abs, of 440-658 nm; λ_em, of 485-715 nm; and ϵ− of 12,200-49,000 M⁻¹cm⁻¹ at polar solvents having polarity values between 1 to 0.355. The more polar the solvent is, the shorter is the absorption wavelength of the dye. Table 2 provides photophysical properties of the compounds (I-a-I-l) in various solvents at a range of relative polarity values; between 1 (water) to 0.355 (acetone).

TABLE 2
Photophysical properties of derivatives I-a-I-l.
	λAbs		ϵem		λAbs	ϵ	λem
Dye	(nm)	(M−1cm−1)	(nm)	Dye	(nm)	(M−1cm−1)	(nm)
Water				MeOH
I-a	460	21200	515	12a	475	22100	525
I-b	438	20700	501	12b	440	19900	490
I-c	508	26300	560	12c	560	27200	610
I-d	502	24900	555	12d	508	24900	600
I-e	520	45800	605	12e	535	46100	670
I-f	515	41200	580	12f	520	40900	648
I-g	503	23300	518	12g	505	23800	591
I-h	478	22900	508	12h	490	22900	580
I-i	510	29800	575	12i	530	30300	665
I-j	470	19200	503	12j	478	18600	500
I-k	440	16400	495	12k	445	17400	495
I-l	475	13300	499	12l	480	12200	500
EtOH				DMSO
I-a	480	21900	525	I-a	490	23500	528
I-b	450	21300	495	I-b	465	20600	501
I-c	565	27100	640	I-c	566	25700	660
I-d	510	28200	605	I-d	520	23600	610
I-e	556	49000	685	I-e	600	44700	715
I-f	548	41200	665	I-f	591	42100	675
I-g	509	23900	625	I-g	499	22950	603
I-h	506	22700	608	I-h	500	20900	595
I-i	544	31000	675	I-i	590	30100	703
I-j	480	17900	495	I-j	480	18400	500
I-k	460	16900	495	I-k	450	17900	501
I-l	485	13900	506	I-l	485	12200	509
DMF				Aceton
I-a	490	21500	520	I-a	90	19900	520
I-b	460	21100	500	I-b	55	18100	505
I-c	555	28100	660	I-c	10	27600	628
I-d	519	25000	610	I-d	90	26300	605
I-e	599	44900	715	I-e	50	47100	702
I-f	590	41100	675	I-f	90	44500	685
I-g	495	22100	603	I-g	00	23200	590
I-h	500	23400	595	I-h	05	22900	590
I-i	588	29800	705	I-i	80	28300	695
I-j	482	19200	503	I-j	60	18700	495
I-k	450	16400	500	I-k	90	15800	515
I-l	485	11100	505	I-l	80	12200	508

Example 4: pH Dependence of Absorption and Emission

The absorption and emission wavelengths of the styryl quinolinium compounds of formula (I) containing OH/NMe₂ groups in the para position of the styryl moiety is pH dependent. A red-shift of the wavelengths of absorption and emission is obtained in basic vs. acidic medium. The styryl quinolinium compounds of formula (I) are further characterized by quantum yield that is dependent on the viscosity of the solvent. The more polar the solvent is, the shorter is the absorption wavelength of the dye. Table 3 provides values of absorption and emission wavelengths of compounds I-a-I-l that as evident depend on the pH. A red-shift of the wavelengths of absorption and emission was obtained in basic vs. acidic medium

TABLE 3
The absorption and emission wavelengths of compounds I-a-I-l
depending on the pH.
	λabs (nm)	λabs (nm)	λem (nm)	λem (nm)
Dye	pH 2	pH 10	pH 2	pH 10
I-a	460	467	515	522
I-b	438	438	501	500
I-c	508	512	560	564
I-d	502	502	555	556
I-e	520	531	605	608
I-f	515	515	580	580
I-g	503	501	518	520
I-h	478	478	508	508
I-i	510	508	575	578
I-j	470	475	503	500
I-k	440	447	495	499
I-l	475	477	499	502

Example 5: Photostability of Compounds

Turning to FIG. 2, the photostability of compounds of the present invention is provided by demonstrating the measured loss of fluorescence intensity of dyes I-a-I-l. FIG. 2 clearly demonstrates that the styryl quinolinium compounds of formula (I) are photostable, for up to 200 minutes of irradiation (96% remaining). The photostability of dyes I-a-I-l was tested by exposure to a light source of a Cary Eclipse Fluorescence Spectrophotometer (λ_ex 500-650 nm), with the exclusion of ambient light. 2 μM of the Compound in phosphate buffer solution were irradiated for 200 min, with an assessment of their fluorescence intensity at five-minute intervals.

Results after a 200-minute irradiation in PBS buffer as provided in FIG. 2 demonstrate in a clear manner that the fluorescence intensity of dyes I-a-I-l was not affected by the irradiation. The remaining emission percentages of the dyes after 200 min were: I-a (89%), I-b (93%), I-c (91%), I-d (88%), I-e (96%), I-f (92%), I-g (95%), I-h (93%), I-i (95%), I-j (88%), I-k (94%) and I-l (87%).

Example 6: Quantum Yield

The quantum yields of compounds of formula (I) of the present invention were found to vary. The fluorescence quantum yields of the compounds (I) were determined relative to rhodamine B in ethanol at 25° C. The quantum yield was calculated according to the following equation:

Φ_F=Φ_RI/I_R*OD_R/OD*η²/η_R

Here, Φ and Φ_R are the fluorescence quantum yield of the sample and the reference, respectively, I and I_R are areas under the fluorescence spectra of the sample and of the reference, respectively, OD and OD_R are the absorption values of the sample and the reference at the excitation wavelength, and η and η_R are the refractive index for the respective solvents used for the sample and the reference. The photostability of all the dyes was tested by exposure to a light source of a Cary Eclipse Fluorescence Spectrophotometer, with the exclusion of ambient light. 2 μM Dye solutions in PBS were irradiated for 200 min, with an assessment of their fluorescence intensity at five-minute intervals.

In particular, the quantum yield of compounds I-a-I-l was found to depend on the viscosity of the solvent. The more polar the solvent is, the shorter is the absorption wavelength of the compound. An example of the quantum yields of the compounds I-a-I-l that were measured in water and glycerol is shown in the Table 4 below:

TABLE 4
Quantum yield in water and glycerol
Compound	Φa (water) %)	Φa (glycerol) (%)
I-a	0.69	2.13
I-b	0.61	1.74
I-c	0.94	2.84
I-d	0.87	3.47
I-e	1.77	5.55
I-f	1.61	4.89
I-g	0.62	1.68
I-h	0.48	1.09
I-i	1.07	3.74
I-j	0.79	2.91
I-k	0.11	0.31
I-l	0.28	0.92

The toxicity of the compounds on T lymphocytes was evaluated by flow cytometry. Cell death was measured by fluorescence-activated cell sorting (FACS), which is a powerful and precise tool for measurement of a single cell within a population. The cell death was checked before adding the compounds and 3 hours after the addition of the dyes. Compounds I-c, I-i and I-e were not toxic for a period of 3 hours.

The styryl quinolinium compounds of formula (I) stain the nucleoli and the cytoplasm of fixed and living cells. Preferably, the compounds bind to rRNA in the nucleolus and the cytoplasm. In some embodiments, does not stain nuclear dsDNA and binds histones-free DNA extract. The styryl quinolinium compounds of formula (I) may stain the nucleoli rapidly, in about 150 seconds.

Staining and Probing of Cells.

General

Cell Culture

Human U2OS cells were cultured under standard conditions at 37° C., 5% CO₂, in low glucose Dulbecco's modified Eagle's medium (DMEM, Biological Industries, Israel) containing 10% fetal bovine serum (FBS, HyClone), and 4 mM Glutamine, 100 IU/mL Penicillin, and 100 μg/mL Streptomycin (Biological Industries). HeLa, HEK293, and mouse embryonic fibroblasts (MEF) were maintained in high glucose DMEM containing 10% FBS, and 100 IU/mL Penicillin, and 100 μg/mL Streptomycin. For RNase digestion, cells were treated with 5 μg/ml actinomycin D (Sigma) for 3 hrs, then fixed in ice cold methanol for 2 min, and digested with RNase (100 mg/ml in PBS with 3 mM MgCl₂, Sigma) for 45 min at room temperature. For DNase treatment, cells were first fixed in ice cold methanol for 2 min, and then incubated (100 mg/ml, 5 mM MgCl₂) for 2 hrs at room temperature. Nuclei were counterstained with 1 μM Hoechst 33342 (Sigma) and coverslips were mounted in mounting medium.

Immunofluorescence

Cells were grown on coverslips, washed with PBS and fixed for 20 min in 4% PFA. Cells were then permeabilized in 0.5% Triton X-100 for 2.5 min. Cells were washed twice with PBS and blocked with 5% BSA for 20 min, and immunostained for 1 hr with a primary antibody. After three washes with PBS, the cells were incubated for 1 hr with secondary fluorescent antibodies. Primary antibodies: mouse anti-G3BP1 (Abcam) and rabbit anti-fibrillarin (Abcam). Secondary antibodies: Alexa Fluor 488 goat anti-mouse (Abcam) Alexa Flour 488 goat anti-rabbit (Abcam). Cells were then stained with compound I-e (10 μg/mL). Nuclei were counterstained with 1 μM Hoechst 33342 (Sigma) and coverslips were mounted in mounting medium.

Fluorescence Microscopy

For spectral scanning, an inverted Leica SP8 scanning confocal microscope, driven by the LASX software (Leica Microsystems, Mannheim, Germany) and equipped with a super-continuum white light laser, was used. A lambda-lambda scan was performed with the software, with a PL APO 63x/1.40 OIL objective. After export, images were analysed with Cell Profiler to measure the intensity of the images, and data was exported to Excel and plotted. For live cell imaging, a Leica DMI8 wide-field inverted microscope was used, equipped with a Leica sCMOS camera and CO₂/incubation system. Cells were imaged every 5 seconds for the short time course, and every 15 min for the longer time course. Wide-field fluorescence images of fixed cells were obtained using the Cell{circumflex over ( )}R system based on an Olympus IX81 fully motorized inverted microscope (60× PlanApo objective, 1.42 NA) fitted with an Orca-AG CCD camera (Hamamatsu) driven by the Cell® software. ImageJ software was used when measuring the signal-to-noise ratio graph by acquiring the intensity of several pixels from each time point.

RNA Extraction

Total RNA was produced by using Tri-Reagent (Sigma), and DNA was removed using Turbo-DNase free kit (Invitrogen). 2 μg of total RNA extracts from HeLa cells were separated by electrophoresis in a 1% agarose gel. The rRNA 28S and 18S bands were extracted separately from the smeared mRNA in the gel. RNAs were cleaned with a Nucleospin gel and PCR clean up kit (Macherey-Nagel).

DNA and Chromatin Protein Extraction

Genomic DNA lacking nucleosomes was purified from HEK293 cells using the TIANamp Genomic kit (TIANGEN, China). Genomic DNA including nucleosomes was purified from HEK293T cells as follows. Cells were washed with PBS and collected at 500×g for 5 min and then lysed in Nonidet P-40 lysis buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10% glycerol and 1% Nonidet P-40) with a protease inhibitor cocktail (1:100 dilution) at 4° C. for 5 min. After centrifugation at 15,000×g for 5 min, the pellets were collected and washed with Nonidet P-40 lysis buffer. Then Nonidet P-40 lysis buffer with micrococal nuclease (1:100 BioLabs), 5 mM CaCl₂ and a protease inhibitor cocktail (1:100 dilution) were added to the pellets and incubated at 25° C. for 10 min. 10 mM EGTA was added to stop the reaction. After centrifugation at 15,000×g for 15 min, the supernatant containing the chromatin were collected.

Example 7: Compound I-e Binds RNA in Solution

Binding of dye I-e to RNA was tested by adding this dye to an RNA solution extracted from human cells (as described above) and evaluating the increase in fluorescence intensity. Notably, a significant, 3.1-fold increase in fluorescence intensity was observed once compound I-e interacted with total RNA extract. FIG. 3 shows the Emission spectra of probe I-e with RNA in water. Compound I-e alone, RNA solution+Compound I-e, Compound I-e+RNA solution+RNASE, control, water+RNASE, control.

Compound I-e Stains Fixed Cells

The fluorescent properties of the compound I-e in cells were analyzed. Compound I-e was applied to fixed HeLa cells. A lambda-lambda scan, using a scanning confocal microscope equipped with a supercontinuum laser and tunable detection, was done to determine the excitation-emission spectrum of the compound. The peak fluorescent intensity was at the emission range of 647-667 nm and optimal excitation at 570 nm. This clearly showed the detection of the cells as shown in FIGS. 4A-4B. Spectrum scan shows peak of excitation/emission. FIG. 4A shows that following the addition of Compound I-e to HeLa cells these were scanned as described in the Methods section (above). The strongest excitation, at the emission range of 647-667 nm was found to be 570 nm. Bar=10 μm. FIG. 4B demonstrates the analysis of images as described in the Methods (above) and the image emission intensities were plotted. The excitation that resulted in the strongest emission was found to be at 570 nm. FIG. 4C provides a 2D heat map exported from acquisition software showing the excitation vs. the emission profiles. FIG. 4D demonstrates that Compound I-e localizes to nucleoli in treated cells. Nucleoli are identified by staining with an antibody to the nucleolar marker fibrillarin (green). Hoechst DNA stain is in cyan. No I-e signal is detected in untreated cells. Bar=10 μm.

Example 8: Staining of Cells

Staining Living Cells with Compound I-e

Compound I-e is highly cell permeable. Within 30 seconds the compound was detected in the cytoplasm, and within 150 seconds the compound stained nucleoli (FIG. 5A). Additionally, compound I-e could be applied to living cells for over 24 hours without affecting viability, cell morphology, or proliferation as cell divisions continuously occurred indicating that compound I-e is not toxic to cells (FIG. 5B). Notably, compound I-e remained fluorescently stable, bright and specifically bound to nucleoli (FIG. 5B), with a high signal-to-noise ratio (FIG. 5C).

Example 9: Staining Fixed Living Cells

Staining Fixed and Living Cells with Compound I-e

The binding of compound I-e to nucleic acids within cells was investigated. To this end, fixed and stained cells were treated with either deoxyribonuclease (DNase) or ribonuclease (RNase) to remove DNA or RNA from cells, respectively (FIG. 6). While with the treatment with DNase no considerable loss of fluorescence signal was observed (FIG. 6A), after RNase treatment, cytoplasmic and nucleolar fluorescence signal disappeared in comparison to untreated cells (FIG. 6B). These digestion experiments validated the preference of compound I-e to attach to RNA since cells in which the RNA was removed by RNase, did not stain anymore. When the cells were treated with actinomycin D, an inhibitor of RNA polymerase II that transcribes mRNA (and not rRNA), there was not much change in the intensity of the dye in the cells, suggesting that the dye preferably binds to rRNA in the nucleolus and the cytoplasm.

The interaction of compound I-e with rRNA was tested, nucleolar rRNA transcription was inhibited by inhibiting RNA polymerase I activity with two specific inhibitors (FIG. 7A). Following these treatments, the fluorescent signal throughout the nucleoli and the cytoplasm was significantly reduced. Further the assembly of cytoplasmic stress granules that contain mRNAs but not rRNAs was induced. This way foci containing mRNAs were formed in the cytoplasm, which might be detectable by the compound if it binds mRNA. Stress granules were formed by the addition of arsenite to the cells. However, the compound did not localize with stress granules, implying that compound I-e is not binding to cytoplasmic mRNA (FIG. 7B).

Example 10: Preference to Secondary Structures in the rRNA

Compound I-e was found to have preference to secondary structures in the rRNA:

rRNA and mRNA were purified from total cell RNA extracts. The binding of the compound to these RNAs under two conditions was examined. At room temperature the secondary structures are preserved, and strong binding to rRNA was observed (FIG. 8) as measured by fluoresce (increase of fluorescence). Once the secondary structures of the RNA were destroyed by heating the RNAs to 90° C., the binding to the rRNA was lost (no increase in fluorescence was observed), indicating that indeed compound I-e binds the complex secondary structures of rRNA.

Example 11: Binding of Compound I-e to Histones-Free DNA Extract

Compound I-e binds secondary structures in the rRNA that are probably double stranded (ds) however nuclear dsDNA was not stained as well since histones forming the nucleosomes on the DNA in cells might mask the binding of the dye. DNA from cells, with or without histones, was extracted. Compound I-e did not bind to DNA with histones and could bind only to DNA extract in which the histones were removed (FIG. 9).

Claims

1. A compound of formula (I):

2. The compound of claim 1, wherein R1 is H, F, Cl, or OR, R2 is H, F, Cl, OR, NO2 or CN, R3 is H, OR, NHR or NR2, R4 is H, F, Cl, CN, NO2, R5 is H, F, Cl; R being H, or straight or branched C1-6alkyl and X is I.

3. The compound of claim 1, wherein R1 is H, F, or OH, R2 is H, F, OR, NO2 or CN, R3 is H, OR, NHR or NR2, R4 is H, F, CN or NO2, R5 is H or F; R being H, or straight or branched C1-6alkyl and X is I.

4. A compound according to claim 1, selected from: (E)-2-(4-hydroxystyryl)-1,4-dimethylpyridinium iodide (I-a);(E)-2-(4-Methoxystyryl)-1,4-dimethylpyridinium iodide (I-b);(E)-2-(3-Flouro-4-hydroxystyryl)-1,4-dimethylpyridinium iodide (I-c);(E)-2-(3-Flouro-4-methoxystyryl)-1,4-dimethylpyridinium iodide (I-d);(E)-2-(3, 5-Difluoro-4-hydroxystyryl)-1,4-dimethylpyridinium iodide (I-e);(E)-2-(3, 5-Difluoro-4-methoxystyryl)-1,4-dimethylpyridinium iodide (I-f);(E)-2-(2,3,5,6-Tetrafluoro-4-hydroxystyryl)-1,4-dimethylpyridinium iodide (I-g);(E)-2-(2,3,5,6-Tetrafluoro-4-methoxystyryl)-1,4-dimethylpyridinium iodide (I-h);(E)-2-(4-Dimethylaminostyryl)-1,4-dimethylpyridinium iodide (I-i);(E)-2-(3,4-Dihydroxystyryl)-1,4-dimethylpyridinium iodide (I-j);(E)-2-(2-Hydroxy-3-methoxy-5-nitrostyryl)-1,4-dimethylpyridinium iodide (I-k); and(E)-2-(4-Hydroxy-3-nitrostyryl)-1,4-dimethylpyridinium iodide (I-l).

5. A process for the preparation of compound of formula (I) comprising: reacting a compound of formula (II)

6. The process according to claim 5, wherein the base is a nitrogen base selected from pyridine, piperidine, the C1-4alcohol is selected from methanol, ethanol, propanol, isopropanol, butanol, sec-butyl alcohol, tert-butyl alcohol and heating is conducted under microwave irradiation.

7. A of formula (I):

8. A compound of claim 7 for use as a fluorescent probe in imaging rRNA.

9. A compound of claim 7 for use in imaging rRNA.

10. The compound for the use of claim 7, selected from: (E)-2-(4-hydroxystyryl)-1,4-dimethylpyridinium iodide (I-a);(E)-2-(4-Methoxystyryl)-1,4-dimethylpyridinium iodide (I-b);(E)-2-(3-Flouro-4-hydroxystyryl)-1,4-dimethylpyridinium iodide (I-c);(E)-2-(3-Flouro-4-methoxystyryl)-1,4-dimethylpyridinium iodide (I-d);(E)-2-(3, 5-Difluoro-4-hydroxystyryl)-1,4-dimethylpyridinium iodide (I-e);(E)-2-(3, 5-Difluoro-4-methoxystyryl)-1,4-dimethylpyridinium iodide (I-f);(E)-2-(2,3,5,6-Tetrafluoro-4-hydroxystyryl)-1,4-dimethylpyridinium iodide (I-g);(E)-2-(2,3,5,6-Tetrafluoro-4-methoxystyryl)-1,4-dimethylpyridinium iodide (I-h);(E)-2-(4-Dimethylaminostyryl)-1,4-dimethylpyridinium iodide (I-i);(E)-2-(3,4-Dihydroxystyryl)-1,4-dimethylpyridinium iodide (I-j);(E)-2-(2-Hydroxy-3-methoxy-5-nitrostyryl)-1,4-dimethylpyridinium iodide (I-k); and(E)-2-(4-Hydroxy-3-nitrostyryl)-1,4-dimethylpyridinium iodide (I-l).

11. The compound of claim 10 being I-e.

12. A method for imaging rRNA comprising adding a compound of claim 1 to a sample containing rRNA, and imaging the fluorescence.

13. The method according to claim 12, wherein the compound is of formula I-e.