TISSUE-TARGETING COMPLEX

Abstract

A tissue-targeting complex is described that comprises at least one targeting element [A-L-Q.sup.+-Alk B-] in which A is an amino-containing anchor element, L a linker, Q.sup.+ a quaternary ammonium group, B.sup.- an ophthalmologically acceptable counterion, and Alk an alkyl chain having 4 to 12 carbon atoms; at least one chromophore, and optionally a carrier molecule.

Claims

1. A tissue-targeting complex comprising a) at least one targeting element [A-L-Q.sup.+-Alk B.sup.-] in which A is an anchor group, L a linker, Q.sup.+ a quaternary ammonium group, B.sup.- an ophthalmologically acceptable counterion, and Alk an alkyl chain having a chain length of 4 to 12 carbon atoms; b) at least one chromophore and/or at least one carrier molecule; for binding to ocular tissue.

2. The tissue-targeting complex as claimed in claim 1, characterized in that the anchor group is a heteroatom or a functional group.

3. The tissue-targeting complex as claimed in claim 1, characterized in that the anchor group is an amino group -(R.sup.1)N- in which R.sup.1 is H or a C.sub.1-C.sub.4 alkyl group and the linker is -C.sub.1-C.sub.6 alkyl.

4. The tissue-targeting complex as claimed in claim 1, characterized in that -Q.sup.+- is -(R.sup.2)(R.sup.3)N.sup.+- in which R.sup.2 and R.sup.3 are each independently C.sub.1-C.sub.4 alkyl radicals.

5. The tissue-targeting complex as claimed in claim 1, characterized in that the counterion B.sup.- is a halide ion, hydrogen phosphate ion or hydrogen sulfate ion.

6. The tissue-targeting complex as claimed in claim 1, characterized in that the chromophore is an ophthalmologically tolerated, water-soluble dye.

7. The tissue-targeting complex as claimed in claim 1, characterized in that the chromophore is a compound of the formula I ##STR00020## wherein each of the radicals R.sup.10, R.sup.11, R.sup.12, and R.sup.13 is independently hydrogen, C.sub.1-C.sub.4 alkyl radicals, an anchor group as defined in claim 1 or a linker molecule, each of the radicals R.sup.14, R.sup.15, R.sup.16, and R.sup.17 is independently hydrogen, a C.sub.1-C.sub.4 alkyl radical or a linker unit.

8. The tissue-targeting complex as claimed in claim 1, characterized in that the chromophore is a compound having formula I in which R.sup.10 and R.sup.11 are each a targeting element and R.sup.12, R.sup.13, R.sup.14, R.sup.15, R.sup.16, and R.sup.17 are each hydrogen.

9. The tissue-targeting complex as claimed in claim 6, characterized in that R10 and R11 are each a targeting element, one of radicals R12, R13, R14, R15, R16, and R17 is a linker unit, and the remaining radicals are each hydrogen.

10. The tissue-targeting complex as claimed in claim 1, characterized in that the complex includes at least one carrier molecule, wherein at least one chromophore and at least one targeting element are attached to the carrier molecule.

11. The tissue-targeting complex as claimed in claim 1, characterized in that the targeting element and chromophore are each independently attached to the carrier molecule.

12. The tissue-targeting complex as claim 1, characterized in that at least one targeting element is attached to a chromophore, wherein the carrier molecule includes chromophores and targeting elements attached independently of one another and/or attached chromophores with carrier molecules attached thereto.

13. The tissue-targeting complex as claimed in claim 1, characterized in that the carrier molecule is a water-soluble, ophthalmologically acceptable polymer bearing functional groups for the attachment of chromophore units and/or targeting units.

14. The tissue-targeting complex as claimed in claim 1, characterized in that the carrier molecule is a linear, branched, or bead-type, water-compatible polymer.

15. The tissue-targeting complex as claimed in claim 1, characterized in that the carrier molecule is a homopolymer or copolymer derived from vinylamine.

16. The tissue-targeting complex as claimed in claim 1 for binding to ocular membranes.

17. The tissue-targeting complex as claimed in claim 1 for staining ocular tissue, in particular ocular membranes.

18. Use of the tissue-targeting complex as claimed in claim 1 for binding to ocular membranes and/or for staining ocular membranes, such as epiretinal membranes (ERM) or the inner limiting membrane (ILM).

Description

[0063] The drawings serve to illustrate the invention, wherein

[0064] FIG. 1 shows a graphical plot of the UV/vis spectroscopy data obtained for the BBG reference (blue) and BBG MS solution (red) at concentrations of 0.005 to 0.08 mg/mL.

[0065] FIG. 2 shows a graphical plot of the UV/vis spectroscopy data obtained for the 7a and 7b references (blue and red) and 7a and 7b MS solutions (purple and green) at concentrations of 0.005 to 0.16 mg/mL.

[0066] FIG. 3 shows a graphical plot of the UV/vis spectroscopy data obtained for the 7c and 7d references (blue and red) and 7c and 7d MS solutions (purple and green) at concentrations of 0.005 to 0.16 mg/mL.

[0067] FIG. 4 shows a graphical plot of the UV/vis spectroscopy data obtained for the ICG reference (blue) and ICG MS solution (red) at concentrations of 0.005 to 0.04 mg/mL.

EXAMPLES

[0068] The invention is further illustrated by the examples below, without being limited by them. All concentrations given in the examples refer to mg of substrate per mL of total solution. Unless otherwise stated, all reactions were carried out at room temperature, i.e. approximately 25° C.

[0069] The following procedures and conditions were employed for the analyses:

.SUP.1.H Nuclear Magnetic Resonance Spectroscopy (.SUP.1.H NMR Spectroscopy)

[0070] 300 MHz and 600 MHz NMR were recorded at room temperature using a Bruker Avance III FT-NMR spectrometer. Chemical shifts are reported with reference to the signal of a deuterated solvent as internal standard. Spin multiplicities have been abbreviated to s (singlet), d (doublet), dd (doublet of doublets), t (triplet) or m (multiplet).

.SUP.13.C Nuclear Magnetic Resonance Spectroscopy (.SUP.13.C NMR Spectroscopy)

[0071] Measurements were likewise performed on a Bruker Avance III FT-NMR spectrometer at 75 MHz and 150 MHz and at room temperature. Chemical shifts are reported with reference to the signal of a deuterated solvent as internal standard.

MALDI-TOF-MS

[0072] Measurements were recorded using a Bruker Ultraflex TOF (time of flight) mass spectrometer. The instrument is operated using a 337 nm nitrogen laser, both in linear mode and in reflector mode. The samples were dissolved in a suitable solvent and transferred using 2-(4-hydroxyphenylazo)benzoic acid (HABA).

ESI-MS

[0073] Measurements were recorded using a Finnigan LCQ Deca ion-trap API mass spectrometer. The substances to be determined were dissolved in a suitable solvent at a concentration of 1 mg/mL. Ionization was by electrospray ionization.

LC-MS

[0074] Measurements were performed using an Agilent Technologies 6120 instrument with an absorption detector (214 nm) in combination with an Agilent quadrupole mass spectrometer. A 1.8 .Math.m (2.1 × 50 mm) Agilent SB-C.sub.18 column was used, with H.sub.2O:MeCN + 0.1% formic acid as solvent mixture at room temperature. The flow rate was 0.4 mL/min with a solvent mixture of 95:5 H.sub.2O:MeCN and 5:95 H.sub.2O:MeCN.

GC-MS

[0075] Measurements were recorded using a Thermo Finnigan Trace DSQ (dual-stage quadrupole). Ionization of the samples was by electron impact ionization (EI).

FTIR Spectroscopy

[0076] FTIR spectra were recorded at room temperature using a Nicolet 6700 FTIR spectrometer with a LOT ATR attachment. The wavenumber scale was calibrated using a HeNe laser. Measurements were recorded over a 4000-300 cm.sup.-1 range.

UV/VIS Spectroscopy

[0077] UV/vis spectra were recorded using an Analytik Jena AG Specord® 210 Plus double-beam spectrometer. Measurements were performed at room temperature between 300 and 800 nm using a quartz glass cuvette with a path length of 1 cm. Evaluation was with the aid of Analytik Jena AG WinAspect Plus software.

Thin-Layer Chromatography

[0078] Thin-layer chromatography was carried out using Merck plates (silica gel 60 F.sub.254 on aluminum foil).

HPLC

[0079] RP-HPLC (reversed-phase high performance liquid chromatography) measurements were recorded using an Agilent 1200 with absorption and fluorescence detector. An Agilent Zorbax Eclipse XDB-C.sub.18 column (4.6 × 100 mm) at a flow rate of 1 mL/min at 60° C. was used. A binary eluent mixture of H.sub.2O and MeCN containing 0.1% trifluoroacetic acid was employed. The measurements were performed by gradient elution over a period of 10 min from acetonitrile/water 5:95 to 95:5.

Example 1

Synthesis of 1,4-difluoroanthraquinone (3)

[0080] 13.4 g (0.9 mol) of phthalic anhydride (1), 48.8 g (0.37 mol) of AlCl.sub.3, and 108 mL (2 mol) of 1,4-difluorobenzene (2) are heated under reflux for 48 h. The excess 1,4-difluorobenzene is then recovered by distillation. To the brown residue is added 400 mL of 1 N hydrochloric acid and the residue is extracted with three 600 mL portions of chloroform. The combined organic phases are concentrated under reduced pressure to approximately 50 mL. The intermediate product is then precipitated with at least 100 mL of low-boiling petroleum ether. The brownish solid is filtered off and then dried. (Yield: 66.7% of theory.) The solid is placed in 200 g of polyphosphoric acid and heated at 140° C. for 2 h. After cooling to room temperature, the viscous reaction mixture is added to 1 L of ice-water and extracted with three 400 mL portions of dichloromethane. The organic phase is concentrated under reduced pressure and the product (3) can then be isolated by column chromatography purification on silica gel (hexane: ethyl acetate [7:1]).

##STR00002##

[0081] Yield: 13.2 g (54.1 mmol) 60.1% of theory. (literature = 50%)

[0082] FTIR (diamond): v= 3084 (s, υ.sub.c=c), 1678 (s, υ.sub.c=o), 1587 (m, υ.sub.c=c), 1249 (vs, υ.sub.C-F), 719 (s, δ.sub.Ar-H) cm.sup.-1.

[0083] .sup.1H NMR (300 MHz, CDCl.sub.3, RT): δ = 8.20-8.29 (m, 2H, Ar—H.sup.3,6), 7.76-7.85 (m, 2H, Ar—H.sup.1,2), 7.43-7.53 (m, 2H, Ar—H.sup.11,12) ppm.

[0084] .sup.13C NMR (300 MHz, DMSO-d.sub.6, RT): δ= 180.23 (C.sup.9,17), (158.55, 155.10) (C.sup.10,13), 134.40 (C.sup.1,2), 133.04 (C.sup.4,5), 126.23 (C.sup.3,6), 124.6-125.25 (C.sup.7,8), 121.43-121.60 (C.sup.11,12) ppm.

[0085] EI-MS m/z: 244 [M]

Example 2

Synthesis of 1,4-bis[[2-(dimethylamino)propyl]amino]anthraquinone (5)

[0086] 3.0 g (12.3 mmol) of 1,4-difluoroanthraquinone (3) is heated at approximately 100° C. with 3.1 mL (24.6 mmol) of N,N-dimethylaminopropylamine (4) in 25 mL of DMSO under a nitrogen atmosphere. After stirring for 24 h, the reaction mixture is cooled to room temperature and diluted with water. The blue crude product is extracted with CHCl.sub.3, concentrated under reduced pressure, and purified by column chromatography on silica gel (CH.sub.2Cl.sub.2:MeOH:NEt.sub.3 [3:4:0.1]).

##STR00003##

[0087] Yield: 2.1 g (5.2 mmol) 42.3% of theory.

[0088] FTIR (diamond): v= 3425 (w, υ.sub.NH), 3068 (w, υ.sub.Ar-H), 2948 (m, υ.sub.CH2), 2859 (m, υ.sub.CH2), 2787 (m, υ).sub.(N-CH3)C-H), 2766 (m, υ(.sub.N-CH3).sub.N-C), 1642 (w, υ.sub.c=o), 1591 (m, υ.sub.c=c), 1567 (m, υ.sub.c=c), 1556 (s, υ.sub.c=c), 1457 (m, δ.sub.CH2,CH3), 731 (s, υ.sub.Ar-H) cm.sup.-1.

[0089] .sup.1H NMR (300 MHz, CDCl.sub.3, RT): δ = 10.83 (t, .sup.3J.sub.H,H=5.35 Hz, 2H, NH.sup.15,16), 8.34 (dd, .sup.3J= 5.92, .sup.4J= 3.27 Hz, 2H, Ar—H.sup.3,6), 7.69 (dd, .sup.3J= 5.91, .sup.4J= 3.31 Hz, 2H, Ar—H.sup.1,2), 7.30 (s, 2H, Ar—H.sup.11,12), 3.48 (m, 4H, CH.sub.2.sup.19,20), 2.45 (t, .sup.3J=6.94 Hz, 4H, CH.sub.2.sup.22,27), 2.27 (s, 12H, NMe.sub.2.sup.24,24,29,30), 1.92 (t, .sup.3J= 7.1 Hz, 4H, CH.sub.2.sup.21,26) ppm.

[0090] .sup.13C NMR (75 MHz, DMSO-d.sub.6, RT): δ= 182.41 (C.sup.9,17), 146.42 (C.sup.10,13), 134.70 (C.sup.1,2), 132.07 (C.sup.4,5), 126.12 (C.sup.3,6), 123.80 (C.sup.7,8), 109.85 (C.sup.11,12), 57.78 (C.sup.22,27), 45.67 (C.sup.24,25,29,30), 41.03 (C.sup.19,20), 28.05 (C.sup.21,26) ppm.

[0091] ESI-MS m/z: 205.3 [M+2H].sup.2+

[0092] Elemental analysis: theoretical values: C: 70.56; H: 7.90; N: 13.71 [0093] analysis results: C: 70.65; H: 7.70; N: 13.58

Example 3

Synthesis of N,N′=(((9,10-dioxo-9,10-dihydroanthracene-1,4-diyl)bis(azanediyl))bis(propane-3,1-diy1))bis(N,N-dimethylbutan-1-aminium) bromide (7a)

[0094] 0.5 g (1.23 mmol) of 1,4-bis[[2-(dimethylamino)propyl]amino]anthraquinone (5) is heated at reflux with 1.68 g (1.3 mL, 12.25 mmol) of 1-bromobutane (6a) in approximately 5 mL of acetone for 24 h. After cooling the solution to room temperature, the product is completely precipitated in hexane, filtered off, and washed with hexane. The blue solid is dried in high vacuum.

##STR00004##

[0095] Yield: 0.81 g (1.19 mmol) 97% of theory.

[0096] FT-IR (diamond): v= 3404 (br, υ.sub.NH), 3006 (w, υ.sub.Ar-H), 2958 (m, υ.sub.CH2), 2871 (m, υ.sub.CH2), 1641 (w, υ.sub.c=o), 1607 (w, υ.sub.c=c), 1594 (m, υ.sub.c=c), 1572 (s, υ.sub.c=c), 1519 (m, υ.sub.c=c), 1467 (m, δ.sub.CH2,CH3), 724 (s, υ.sub.Ar-H) cm.sup.-1.

[0097] .sup.1H NMR (300 MHz, DMSO-d.sub.6, RT): δ[ppm]= 10.79 (t, .sup.3J= 5.82 Hz, 2H, NH.sup.15,16), 8.24 (dd, .sup.3J= 5.85 Hz, .sup.4J= 3.4 Hz, 2H, Ar—H.sup.3,6), 7.83 (m, 2H, Ar—H.sup.1,2), 7.57 (s, 2H, Ar—H.sup.11,12), 3.55 (m, 4H, CH.sub.2.sup.19,20), 3.44 (m, 4H, CH.sub.2.sup.22,26), 3.31 (m, 4H, CH.sub.2.sup.24,28), 3.07 (s, 12H, NMe.sub.2.sup.35-38), 2.09 (m, 4H, CH.sub.2.sup.21,25), 1.64 (m, 4H, CH.sub.2.sup.29,32), 1.29 (m, 4H, CH.sub.2.sup.30,33), 0.91 (t, .sup.3J= 7.3 Hz 6H, CH.sub.3.sup.31,34) ppm.

[0098] .sup.13C NMR (75 MHz, DMSO-d.sub.6, RT): δ= 181.12 (C.sup.9,17), 145.63 (C.sup.10,13), 133.72 (C.sup.1,2), 132.55 (C.sup.4,5), 125.67 (C.sup.3,6), 124.52 (C.sup.7,8), 108.84 (C.sup.11,12), 62.89 (C.sup.24,28), 60.62 (C.sup.22,26), 50.24 (C.sup.35,36,37,38), 39.5 (C.sup.19,20 under the DMSO-d.sub.6 signal), 23.67 (C.sup.29,32), 22.73 (C.sup.21,25), 19.13 (C.sup.30,33), 13.40 (C.sup.31,34) ppm.

[0099] LC-MS m/z: 261.2 [M-2Br].sup.2+

[0100] HPLC: 97.07% 6.03 min

Example 4

Synthesis of N,N′-(((9,10-dioxo-9,10-dihydroanthracene-1,4-diyl)bis(azanediyl))bis(propane-3,1-diyl))bis(N,N-dimethyloctan-1-aminium) bromide (7b)

[0101] 0.65 g (1.33 mmol) of 1,4-bis[[2-(dimethylamino)propyl]amino]anthraquinone (5) is heated at reflux with 2.6 g (2.3 mL, 13.3 mmol) of 1-bromooctane (6b) in approximately 5 mL of acetone for 24 h. After cooling the solution to room temperature, the product is completely precipitated in hexane, filtered off, and washed with hexane. The blue solid is dried in high vacuum.

##STR00005##

[0102] Yield: 0.99 g (1.25 mmol) 94% of theory.

[0103] FT-IR (diamond): v= 3419 (br, υ.sub.NH), 3004 (w, υ.sub.Ar-H), 2953 (m, υ.sub.CH2), 2923 (m, υ.sub.CH2), 2854 (m, υ.sub.CH2), 1645 (w, υ.sub.c=o), 1595 (w, υ.sub.c=c), 1576 (m, υ.sub.c=c), 1588 (s, υ.sub.c=c), 1467 (m, δ.sub.CH2,CH3), 717 (s, υ.sub.Ar-H) cm.sup.-1.

[0104] .sup.1H NMR (300 MHz, DMSO-d.sub.6, RT): δ[ppm]= 10.79 (t, .sup.3J= 5.84 Hz, 2H, NH.sup.15,16), 8.25 (dd, .sup.3J= 5.90 Hz, 4J=3.30 Hz, 2H, Ar—H.sup.3,6), 7.82 (m, 2H, Ar—H.sup.1,2), 7.57 (s, 2H, Ar—H.sup.11,12), 3.55 (m, 4H, CH.sub.2.sup.19,20), 3.43 (m, 4H, CH.sub.2.sup.22,26), 3.29 (m, 4H, CH.sub.2.sup.24,28), 3.06 (s, 12H, NMe.sub.2.sup.35-38), 2.09 (m, 4H, CH.sub.2.sup.21,25), 1.64 (m, 4H, CH.sub.2.sup.29,32), 1.12-1.31 (m, 20H, CH.sub.2.sup.30,31,33,34,39-41,43-45), 0.82 (m, 6H, CH.sub.3.sup.42,46) ppm.

[0105] .sup.13C NMR (75 MHz, DMSO-d.sub.6, RT): δ= 181.10 (C.sup.9,17), 145.63 (C.sup.10,13), 133.74 (C.sup.1,2), 132.56 (C.sup.4,5), 125.71 (C.sup.3,6), 124.56 (C.sup.7,8), 108.84 (C.sup.11,12), 62.85 (C.sup.24,28), 60.38 (C.sup.22,26), 50.32 (C.sup.35-38), 39.5 (C.sup.19,20 under the DMSO-d.sub.6 signal), 31.16 (C.sup.40,44), 28.50 (C.sup.31,34,39,43), 25.80 (C.sup.30,33), 22.73 (.sub.C.sup.29,32), 22.02 (C.sup.21,25), 21.71 (C.sup.41,45), 13.94 (C.sup.42,46) ppm.

[0106] LC-MS m/z: 317.3 [M-2Br].sup.2+

[0107] HPLC: 96.98% 7.75 min

Example 5

Synthesis of N,N′=(((9,10-dioxo-9,10-dihydroanthracene-1,4-diyl)bis(azanediyl))bis(propane-3,1-diyl))bis(N,N-dimethyldecan-1-aminium) Bromide (7c)

[0108] 0.33 g (0.81 mmol) of 1,4-bis[[2-(dimethylamino)propyl]amino]anthraquinone (5) is heated at reflux with 1.69 mL (8.1 mmol) of 1-bromodecane (6c) in 5 mL of acetone for approximately 48 h. After cooling the solution to room temperature, the product is completely precipitated in hexane, filtered off, and washed with hexane. The blue solid is dried in high vacuum.

##STR00006##

[0109] Yield: 0.68 g (0.8 mmol) 99% of theory.

[0110] FT-IR (diamond): v= 3419 (br, υ.sub.NH), 3007 (w, υ.sub.Ar-H), 2948 (m, υ.sub.CH2), 2920 (m, υ.sub.CH2), 2852 (m, υ.sub.CH2), 1639 (w, υ.sub.c=o), 1595 (m, υ.sub.c=c), 1579 (m, υ.sub.c=c), 1562 (s, υ.sub.c=c), 1467 (m, δ.sub.CH2,CH3), 731 (s, υ.sub.Ar-H) cm.sup.-1.

[0111] .sup.1H NMR (300 MHz, DMSO-d.sub.6, RT): δ= 10.79 (t, .sup.3J= 5.73 Hz, 2H, NH.sup.15,16), 8.24 (dd, .sup.3J= 5.88 Hz, .sup.4J= 3.31 Hz, 2H, Ar—H.sup.3,6), 7.82 (m, 2H, Ar—H.sup.1,2), 7.57 (s, 2H, Ar—H.sup.11,12), 3.55 (m, 4H, CH.sub.2.sup.19,20), 3.43 (m, 4H, CH.sub.2.sup.22,26), 3.29 (m, 4H, CH.sub.2.sup.24,28), 3.06 (s, 12H, NMe.sub.2.sup.35-38), 2.08 (m, 4H, CH.sub.2.sup.21,25), 1.63 (m, 4H, CH.sub.2.sup.29,32), 1.12-1.32 (m, 28H, CH.sub.2.sup.30,31,33,34,39-43,45-49), 0.83 (m, 6H, CH.sub.3.sup.44,50) ppm.

[0112] .sup.13C NMR (75 MHz, DMSO-d.sub.6, RT): δ= 181.17 (C.sup.9,17), 145.70 (C.sup.10,13), 133.79 (C.sup.1,2), 132.62 (C.sup.4,5), 125.77 (C.sup.3,6), 124.62 (C.sup.7,8), 108.90 (C.sup.11,12), 62.87 (C.sup.24,28), 60.41 (C.sup.22,26), 50.39 (C.sup.35-38), 39.5 (C.sup.19,20 under the DMSO-d.sub.6 signal), 31.31 (C.sup.42,48), 28.57-28.94 (C.sup.31,34,39-.sup.41,45-.sup.47), 25.82 (C.sup.30,33), 22.75 (C.sup.29,32), 22.11 (C.sup.21,25), 21.74 (C.sup.43,49), 13.98 (C.sup.44,50) ppm.

[0113] LC-MS m/z: 345.4 [M-2Br].sup.2+

[0114] HPLC: 96.92% 8.57 min

[0115] Elemental analysis: theoretical values: C: 62.11; H: 8.77; N: 6.58 [0116] analysis results: C: 61.87; H: 8.72; N: 6.42

Example 6

Synthesis of N,N′(((9,10-dioxo-9,10-dihydroanthracene-1,4-diyl)bis(azanediyl))bis(propane-3,1-diyl))bis(N,N-dimethyldodecan-1-aminium) Bromide (7d)

[0117] 0.35 g (0.86 mmol) of 1,4-bis[[2-(dimethylamino)propyl]amino]anthraquinone (5) is heated at reflux with 0.85 g (0.89 mL, 3.43 mmol) of 1-bromododecane (6d) in approximately 2 mL of acetone for 24 h. After cooling the solution to room temperature, the product is completely precipitated in hexane, filtered off, and washed with hexane. The blue solid is dried in high vacuum.

##STR00007##

[0118] Yield: 0.71 g (0.79 mmol) 91% of theory.

[0119] FT-IR (diamond): v= 3425 (br, υ.sub.NH), 3006 (w, υ.sub.Ar-H), 2948 (m, υ.sub.CH2), 2919 (s, υ.sub.CH2), 2851 (m, υ.sub.CH2), 1639 (w, υ.sub.c=o), 1595 (m, υ.sub.c=c), 1580 (m, υ.sub.c=c), 1562 (s, υ.sub.c=c), 1468 (m, δ.sub.CH2,CH3), 731 (s, υ.sub.Ar-H) cm.sup.-1.

[0120] .sup.1H NMR (300 MHz, DMSO-d.sub.6, RT): δ= 10.78 (m, 2H, NH.sup.15,16), 8.25 (m, 2H, Ar—H.sup.3,6), 7.82 (m, 2H, Ar—H.sup.1,2), 7.56 (s, 2H, Ar—H.sup.11,12), 3.55 (m, 4H, CH.sub.2.sup.19,20), 3.21-3.46 (m, CH.sub.2.sup.22,26,24,28 obscured by the H.sub.2O signal), 3.05 (s, 12H, NMe.sub.2.sup.35-38), 2.08 (m, 4H, CH.sub.2.sup.21,25), 1.63 (m, 4H, CH.sub.2.sup.29,32), 1.11-1.32 (m, 36H, CH.sub.2.sup.30,31,33,34,39-51,53), 0.84 (m, 6H, CH.sub.3.sup.52,54) ppm.

[0121] .sup.13C NMR (75 MHz, DMSO-d.sub.6, RT): δ= 181.17 (C.sup.9,17), 145.65 (C.sup.10,13), 133.77 (C.sup.1,2), 132.61 (C.sup.4,5), 125.74 (C.sup.3,6), 124.54 (C.sup.7,8), 108.89 (C.sup.11,12), 62.80 (C.sup.24,28), 60.38 (C.sup.22,26), 50.38 (C.sup.35-38), 39.5 (C.sup.19,20 under the DMSO-d.sub.6 signal), 31.29 (C.sup.44,50), 28.53-29.1 (C.sup.31,34,39-43,45-49), 25.78 (C.sup.30,33), 22.70 (C.sup.29,32), 22.09 (C.sup.21,25), 21.69 (C.sup.51,53), 13.96 (C.sup.52,54) ppm.

[0122] LC-MS m/z: 373.4 [M-2Br].sup.2+

[0123] HPLC: 96.44% 9.79 min

Example 7

[0124] An ILM model substrate that is similar to the ILM was used to test the binding capacity of known dyes and of staining complexes according to the invention. Silk granules have been found to show good suitability as an ILM model substrate. To examine the binding of various dyes to the ILM model substrate, UV/vis measurements of the dyes Brilliant Blue G (BBG), compounds 7a-d, and Indocyanine green (ICG) were performed at various concentrations both before and after treatment with the model substrate. The aim of these measurements is to compare the binding strength of the individual dyes. The results are shown below and are each depicted in graph form in FIGS. 1 to 4.

[0125] Stock solutions of the dyes with a concentration of 5 mg/mL were prepared using phosphate-buffered saline (PBS) at physiological pH 7.4. Because of the poor solubility of ICG in saline solutions, double-distilled water was used. The concentrations prepared are shown in the Table.

TABLE-US-00001 Preparation of dye solutions for the UV/vis-monitored study of binding to ILM model substrate Concentration [mg/mL] Composition of dye solution 0.005 6000 .Math.L PBS + 6 .Math.L stock solution 0.01 5994 .Math.L PBS + 12 .Math.L stock solution 0.02 5982 .Math.L PBS + 24 .Math.L stock solution 0.04 5958 .Math.L PBS + 48 .Math.L stock solution 0.08 5910 .Math.L PBS + 96 .Math.L stock solution 0.16 5814 .Math.L PBS + 192 .Math.L stock solution

[0126] To ensure measurement is as accurate as possible, the dye-model substrate solutions and the references were not prepared separately, but likewise used as stock solutions. This was done by preparing two and a half times the amount required as shown in Table 1.

[0127] Subsequently, 6 mg of ILM model substrate plus 6 mL of dye solution and a reference solution comprising only 6 mL of dye solution were stirred for exactly 10 minutes and then centrifuged together for 8 minutes at 6000 rpm. The solutions were photographed and the carefully decanted supernatants analyzed by UV/vis spectroscopy. The ratios of the absorption maxima λ.sub.max with the model substrate to the absorption maximum λ.sub.max of the reference were calculated. This allowed the binding strengths of the individual dyes to be compared. Graphical plots of the results are shown in FIG. 1. The tabulated summary of the data obtained is shown below.

A) UV/Vis Measurements of the Vital Dye Brilliant Blue G (BBG) (Comparison)

[0128] As previously explained, Brilliant Blue G is a dye commonly used in chromovitrectomy which selectively stains the inner limiting membrane. This means that good staining and a consequent pronounced decrease in its absorption maximum after treatment with a model substrate would also be expected in binding studies with a model substrate.

[0129] As can be demonstrated by FIG. 1, the expected good binding to the model substrate is confirmed. Only the strongest absorption maximum, which is responsible for imparting the blue color, was considered here. The maximum of the references is at 585 nm here. The samples treated with the model substrate show a slight shift in the maximum which, as the concentration increases, approaches that of the reference. The absorption of the references is considerably more intense than that of the samples treated with the model substrate. This suggests a good interaction between the dye and the model substrate. Table 2 shows the absorption maxima of each dye solution. The ratio of λ.sub.max with the model substrate to λ.sub.max of the associated reference is confirmed by the graphical plot of the binding strength. At a concentration of 0.005 mg/mL, the sample treated with model substrate shows only 24% of the reference maximum. This equates to 76% binding of the dye. Binding decreases as the concentration of the solution increases. At a concentration of 0.08 mg/mL, binding of only 48% of the dye can be demonstrated.

TABLE-US-00002 UV/vis spectroscopy data obtained for the BBG reference and BBG model substrate (MS) solution at concentrations of 0.005 to 0.08 mg/mL Concentration [mg/mL] λ.sub.max Reference (585 nm) λ.sub.max With MS (x nm) Ratio λ.sub.max with MS and reference 0.005 0.1241 0.0299 (603 nm) 24% 0.01 0.3755 0.1217 (595 nm) 32% 0.02 0.835 0.3672 (589 nm) 44% 0.04 1.6741 0.8006 (587 nm) 48% 0.08 3.2859 2.0313 (585 nm) 62%

B) UV/Vis Measurements of Anthraquinone Dyes 7a-d (According to the Invention)

[0130] The anthraquinone dyes 7a-d according to the invention were likewise tested in respect of their strength of binding to the model substrate. Because of the lower absorption maximum compared with the known BBG, 6 concentrations from 0.005 mg/mL to 0.16 mg/mL were measured in this case. Graphical plots of the UV/vis spectroscopy data obtained are shown in FIGS. 2 and 3.

[0131] FIGS. 2 and 3 demonstrate clearly the very good binding of anthraquinone dyes 7b-d to the model substrate. Since the anthraquinone dyes have two absorption maxima, the graphs show two reference lines and two sample lines. The absorption maxima are at 588 nm and 632-635 nm. The reference absorption maxima here are well above those of the values treated with model substrate. Higher concentrations could not be recorded, because the absorption of the reference was too high. Table 3 shows the ratios of the λ.sub.max values of the MS samples in relation to the corresponding references. The exact measured data obtained for the UV/vis spectroscopy investigations are shown in Tables 4 to 7. As the length of the alkyl radicals increases, the interaction of the dyes with the model substrate is heightened. Whereas compound 7a with a butyl radical has λ.sub.max ratios only of between approximately 50 to 80%, improved values can be achieved with dyes 7b-d. What is however clear is that, as the length of the alkyl radical increases, the extinction coefficient of the dye also decreases. This is a consequence both of the molecular weight of the compound and of the increasing hydrophobic character.

TABLE-US-00003 UV/vis spectroscopy data obtained for the 7 references and 7 MS solution at concentrations of 0.005 to 0.16 mg/mL Concentration [mg/mL] 7a 7b 7c 7d Ratio λ.sub.max with MS and reference 588 nm 632 nm 588 nm 634 nm 588 nm 635 nm 588 nm 634 nm 0.005 54% 54% 11% 9% 48% 37% 12% 13% 0.01 52% 52% 10% 8% 21% 17% 7% 8% 0.02 54% 55% 6% 6% 7% 7% 3% 4% 0.04 57% 58% 8% 8% 4% 5% 3% 3% 0.08 67% 69% 18% 19% 8% 10% 1% 2% 0.16 76% 78% 32% 34% 13% 17% 18% 18%

[0132] Table 4: UV/vis spectroscopy data obtained for the 7a references and 7a MS solutions at concentrations of 0.005 to 0.16 mg/mL

##STR00008##

TABLE-US-00004 Concentration [mg/mL] λ.sub.max Reference (588 nm) λ.sub.max With MS (588 nm) λ.sub.max Reference (632 nm) λ.sub.max With MS (632 nm) Ratio λ.sub.max with MS and reference 588 nm 632 nm 0.005 0.0932 0.0502 0.1159 0.0624 54% 54% 0.01 0.2000 0.1032 0.2448 0.1268 52% 52% 0.02 0.3868 0.2103 0.4640 0.2560 54% 55% 0.04 0.7611 0.4333 0.8862 0.5177 57% 58% 0.08 1.500 1.0086 1.6571 1.1524 67% 69% 0.16 2.8594 2.1625 2.9961 2.3477 76% 78%

[0133] Table 5: UV/vis spectroscopy data obtained for the 7b references and 7b MS solutions at concentrations of 0.005 to 0.16 mg/mL

##STR00009##

TABLE-US-00005 Concentration [mg/mL] λ.sub.max Reference (588 nm) λ.sub.max With MS (588 nm) λ.sub.max Reference (634 nm) λ.sub.max With MS (634 nm) Ratio λ.sub.max with MS and reference 588 nm 634 nm 0.005 0.0733 0.0078 0.0930 0.0082 11% 9% 0.01 0.1508 0.0150 0.1890 0.0159 10% 8% 0.02 0.3142 0.0201 0.3881 0.0232 6% 6% 0.04 0.6307 0.0496 0.7606 0.0609 8% 8% 0.08 1.2728 0.2326 1.4791 0.2873 18% 19% 0.16 2.4402 0.7719 2.6985 0.9204 32% 34%

[0134] Table 6: UV/vis spectroscopy data obtained for the 7c references and 7c MS solutions at concentrations of 0.005 to 0.16 mg/mL

##STR00010##

TABLE-US-00006 Concentration [mg/mL] λ.sub.max Reference (588 nm) λ.sub.max With MS (588 nm) λ.sub.max Reference (635 nm) λ.sub.max With MS (635 nm) Ratio λ.sub.max with MS and reference 588 nm 635 nm 0.005 0.0503 0.0243 0.0633 0.0234 48% 37% 0.01 0.124 0.0267 0.1476 0.0261 21% 17% 0.02 0.2708 0.0205 0.2786 0.0216 7% 7% 0.04 0.5298 0.0218 0.5068 0.0257 4% 5% 0.08 1.0636 0.0799 0.9575 0.0977 8% 10% 0.16 2.2754 0.2842 1.9492 0.3300 13% 17%

[0135] Table 7: UV/vis spectroscopy data obtained for the 7d references and 7d MS solutions at concentrations of 0.005 to 0.16 mg/mL

##STR00011##

TABLE-US-00007 Concentration [mg/mL] λ.sub.max Reference (588 nm) λ.sub.max With MS (588 nm) λ.sub.max Reference (634 nm) λ.sub.max With MS (634 nm) Ratio λ.sub.max with MS and reference 588 nm 634 nm 0.005 0.0369 0.0043 0.0304 0.0041 12% 13% 0.01 0.1045 0.0074 0.0845 0.0071 7% 8% 0.02 0.2440 0.0081 0.1949 0.0080 3% 4% 0.04 0.3649 0.0101 0.2895 0.0100 3% 3% 0.08 0.8039 0.0120 0.6368 0.0120 1% 2% 0.16 1.8155 0.3218 1.4306 0.2580 18% 18%

C) UV/Vis Measurements of the Vital Dye Indocyanine Green (ICG)

[0136] Indocyanine green is one of the most important dyes in chromovitrectomy, particularly in the field of ILM peeling. The dye does, however, give rise to some problems and also to handling difficulties. Although it has long since been used for staining the inner limiting membrane and epiretinal membranes, studies have shown the dye to have numerous disadvantages. In addition to postoperative deterioration of vision, low stability in the presence of light is a serious problem when working with the dye. Moreover, ICG cannot be measured or used in phosphate-buffered saline, as this causes the compound to precipitate. This means that the subsequent measurements also had to be performed in double-distilled water, since measurements in buffer solution showed only settling-out of the dye after centrifugation. Graphical plots of the UV/vis data obtained are shown in FIG. 4.

[0137] As is clear from the data presented, ICG showed some problems in the present measurements too. Whereas the dye showed virtually no absorption up to a concentration of 0.02 mg/mL, a concentration of 0.04 mg/mL was sufficient for Indocyanine green to attain an absorption of 3.5822 and thus the maximum possible measured value. Use of a higher concentration was not possible, because of the excessively high absorption and consequent inaccurate measurement. As can be seen from Table 8, although the ratios of the λ.sub.max values of the reference and of the model substrate-dye solution revealed positive binding of the dye to the model substrate, the results are subject to substantial variation within the margin of error due to the low concentration. This may be a consequence both of the weak absorption at low concentrations and of photolytic decomposition. During the measurement method performed, it was not possible to ensure the complete exclusion of light.

TABLE-US-00008 UV/vis spectroscopy data obtained for the ICG reference and ICG MS solution at concentrations of 0.005 to 0.04 mg/mL Concentration [mg/mL] λ.sub.max Reference (779 nm) λ.sub.max With MS (780 nm) Ratio λ.sub.max with MS and reference 0.005 0.0226 0.0106 47% 0.01 0.0162 0.0154 95% 0.02 0.0477 0.0204 42% 0.04 3.5822 3.0389 85%

Example 8

[0138] Some example syntheses and schemes for the preparation of the dyes are shown below. The 6-methyl-substituted derivative of 1,4-difluoroanthraquinone was used, which was synthesized from 4-methylphthalic anhydride and 1,4-difluorobenzene and then had to undergo oxidation. This method permits modification of the side groups without potential side reactions due to the acid. It also affords the possibility of a simplified purification. Subsequent oxidation of the methyl group in the 6-position should result in the desired acid group. The synthesis of 1,4-difluoro-6-methylanthraquinone (14) is shown in Scheme 1.

##STR00012##

Scheme 1: Synthesis of 1,4-difluoro-6-methylanthraquinone (14)

[0139] The reaction was carried out in analogous manner to the synthesis of 1,4-difluoroanthraquinone (1). After purification by column chromatography, the successful synthesis of compound 14 in low yields of around 20% was verified by 1H NMR, 13C NMR, and IR spectroscopy, and by elemental analysis. This was followed by modification with N,N-dimethylaminopropylamine (4) to give the difunctionalized product 1,4-bis((3-(dimethylamino)propyl)amino)-6-methylanthraquinone (15) (Scheme 2). After purification of the product mixture by column chromatography, compound 15 was verified unambiguously by 1H NMR. In this case too, the protons vicinal to fluorine give rise to a singlet after successful conversion to dye 15.

##STR00013##

Scheme 2: Reaction of 1,4-difluoro-6-methylanthraquinone (14) with N,N-dimethylaminopropylamine (4) to dye 10

[0140] Because the higher selectivity of the quaternary anthraquinone dye having a C10-substituent was already known at this time, quaternization of the doubly modified 1,4-difluoro-6-methylanthraquinone 15 was carried out only with 1-bromodecane. Scheme 3 shows the reaction carried out.

##STR00014##

Scheme 3: Reaction of the difunctionalized 1,4-difluoro-6-methylanthraquinone (15) with 1-bromodecane (6c)

[0141] The successful synthesis of anthraquinone dye 16 was verified unambiguously by.sup.1H NMR. The success of the synthesis is indicated by the shift of the signals for the amine-bound methyl groups from 2.21 ppm to 3.04 ppm. Subsequent oxidation of the methyl group in compound 16 by potassium permanganate with sodium carbonate and the phase-transfer catalyst “Aliquat 336” in dichloromethane/water (1:1) was unsuccessful. A further attempt to obtain the carboxyl-modified anthraquinone compound was accordingly made directly from trimellitic anhydride (17) and 1,4-difluorobenzene (1). The reaction carried out is shown in Scheme 4. 5,8-Difluoro-9,10-dioxo-9,10-dihydroanthracene-2-carboxylic acid (18) was verified by 1H NMR spectroscopy and by mass spectrometry.

##STR00015##

Scheme 4: Synthesis of the dye 5,8-difluoro-9,10-dioxo-9,10-dihydroanthracene-2-carboxylic acid, modified with an acid group in the 6-position (18)

[0142] The further conversion to 19 was carried out in analogous manner to the earlier approach by reaction with N,N-dimethylaminopropylamine (4) and is shown in Scheme 5.

##STR00016##

Scheme 5: Reaction of the carboxyl-modified 1,4-difluoroanthraquinone (18) with N,N-dimethylaminopropylamine (4) to give 5,8-bis((3-(dimethylamino)propyl)amino)-9,10-dioxo-9,10-dihydroanthracene-2-carboxylic acid

[0143] The successful synthesis of the anthraquinone dye 5,8-bis((3-(dimethylamino)propyl)amino)-9,10-dioxo-9,10-dihydroanthracene-2-carboxylic acid (19) is confirmed by 1H NMR spectroscopy and mass spectrometry analyses. The final step to incorporate the positive charge by reaction with 1-bromodecane was unsuccessful. Numerous side reactions interfere with the reaction, preventing product formation. Subsequent purification and isolation of the product is likewise made more difficult by the positive charge. This meant it was not possible to successfully verify the target structure.

Example 9 Polymeric Anthraquinone Dyes

[0144] The synthesis of additional polymeric structures based on the compound N,N′-(((anthraquinone-1,4-diyl)bis(azanediyl))bis(propane-3,1-diyl))bis(N,N-dimethyldecan-1-aminium) bromide (7c) was attempted. When used in the body, polymers prevent diffusion into deeper cell layers, thereby preventing toxic effects. Because of the high reactivity of difluoroanthraquinone toward primary amines, polyvinylamine (PVA) was used as the polymeric base structure. To attach the dye to the polymer, a monofunctionalized derivative was accordingly used that is formed as a side product in the synthesis of compound 5 and can be isolated in clean form through purification by column chromatography. Use of equimolar amounts of the two starting materials as shown in Scheme 6 allows compound 20 to be isolated in good yields.

##STR00017##

Scheme 6: Synthesis of the monofunctionalized dye 1-((3-(dimethylamino)propyl)amino)-4-fluoroanthraquinone (20)

[0145] The successful synthesis of compound 20 was verified by 1H NMR and .sup.13C NMR spectroscopy. By comparison with the difunctionalized derivative, the aromatic protons vicinal to fluorine can be shown unambiguously to give rise to two signals, as they are not chemically equivalent. In addition, the signals show higher multiplicity, as coupling to the fluorine atom can likewise be observed. In the .sup.1H NMR spectrum, the hydrogen atom vicinal to the fluorine atom shows two .sup.3J couplings, whereas the second hydrogen atom gives rise both to a .sup.3J coupling and a .sup.4J coupling. This allows unambiguous assignment of the signals.

[0146] Further reaction was in analogous manner to compounds 7a-d. This was accomplished by reacting the monofunctionalized dye 20 with 1-bromodecane (6c) in acetone. Here too, the product precipitates in the reaction and can be separated off by filtration. The successful synthesis was verified by .sup.1H NMR spectroscopy. The reaction is shown in Scheme 7.

##STR00018##

Scheme 7: Synthesis of monofunctionalized dye 21

[0147] Attachment to a polymeric structure was achieved by virtue of the free fluorine atom in compound 21 via the primary amines in the polyvinylamine (PVA). The reaction is carried out in THF/H.sub.2O containing traces of triethylamine, to prevent possible protonation of the amino groups. The polymer-analogous attachment to 21 via the amine means that the polymer formed is subsequently blue. The reaction scheme is shown below.

##STR00019##

Scheme 8: Reaction of monofunctionalized compound 21 with PVA (22)

[0148] The anthraquinone dye was incorporated into the polymer in contents of 10% and 20% by weight. The course of the reaction can be monitored with the naked eye, through the color change brought about by the substitution, and by thin-layer chromatography. The incomplete conversion means that the polymer still contains free amino groups, which interfere both with binding to the tissue to be stained and with GPC monitoring of the molecular weight. No further methods of analysis were therefore employed. The evaluation of binding strength is shown in section 3.1.2.