HYDROPHILIC AND PARTICULARLY WATER SOLUBLE DBOV-DERIVATIVES

Abstract

The present invention relates to a compound having the general formula (1) wherein in the general formula (1) the residues R.sub.1 to R.sub.8 and Ar are the same or different and are independently from each other selected from the group consisting of hydrogen, unsubstituted hydrocarbon groups, substituted hydrocarbon groups and inorganic groups, wherein at least one of the residues R.sub.1 to R.sub.8 and Ar is a hydrophilic group, such as for instance a group comprising one or more polyethylene groups.

##STR00001##

Claims

1. A compound having the general formula (1): ##STR00014## wherein in the general formula (1) R.sub.1 to R.sub.8 and Ar are the same or different and are independently from each other selected from the group consisting of hydrogen, unsubstituted hydrocarbon groups, substituted hydrocarbon groups and inorganic groups, wherein at least one of R.sub.1 to R.sub.8 and Ar is a hydrophilic group.

2. The compound in accordance with claim 1, wherein in the general formula (1) at least one of R.sub.1 to R.sub.8 and Ar is a hydrophilic group, which is selected from the group consisting of anionic groups, cationic groups and non-charged groups comprising one or more groups selected from inorganic acid groups, organic acid groups, ester groups, amide groups, hydroxy groups, thiol groups, amino groups, aldehyde groups, ketone groups, acryl groups, ether groups, thioether groups and arbitrary combinations thereof.

3. The compound in accordance with claim 1, wherein in the general formula (1) at least one of R.sub.1 to R.sub.8 and Ar is a hydrophilic group, which is selected from the group consisting of quaternary amino groups, iminium salt groups, pyrrolidinium salt groups, pyrrolium salt groups, pyrazolidinum salt groups, pyrazolidinum salt groups, imidazolium salt groups, imidazolidinum salt groups, piperidinium salt groups, pyridinium salt groups, piperazinium salt groups, morpholinium salt groups, thiomorpholinium salt groups, oxazine salt groups, thiazine salt groups, indolinium salt groups, indole salt groups, sulfate groups, phosphate groups, nitrate groups, sulfonate groups, carboxylic acid groups, sulfonic acid groups, sulfenic acid groups, sulfinic acid groups, phosphonic acid groups, phosphenic acid groups, phosphinic acid groups, sugar groups, ester groups, amide groups, hydroxy groups, thiol groups, amino groups, aldehyde groups, ketone groups, acryl groups, ether groups, thioether groups, sulfhydryl groups, glycosidic linkages, peptide bonds, triazol groups and arbitrary combinations thereof.

4. The compound in accordance with claim 3, wherein in the general formula (1) at least one of R.sub.1 to R.sub.8 and Ar is a hydrophilic group, which is selected from the group consisting of quaternary amino groups, iminium salt groups, pyridinium salt groups, sulfate groups, sulfonate groups, carboxylic acid groups, sulfonic acid groups, ester groups, amide groups, ether groups, triazol groups and arbitrary combinations thereof.

5. The compound in accordance with claim 3, wherein in the general formula (1) at least one of R.sub.1 to R.sub.8 and Ar is a hydrophilic group, which is selected from polyalkylene glycol groups having a repeating number of 2 to 20 alkoxy units, groups comprising one to five polyalkylene glycol groups with each having a repeating number of 2 to 20 alkoxy units, sulfonate groups, quaternary ammonium groups, one or more amide groups, one or more imine groups and one or more pyridinium groups.

6. The compound in accordance with claim 3, wherein in the general formula (1) at least one of R.sub.1 to R.sub.8 and Ar is a phenyl group, which is substituted with two to five polyalkylene glycol groups.

7. The compound in accordance with claim 1, wherein in the general formula (1) at least two of R.sub.1 to R.sub.8 and Ar are a hydrophilic group.

8. The compound in accordance with claim 1, which has a solubility in water at 23° C. of at least 0.01 g/l.

9. The compound in accordance with claim 1, wherein in the general formula (1) those of R.sub.1 to R.sub.8 and Ar, which are not an aforementioned hydrophilic group, are the same or different and are independently from each other selected from the group consisting of hydrogen, unsubstituted alkyl groups, substituted alkyl groups, unsubstituted alkenyl groups, substituted alkenyl groups, unsubstituted alkynyl groups, substituted alkynyl groups, unsubstituted alkoxy groups, substituted alkoxy groups, unsubstituted cycloalkyl groups, substituted cycloalkyl groups, unsubstituted aryl groups, substituted aryl groups, unsubstituted aralkyl groups, substituted aralkyl groups, unsubstituted hetaryl groups, substituted hetaryl groups, azide groups, and groups formed in that two of adjacent R.sub.1 to R.sub.8 and Ar are linked with each other to form an aromatic, heteroaromatic, cyclic or heterocyclic group.

10. The compound in accordance with claim 9, wherein in the general formula (1) those of R.sub.1 to R.sub.8 and Ar, which are not an aforementioned hydrophilic group, are the same or different and are independently from each other selected from the group consisting of hydrogen, unsubstituted linear or branched C.sub.1-30-alkyl groups, unsubstituted C.sub.3-30-cycloalkyl groups, phenyl groups, naphthyl groups, anthryl groups, pyrenyl groups, azide groups, polyethylene groups with a repeating number of 2 to 20 ethoxy units, phenyl ethylene, triisopropylsilyl ethynyl, trimethylsilyl ethynyl, and groups formed in that two of adjacent R.sub.1 to R.sub.8 and Ar are linked with each other to form an aromatic, heteroaromatic, cyclic or heterocyclic group.

11. The compound in accordance with claim 1, wherein in the general formula (1): R.sub.1 is hydrogen or a C.sub.1-20-alkyl group, R.sub.3 and R.sub.8 are a hydrophilic group, R.sub.2 and R.sub.4 to R.sub.7 are hydrogen and Ar is aryl, a C.sub.1-15-alkyl group or a trialkylsilyl alkynyl group.

12. The compound in accordance with claim 1, wherein in general formula (1) Ar is selected from the group consisting of phenyl, trifluorphenyl, 1,5-dimethylphenyl, mesitylene, triisopropylsilyl ethynyl, trimethylsilyl ethynyl, phenylsilyl ethynyl and C.sub.1-15-alkyl groups.

13. The compound in accordance with claim 1, wherein in the general formula (1) at least one of R.sub.1 to R.sub.8 and Ar is a group with a terminal alkyne group.

14. Use of a compound in accordance with claim 1 for optical super-resolution microscopy, confocal microscopy (e.g., linear [single-photon] and/or nonlinear [multi-photon] imaging), wide field microscopy, fluorescence-lifetime imaging microscopy (FLIM), fluorescence resonance energy transfer (FRET) microscopy, FLIM-FRET microscopy, fluorescence anisotropy, fluorescence correlation spectroscopy (FCS), light-emitting devices.

15. The use in accordance with claim 14, wherein the compound is used in the high-resolution microscopy as fluorescent marker, and wherein the compound is used in photon tunneling microscopy (PTM), near-field optical random mapping (NORM) microscopy, structured illumination microscopy (SIM), spatially modulated illumination (SMI), ground state depletion (GSD), saturated structured illumination microscopy (SSIM), super-resolution optical fluctuation imaging (SOFI), omnipresent Localization Microscopy (OLM), stimulated emission depletion microscopy (STED) or single-molecule localization microscopy (SMLM), such as photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), ground state depletion individual molecule return (GSDIM), binding activated localization microscopy (BALM) or fluorescence photo-activation localization microscopy (FPALM).

16. A compound in accordance with claim 1 for use in photothermal therapy.

Description

[0052] In accordance with still a further aspect, the present invention relates to the aforementioned compound for use in photothermal therapy. Subsequently, the present invention is further described by means of illustrative, but not limiting examples and figures.

[0053] FIG. 1 shows a reaction scheme for synthesizing symmetric dibenzo[hi,st]ovalenes to be used in accordance with an embodiment of the present invention.

[0054] FIG. 2 shows a reaction scheme for converting a symmetric dibenzo[hi,st]ovalene into an asymmetric dibenzo[hi,st]ovalene to be used in accordance with another embodiment of the present invention.

[0055] FIG. 3 show the absorption and emission spectra of the compound prepared in example 1 (Abs: absorbance; PI: PL-emission).

[0056] FIG. 4 shows the results of a cytotoxicity test DBOV-Mes-OTEG synthesized in example 1 in living cells.

EXAMPLE 1—(SYNTHESIS OF PEGYLATED 6,14-DIMESITYLDIBENZO[HI,ST]OVALENE)

[0057] (Synthesis of 6,6′-diiodo-[5,5′-bichrysene]-3,3′-dicarbaldehyde)

[0058] In accordance with the reaction schemes shown in FIGS. 1 and 2, 6,6′-diiodo-[5,5′-bichrysene]-3,3′-dicarbaldehyde, which is compound 6 with all residues R.sub.1 to R.sub.7 being hydrogen atoms, was prepared starting from compound 5, in which all residues R.sub.1 to R.sub.3 are hydrogen atoms. The synthesis thereof is e.g. described in Nano Letters, 2017, volume 17, pages 5521-5525. More specifically, to a solution of compound 5 (2.0 g, 3.9 mmol) dissolved in anhydrous dichloromethane (240 mL) was added ICI (8.58 mmol, 8.58 mL, 1 M in dichloromethane). After stirring at room temperature for 2 hours, the excess ICI was quenched by addition of saturated aqueous Na.sub.2S.sub.2O.sub.3 solution (50 mL). The organic phase was separated, washed with brine (50 mL), dried over Na.sub.2SO.sub.4 and evaporated. The residual solid was recrystallized with dichloromethane and methanol.

[0059] After filtration, the product (2.2 g, 76%) was obtained as white solid. The product had the following characteristics:

[0060] Mp: >400° C.; .sup.1H NMR (300 MHz, Methylene Chloride-d.sub.2) δ9.18 (d, J=9.2 Hz, 2H), 9.00 (d, J=8.5 Hz, 2H), 8.72 (s, 2H), 8.53-8.42 (m, 4H), 8.28 (d, J=9.1 Hz, 2H), 8.04 (d, J=8.3 Hz, 2H), 7.98-7.88 (m, 2H), 7.86-7.74 (m, 4H); .sup.13C NMR (75 MHz, Methylene Chloride-d.sub.2) δ191.8, 150.1, 137.4, 135.3, 134.6, 134.5, 133.7, 132.1, 130.9, 130.5, 130.4, 130.2, 129.7, 129.5, 129.3, 125.7, 124.4, 123.7, 114.1; FD-MS (8 kV): m/z 762.2; HRMS (MALDI-TOF): m/z Calcd for C.sub.38H.sub.20I.sub.2O.sub.2: 761.9553 [M].sup.+, found: 761.9553 (error=0 ppm).

[0061] Thus, it was shown that the product had the formula:

##STR00009##

(Synthesis of 5,14-diformylbenzo[a]dinaphtho[2,1,8-cde:1′,2′,3′,4′-ghi]perylene)

[0062] In accordance with the reaction scheme shown in FIG. 1, 5,14-diformylbenzo[a]dinaphtho[2,1,8-cde:1′,2′,3′,4′-ghi]perylene, which is compound 7 with all residues R.sub.1 to R.sub.7 being hydrogen atoms, was prepared starting from compound 6 prepared as described above. More specifically, to a 3 L cylindrical quartz reactor containing 6,6′-diiodo-[5,5′-bichrysene]-3,3′-dicarbaldehyde (300 mg, 0.394 mmol) was added a mixture of acetone (600 mL) and triethylamine (6 mL). Then the mixture was degassed by bubbling with Ar for 20 minutes. After that, the reaction mixture was stirred and irradiated at room temperature in a photoreactor equipped with six 300 nm wavelength UV lamps with strong stirring for 2 hours.

[0063] After cooling down to room temperature, the solvent was evaporated and the residue was purified by column chromatography (n-hexane:ethyl acetate=4:1) to give the product (170 mg, 86% yield) as red solid. The product had the following characteristics, which was further confirmed by X-ray single crystal structure analysis. Mp: >400° C.; .sup.1H NMR (300 MHz, 1,1,2,2-tetrachloroethane-d.sub.2) δ9.46 (s, 2H), 9.01 (d, J=9.1 Hz, 2H), 8.90 (d, J=8.4 Hz, 2H), 8.52-8.42 (m, 4H), 8.34 (t, J=9.1 Hz, 4H), 7.83-7.72 (m, 2H), 7.64 (dd, J=8.1, 1.1 Hz, 2H); .sup.13C NMR (75 MHz, C.sub.2D.sub.2Cl.sub.4) δ 190.8, 133.7, 131.7, 131.6, 130.6, 128.5, 128.2, 127.9, 127.5, 127.1, 127.1, 125.9, 124.9, 124.4, 124.0, 123.9, 123.2, 121.2, 120.6; FD-MS (8 kV): m/z 506.9; HR MS (MALDI-TOF): m/z Calcd for C.sub.38H.sub.18O.sub.2: 506.1307 [M].sup.+, found: 506.1288 (error=−3.7 ppm).

[0064] Thus, it was shown that the product had the formula:

##STR00010##

(Synthesis of 6,14-dimesityldibenzo[hi,st]ovalene)

[0065] To a solution of 5,14-diformylbenzo[a]dinaphtho[2,1,8-cde:1′,2′,3′,4′-ghi]perylene (7) (5 mg, 10 μmol) in anhydrous THF (5 mL) was added mesitylmagnesium bromide (0.15 mL, 150 μmol, 1.0 M in ether) dropwise under the protection of Ar. The mixture was stirred at room temperature for 2 h. After completion of the reaction, the yellow green colored solution was poured into saturated aqueous solution of NH.sub.4Cl (10 mL) and then extracted with ethyl acetate (20 mL) for 3 times. The combined organic layers were washed with brine (30 mL), dried over Na.sub.2SO.sub.4, and evaporated. After drying under vacuum using an oil pump for 2 h, the residue was redissolved in anhydrous dichloromethane (50 mL) and BF.sub.3.OEt.sub.2 (0.5 mL) was added using a syringe. The mixture was stirred at room temperature for 2 h. After completion of the reaction, the mixture was poured into saturated NaHCO.sub.3 solution (10 mL). The organic phase was separated and dried over Na.sub.2SO.sub.4. The solvents were evaporated under reduced pressure and the residue was purified by column chromatography over silica gel (eluent: n-hexane/DCM=10: 1) to give 6,14-dimesityldibenzo[hi,st]ovalene (4 mg, 56%) as blue powder. TLC R.sub.f=0.6 (n-hexane/ethyl acetate=10: 1).

[0066] The product had the following characteristics:

[0067] Mp: >400° C.; .sup.1H NMR (700 MHz, o-dichlorobenzene-d.sub.4) δ 9.33 (d, J=7.8 Hz, 2H), 9.04 (d, J=7.2 Hz, 2H), 8.44 (d, J=7.4 Hz, 2H), 8.00 (d, J=9.1 Hz, 2H), 7.98 (d, J=8.3 Hz, 2H), 7.89 (t, J=7.5 Hz, 2H), 7.78 (d, J=9.0 Hz, 2H), 7.19 (s, 4H), 2.50 (s, 6H), 1.99 (s, 12H); FD-MS (8 kV): m/z 709.5; HRMS (MALDI-TOF): m/z Calcd for C.sub.56H.sub.36: 708.2817 [M].sup.+, found: 708.2814.

[0068] Thus, it was shown that the product had the formula:

##STR00011##

(Synthesis of 3,11-dibromo-6,14-dimesityldibenzo[hi,st]ovalene)

[0069] To a solution of 6,14-dimesityldibenzo[hi,st]ovalene (14 mg, 0.020 mmol) dissolved in tetrahydrofuran (70 mL) was added N-bromosuccinimide (NBS) (14 mg, 0.079 mmol). The resulting mixture was stirred at room temperature for 2 h. The solvent was evaporated and the residue was purified by column chromatography to give the product (14 mg, 84%) as blue solid. The product had the following characteristics:

[0070] Mp: >400° C.; .sup.1H NMR (700 MHz, THF-d.sub.8) δ 10.67 (d, J=8.3 Hz, 2H), 8.72 (d, J=8.5 Hz, 2H), 8.34 (d, J=8.8 Hz, 2H), 8.29 (d, J=9.2 Hz, 2H), 7.87 (d, J=9.0 Hz, 2H), 7.85 (d, J=8.8 Hz, 2H), 7.27 (s, 4H), 1.93 (s, 12H); MS (MALDI-TOF): m/z Calcd for C.sub.54H.sub.30Br.sub.2: 864.10 [M].sup.+, found: 864.06.

[0071] Thus, it was shown that the product had the formula:

##STR00012##

(Synthesis of 3,4,5-tris(2,5,8,11-tetraoxatridecan-13-yloxy)phenyl substituted 6,14-dimesityldibenzo[hi,st]ovalene)

[0072] To a Schlenk tube equipped with a stirring bar was added dibrominated 6,14-dimesityldibenzo[hi,st]ovalene (3.0 mg, 3.5 μmol), 3,4,5-tris(2,5,8,11-tetraoxatridecan-13-yloxy)phenyl boronic acid pinacol ester (12 mg, 14 μmol), Pd(PPh.sub.3).sub.4 (1.6 mg, 1.4 μmol) and K.sub.2CO.sub.3 (4.8 mg, 35 μmol). The reaction tube was evacuated and backfilled with Ar for three times before a mixture of toluene/EtOH/H.sub.2O=2 mL/0.5 mL/0.5 mL was added. The mixture was degassed by three times freeze-pump-thaw cycles and heated at 90° C. overnight. After cooling down to room temperature, the reaction solution was extracted with ethyl acetate, washed with brine, dried over Na.sub.2SO.sub.4 and evaporated.

[0073] The residue was purified by column chromatography (ethyl acetate: MeOH=10:1 to 1:1) to give the product (5 mg, 69%) as blue oil. The product had the following characteristics:

[0074] .sup.1H NMR (250 MHz, Deuterium Oxide) δ 8.95 (d, J=8.6 Hz, 1H), 8.24 (d, J=8.7 Hz, 1H), 8.09 (d, J=9.3 Hz, 1H), 7.95 (s, 2H), 7.73 (d, J=9.2 Hz, 1H), 7.26 (s, 2H), 7.01 (s, 2H), 4.31 (t, J=5.2 Hz, 2H), 3.93-3.84 (m, 2H), 3.75 (d, J=5.5 Hz, 8H), 3.68-3.31 (m, 84H), 3.27 (s, 8H), 3.19 (s, 6H), 1.96 (s, 6H). MS (MALDI-TOF): m/z Calcd for C.sub.122H.sub.152O.sub.30: 2097.04 [M].sup.+, found: 2097.06

[0075] Thus, it was shown that the product had the formula:

##STR00013##

[0076] The solubility of this compound in water was evaluated at room temperature and was found to be 0.1 g/l.

[0077] The absorption and emission spectra of this compound dissolved in water in a concentration of 10.sup.−6 mol/L are shown in FIG. 3 (UV-vis: absorbance; PI: PL-emission). The excitation wavelength for the PL measurement was 592 nm.

EXAMPLE 2—(EVALUATING THE WATER SOLUBLE COMPOUND SYNTHESIZED IN EXAMPLE 1 FOR CELL IMAGING)

[0078] 3,4,5-tris(2,5,8,11-tetraoxatridecan-13-yloxy)phenyl substituted 6,14-dimesityldibenzo[hi,st]ovalene (subsequently abbreviated as DBOV-Mes-OTEG) synthesized in example 1 was evaluated concerning its properties.

[0079] More specifically, the cytotoxicity of DBOV-Mes-OTEG was tested in living cells. The results are shown in FIG. 4. The test showed that the water soluble DBOV-Mes-OTEG does not show any significant toxicity to cells in a concentration of 1 μM. Moreover, the uptake of DBOV-Mes-OTEG into living cells was investigated by imaging 21 hours with low laser intensity at the spinning disk confocal microscopy (Visitron). The test revealed that DBOV-Mes-OTEG was taken into the cytoplasm at the beginning of the incubation and that its location was after 21 hours incubation in the nucleus and nuclear membrane.

[0080] After the aforementioned live cell imaging, the samples were fixed and imaged in phosphate-buffered saline (PBS) without special imaging buffer. The test revealed that DBOV-Mes-OTEG was able to be imaged in the blinking mode under continuous exposure with high laser intensity 10 kW/cm.sup.2 for 90 min. Furthermore, no significant decrease of blinking signals of DBOV-Mes-OTEG were detected. In addition, blinking signals could also be imaged with low laser intensity of 240 W/cm.sup.2 and this reveals the possibility of living cell imaging with DBOV-Mes-OTEG.