Acetalanthraquinone derivatives useful for cellular staining

Abstract

The present invention provides di-substituted acetalanthraquinone-based compounds of the Formulae (l)-(IV) that are useful for cellular staining and for light-based detection of biological material, e.g. fluorescence-based detection of biological material. These compounds may be used in hydrolysed or non-hydrolysed form. Also provided is a fluorescent complex that comprises a nucleic acid and an acetalanthraquinone-based compound of the invention; and a method of staining a biological sample comprising cells or other biological material containing nucleic acid, which method comprises contacting the biological sample with an acetalanthraquinone-based compound of the invention. In the Formulae (l)-(IV), A and B are each independently of formula —R.sup.aCH.sub.2CH(OR.sup.b).sub.2; or one of A and B is of formula —R.sup.aCH.sub.2CH(OR.sup.b).sub.2 and the other one is of formula: —NR.sup.b.sub.2. ##STR00001##

Claims

1. A compound of any one of Formulae (I), (Ib), (Id), (II), (III) or (IV), as defined below: ##STR00040## wherein n and m are independently selected from 1, 2, 3, 4, 5, and 6, ##STR00041## wherein m is 1, 2, 3, 4 or 5, ##STR00042## wherein n and m are the same and selected from 1, 2, 3, 4, 5, and 6, ##STR00043## wherein n and m are independently selected from 1, 2, 3, 4, 5, and 6, ##STR00044## wherein n and m are independently selected from 1, 2, 3, 4, 5, and 6, ##STR00045## wherein R.sub.1 and R.sub.2, when present, are each independently selected from —OH or H, or CH.sub.3, halo or OR, wherein R is selected from —CH.sub.3, —CH.sub.2OCH.sub.3, —CH(CH.sub.3).sub.2, —COCH.sub.3, —CH.sub.2CH═CH.sub.2, benzene and benzyl; and either: (i) A and B are each independently of formula: —R.sup.aCH.sub.2CH(OR.sup.b).sub.2; or (ii) one of A and B is of formula: —R.sup.aCH.sub.2CH(OR.sup.b).sub.2 and the other one is of formula: —NR.sup.b.sub.2; wherein the or each R.sup.a is independently selected from —CH.sub.2—, —C(═O)NH—, —NHC(═O)—, —C(═O)—, —C(═CH.sub.2)—, —C(═CHCH.sub.3)—, —N(CH.sub.3)—, and —N(alkylaryl)- and each R.sup.b is independently selected from —CH.sub.3, —CH.sub.2CH.sub.3, —CH(CH.sub.3).sub.2, and —CH.sub.2CH═CH.sub.2.

2. The compound of claim 1, wherein R.sub.1 and R.sub.2 are the same.

3. The compound of claim 2, wherein R.sub.1 and R.sub.2 are both H.

4. The compound of claim 2, wherein R.sub.1 and R.sub.2 are both OH.

5. The compound of claim 1, wherein the or each R.sup.a is independently selected from —CH.sub.2—, —C(═O)NH—, —NHC(═O)—, —C(═O)—, —C(═CH.sub.2)—, and —C(═CHCH.sub.3)—.

6. The compound of claim 5, wherein the or each R.sup.a is independently selected from —CH.sub.2—, —C(═O)NH—, and —NHC(═O)—.

7. The compound of claim 1, wherein the or each R.sup.b is independently selected from —CH.sub.3, —CH.sub.2CH.sub.3, and —CH.sub.2CH═CH.sub.2.

8. The compound of claim 7, wherein the or each R.sup.b is —CH.sub.3.

9. The compound of claim 1, wherein A and B are each independently of formula: R.sup.aCH.sub.2CH(OR.sup.b).sub.2; and m and n are the same.

10. The compound of claim 9, wherein m and n are 1 or 2.

11. The compound of claim 1, wherein A and B are each independently of formula: R.sup.aCH.sub.2CH(OR.sup.b).sub.2; and A and B have the same R.sup.a group.

12. The compound of claim 11, wherein each R.sup.a is selected from —CH.sub.2—, —C(═O)NH—, and —NHC(═O)—.

13. The compound of claim 1, wherein A and B are each independently of formula: —R.sup.aCH.sub.2CH(OR.sup.b).sub.2; and A and B have the same R.sup.b group.

14. The compound of claim 13, wherein each R.sup.b is —CH.sub.3.

15. The compound of claim 1, wherein A and B have the formula: —R.sup.aCH.sub.2CH(OR.sup.b).sub.2; and A and B are the same.

16. The compound of claim 1, wherein A is of formula: —NR.sup.b.sub.2 and B is of formula —R.sup.aCH.sub.2CH(OR.sup.b).sub.2, m is 1 or 2 or 3 or 4 or 5 and n is 1 or 2 or 3.

17. The compound of claim 16, wherein m is 1 or 2 or 3 or 4 or 5 and n is 2.

18. The compound of claim 1, wherein one of A and B is of formula: —R.sup.aCH.sub.2CH(OR.sup.b).sub.2 and the other one is of formula: —NR.sup.b.sub.2; and A and B have the same R.sup.b group.

19. The compound of claim 18, wherein R.sup.b is CH.sub.3.

20. A fluorescent complex comprising a nucleic acid and a compound as defined in claim 1.

21. A method of analysing a biological sample comprising cells, or other biological material containing nucleic acid, the method comprising the steps of: a) preparing a biologically compatible solution containing a compound as defined in claim 1; b) treating a biological sample with the biologically compatible solution; and c) detecting a spectroscopic property associated with the absorption of electromagnetic radiation by the compound.

22. The method of claim 21 wherein the spectroscopic property associated with absorption of electromagnetic radiation by the compound is: (i) fluorescence, or (ii) a colorimetric property, or (iii) a photoacoustic property, or (iv) a Raman scattering property.

23. A method of staining a biological sample comprising cells or other biological material containing nucleic acid, which method comprises contacting the biological sample with a compound as defined in claim 1.

24. The method of claim 23, wherein the method comprises contacting the biological sample with the compound and then: carrying out a fluorescence-based detection step; or carrying out a Raman scattering-based detection step; or carrying out a photoacoustic-based detection step.

25. The method of claim 23, which comprises the steps of: a) preparing a biologically compatible solution containing the compound for contacting the biological sample; b) treating a biological sample with the biologically compatible solution for a time sufficient to cause staining; c) illuminating the stained sample with a light source sufficient to excite the compound; and d) detecting the emitted fluorescence from the sample using a fluorescence detection instrument.

26. A method of quantifying nucleic acid content in live cells, comprising: combining a compound as defined in claim 1 with a sample to form a mixture, wherein the sample comprises a nucleic acid molecule; incubating the mixture for a sufficient amount of time for the compound to associate with the nucleic acid in the sample; illuminating the incubated sample with an appropriate wavelength to generate a detectable optical response or photoacoustic response; and detecting the presence of nucleic acid in the sample by flow cytometric analysis, image cytometry analysis, image analysis including, or high content image analysis.

27. A kit for carrying out an assay, which includes a compound as defined in claim 1, and further including one or more of the following: buffering agents, sample preparation reagents, additional detection reagents, organic solvent, other fluorescent detection probes, standards, microspheres, specific cell lines, antibodies and/or instructions for carrying out an assay.

Description

(1) Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

(2) FIG. 1A shows absorption (line) and fluorescence (dots) spectra of compounds BST-108, BST-109, BST-110 and BST-111. The fluorescence spectra was obtained at excitation wavelengths of 610 nm; 584 nm; 583 nm; 610 nm respectively, representing peak excitation wavelengths.

(3) FIG. 1B shows absorption (line) and fluorescence (dots) spectra of compounds BST-101, BST-100. BST-102 and BST-106. The fluorescence spectra was obtained at excitation wavelengths of 540 nm; 584 nm; 585 nm; 584 nm respectively, representing peak excitation wavelengths.

(4) FIG. 2 shows staining of fixed cells. The figure shows fluorescence microscopic images of α-2 OS cells stained for 1 hour with different compounds according to the present invention and with DRAQ5 as a reference.

(5) FIG. 3 shows staining of live cells. The figure shows fluorescent microscopic images of A549 cells stained with different compounds according to the present invention and with DRAQ5 as a reference.

(6) FIG. 4 shows fluorescence analysis via direct confocal imaging of compounds BST-100, BST-101 and BST-106.

(7) FIG. 5 shows fluorescence analysis via direct confocal imaging of compounds BST-108, BST-109.

(8) FIG. 6 shows analysis of A549 cells treated with compounds BST-101, BST-109, BST-110 after 72 hours, in terms of (a) total signal of probe per cell, (b) cell cycle distribution across the cell compartments G1, S and G2, and (c) cell viability.

(9) FIG. 7 shows K-S statistical analysis of compounds BST-106, BST-108, BST-111, and hydrolysed forms thereof, uptake in Aldefluors buffer.

EXAMPLES

(10) Production of the Compounds of the Invention

(11) Compounds of Formula (I)-(IV) can be synthesized in accordance with, or in analogy to, the general routes described in the below Schemes:

(12) ##STR00010##

(13) ##STR00011##

(14) ##STR00012##

(15) The compounds of Formula (I)-(IV) can be obtained by following the processes described below. Unless otherwise stated, in the methods described below the meanings of the substituent groups are the meanings described above with regard to a compound of Formula (I)-(IV).

(16) The following compounds according to the invention were prepared and tested in the examples:—

(17) ##STR00013## ##STR00014##

Example 1—Synthesis of Symmetrical 1,4-Di-Substituted-Anthraquinones (Formula (I))

(18) The following specific compounds were synthesized:

Example 1a

1,4-Bis(5,5-dimethoxypentylamino)anthracene-9,10-dione (BST-101)

(19) ##STR00015##

(20) To a stirred solution of 1,4-difluoro-anthraquinone (74 mg, 0.29 mmol) in pyridine (1 mL) was added 5,5-dimethoxypentanamine (261 mg, 1.72 mmol) and the resulting solution was stirred at 70° C. for 3 h. The solvent was evaporated under reduced pressure and the reaction mixture was purified by flash chromatography using gradient eluent (EtOAc:PE, 0:100.fwdarw.3:7) to yield the title compound as a dark blue powder (62 mg, 44%). δ.sub.H (CDCl.sub.3, 400 MHz) 8.24 (2H, dd, J 3.5, 6.0 Hz), 7.6 (2H, dd, J 3.5, 6.0 Hz), 7.14 (2H, s), 4.32 (2H, t, J 5.6), 3.62 (4H, t, J 6.5 Hz), 3.36 (4H, t, J 5.5 Hz), 3.25 (12H, s, O(CH.sub.3).sub.2), 1.75-1.45 (12H, m); δ.sub.C (CDCl.sub.3, 100 MHz) 181.3, 145, 133.5, 130.7, 125.3, 122.1, 108.7, 103.4, 51.7, 41.2, 30.8, 28.5, 21.1; m/z (ES+) 499 (M+H, 100).

Example 1b

3,3′-(9,10-Dioxo-9,10-dihydroanthracene-1,4-diyl)bis(azanediyl)bis(N-(2,2-dimethoxyethyl)propanamide) (BST-102)

(21) ##STR00016##

(22) To a stirred solution of 1,4-difluoro-anthraquinone (80 mg, 0.33 mmol) in pyridine (1 mL) was added 3-amino-N-(2,2-dimethoxyethyl)propanamide (345.7 mg, 1.96 mmol) and the resulting solution was stirred at 70° C. for 4 h. The solvent was evaporated under reduced pressure and the reaction mixture was purified by flash chromatography using gradient eluent (CH.sub.3OH:CH.sub.2Cl.sub.2, 1:99.fwdarw.4:9 to yield the title compound as a dark blue powder (107 mg, 59%). δ.sub.H (CDCl.sub.3, 400 MHz) 8.15 (2H, dd J 3.5, 6.0 Hz), 7.6 (2H, dd J 3.5, 6.0 Hz), 7.12 (2H, s), 4.32 (2H, t, J 5.6 Hz), 3.61 (4H, t, J 6.5 Hz), 3.37 (4H, t, J 5.6 Hz), 3.28 (12H, s, O(CH.sub.3).sub.2), 2.53 (4H, t, J 6.5 Hz); δ.sub.C (CDCl.sub.3, 100 MHz) 182.2, 170.8, 145.6, 134.2, 132.1, 126.0, 123.3, 110.3, 102.8, 54.2, 41.2, 36.5, 39.1; m/z (ES+) 557 (M+H, 100).

Example 1c

N,N′-(2,2′-(9,10-Dioxo-9,10-dihydroanthracene-1,4-diyl)bis(azanediyl)bis(ethane-2,1-diyl))bis(3,3-dimethoxypropanamide) (BST-103)

(23) ##STR00017##

(24) To a stirred solution of 1,4-difluoro-anthraquinone (80 mg, 0.33 mmol) in pyridine (1 mL) was added N-(2-aminoethyl)-3,3-dimethoxypropanamide (346 mg, 1.96 mmol) and the resulting solution was stirred at 70° C. for 4 h. The solvent was evaporated under reduced pressure and the reaction mixture was purified by flash chromatography using gradient eluent (CH.sub.3OH:CH.sub.2Cl.sub.2, 1:99.fwdarw.4:9). After the chromatographic purification residual acidic aliphatic impurities were removed by washing with 0.1 M NaOH (2×10 mL), and brine (1×10 mL) before being dried over MgSO.sub.4. After filtration, the solvent was concentrated in vacuo to yield the title compound as a dark blue powder (25 mg, 29%). δ.sub.H (CDCl.sub.3, 400 MHz) 8.10 (2H, dd, J 3.5, 6.0 Hz), 7.57 (2H, dd, J 3.0, 5.5 Hz), 7.06 (1H, br s, NH), 6.98 (2H, s), 5.20 (2H, s), 4.69 (2H, t, J 5.6 Hz), 3.51 (4H, t, J 6.0 Hz), 3.25 (12H, s, O(CH.sub.3).sub.2), 2.60 (4H, d, J 5.6 Hz); δ.sub.C (CDCl.sub.3, 100 MHz) 182.2, 169.9, 168.3, 134.2, 132.1, 126.0, 123.2, 109.9, 102.3, 54.3, 42.1, 40.9, 39.2; m/z (ES+) 557 (M+H, 100).

Example 2—Synthesis of Non-Symmetrical 1,4-Di-Substituted-Anthraquinones (Formula (I))

(25) The following specific compounds were synthesized:

Example 2a

1-(5,5-Dimethoxypentylamino)-4-(2-(dimethylamino)ethylamino) anthracene-9,10-dione (BST-106)

(26) ##STR00018##

(27) To a stirred solution of 1-(2-(dimethylamino)ethylamino)-4-fluoroanthracene-9,10-dione (60 mg, 0.19 mmol) in pyridine (1 mL) was added 5,5-dimethoxypentan-1-amine (113 mg, 0.77 mmol) and the resulting solution was stirred at 70° C. for 4 h. The solvent was evaporated under reduced pressure and the reaction mixture was purified by flash chromatography using gradient eluent (CH.sub.3OH:CH.sub.2Cl.sub.2, 1:99.fwdarw.4:9) to yield the title compound as a dark blue powder (59 mg, 70%). δ.sub.H (CDCl.sub.3, 400 MHz) 10.7 (2H, br s, NH), 8.3 (2H, dd, J 4.0, 5.5 Hz), 7.6 (2H, dd, J 4.0, 5.5 Hz), 7.3 (2H, dd, J 6.0, 15.6 Hz), 4.32 (1H, t, J 5.6 Hz), 3.53 (2H, t, J 5.6 Hz), 3.33 (2H, t, J 6.5 Hz), 3.26 (6H, s), 2.72 (2H, t, J 6.5 Hz), 2.33 (6H, s), 1.7 (2H, m), 1.63 (2H, m), 1.42 (2H, m); δ.sub.C (CDCl.sub.3, 100 MHz) 182.7, 182.4, 146.6, 145.6, 134.5, 134.4, 132.1, 132.0, 126.2, 126.0, 123.6, 123.5, 110.2, 109.8, 104.4, 58.4, 52.9, 45.4, 45.39, 42.8, 40.7, 32.3, 29.5, 22.3; m/z (ES+) 440 (M+H, 100).

Example 2b

N-(2,2-Dimethoxyethyl)-3-(4-(2-(dimethylamino)ethylamino)-9,10-dioxo-9,10-dihydroanthracen-1-ylamino)propanamide (BST-109)

(28) ##STR00019##

(29) To a stirred solution of 1-(2-(dimethylamino)ethylamino)-4-fluoroanthracene-9,10-dione (60 mg, 0.19 mmol) in pyridine (1 mL) was added 3-amino-N-(2,2-dimethoxyethyl)propanamide (135 mg, 0.77 mmol) and the resulting solution was stirred at 70° C. for 6 h. The solvent was evaporated under reduced pressure and the reaction mixture was purified by flash chromatography using gradient eluent (CH.sub.3OH:CH.sub.2Cl.sub.2, 1:99.fwdarw.6:94) to yield the title compound as a dark blue powder (56 mg, 70%). δ.sub.H (CDCl.sub.3, 400 MHz) 10.6 (2H, br s, NH), 8.2 (2H, d, J 6.0 Hz), 7.5 (2H, d, J 5.5 Hz), 5.95 (1H, br s, NH), 4.37 (1H, t, J 5.3 Hz), 3.71, (2H, t, J 5.5 Hz), 3.5 (2H, t, J 5.5 Hz), 3.3 (2H, t, J 6.5 Hz), 3.25 (6H, s), 2.76 (2H, t, J 6.5 Hz), 2.54 (2H, t, J 5.5 Hz), 2.41 (6H, s); δ.sub.C (CDCl.sub.3, 100 MHz) 181.7, 181.7, 169.7, 144.7, 144.5, 133.3, 131.1, 125.1, 124.9, 122.5, 122.3, 109.2, 109.2, 101.5, 57.0, 53.4, 44.1, 40.0, 39.2, 38.1, 35.8; m/z (ES+) 469 (M+H, 100).

Example 2c

N-(2-(4-(2-(dimethylamino)ethylamino)-9,10-dioxo-9,10-dihydroanthracen-1-ylamino)ethyl)-3,3-dimethoxypropanamide (BST-110)

(30) ##STR00020##

(31) To a stirred solution of 1-(2-(dimethylamino)ethylamino)-4-fluoroanthracene-9,10-dione (50 mg, 0.16 mmol) in pyridine (1 mL) was added N-(2-aminoethyl)-3,3-dimethoxypropanamide (112 mg, 0.64 mmol) and the resulting solution was heated under reflux for 4 h. The solvent was evaporated under reduced pressure and the reaction mixture was purified by flash chromatography using gradient eluent (CH.sub.3OH:CH.sub.2Cl.sub.2, 1:99.fwdarw.6:94) to yield the title compound as a dark blue powder (45 mg, 60%). δ.sub.H (CDCl.sub.3, 400 MHz); 10.7 (1H, br s, NH), 10.6 (1H, br s, NH), 8.2 (2H, dq, J 4.5, 9.0 Hz), 7.64 (2H, dd, J 2.0, 4.0 Hz), 7.2 (2H, dd, J 9.6, 25.2 Hz), 6.71 (1H, NH br s), 4.6 (1H, t, J 5.5 Hz), 3.5 (2H, t, J 5.5 Hz), 3.2 (6H, s), 2.73 (2H, t, J 6.5 Hz), 2.59 (2H, t, J 6.5 Hz), 2.43 (2H, t, J 5.5 Hz), 2.37 (6H, s); δ.sub.C (CDCl.sub.3, 100 MHz) 182.7, 182.4, 169.8, 169.2, 169.1, 146.0, 145.9, 134.4, 134.3, 132.2, 126.0, 125.9, 123.4, 110.0, 102.3, 102.2, 101.9, 58.3, 54.1, 51.4, 45.4, 41.9, 40.7, 39.4, 37.0; m/z (ES+) 469 (M+H, 100).

Example 3—Synthesis of Symmetrical 1,4-di-substituted-5,8-dihydroxy-anthraquinones (Formula (I))

(32) The following specific compounds were synthesized:

Example 3a

1,4-Bis(5,5-dimethoxypentylamino)-5,8-dihydroxyanthracene-9,10-dione (BST-100)

(33) ##STR00021##

(34) To a stirred solution of 1,4-difluoro-5,8-dihydroxyanthracene-9,10-dione (47 mg, 0.17 mmol) in pyridine (1 mL) was added 5,5-dimethoxypentan-1-amine (200 mg, 1.36 mmol) and the resulting solution was heated under reflux for 3 h. The reaction mixture was diluted with CH.sub.2Cl.sub.2 (20 mL) and washed with cold brine (2×10 mL). The organic solution was dried with MgSO.sub.4, filtered and the remaining solution was concentrated in vacuo. The crude solid was purified by flash chromatography using gradient eluent (EtOAc:PE, 1:9.fwdarw.4:1) to yield the title compound as a dark blue powder (47 mg, 52%). δ.sub.H (CDCl.sub.3, 400 MHz) 13.14 (2H, s), 10.28 (2H, br s), 6.99 (2H, s), 6.96 (2H, s), 4.33 (2H, t, J 5.6 Hz), 3.27 (12H, s, O(CH.sub.3).sub.2), 1.73-1.44 (8H, m), 1.19 (4H, s); δ.sub.C (CDCl.sub.3, 100 MHz) 183.8, 154.3, 145.4, 123.5, 122.7, 114.4, 107.6, 103.3, 51.8, 41.8, 28.3, 21.67, 13.1; m/z (ES+) 531 (M+H, 100).

Example 3b

3,3′-(5,8-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-1,4diyl)bis(azanediyl)bis(N-(2,2-dimethoxyethyl)propanamide) (BST-104)

(35) ##STR00022##

(36) To a stirred solution of 1,4-difluoro-5,8-dihydroxyanthracene-9,10-dione (80 mg, 0.29 mmol) in pyridine (1 mL) was added 3-amino-N-(2,2-dimethoxyethyl) propanamide (306. mg, 1.74 mmol) and the resulting solution was heated under reflux for 3 h. The solvent was evaporated under reduced pressure and the reaction mixture was purified by flash chromatography using gradient eluent (CH.sub.3OH:CH.sub.2Cl.sub.2, 1:99.fwdarw.1:9) to yield the title compound as a dark blue powder (112 mg, 66%). δ.sub.H (DMSO, 400 MHz) 13.4 (2H, s, OH), 10.6 (2H, br s, NH), 8.15 (2H, br s, NH), 7.47 (2H, s), 7.11 (2H, s), 4.32 (2H, t, J 5.55 Hz), 3.73 (4H, q, J 5.5 Hz), 3.30 (12H, s, O(CH.sub.3).sub.2), 3.25 (4H, t, J 5.5 Hz), 2.43 (4H, m); δ.sub.C (DMSO, 100 MHz) 183.3, 170.2, 154.5, 146.8, 125.4, 124.3, 114.9, 107.2, 102.1, 53.2, 40.5, 38.9, 35.2; m/z (ES+) 589 (M+H, 100).

Example 3c

N,N′-(2,2′-(5,8-Dihydroxy-9,10-dioxo-9,10-dihydroanthracene-1,4-diyl)bis(azanediyl)bis(ethane-2,1-diyl))bis(3,3-dimethoxypropanamide) (BST-105)

(37) ##STR00023##

(38) To a stirred solution of 1,4-difluoro-5,8-dihydroxyanthracene-9,10-dione (71 mg, 0.26 mmol) in pyridine (1 mL) was added N-(2-aminoethyl)-3,3-dimethoxypropanamide (272 mg, 1.54 mmol) and the resulting solution was heated under reflux for 4 h. The solvent was evaporated under reduced pressure and the reaction mixture was purified by flash chromatography using gradient eluent (CH.sub.3OH:CH.sub.2Cl.sub.2, 1:99.fwdarw.1:9) to yield the title compound as a dark blue powder (10 mg, 37%). δ.sub.H (DMSO, 400 MHz) 13.5 (2H, s, OH), 10.7 (2H, br s, NH), 8.13 (2H, br s, NH), 7.64 (2H, s), 7.21 (2H, s), 4.72 (2H, t, J 5.5 Hz) 3.53 (4H, q, J 5.5 Hz), 3.35 (4H, t, J 5.5 Hz), 3.25 (12H, s, O(CH.sub.3).sub.2); δ.sub.C (DMSO, 100 MHz) 182.3, 172.2, 154.5, 140.9, 124.4, 124.3, 115.9, 108.5, 103.1, 53.7, 44.5, 38.9, 34.2; m/z (ES+) 589 (M+H, 100).

Example 3d

1,4-bis((2-((2,2-dimethoxyethyl)amino)ethyl)amino)-5,8-dihydroxyanthracene-9,10-dione (BST-200)

(39) ##STR00024##

(40) To a stirred solution of 1,4-difluoro-5,8-dihydroxyanthracene-9,10-dione (50 mg, 0.18 mmol) in pyridine (1 mL) was added N-(2,2-dimethoxyethyl)ethane-1,2-diamine (205 mg, 1.38 mmol) and the resulting solution was heated under reflux for 4 h. The solvent was evaporated under reduced pressure and the reaction mixture was purified by flash chromatography using gradient eluent (CH.sub.3OH:CH.sub.2Cl.sub.2, 1:99.fwdarw.4:9) to yield the title compound as a dark blue powder (45 mg, 47%). δ.sub.H (CDCl.sub.3, 400 MHz) 13.37 (2H, s br), 10.32 (2H, t, J 5H), 7.00 (2H, s), 6.97 (2H, s), 4.43 (2H, t, J 5.6 Hz), 3.39 (4H, q, J 5.6 Hz), 3.34 (12H, s), 2.93 (4H, t, J 6 Hz), 2.76 (4H, d, J 5.6 Hz); δ.sub.C (CDCl.sub.3, 101 MHz) 185.23, 155.41, 146.41, 124.71, 123.68, 115.35, 109.12, 103.94, 54.17, 51.03, 48.77, 43.01; m/z (ES+) 533 (M+H, 100). Anal. calculated for C.sub.26H.sub.36N.sub.4O.sub.8: C, 58.63; H, 6.81; N, 10.52. Found: C, 57.23; H, 6.46; N, 10.21.

Example 3e

1,4-bis((6-((2,2-dimethoxyethyl)amino)hexyl)amino)-5,8-dihydroxyanthracene-9,10-dione) (BST-300)

(41) ##STR00025##

(42) To a stirred solution of 1,4-difluoro-5,8-dihydroxyanthracene-9,10-dione (33 mg, 0.12 mmol) in pyridine (1 mL) was added N-(2,2-dimethoxyethyl)hexane-1,6-diamine (200 mg, 0.99 mmol) and the resulting solution was stirred at 70° C. for 2 h. The solvent was evaporated under reduced pressure and the reaction mixture was purified by flash chromatography using gradient eluent (CH.sub.3OH:CH.sub.2Cl.sub.2, 1:99.fwdarw.4:9 to yield the title compound as a dark blue powder (45 mg, 58%). δ.sub.H (CDCl.sub.3, 400 MHz) 13.45 (2H, s), 10.35 (2H, t, J 5H), 7.09 (2H, s), 7.04 (2H, s), 4.42 (2H, t, J 5.6 Hz), 3.32 (18H, s), 2.67 (4H, d, J 5.6 Hz), 2.58 (4H, m), 1.68 (4H, m), 1.45 (6H, m), 1.37 (6H, m); δ.sub.C (CDCl.sub.3, 101 MHz) 185.18, 155.44, 146.62, 124.70, 123.88, 115.48, 108.85, 103.81, 54.05, 51.18, 49.86, 42.98, 29.89, 29.51, 27.02, 27.00; m/z (ES+) 545 (M+H, 100). Anal. calculated for C.sub.34H5.sub.2N.sub.4O.sub.8: C, 63.33; H, 8.13; N, 8.69. Found: C, 63.05; H, 7.98; N, 8.48.

Example 3f

1,4-bis((2-((2,2-dimethoxyethyl)(methyl)amino)ethyl)amino)-5,8-dihydroxyanthracene-9,10-dione (BST-201)

(43) ##STR00026##

(44) To a stirred solution of 1,4-bis((2-((2,2-dimethoxyethyl)amino)ethyl)amino)-5,8-dihydroxyanthracene-9,10-dione (134 mg, 0.25 mmol) in DMF (3 mL) was added DIPEA (131 μL, 0.75 mmol) and methyl iodide (47 μL, 0.75 mmol) before the resulting solution was stirred at room temperature overnight. The solution was washed with saturated NaHCO.sub.3 (2×10 mL) and the organic layer was dried with MgSO.sub.4. The solvent was evaporated under reduced pressure and the reaction mixture was purified by flash chromatography using gradient eluent (CH.sub.3OH:CH.sub.2Cl.sub.2, 1:99.fwdarw.6:94) to yield the title compound as a dark blue powder (63 mg, 45%). δ.sub.H (CDCl.sub.3, 400 MHz) 13.61 (2H, s, OH), 10.47 (2H, t, NH, J=5.5. Hz), 7.24 (2H, s), 7.14 (2H, s), 4.59 (2H, t, J 5.5 Hz) 3.51 (4H, q, J 5.5 Hz), 3.43 (12H, s, O(CH.sub.3).sub.2), 2.85 (4H, t, J 5.5 Hz), 2.67 (4H, d, J 5.5 Hz), 2.44 (6H, s); δ.sub.C (CDCl.sub.3, 100 MHz) 185.58, 155.51, 146.32, 124.89, 124.02, 115.42, 109.31, 103.53, 59.54, 56.93, 53.84, 43.11, 41.04; m/z (ES+) 561 (M+H, 100).

Example 3g

1,4-bis((2-(benzyl(2,2-dimethoxyethyl)amino)ethyl)amino)-5,8-dihydroxyanthracene-9,10-dione (BST-202)

(45) ##STR00027##

(46) To a stirred solution of 1,4-bis((2-((2,2-dimethoxyethyl)amino)ethyl)amino)-5,8-dihydroxyanthracene-9,10-dione (134 mg, 0.25 mmol) in CH.sub.2Cl.sub.2 (10 mL) was added DIPEA (174 μL) and benzyl bromide (118 μL) before the resulting solution was stirred at room temperature overnight. The solution was washed with saturated NaHCO.sub.3 (2×10 mL) and the organic layer was dried with MgSO.sub.4. The solvent was evaporated under reduced pressure and the reaction mixture was purified by flash chromatography using gradient eluent (CH.sub.3OH:CH.sub.2Cl.sub.2, 1:99.fwdarw.6:94) to yield the title compound as a dark blue powder (124 mg, 93%). δ.sub.H (CDCl.sub.3, 400 MHz) 13.65 (2H, s, OH), 10.46 (2H, t, NH), 7.37 (4H, d, J 7.2 Hz), 7.17 (6H, m), 7.09 (2H, s), 6.99 (2H, s), 4.40 (2H, t, J 5.5 Hz), 3.69 (4H, s), 3.38 (4H, q, J 6 Hz), 3.25 (12H, s, O(CH.sub.3).sub.2), 2.84 (4H, t, J 6 Hz), 2.49 (4H, d, J 5 Hz); δ.sub.C (CDCl.sub.3, 100 MHz) 185.4, 155.5, 146.3, 139.1, 128.9, 128.3, 127.2, 124.8, 124.1, 115.5, 109.2, 104.6, 60.1, 56.0, 54.2, 53.5, 41.0; m/z (ES+) 713 (M+H, 100).

Example 4—Synthesis of non-symmetrical 1,4-di-substituted-5,8-dihydroxy-anthraquinones (Formula (I))

(47) The following specific compounds were synthesized:

Example 4a

1-(5,5-Dimethoxypentylamino)-4-(2-(dimethylamino)ethylamino)-5,8-dihydroxyanthracene-9,10-dione (BST-107)

(48) ##STR00028##

(49) To a stirred solution of 1-(2-(dimethylamino)ethylamino)-4-fluoro-5,8-dihydroxyanthracene-9,10-dione (60 mg, 0.17 mmol) in pyridine (1 mL) was added 5,5-dimethoxypentan-1-amine (102.61 mg, 0.7 mmol) and the resulting solution was stirred at 70° C. for 6 h. The solvent was evaporated under reduced pressure and the reaction mixture was purified by flash chromatography using gradient eluent (CH.sub.3OH:CH.sub.2Cl.sub.2, 1:99.fwdarw.6:94) to yield the title compound as a dark blue powder (40 mg, 49%). δ.sub.H (CDCl.sub.3, 400 MHz) 13.3 (2H, brs, OH), 10.3 (2H, br s, NH), 7.16-6.96 (4H, m), 4.32 (1H, t, J 5.5 Hz), 3.45 (2H, t, J 6.5 Hz), 3.21 (6H, s), 2.74 (2H, t, J 6.5 Hz), 2.45 (6H, s), 1.74-1.42 (6H, m); δ.sub.C (CDCl.sub.3, 100 MHz) 185.6, 185.2, 155.5, 155.46, 146.6, 145.9, 125.0, 124.8, 123.9, 123.7, 115.3, 115.2, 109.4, 108.8, 104.4, 57.7, 52.9, 45.0, 42.9, 40.2, 32.2, 29.4, 22.2; m/z (ES+) 472 (M+H, 100).

Example 4b

N-(2,2-Dimethoxyethyl)-3-(4-(2-(dimethylamino)ethylamino)-5,8-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-1-ylamino)propanamide (BST-108)

(50) ##STR00029##

(51) To a stirred solution of 1-(2-(dimethylamino)ethylamino)-4-fluoro-5,8-dihydroxyanthracene-9,10-dione (60 mg, 0.17 mmol) in pyridine (1 mL) was added 3-amino-N-(2,2-dimethoxyethyl)propanamide (123 mg, 0.7 mmol) and the resulting solution was stirred at 70° C. for 6 h. The solvent was evaporated under reduced pressure and the reaction mixture was purified by flash chromatography using gradient eluent (CH.sub.3OH CH.sub.2Cl.sub.2, 1:99.fwdarw.6:94) to yield the title compound as a dark blue powder (79 mg, 90%). δ.sub.H (CDCl.sub.3, 400 MHz) 13.3 (2H, brs, OH), 10.1 (2H, br s, NH), 6.93 (2H, d, J 6.0 Hz), 6.82 (2H, t, J 6.0 Hz), 6.23 (1H, br s, NH), 4.37 (1H, t, J 5.7 Hz), 3.45 (2H, t, J 6.5 Hz), 3.41 (2H, t, J 5.2 Hz), 3.31 (6H, s), 2.64 (2H, t, J 6.5 Hz), 2.52 (2H, t, J 5.2 Hz), 2.33 (6H, s); δ.sub.C (CDCl.sub.3, 100 MHz) 184.9, 184.8, 170.6, 155.3, 155.2, 146.1, 145.9, 124.7, 124.5, 123.6, 123.3, 115.2, 115.1, 108.9, 108.7, 102.5, 58.1, 54.3, 45.5, 41.1, 40.8, 39.2, 36.5; m/z (ES+) 501 (M+H, 100).

Example 4c

N-(2-(4-(2-(Dimethylamino)ethylamino)-5,8-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-1-ylamino)ethyl)-3,3-dimethoxypropanamide (BST-111)

(52) ##STR00030##

(53) To a stirred solution of 1-(2-(dimethylamino)ethylamino)-4-fluoro-5,8-dihydroxyanthracene-9,10-dione (54 mg, 0.16 mmol) in pyridine (1 mL) was added N-(2-aminoethyl)-3,3-dimethoxypropanamide (111 mg, 0.63 mmol) and the resulting solution was stirred at 70° C. for 6 h. The solvent was evaporated under reduced pressure and the reaction mixture was purified by flash chromatography using gradient eluent (CH.sub.3OH:CH.sub.2Cl.sub.2, 1:99.fwdarw.5:95) to yield the title compound as a dark blue powder (45 mg, 60%). δ.sub.H (CDCl.sub.3, 400 MHz) 13.3 (2H, br s, OH), 10.2 (2H, br s, NH), 6.92 (2H, t, J 5.9 Hz), 6.82 (2H, t, J 5.9 Hz), 6.2 (1H, br s, NH), 4.62 (1H, t, J 5.5 Hz), 3.47 (2H, q, J 6.4 Hz), 3.41 (2H, t, J 5.5 Hz), 3.33 (6H, s), 2.63-2.54 (4H, m), 2.52 (2H, t, J 5.5 Hz), 2.27 (6H, s); δ.sub.C (CDCl.sub.3, 100 MHz) 184.8, 184.5, 169.8, 169.2, 155.2, 155.1, 146.2, 124.5, 124.2, 123.6, 123.4, 115.2, 115.1, 108.8, 108.5, 102.2, 58.1, 51.4, 45.5, 40.8, 40.8, 37.0, 36.8; m/z (ES+) 501 (M+H, 100).

Example 4d

1-((2-((2,2-dimethoxyethyl)amino)ethyl)amino)-4-((2-(dimethylamino)ethyl)amino)-5,8-dihydroxyanthracene-9,10-dione (BST-302)

(54) ##STR00031##

(55) To a stirred solution of 1-(2-(dimethylamino)ethylamino)-4-fluoro-5,8-dihydroxyanthracene-9,10-dione (30 mg, 0.09 mmol) in pyridine (1 mL) was added N-(2,2-dimethoxyethyl)ethane-1,2-diamine (125 mg, 0.84 mmol) and the resulting solution was stirred at 70° C. for 2 h. The solvent was evaporated under reduced pressure and the reaction mixture was purified by flash chromatography using gradient eluent (CH.sub.3OH:CH.sub.2Cl.sub.2, 1:99.fwdarw.4:9 to yield the title compound as a dark blue powder (37 mg, 90%). δ.sub.H (CDCl.sub.3, 400 MHz) 13.36 (2H, s), 10.25 (1H, t, J 5H), 10.22 (1H, t, J 5H), 6.97 (2H, s), 6.89 (2H, s), 4.43 (1H, t, J 5 Hz), 3.34 (10H, m), 2.91 (2H, t, J 6 Hz), 2.75 (2H, d, J 6 Hz), 2.58 (2H, t, J 6 Hz), 2.26 (6H, s); δ.sub.C (CDCl.sub.3, 101 MHz) 185.29, 184.93, 155.29, 146.30, 146.21, 124.52, 124.43, 123.58, 123.55, 115.38, 108.96, 108.84, 103.92, 58.32, 54.15, 51.02, 50.62, 45.62, 42.96, 41.11; m/z (ES+) 473 (M+H, 100); Anal. calculated for C.sub.24H.sub.32N.sub.4O.sub.6: C, 61.04; H, 6.83; N, 11.86. Found: C, 61.15; H, 6.78; N, 11.81.

Example 4e

1-((3-((2,2-dimethoxyethyl)amino)propyl)amino)-4-((2-(dimethylamino)ethyl)amino)-5,8-dihydroxyanthracene-9,10-dione (BST-303)

(56) ##STR00032##

(57) To a stirred solution of 1-(2-(dimethylamino)ethylamino)-4-fluoro-5,8-dihydroxyanthracene-9,10-dione (30 mg, 0.09 mmol) in pyridine (1 mL) was added N-(2,2-dimethoxyethyl)propane-1,3-diamine (129 mg, 0.80 mmol) and the resulting solution was stirred at 70° C. for 2 h. The solvent was evaporated under reduced pressure and the reaction mixture was purified by flash chromatography using gradient eluent (CH.sub.3OH:CH.sub.2Cl.sub.2, 1:99.fwdarw.4:9 to yield the title compound as a dark blue powder (34 mg, 70%). δ.sub.H (CDCl.sub.3, 400 MHz) 13.45 (1H, s), 13.35 (1H, s), 10.27 (1H, t, J 5H), 10.23 (1H, t, J 5H), 6.96 (2H, s), 6.89 (2H, s), 4.48 (1H, t, J 5 Hz), 3.32 (10H, m), 2.74 (2H, t, J 6 Hz), 2.70 (2H, d, J 6 Hz), 2.58 (2H, t, J 6 Hz), 2.28 (6H, s), 1.83 (2H, m, J 6 Hz); δ.sub.C (CDCl.sub.3, 101 MHz) 184.83, 155.30, 155.24, 146.43, 146.21, 124.40, 124.35, 123.60, 123.55, 115.44, 108.75, 108.68, 103.89, 58.36, 54.09, 51.27, 47.33, 45.66, 41.17, 40.93, 29.97; m/z (ES+) 487 (M+H, 100); Anal. calculated for C.sub.25H.sub.34N.sub.4O.sub.6: C, 61.75; H, 7.04; N, 11.51. Found: C, 60.76; H, 7.50; N, 11.14.

Example 4f

1-((4-((2,2-dimethoxyethyl)amino)butyl)amino)-4-((2-(dimethylamino)ethyl)amino)-5,8-dihydroxyanthracene-9,10-dione (BST-304)

(58) ##STR00033##

(59) To a stirred solution of 1-(2-(dimethylamino)ethylamino)-4-fluoro-5,8-dihydroxyanthracene-9,10-dione (41 mg, 0.12 mmol) in pyridine (1 mL) was added N-(2,2-dimethoxyethyl)butane-1,4-diamine (140 mg, 0.79 mmol) and the resulting solution was stirred at 70° C. for 2 h. The solvent was evaporated under reduced pressure and the reaction mixture was purified by flash chromatography using gradient eluent (CH.sub.3OH:CH.sub.2Cl.sub.2, 1:99.fwdarw.4:9 to yield the title compound as a dark blue powder (46 mg, 92%). δ.sub.H (CDCl.sub.3, 400 MHz) 13.42 (1H, s), 13.35 (1H, s), 10.24 (1H, t, J 5H), 10.18 (1H, t, J 5H), 6.97 (2H, s), 6.85 (2H, s), 4.41 (1H, t, J 5.2 Hz), 3.32 (10H, m), 3.22 (2H, q, J 5.2 Hz), 2.68 (2H, d, J 5.2 Hz), 2.64 (2H, t, J 7.2 Hz), 2.56 (2H, t, J 7.2 Hz), 2.27 (6H, s), 1.68 (2H, m), 1.57 (2H, m); δ.sub.C (CDCl.sub.3, 101 MHz) 184.82, 184.75, 155.28, 155.23, 146.36, 146.17, 124.35, 124.32, 123.60, 123.46, 115.44, 108.75, 108.60, 103.91, 58.36, 54.06, 51.25, 49.56, 45.67, 42.87, 41.16, 27.66, 27.30; m/z (ES+) 501 (M+H, 100). Anal. calculated for C.sub.26H.sub.36N.sub.4O.sub.6: C, 62.42; H, 7.25; N, 11.20. Found: C, 61.99; H, 7.64; N, 10.99.

Example 4g

1-((5-((2,2-dimethoxyethyl)amino)pentyl)amino)-4-((2-(dimethylamino)ethyl)amino)-5,8-dihydroxyanthracene-9,10-dione (BST-305)

(60) ##STR00034##

(61) To a stirred solution of 1-(2-(dimethylamino)ethylamino)-4-fluoro-5,8-dihydroxyanthracene-9,10-dione (30 mg, 0.09 mmol) in pyridine (1 mL) was added N-(2,2-dimethoxyethyl)pentane-1,5-diamine (122 mg, 0.64 mmol) and the resulting solution was stirred at 70° C. for 2 h. The solvent was evaporated under reduced pressure and the reaction mixture was purified by flash chromatography using gradient eluent (CH.sub.3OH:CH.sub.2Cl.sub.2, 1:99.fwdarw.4:9 to yield the title compound as a dark blue powder (39 mg, 76%). δ.sub.H (CDCl.sub.3, 400 MHz) 13.40 (1H, s), 13.33 (1H, s), 10.21 (1H, t, J 5H), 10.14 (1H, t, J 5H), 6.95 (2H, s), 6.80 (2H, m), 4.39 (1H, t, J 5.2 Hz), 3.31 (10H, m), 3.16 (2H, m), 2.67 (2H, d, J 5.2 Hz), 2.58 (4H, m), 2.27 (6H, s), 1.66 (2H, m), 1.51 (2H, m), 1.42 (2H, m); δ.sub.C (CDCl.sub.3, 101 MHz) 184.65, 184.57, 155.22, 155.15, 146.36, 146.13, 124.18, 123.53, 123.39, 115.45, 115.44, 108.61, 108.43, 103.90, 58.34, 54.02, 51.27, 49.89, 49.82, 45.66, 42.86, 41.12, 29.86, 29.40, 24.86; m/z (ES+) 515 (M+H, 100). Anal. calculated for C.sub.27H.sub.38N.sub.4O.sub.6: C, 63.06; H, 7.45; N, 10.89. Found: C, 62.02; H, 7.18; N, 10.45.

Example 4h

1-((6-((2,2-dimethoxyethyl)amino)hexyl)amino)-4-((2-(dimethylamino)ethyl)amino)-5,8-dihydroxyanthracene-9,10-dione (BST-301)

(62) ##STR00035##

(63) To a stirred solution of 1-(2-(dimethylamino)ethylamino)-4-fluoro-5,8-dihydroxyanthracene-9,10-dione (30 mg, 0.09 mmol) in pyridine (1 mL) was added N-(2,2-dimethoxyethyl)hexane-1,6-diamine (103 mg, 0.51 mmol) and the resulting solution was stirred at 70° C. for 2 h. The solvent was evaporated under reduced pressure and the reaction mixture was purified by flash chromatography using gradient eluent (CH.sub.3OH:CH.sub.2Cl.sub.2, 1:99.fwdarw.4:9 to yield the title compound as a dark blue powder (30 mg, 63%). δ.sub.H (CDCl.sub.3, 400 MHz) 13.43 (1H, s), 13.39 (1H, s), 10.32 (1H, t, J 5H), 10.26 (1H, t, J 5H), 7.01 (2H, s), 6.99 (2H, d, J 1.6 Hz), 4.40 (1H, t, J 5.6 Hz), 3.38 (2H, q, J 1.6 Hz), 3.32 (6H, s), 3.27 (2H, q, J 1.6 Hz), 2.67 (2H, d, J 5.6 Hz), 2.57 (4H, m), 2.28 (6H, s), 1.68 (2H, m), 1.46 (4H, m), 1.37 (3H, m); δ.sub.C (CDCl.sub.3, 101 MHz) 185.10, 185.08, 155.41, 155.34, 146.55, 146.25, 124.59, 123.78, 123.67, 115.47, 115.44, 108.99, 108.80, 103.92, 58.40, 54.03, 51.28, 49.91, 45.66, 42.96, 41.23, 30.02, 29.49, 27.05, 27.03; m/z (ES+) 529 (M+H, 100); Anal. calculated for C.sub.28H.sub.40N.sub.4O.sub.6: C, 63.66; H, 7.63; N, 10.60. Found: C, 62.05; H, 7.45; N, 10.80.

Example 4i

1-((2,2-dimethoxyethyl)amino)-4-((2-(dimethylamino)ethyl)amino)-5,8-dihydroxyanthracene-9,10-dione (BST-203)

(64) ##STR00036##

(65) To a stirred solution of 1-(2-(dimethylamino)ethylamino)-4-fluoro-5,8-dihydroxyanthracene-9,10-dione (35 mg, 0.10 mmol) in pyridine (1 mL) was added 2,2-dimethoxyethanamine (120 mg, 1.14 mmol) and the resulting solution was stirred at 90° C. for 1 h. The solvent was evaporated under reduced pressure and the reaction mixture was purified by flash chromatography using gradient eluent (CH.sub.3OH:CH.sub.2Cl.sub.2, 1:99.fwdarw.5:95) to yield the title compound as a dark blue powder (27 mg, 63%). δ.sub.H (CDCl.sub.3, 400 MHz) 13.45 (1H, s, OH), 13.42 (1H, s, OH), 10.38 (1H, t, NH, J 5 Hz), 10.32 (1H, t, NH, J 5 Hz), 7.13 (1H, d, J 10 Hz), 7.07 (1H, d, J 10 Hz), 7.05 (2H, s), 4.69 (1H, t, J 5 Hz), 3.72 (2H, m), 3.58 (2H, m), 3.46 (2H, t, J 5.5), 3.41 (2H, q, J 5.5), 2.59 (2H, t, J 5.5), 2.28 (6H, s), 1.20 (6H, t, J 6); δ.sub.C (CDCl.sub.3, 100 MHz) 185.72, 185.48, 155.51, 155.49, 146.42, 146.23, 125.05, 124.89, 124.11, 123.67, 115.35, 109.49, 109.15, 101.20, 62.96, 58.34, 45.73, 41.22, 15.42; m/z (ES+) 458 (M+H, 100).

Example 5—Synthesis of Symmetrical 1,5-di-substituted-anthraquinones (Formula (II))

(66) The following specific compound was synthesized:

Example 5a

1,5-bis((5,5-dimethoxypentyl)amino)anthracene-9,10-dione (BST-401)

(67) ##STR00037##

(68) To a stirred solution of 1,5-dichloroanthraquinone (50 mg, 0.18 mmol) in pyridine (2 mL) was added of 5,5-dimethoxypentan-1-amine (725 mg, 1.80 mmol) and the resulting solution was stirred at 90° C. for 96 hours. The solvent was evaporated under reduced pressure and the reaction mixture was purified by flash chromatography using gradient eluent (CH.sub.3OH:CH.sub.2Cl.sub.2, 0:100.fwdarw.40:90) to yield the title compound as a purple solid (15.7 mg, 17.5%). δ.sub.H (CDCl.sub.3, 400 MHz)) δ 9.64 (t, J=4.8 Hz, 2H, NH.sub.2), 7.46 (m, 4H, Ar—H), 6.88 (d, J=6.4 Hz, 2H, Ar—H), 4.32 (t, J=5.6 Hz, 2H, CH), 3.26 (s, 12H, OCH.sub.3), 3.24 (m, 4H, NHCH.sub.2), 1.71 (m, 4H, NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.62 (m, 4H, NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.51 (m, 4H, NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH); δ.sub.C (CDCl.sub.3, 101 MHz) δ 185.47, 151.46, 136.31, 135.25, 116.37, 114.68, 112.88, 104.36, 52.84, 42.84, 32.26, 28.99, 22.34. HRMS Found 499.2796, C.sub.28H.sub.38N.sub.2O.sub.6 req. 499.2803.

Example 6—Synthesis of Symmetrical 1,8 di-substituted-anthraquinones (Formula (IV))

(69) The following specific compound was synthesized:

Example 6a

1,8-bis((5,5-dimethoxypentyl)amino)anthracene-9,10-dione (BST-400)

(70) ##STR00038##

(71) To a stirred solution of 1,8-dichloroanthraquinone (50 mg, 0.18 mmol) in pyridine (2 mL) was added of 5,5-dimethoxypentan-1-amine (726 mg, 1.80 mmol) and the resulting solution was stirred at 90° C. for 72 hours. The solvent was evaporated under reduced pressure and the reaction mixture was purified by flash chromatography using gradient eluent (CH.sub.3OH:CH.sub.2Cl.sub.2, 0:100.fwdarw.40:90) to yield the title compound as a purple solid (22.4 mg, 25%). δ.sub.H (CDCl.sub.3, 400 MHz) δ 9.54 (t, J=4.8 Hz, 2H, NH.sub.2), 7.46 (d, J=6.8 Hz, 2H, Ar—H), 7.43-7.36 (m, 2H, Ar—H), 6.94 (d, J=8.3 Hz, 2H, Ar—H), 4.35 (t, J=5.6 Hz, 2H, CH), 3.27 (s, 12H, OCH.sub.3), 3.26-3.22 (m, 4H, NHCH.sub.2), 1.73 (dt, J=14.8, 7.5 Hz, 4H, NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.64 (dd, J=15.3, 6.0 Hz, 4H, NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH), 1.51-1.42 (m, 4H, NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH); δ.sub.C (CDCl.sub.3, 101 MHz) δ 189.04 & 184.73, 151.13, 134.36, 134.14, 117.64, 114.88, 114.34, 104.33, 52.78, 42.99, 32.25, 29.04, 22.35. HRMS Found 499.2799, C.sub.28H.sub.38N.sub.2O.sub.6 req. 499.2803.

Example 7—Synthesis of Symmetrical 2,6-di-substituted-anthraquinones (Formula (III))

(72) The following specific compound can be synthesized under the same conditions described above for BST-400 but using 2,6-dichloroanthraquinone as starting material:

Example 7a

2,6-bis((5,5-dimethoxypentyl)amino)anthracene-9,10-dione (BST-402)

(73) ##STR00039##

(74) To a stirred solution of 2,6-dichloroanthraquinone (50 mg, 0.18 mmol) in pyridine (2 mL) was added of 5,5-dimethoxypentan-1-amine (726 mg, 1.80 mmol) and the resulting solution was stirred at 90° C. for 72 hours. The solvent was evaporated under reduced pressure and the reaction mixture was purified by flash chromatography using gradient eluent (CH.sub.3OH:CH.sub.2Cl.sub.2, 0:100.fwdarw.10:90) to yield the title compound.

(75) Testing of the Compounds

(76) A. Absorption and Fluorescence Spectra

(77) The compounds were exited at 540 nm and at the wavelength of their absorption peak 1 (λ1 nm). The far-red fluorescent DNA dye DRAQ5 was used as reference.

(78) Materials and Methods for the Determination of the Fluorescence Excitation and Emission Characteristics of Compounds

(79) Compounds were dissolved in DMSO (Sigma) to 20 mM stock concentration. DRAQ5 (Biostatus Ltd) was used as a control anthraquinone agent dissolved in deionised water. Working solutions were prepared in PBS (phosphate buffered saline; Sigma) or HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; Sigma) at 20 or 40 μM. Probe solutions (20 μM), respectively in PBS or HEPES, were pipetted into a 1 mL quartz cuvette (Thermo Fisher Scientific). After obtaining baseline spectra in relevant buffer to zero the instrument, absorbance measurements were obtained using a Beckmann Coulter DU 800 UV/Visible spectrophotometer. The excitation wavelengths were scanned from 280 to 800 nm. Fluorescent spectral scans were performed using a Perkin Elmer LS-50B Luminescence spectrophotometer. To obtain emission scans, the excitation wavelength was set accordingly to the maximum values for each agent or another designated excitation wavelength that matches typical laser lines; and the emission wavelength were scanned from 400 nm to 900 nm in 1 nm increments. A Xenon flash lamp was used as the light source, with the lamp energy set to high. For each data point ten measurements were acquired with the photomultiplier tube sensitivity set at 125.

(80) Typical absorption spectra and fluorescence spectra of compounds BST-108, BST-109, BST-110 and BST-111 and BST-100, BST-101 and BST-106 can be seen in FIG. 1A and FIG. 1B respectively.

(81) Compounds were excited at a wavelength corresponding to the first absorbance peak 1 (FIG. 1A and FIG. 1B show absorption (line) and fluorescence (dots) spectra of compounds; both are normalised to peak value.

(82) The absorption and fluorescence spectra show that the compounds of the invention have absorption and emission in the long-wavelength spectral region (>650 am).

(83) Table 1 shows the summarised fluorescence emission data for tested compounds in PBS and HEPES.

(84) TABLE-US-00001 TABLE 1 λ1 nm 540 nm λ1 nm Compound (Ex in PBS) (Ex in PBS) (Ex in HEPES) DRAQ5 >665 635 >665  BST-104 684 712 726 BST-108 685 692 684 BST-111 684 684 684 BST-200 688 690 684 BST-201 690.5 684 684 BST-202 — — 680 BST-203 678 690 696 BST-100 747.5 — 740 BST-101 — 712 — BST-102 661 670 663 BST-106 660 670 664 BST-109 666 660 660 BST-110 665.5 662 660

(85) B. DNA Affinity

(86) DNA melting temperature (variation in Tm) was used to assess interaction between CT-DNA and compounds of Formula (I). Studies were carried out using a thermal DNA denaturation assay.

(87) Materials and Methods for the Assessment of DNA-Binding Affinity Via DNA Melting Studies Using Calf Thymus DNA (CT-DNA)

(88) Melting curve assays for DNA binding analysis of compounds were constructed as follows:

(89) CT-DNA melting buffer was prepared by making up a 1 L solution A of (10 mM NaH.sub.2PO.sub.4 and 1 mM Na.sub.2EDTA) and a separate 1 L solution B (10 mM Na.sub.2HPO.sub.4 and 1 mM Na.sub.2EDTA), and then solution A was added to solution B until pH 7.0 was achieved. Calf thymus DNA (CT-DNA, Sigma) solutions were prepared for use by dissolving 21-23 mg in 20 mL of the CT-DNA melting buffer and stored at 4° C. Mitoxantrone (Sigma) was solubilized in DMSO, diluted to desired concentrations in 10 mM Tris-HCl buffer (pH 7.4) and used as control. Its concentration was determined spectrophotometrically using a molar extinction coefficient of 8,360 M.sup.−1 cm.sup.−1 at 608 nm (Enache, M. and Volanschi, E. 2010. Spectral characterisation of self-association of antitumor drug mitoxantrone Revue Roumaine De Chimie 55(4), pp. 255-+).

(90) The CT-DNA thermal melting assay mixtures were prepared by adding sufficient CT-DNA stock solution to CT-DNA melting buffer, to ensure the preparation of a 50 μM CT-DNA solution. Compound stock solution was then added to the 50 μM CT-DNA solution, to ensure the final concentration of compound in the mixture was 10 μM.

(91) All UV absorbance experiments were conducted on a Cary 400Bio Spectrophotometer (Varian, Agilent) equipped with a Peltier Temperature Controller. This was attached to a Cary Thermostatable Multicell Block, to provide stable, continuous temperature control. A pair of masked quartz 1.2 mL microcells cells with a 4 mm width, and 10 mm path length was used for all absorbance studies. 1.2 mL of CT-DNA melting buffer was added to the reference cuvette and 1.2 mL of DNA-anthraquinone mixture was added to the reaction cuvette. Absorbance versus temperature profiles were measured at 260 nm with melting temperature (Tm) measurements initiated at 40° C. The temperature of the test solution was increased at 1° C./min till 95° C. was reached. Melting curves generated by computer software were printed out and the melting points were determined using the published graphical method by Brown and Fox (1997; Brown, P. M. and Fox, K. R. 1997. Foot-printing studies with nucleosome-bound DNA. Methods in Molecular Biology; Drug-DNA interaction protocols. Vol. 90. pp. 81-93). The Tm was the mean of two separate determinations within 0.2° C.

(92) The assay was performed initially by incubating 50 μM of calf thymus DNA (CT-DNA), in the presence of 10 μM of mitoxantrone, to validate the assay and to have a positive control for comparison. 10 μM solutions of compound counterparts were incubated with 50 μM CT-DNA.

(93) Table 2 shows results of CT-DNA/compound Formula (I) melting temperatures before and after acidic hydrolysis.

(94) TABLE-US-00002 TABLE 2 Hydrolysed product of Compound Compound Tm (° C.) compound Tm (° C.) Mitoxantrone >95 — BST-101 69.0 69.7 BST-107 74.0 71.9 BST-102 69.0 70.0 BST-103 69.4 70.1 BST-104 69.6 ** BST-105 69.0 ** BST-106 72.1 * BST-108 76.4 * BST-109 72.0 72.9 BST-110 72.0 71.6 BST-111 71.6 74.6 BST-200 74.2 70.4 BST-201 70.8 * BST-202 70.0 70.0 BST-203 73.0 72.6 * Excess of interference, hence any calculation of Tm was inaccurate. ** Solubility issues in the working buffer, therefore no experiment was performed.

(95) C. Cell Studies—Labelling and Loading

(96) Methodology: Labelling of Fixed and Live Cells

(97) Cells were seeded in a 24 well coverslip bottomed-plate, cultured overnight in McCoys media. 24 or 48 hours later the cells were washed gently with 3×2 mL PBS per well. The PBS was removed and 0.5 mL of 4% paraformaldehyde solution [a 4% paraformaldehyde solution in PBS was made fresh each time from a 16% stock solution (Thermo Fisher Scientific)] was added in each well and cells were incubated for 30 min. The paraformaldehyde was removed and cells were washed 3×2 mL PBS per well. 1 mL of PBS or HEPES was added to each well.

(98) Before analysis, cells were incubated at 37° C. in the dark for the period designated. Samples were analysed directly without any further washing step. For live cell labelling cells were seeded, plated in a similar way, into a similar multi-well dish for optimal optical performance and could be used across all microscope platforms.

(99) This basic protocol was the standard for testing the patterns of AAQ agents at 20 μM, compared to 20 μM of DRAQ5 as the benchmark reference (both in terms of cellular location and ability to detect the red fluorescence).

(100) a) Labelling—Fixed Cells

(101) Low resolution wide-field fluorescence microscopy was used to evaluate cell labelling in fixed human osteosarcoma U-2 OS cells. The system was an Axiovert 100 microscope (Carl Zeiss, Welwyn Garden City, UK) ×10 0.3 NA air objective lens and an ORCA-ER CCD camera (Hamamatsu, Reading, UK). Illumination was controlled by a shutter in front of the transmission lamp. Image capture was controlled using Metamorph software. This evaluates the ability of the compounds to label cellular compartments without active uptake or efflux prohibiting delivery into the given cell. Image collection was performed after 1 h of exposure to probes in PBS. The results are shown in FIG. 2. The calibration bar represents 50 μm.

(102) Compounds BST-101 and BST-106 showed diverse staining when put into cells. BST-101 and BST-106 have a diffuse cytoplasmic staining, with some perinuclear and nucleoli labelling. Moreover, very bright background was observed in BST-106 compared to BST-101.

(103) Compounds BST-108 and BST-109 showed heterogeneity in cytoplasmic staining and positive spots in the nuclei due to DNA binding. Moreover, BST-110 and BST-111 displayed very high background fluorescence, especially in BST-110, with cytoplasmic staining and heterogeneity on labelling of mitotic figures.

(104) A very bright fluorescence was seen for BST-200, with cytoplasmic staining, good object recognition, and some heterogeneity on nuclear staining.

(105) b) Loading—A549 Live Cells

(106) Low resolution wide-field fluorescence microscopy was used to evaluate cell uptake and localisation of the probe compounds. The system was an Axiovert 100 microscope (Carl Zeiss, Welwyn Garden City, UK) ×10 0.3 NA air objective lens and an ORCA-ER CCD camera (Hamamatsu, Reading, UK). Illumination was controlled by a shutter in front of the transmission lamp. Image capture was controlled using Metamorph software. Human non-small cell lung cancer A549 cell line was used as the cell model to determine whether variation of the probe loading and localisation occurred in cell population sustaining a “side population” (SP) (Smith P J, Wiltshire M, Chappell S C, Cosentino L, Burns P A, Pors K, Errington R J. Kinetic analysis of intracellular Hoechst 33342—DNA interactions by flow cytometry: misinterpretation of side population status? Cytometry A 2013; 83:161-9) with a known capacity to efflux xenobiotic molecules, including the DNA dye Hoechst 33342.

(107) Cells were incubated for 30 min with each compound and then images were collected at a 30 ms exposure time using low resolution wide-field fluorescence microscopy. Using an Axiovert 100 microscope (Carl Zeiss, Welwyn Garden City, UK) ×10 0.3 NA air objective lens and an ORCA-ER CCD camera (Hamamatsu, Reading, UK). Illumination was controlled by a shutter in front of the transmission lamp. Image capture was controlled using Metamorph software. The results are shown in FIG. 3. The calibration bar represents 50 μm.

(108) As before, BST-101 and BST-106 showed heterogeneous cytoplasmic staining, with BST-106 in particular displaying a high background. The BST-108, BST-109 and BST-110 analogues revealed very bright cellular fluorescence, whereas BST-111 has a greater contrast and diffuse cellular staining.

(109) BST-200 showed heterogeneity on cytoplasmic load on, with bright fluorescence signal and staining of mitotic figures, whereas BST-201 had perinuclear accumulation.

(110) c) Confocal Laser Scanning Microscopy (CLSM) Analysis in the U-2 OS Cell Line

(111) Confocal laser scanning microscopy (CLSM) was used to analyse the localisation of compounds BST-100, BST-101 and BST-106 with the U-2 OS cells, as it permitted the acquisition of volume limited optical sections through the cell and media. Excitation (637 nm) and emission (>665 nm) values for the probe analysis were the optimal conditions. The data sets obtained were processed and assembled into a single maximum projection image.

(112) The patterns were analysed after 30 min incubation at different z depths and eliminating the solution background. The nucleus/cytoplasm ratio was calculated to quantify the localisation of the probe per cell.

(113) The results are shown in FIG. 4. A Radiance CLSM (BioRad Microscience) was used attached to an inverted Nikon microscope. Image acquisition was performed using excitation (637 nm) and emission (>665 nm) wavelengths. The histogram plot reports the nucleus/cytoplasm index to determine DNA binding in live cells. The calibration bar represents 10 μm.

(114) Optical sectioning enabled detection of low levels of probe in the perinuclear region, with probable overload in Golgi body and large vesicles for BST-100 and BST-101. The accumulation area appeared similarly to the MTOC (microtubule organizing centre); however BST-106 gave an additional nuclear signal.

(115) The compounds BST-108, BST-109 were analysed in the same way. The results are shown in FIG. 5.

(116) Analysis of BST-108, BST-109, BST-110 and BST-111 showed a strong nuclear uptake and perinuclear staining, with possible ER accumulation. A whole cell staining was a specific feature of BST-110 and BST-109, whereas BST-111 was mainly loaded in the nuclear compartment.

(117) D. Consequences of AAQ Retention and Long Term Effects on Cell Viability

(118) Timelapse imaging, in accordance with Errington R J et al, Time-lapse microscopy approaches to track cell cycle and lineage progression at the single-cell level, Curr Protoc Cytom. 2013 April; Chapter 12: Unit 12.4, was carried out in relation to cells with exposure to probe compounds.

(119) In this regard, A549 cells were seeded in a 24 well coverslip bottomed-plate, cultured overnight in McCoys media for 24 or 48 hours. The plates were placed onto an inverted automated microscope, with the capacity for acquiring an image sequence of different wells (therefore exposure to different agent labelling). An Axiovert 100 microscope (Carl Zeiss, Welwyn Garden City, UK) was fitted with an incubator for 37° C./5% CO2 maintenance (Solent Scientific, Portsmouth, UK), and an ORCA-ER CCD camera (Hamamatsu, Reading, UK). Illumination was controlled by a shutter in front of the transmission lamp, and an x, y positioning stage with separate z-focus (Prior Scientific, Cambridge, UK) controlled multi-field acquisition. Image capture was controlled using Metamorph software.

(120) Cells were exposed to a constant dose (0-5 μM) or a pulsed dose of 0 or 20 μM agent for 15 minutes followed by washout with warmed full media.

(121) All images were collected with a ×10 (PH1) apochromat objective lens providing a field size of 500 times 500 microns. In this case a phase contrast image was collected every 30 minutes for 72 hours. Single cell growth and expansion can be tracked over time. The cells were harvested and run through a flow cytometry (typically a FACS Calibur 635 nm excitation/661/16 emission).

(122) a) Constant Exposure to Probes

(123) A549 cell lines were monitored using time-lapse collection and lineage progression, in constant exposure to compounds of the invention. The effects on cells were also determined.

(124) In this regard, A549 cells were treated with probe compounds BST-101, BST-109, BST-110 and put on the time-lapse. Non-fluorescent images were collected over time. 72 hours later the cells were harvested and assayed using conventional flow cytometry and conventional methods for fluorescence intensity measurements to obtain relative fluorescence intensity (for example in units of channel number) and electronic gating (for example to determine the relative frequency of events) understood by those practised in the art, for (a) total signal of probe per cell determined by relative fluorescence intensity (arb. Units) and (b) cell cycle distribution across the cell compartments G1, S and G2 based on relative DNA content determined fluorometrically, and (c) cell viability determined by % total cells showing vital dye exclusion.

(125) The results are shown in FIG. 6.

(126) Analysis showed good viability with constant exposure and probe the capacity of maintaining cell labelling as well as cell expansion. All doses seemed to be non-perturbing to the cell cycle.

(127) Within 24 hours the BST-109 and BST-110 are predominantly in a membrane compartment of the cytoplasm. The nucleus is now a low signal—the nuclear/cytoplasmic ratio is reversed.

(128) b) Pulse Exposure to Probes

(129) A549 cells were loaded with 20 uM BST-101, BST-109 or BST-110 for 15 mins followed by washout. Cells were then time-lapsed and reimaged after T=72 h, as before,

(130) Analysis showed BST-101 in A549 cells dividing, and signal retained in the same membrane compartment as at the start. BST-109 in A549 cells showed dividing, and signal retained but now relocated in a non-nuclear compartment perinuclear. BST-110 in A549 cells gave dividing, and signal now relocated in a non-nuclear compartment perinuclear.

(131) c) Cellular Viability (Proliferation)

(132) The viability of A549 or MCF7 in response to compounds was determined using the MTT (3-(4,5-dimethylthiazolyl-2-)-2, 5-diphenyltetrazolium bromide) assay according to the protocol set out in Mosmann T., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of immunological methods 1983; 65:55-63.

(133) Initially, cells were seeded into a rounded-bottom 96 well plate (Corning) at six different cell seeding densities (0.5×104, 1×104, 2×104, 3×104, 4×104, and 5×104) per mL of R10 media (cells/mL) in order to optimise the assay and establish the most suitable seeding assay. Concentration of 1×104 cells/mL was determined to be the optimum seeding density for both cell lines for 96 h total incubation. A control lane contained supplemented R10 only. The 96 well plates were incubated overnight at 37° C., 5% CO2 and 100% humidity to allow cell adhesion. Cells were then treated respectively with novel AAQs (0.25-100 μM) or growth inhibition (IC.sub.50) of compounds of the invention was studied against MCF7 and A549 cancer cell lines. Compounds were tested with a concentration range between 0.25 and 100 μM for 72 h and doxorubicin, as positive cytotoxic control, was tested in parallel at the same doses.

(134) The IC.sub.50 values are shown in Tables 3 and 4.

(135) TABLE-US-00003 TABLE 3 Compound MCF7 (μM) R.sup.2 value A549 (μM) R.sup.2 value Doxorubicin 0.028 0.1831 0.05 0.1759 BST-101 0.750 0.7865 >100 0.9034 BST-102 1.75 0.6174 >100 0.2725 BST-106 11.3 0.5697 11.7 0.568 BST-107 1.66 0.5541 17.5 0.9475 BST-108 1.44 0.4437 1.7 0.7563 BST-109 15.70 0.7976 22.5 0.7371 BST-110 19.5 0.7258 22.3 0.9473 BST-111 1.8 0.757 38.2 0.9546

(136) TABLE-US-00004 TABLE 4 Compound MCF-7 (μM) R.sup.2 value A549 (μM) R.sup.2 value BST-101 hydrolysed 56.73* 0.9434 >100    0.9621 BST-102 hydrolysed  <0.250* 0.8793 >100*   0.8352 BST-106 hydrolysed 40*   0.933 >100*   0.8502 BST-107 hydrolysed 29.51  0.7825 12.7 0.7826 BST-108 hydrolysed <0.250 0.3528  1.24 0.6786 BST-109 hydrolysed 2.26 0.5933 34   0.8435 BST-110 hydrolysed 20.11  0.723 45.2 0.8345 BST-111 hydrolysed 8.60 0.6085 32.3 0.6153 *Solubility problems, blue crystals appeared after 48 h of treatment. R2 is a statistical measure of how well the regression line approximates the real data points.

(137) Further tests were carried out against A2780, 2780AD and MCP1 cell lines. The results are shown in Table 5.

(138) TABLE-US-00005 TABLE 5 Compound n A2780 2780/cp70 RF MCP1 RF 2780AD RF BST-302 A 1 1.89 ± 0.40 0.60 ± 0.03 0.32  1.16 ± 0.36 0.61 153.9 ± 28.9 81.4 BST-303 A 2 58.6 ± 6.6  10.9 ± 2.0  0.19 21.1 ± 3.8 0.36  570.3 ± 122.1 9.7 BST-304 A 3 82.1 ± 12.7 5.8 ± 1.5 0.07 29.6 ± 5.6 0.36 996.3 ± 15.0 11.8 BST-305 A 4 284..3 ± 33.7.sup.  39.8 ± 4.5  0.14 150.7 ± 7.2  0.53 1489.7 ± 90.9  5.2 BST-301 A 5 695.5 ± 109.2 72.2 ± 5.8  0.10 268.7 ± 54.8 0.39 1912.3 ± 229.2 2.8 BST-200 B 1 964.0 ± 23.0  88.0 ± 14.0 0.09 253.0 ± 5.0  0.26 2758.0 ± 116.0 2.9 BST-300 B 5 7.10 ± 0.36 1.18 ± 0.14 0.17  2.60 ± 0.43 0.37  7.59 ± 0.83 1.07 Cisplatin — — 0.38 ± 0.04 2.04 ± 0.10 5.37  0.48 ± 0.08 1.26  1.73 ± 0.13 4.6

(139) Flow Cytometry

(140) Flow cytometry analysis was performed to evaluate the effect of pre-treatment with HCl to generate an active aldehyde on the cell uptake properties. A549 cells were incubated for 30 min with compounds of Formula (I) and its hydrolysed forms (20 μM) prior to analysis. The rate of uptake was tested in A549 cell culture media and in Aldefluor buffer, containing the MDR-blocker verapamil (Pearce and Bonnet 2007), to evaluate the effect in uptake with/without the Aldefluor assay system. Analysis was conducted in the viable subset of cells, corresponding to ˜95% of the total amount under investigation.

(141) FIG. 7 shows the uptake profiles of BST-106, BST-108, BST-111 compounds and their hydrolysed forms (generated after exposure to 2M HCl for 30 min) in A549 cell line versus Aldefluor buffer. Aldefluor buffer contained verapamil vs. cell media. The cells were incubated with 20 μM of the respective AAQ/HAAQs (30 min) before analysis

(142) Statistical analysis of A549 uptake before/after hydrolysis was also studied. Experiments were performed in cell media versus Aldefluor buffer. Data were processed using FlowJo software (Tree Star). DRAQ5 was used to set the dynamic range and as control dye and empty cells were used to determine auto-fluorescence.

(143) TABLE-US-00006 TABLE 6 A549 sample Solution Median Covariance SD % ile (25) Empty media 15.94 31.29 31.29 13.48 DRAQ5 media 217.4 31.61 31.61 191.27 BST-106 media 101.48 34.58 34.58 84.64 buffer 96.13 32.87 32.87 81.66 BST-106 media 64.9 35.28 35.28 55.56 hydrolysed buffer 102.97 31.38 31.38 86.76 B media 75.48 28.15 28.15 65.32 buffer 67.08 32.09 32.09 57.65 BST-108 media 51.51 33.5 33.5 44.66 hydrolysed buffer 61.44 38.88 38.88 51.25 BST-111 media 72.42 26.78 26.78 62.72 buffer 74.94 28.85 28.85 64.64 BST-111 media 59.33 32.6 32.6 49.93 hydrolysed buffer 57.89 35.36 35.36 48.48 * Low loader cell population (representative of SP)

SUMMARY OF FINDINGS

(144) Spectral and Fluorescence Properties

(145) Although anthraquinones typically have very low quantum yields (as described in Njoh K L et al, Spectral analysis of the DNA targeting bisalkylaminoanthraquinone DRAQ5 in intact living cells, Cytometry A 2006; 69:805-14) this can make them ideal probes for performance as a contrast agent for both fluorescence detection due to cell accumulation and for photoacoustic imaging. Accordingly, low quantum yield molecules such as those described in the present invention, including their hydrolysed and deuterated forms, that also have differential cellular uptake, subcellular localization and persistence profiles can co-display advantageous photoacoustic for use in cell-based assays. AQ-derived molecules can also provide advantageous properties for detection by SERS in cell-based assays.

(146) Absorption spectroscopy and fluorescence spectroscopy was used to show that the compounds of the present invention have far red to near-IR fluorescence properties. Near infrared (NIR) emissive compounds have advantages in the development of fluorescent probes and labelling for bio-imaging in living systems since fluorescence in the long-wavelength region would generate minimum photo-toxicity to biological components, deep tissue penetration and minimal background from auto-fluorescence by bio-molecules.

(147) Therefore, compounds of the invention are useful for analysing biological samples comprising cells, which may be dead, fixed or living or other biological material containing nucleic acid.

(148) Binding Properties

(149) The compounds of the present invention showed intrinsic ability to bind to nucleic acids.

(150) Therefore, compounds of the invention may be used in nuclear discrimination assays.

(151) Labelling Properties of Fixed and Live Cells

(152) The compounds of the invention can be used as nuclear counterstains.

(153) Fixed cell-based and live cell-based imaging assays were used to evaluate cell labelling. Fluorescence of compounds in two fixed and live cell models, U-2 OS and A549 was determined to evaluate their ability to fluorescently label cells with or without the involvement of membrane limiting or active uptake systems.

(154) These analyses showed localization in live cells. Some compounds displayed a nuclear signal greater than membrane signal and therefore they may be use as a live cell labelling ratiometric probes for cell identification. Other compounds showed preferential membrane (Golgi and/or ER) locations and exclusion from nucleus and therefore they may be use as a live cell labelling probes with negative nuclear labelling.

(155) Nuclear counterstains are important tools for many fluorescence-based cell analyses, including but not limited to microscopy, flow cytometry and in-cell western analysis.

(156) The compounds of the invention provide stable fluorophores with structure-dependent subcellular localization and a lack of toxic effects consistent with differential accumulation in endoplasmic reticulum, Golgi or nucleus.

(157) Therefore, labelling experiments showed that compounds of the invention have differential distribution within the cytoplasmic and nuclear compartments thereby providing for pattern based recognition and image-masking methods stained cell nuclei.

(158) The use of the compounds of the present invention as a discriminating or orientating parameter for cell nuclei has been demonstrated for both flow cytometry and confocal laser scanning microscopy.