Antharquinone Compounds as Anti Cancer Compounds

EXAMPLES

General Method 1
Synthesis of the Aminoalkylamino Sidechains—General Method

Synthesis of the 1-(2-aminoethyl)pyrrolidine and piperidine side chains was a two-step procedure. The cyclic secondary amine was alkylated by bromoacetonitrile (dry Et₃N, THF, 45-50° C., 30 min, yield 63-92%) and the nitrile was converted to a primary amine by reduction with LiAlH₄(THF, reflux, 5 h, yield 27-76%). The general method is used for 2-, 3- and 4-hydroxyl piperidines, 2- and 3-hydroxyl pyrrolidines and 2- and 6-bishydroxy-methyl piperidines.

General Method 2
Synthesis of Aminoanthraquinones

Treatment of 1,4-difluoro-5,8-dihydroxyanthraquinone with N,N-dimethylethylenediamine led to a mixture of the di- and monosubstituted anthraquinones from which the intermediate HAQ107 was isolated by flash chromatography (N,N-dimethylethylenediamine, C₅H₅N, 22° C., 24 h, 34%) (Scheme 2, FIG. 1). Pure HAQ107 was then treated with the piperidine or pyrrolidine sidechains to afford the target compounds (HAQ71, HAQ73, HAQ10, HAQ111 and HAQ121) and the non-hydroxylated analogue HAQ148 in good yield after purification (Amine, C₅H₅N, 90° C., 30 min −1 h, 51-65%). HAQ70 and HAQ105, the symmetrically configured anthraquinones, were synthesised in one step by ipso-substitution of both fluoro groups of C with 1-(2-aminoethyl)-piperidine sidechains (C₅H₅N, 90° C., 1 h, 34-37%) (Scheme 3, FIG. 2). Preparation of the chloropropylaminoanthraquinones CAQ74 and CAQ75 were carried out by treating the precursor alcohols (HAQ70 and HAQ71 respectively) with triphenylphosphine-carbon tetrachloride, a commonly employed, complex reagent for conversion of alcohols to corresponding halides (Ph₃P, CCl₄, CH₂Cl₂, reflux, N₂, 5 h, 68-81%).

Synthesis of Hydroxylated N-Acetonitrile-Piperidines and Pyrrolidines
Reference Example 1
3-Hydroxy-piperidin-1-yl-acetonitrile (S1)

BrCH₂CN (3.19 g, 25.82 mmol) was added dropwise to a solution of 3-hydroxypiperidine (6.53 g, 64.55 mmol) in dry THF (25 mL) under N₂, maintaining the temperature between 45-50° C. Following addition of BrCH₂CN, the solution was refluxed for 30 min., before allowing the solution to cool down to room temperature. The solvent was removed in vacuo and the residual oil was purified by flash chromatography using CH₂CI₂: CH₃OH (9:1) as eluent. The title compound was obtained as a straw-coloured oil (3.04 g, 83%). C % (250 MHz; CDCl₃); 1.9-2.15 (m, 3H, CH₂CH₂CH₂, 1×CH₂CH₂CHOH), 2.25 (m, 1H, 1×CH₂CH₂CHOH), 2.55 (d, OH), 2.85-3 (m, 3H, NCH₂CH₂, 1×NCH₂CH), 3.1 (2×d, 1H, 1×NCH₂CH), 3.9 (s, 2H, NCH₂CN) and 4.3 (m, 1H, CH₂CHOH); FAB MS, m/z (M+H)⁺141.

Reference Example 2
3-Hydroxymethyl-piperidin-1-yl-acetonitrile (S2)

The method follows that of S1 using 3-piperidinemethanol (4.79 g, 41.59 mmol), BrCH₂CN (1.99 g, 16.64 mmol) and dry THF (25 mL). The title- compound was yielded as a straw-coloured oil (2.35 g, 92%) after flash chromatography using CH₂Cl₂/CH₃OH (9:1) as eluent. d_H(250 MHz; CDCl₃); 1.05 (m, 1H, CHCH₂OH), 1.5-1.9 (m, 5H, OH, CH₂CH₂CH₂and CH₂CH₂CH), 2.15 (t, 1H, J=10 Hz, 1×NCH₂CH), 2.35 (3×d, 1H, J=4 Hz and J=10 Hz, 1×NCH₂CH₂), 2.7 (m, 1H, 1×NCH₂CH₂), 2.85 (2×d, 1H, J=4 Hz and J=12 Hz, 1×NCH₂CH), 3.52 (s, 2H, NCH₂CN) and 3.45-3.65 (m, 2H, CHCH₂OH); d_c(62.9 MHz; CDCl₃); 24.43, 26.14, 38.55, 46.75, 52.83, 55.73, 65.88 and 114.74 (CN); FAB MS, m/z(M+H)⁺155.

Reference Example 3
4-Hydroxy-piperidin-1-yl-acetonitrile (S3)

BrCH₂CN (28.55 g, 0.238 mmol) was added dropwise to a solution of 4-hydroxypiperidine (24.98 g, 0.216 mol) and Et₃N (33.18 mL, 0.238 mmol) in dry THF (100 mL) under N₂, maintaining the temperature between 45-50° C. After addition of BrCH₂CN, the solution was refluxed for 30 min., before allowing the solution to cool down to room temperature. The title-compound was afforded as a straw-coloured oil (18.97 g, 63%) after purification by flash chromatography using ether/CH₃OH (19:1) as eluent. d_H(250 MHz; CDCl₃); 1.65 (m, 2H, 1×CH₂CH₂CH and 1×CHCH₂CH₂), 1.75 (s, 1H, OH), 1.95 (m, 2H, 1×CH₂CH₂CH, 1×CHCH₂CH₂), 2.45 (m, 2H, 1×NCH₂CH₂and 1×NCH₂CH₂), 2.78 (m, 2H, 1×NCH₂CH₂and 1×NCH₂CH₂), 3.55 (s, 2H, NCH₂CN) and 3.75 (m, 1H, CH₂CHOH); δ_c(62.9 MHz; CDCl₃); 33.96, 46.09, 49.73, 66.58 and 114.71 (CN); IR υ_max/cm⁻¹; 3375 (broad), 2230, 1420, 1330, 1150 and 1060; FAB MS, m/z(M+H)⁺141.

Reference Example 4
2-Hydroxymethyl-piperidin-1-yl-acetonitrile (S4)

The method follows that of S3 using 2-piperidinemethanol (26.50 g, 0.230 mol), BrCH₂CN (30.35 g, 0.253 mol), Et₃N (35.27 mL, 0.253 mol) and dry THF (150 mL). The crude product was purified by flash chromatography using ether/CH₃OH (19:1) as eluent. The title-compound was crystallised from ether yielding cream-coloured crystals (31.46 g, 89%). δ_H(250 MHz; CDCl₃); 1.2-1.85 (m, 6H, NCH₂CH₂CH₂, CH₂CH₂CH₂CH and CH₂CH₂CH), 2.05 (s (broad), 6H, OH), 2.4 (m, 1H, NCH₂CH₂), 2.55 (3×d, 1H. 1×NCH₂CH₂), 2.95 (m, 1H, NCHCH₂), 3.45 (d, 1H, J=17 Hz, 1×NCH₂CN), 3.5 (2×d, 1H, J=3 Hz and 12 Hz, 1×CHCH₂OH), 3.76 (2×d, 1H, J=3 Hz and 12 Hz, CHCH₂OH) and 4.05 (d, 1H, J=17 Hz, 1×NCH₂CN); δ_c(62.9 MHz; CDCl₃); 23.71, 25.25, 28.69, 43.43, 54.04, 60.73, 64.46 and 115.05 (CN); FAB MS, m/z(M+H)⁺155; IR υ_max/cm⁻¹; 3400 (OH, m broad), 2350 and 2240 (CN) and 1650.

Reference Example 5
2-Hydroxymethyl-pyrrolidin-1-yl-acetonitrile (S11)

The method follows that of S3 using 2-pyrrolidinemethanol (24.27 g, 0.241 mol), BrCH₂CN (31.81 g, 0.265 mol), Et₃N (37 mL, 0.265 mol) and dry THF (150 mL). The product was obtained as a straw-coloured oil (21.60 g, 64%) after purification by flash chromatography using ether/CH₃OH (19:1). δ_H(250 MHz; CDCl₃); 1.5-2.0 (m, 5H, CH₂CH₂CH₂, CH₂CH₂CH, OH), 2.7 (m, 1H), 2.85 (m, 1H), 3.05 (m, 1H), 3.45 (2×d, 1H, J=4 Hz and J=11 Hz, 1×CHCH₂OH), 3.65 (2×d, 1H, J=4 Hz and 11 Hz, 1×CHCH₂OH) and 3.75 (d, 2H, NCH₂CN); δ_c(62.9 MHz; CDCl₃); 23.37, 27.45, 40.96, 53.95, 62.43, 63.06 and 115.48(CN); IR υ_max/cm⁻¹; 3375 (NH₂), 3300 (NH₂), 3200 (OH), 2970-2800 (CH₂, CH₃), 1600, 1475, 1375, 1230 and 1060; FAB MS, m/z(M+H)⁺141.

Reference Example 6
3-Hydroxy-pyrrolidin-1-yl-acetonitrile (S12)

The method follows that of S3 using 3-hydroxypyrrolidine (15 g, 0.172 mol), BrCH₂CN (22.67 g, 0.189 mmol) and dry THF (60 mL). The title-compound was yielded as a straw-coloured oil (15.39 g, 71%) after flash chromatography using CH₂Cl₂/CH₃OH (9:1) as eluent. δ_H(250 MHz; CDCl₃); 1.9-2.25 (m, 2H, 2×ring-H), 2.3 (m, 1H, ring-H), 2.65 (d, OH), 2.85-3.05 (m, 3H, 3×ring-H), 3.95 (d, 2H, NCH₂CN) and 4.2 (m, 1H, CH₂CHOH); FAB MS, m/z (M+H)⁺127.

Reference Example 7
2,6-Bis-hydroxymethyl-piperidin-1-yl-acetonitrile (S5)

Step 1. 2,6-Piperidinedimethanol (S15)

2,6-Pyridinedimethanol (15 g, 107.9 mmol) was suspended in glacial acetic acid (250 mL) and hydrogenated at atmospheric pressure and RT for 48 h using PtO₂(1.5 g) as catalyst. The catalyst was then removed by filtration through celite and the acidic solution was concentrated in vacuo to give an oily residue. The oil was diluted with EtOAc (50 mL) and stirred at ice-cold temperature. A saturated solution of brine and concentrated ammonia (pH˜12) was added slowly until the pH was 11-12. The organic phase was then separated from the aqueous phase, which was extracted with EtOAc (3×50 mL). The combined organic phases were dried (MgSO₄) followed by filtration and evaporation of solvent under vacuum yielding a straw-coloured oil (5.21 g, 33%). FAB MS, m/z(M+H)⁺146.

Step 2. The method follows that of S5. 2,6-piperidinedimethanol (3.80 g, 26.21 mmol), iodoacetonitrile (5.24 g, 31.45 mmol), Et₃N (4.38 mL, 31.45 mmol) and dry DMF (20 mL). Purification by flash chromatography using CH₂Cl₂/CH₃OH (9:1) as eluent afforded the title compound as a straw-coloured oil (2.5 g, 53%). FAB MS, m/z(M+H)⁺185.

The iodoacetonitrile was prepared by the use of the Finkelstein reaction. BrCH₂CN (3.77 g, 0.0314 mmol) was added dropwise to a stirred solution of Nal (4.71 g, 0.031 mmol) in acetone. Precipitation of NaBr occurred within a few minutes and indicated that exchange of the halides had taken place. Sodium bromide was filtered off, and the acetone was removed in vacuo. Crude yield (5.24 g, 100%).

Synthesis of Hydroxylated N-2-Aminoethyl Piperidines and Pyrrolidines
Reference Example 8
1-(2-Amino-ethyl)-piperidin-3-ol (S6)

LiAIH₄(2.44 g, 64.2 mmol) was added to dry THF (20 mL) at 0° C. in a three-neck round bottom flask under N₂. The solution was stirred for 15 min. before the 3-hydroxypiperidin-1-yl-acetonitrile (3 g, 21.4 mmol), diluted in dry THF (5 mL), was added slowly via syringe. The reaction mixture was then refluxed 5 h before allowing the solution to cool to RT. Excess of LiAIH₄was destroyed by dropwise addition of 2.4 mL of H₂O, 2.4 mL of NaOH (15%) and finally EtOAc was added dropwise until no effervesence was observed. The formed granular precipitate (lithium hydroxide and aluminium hydroxide) was filtered off and washed several times with CH₂Cl₂and EtOAc. The organic layer was dried (MgSO₄) and the solvent was removed in vacuo to yield a thick yellowish oil. The title-compound was purified by kugelrohr distillation (172° C., 0.05 mbar) and obtained as a straw coloured oil (1.95 g, 63%). δ_H(250 MHz;

CDCl₃); 1.45-1.7 (m, 3H, CH₂CH₂CH₂, 1×CH₂CH₂CHOH), 1.76 (m, 1H, 1×CH₂CH₂CHOH), 1.95 (s, 3H, OH, NH₂), 2.25-2.55 (m, 6H, H₂NCH₂CH₂, NCH₂CH₂, NCH₂CH), 2.75 (t, 2H, H₂NCH₂CH₂), and 3.9 (m, 1H, CH₂CHOH); δ_c(62.9 MHz; CDCl₃); 21.93, 32.01, 38.88, 53.84, 60.80, 61.06 and 66.36; FAB MS, m/z(M+H)⁺145.

Reference Example 9
[1-(2-Amino-ethyl)-3-piperidin-2-yl-]methanol (S7)

The method follows that of S6 using 3-hydroxymethyl-piperidin-1-yl-acetonitrile (2.95 g, 19.1 mmol), LiAlH₄(2.18 g, 57.3 mmol) and dry THF (15 mL). The title compound (2.30 g, 76%) was afforded as a colourless oil by kugelrohr distillation at (164° C., 0.01 mbar). δ_H(250 MHz; CDCl₃); 1.1 (m, 1H, (CHCH₂OH), 1.5-1.9 (m, 7H, OH, NH₂, CH₂CH₂CH₂and CH₂CH₂CH), 1.95 (t, 1H, J=10 Hz, 1×NCH₂CH), 2.1 (3×d, 1H, J=3 Hz and 10 Hz, 1×NCH₂CH₂), 2.4 (t, 2H, J=6 Hz, H₂NCH₂CH₂N), 2.6 (m, 1H, 1×NCH₂CH₂, 2.8 (t, 3H, J=6 Hz, H₂NCH₂CH₂N and 1×NCH₂CH), 3.45-3.65 (m, 2H, CHCH₂OH); δ_c(62.9 MHz; CDCl₃); 24.66, 27.50, 38.19, 38.91, 54.54, 57.43, 61.61 and 66.55; IR υ_max/cm⁻¹; 3375 (broad), 1675, 1625, 1450, 1100 and 1050; FAB MS, m/z(M+H)⁺159.

Reference Example 10
1-(2-Amino-ethyl)-piperndin-4-ol (S8)

The method follows that of S6 using 4-hydroxy-piperidin-1-yl-acetonitrile (18.97 g, 0.136 mol), LiAlH₄(15.48 g, 0.408 mol) and dry THF (150 mL). The title compound (8.56 g, 44%) was afforded as a straw-coloured oil after kugelrohr distillation (178° C., 0.05 mbar). δ_H(250 MHz; CDCl₃); 1.55 (m, 2H, 1×CHCH₂CH₂and 1×CH₂CH₂CH), 1.85 (m, 2H, 1×CHCH₂CH₂and 1×CH₂CH₂CH), 2-2.25 (m, 5H, NCH₂CH₂, NCH₂CH₂and OH), 2.38 (t, 2H, H₂NCH₂CH₂N), 2.72 (t, 4H, H₂NCH₂CH₂N, NH₂) and 3.65 (m, 1H, CH₂CHOH); δ_c(62.9 MHz; CDCl₃); 34.56, 38.98, 51.35, 60.84 and 67.64; FAB MS, m/z(M+H)⁺145; IR υ_max/cm⁻¹; 3375 (broad), 1600, 1460, 1370, 1290 and 1070.

Reference Example 11
[1-(2-Amino-ethyl)-piperidin-2-yl-]methanol (S9)

The method follows that of S6 using 2-hydroxymethyl-piperidin-1-yl-acetonitrile (31.96 g, 0.208 mol), LiAlH₄(23.68 g, 0.624 mol) and dry THF (200 mL). The title compound (8.74 g, 27%) was afforded as straw-coloured oil by kugelrohr distillation (225° C., 0.13 mbar). δ_H(250 MHz; CDCl₃); 1.2-1.75 (m, 6H, CH₂CH₂CH₂, CH₂CH₂CH and CH₂CH₂CH), 2.2 (m, 1H, 1×NCH₂CH₂) 2.35 (m, 2H, H₂NCH₂CH₂N), 2.58 (s (broad), 3H, OH and NH₂), 2.75 (t, 2H, H₂NCH₂CH₂N), 2.85 (3×d, 1H, 1×NCH₂CH₂), 2.92 (m, 1H, NCHCH₂), 3.4 (2×d, 1H, J=4 Hz and 12 Hz, 1×CHCH₂OH) and 3.76 (2×d, 1H, J=4 Hz and 12 Hz, 1×CHCH₂OH; FAB MS, m/z(M+H)⁺159.

Reference Example 12
[1-(2-Amino-ethyl)-piperidin-bis-2,6-yl-]methanol (S10)

The method follows that of S6 using 2,6-bis-hydroxymethyl-piperidin-1-yl-acetonitrile (2.40 g, 13.04 mmol), LiAlH₄(1.49 g, 39.13 mmol) and dry THF (30 mL). The title-compound was afforded as a straw-coloured oil without purification (1.03 g, 42%). It was found necessary to stir the dry destroyed LiAlH₄complex in dry THF for 10 h at 40° C. in order to optimise the yield of the desired di-hydroxylated diamine. FAB MS, m/z(M+H)⁺189.

Reference Example 13
[1-(2-Amino-ethyl)-pyrrolidin-2-yl-]methanol (S13)

The method follows that of S6 using 2-hydroxymethyl-pyrrolidin-1-yl-acetonitrile (19.5 g, 0.139 mol), LiAlH₄(15.84 g, 0.417 mol) and dry THF (150 mL). The title-compound (12.5 g, 63%) was afforded as straw-coloured oil by kugelrohr distillation (142° C., 0.3 mbar). δ_H(250 MHz; CDCl₃); 1.6-1.9 (m, 4H, CH₂CH₂CH₂, CH₂CH₂CH), 1.98 (s (broad), 3H, NH₂, OH), 2.3 (m, 1H), 2.45 (m, 1H), 2.55 (m, 1H), 2.75-2.85 (m, 3H), 3.19 (m, 1H, NCHCH₂), 3.4 (2×d, 1H, J=3 Hz and J=11 Hz, 1×CHCH₂OH), 3.6 (2×d, 1H, J=3 Hz and J=11 Hz, 1×CHCH₂OH); IR υ_max/cm⁻¹; 3375 (NH₂), 3300 (NH₂), 3200 (OH), 2970-2800 (CH₂, CH₃), 1600, 1475, 1375, 1230 and 1060; FAB MS, m/z(M+H)⁺145.

Reference Example 14
1-Amino-ethyl-pyrrolidin-3-ol (S14)

The method follows that of S6 using 3-hydroxy-pyrrolidin-1-yl-acetonitrile (15 g, 0.107 mol), LiAlH₄(10.17 g, 0.268 mol) and dry THF (150 mL). The title compound (9.55 g, 69%) was afforded as a straw-coloured oil and used for the next step without further purification. δ_H(250 MHz; CDCl₃); 1.55-1.85 (m, 2H, 2×ring-H), 2.05 (s, 3H, OH, NH₂), 2.25-2.55 (m, 6H, 4×ring-H and H₂NCH₂CH₂N, 2.9 (t, 2H, H₂NCH₂CH₂N) and 4.05 (m, 1H, CH₂CHOH); FAB MS, m/z(M+H)⁺131.

Preparation of Chromophores
Reference Example 15
1,5-Diamino-4,8-Dihydroxyanthraquinone

In a 2 litre round bottom flask was placed 1,5-diaminoanthracene-9,10-dione (12 g, 50 mmol). Concentrated sulphuric acid (180 g) was added and the mixture was stirred 15 minutes at 0° C. before adding sodium chlorate (14.4 g, 134 mmol) portionwise over 45 minutes. The reaction mixture was allowed to warm up to room temperature where it was left stirred for 3 h before it was poured into 1% solution of chilled sodium hydrogen sulfite (1 L). The precipitated solid was removed by filtration and was washed successively with cold water arid then hot water. After lyophilisation overnight, the product (11.3 g, 83%) was obtained as a crude purple-blue solid. FAB MS, m/z(M+H)⁺271.

Reference Example 16
5,8-Dihydroxyleucoquinizarin (Leuco-1,4,5,8,-tetrahydroxyanthraquinone) (B).

To crude 1,5-diamino-4,8-dihydroxyanthraquinone (11 g, 0.041 mol) was added NaOH (2.5 M, 200 mL) and the suspension was refluxed gently for 3 hours before it was allowed to cool down to room temperature. Sodium hydrogen sulphite (31.74 g, 0.182 mol) was added portionwise and the reaction mixture was refluxed again for 3 hours. It was cooled down to room temperature and was acidified with concentrated hydrochloric acid until pH 3. The precipitate was isolated by filtration and washed successively with cold water and then hot water.. After lyophilisation overnight, the title compound was afforded as a crude brown solid (8 g, 73%). FAB MS, m/z(M+H)⁺251.

Reference Example 17
1,4-Difluoro-5,8-Dihydroxyanthraquinone (C)

A mixture of ground AlCl₃(2.955 g, 22.16 mmol), NaCl (432 mg, 7.39 mmol), 1,4-dihydroxybenzene (224 mg, 2.03 mmol) and 3,6-difluorophthalic dry (340 mg, 1.85 mmol) were stirred vigorously in a round bottom flask and heated to 220° C. in an oil-bath for 3 h. The oil-bath was removed and the reaction quenched by addition of ice and concentrated hydrochloric acid (10 mL). The final aqueous solution was filtered under suction and the precipitated material was washed with water followed by freeze-drying. No further work-up was required, as the brown title-compound (470 mg, 92%) was pure as observed by TLC and confirmed by NMR and MS: δ_H(250 MHz; CDCl₃); 7.3 (s, 2H), 7.55 (m, 2H), 12.9 (s, 2H); FAB MS, m/z(M+H)⁺277.

Reference Example 18
1-[[2-(Dimethylamino)ethyl]amino]-4-fluoro-5,8-dihydroxyanthracene-9,10-dione (HAQ107)

1,4-Difluoro-5,8-dihydroxyanthraquinone (0.50 g, 1.812 mmol) N,N-dimethylethylenediamine (0.16 g, 1.812 mmol) and pyridine (3 mL) were stirred for 24 h at RT. The mixture was quenched in cold brine (50 mL) and left for 3 hours before the crude product was isolated by filtration. The crude product was chromatographed using a gradient elution from 1 to 5% MeOH in CH₂Cl₂. The product HAQ107 was afforded as a purple powder (0.24 g, 38%). δ_H(250 MHz; DMSO); 2.35 (s, 6H, 2×NCH₃) 2.7 (t, 2H, HNCH₂CH₂N), 3.5 (q,2H, HNCH₂CH₂N), 7.1 (s, 1H), 7.1 (s,1H), 7.4 (s, 2H, C(6)H and C(7)H), 10.75 (t, 2H, C(1)NH and C(4)NH) and 13.4 (s, 2H, C(5)OH and C(8)OH); δ_c(62.9 MHz; DMSO); FAB MS, m/z(M+H)⁺344.

Chromophore Substitution Reactions
Examples 1 and 2
Amination of Leucoquinizarin and 5,8-Dihydroxyleucoquinizarin 1-{[(2-Dimethylamino)ethyl]amino}-4-{[2-(3-hydroxypiperidin-1-yl)ethyl]amino}-anthracene-9,10-dione (HAQ22) and 1,4-Bis-{[2-(3-hydroxypiperidin-1-yl)ethyl]-amino}-anthracene-9,10-dione (HAQ24)

N-N-dimethylethylenediamine (0.92 g, 10.42 mmol) and 1-(2-aminoethyl)-3-piperidin-3-ol (1.50 g, 10.42 mmol) in EtOH (5 mL) were simultaneously added to a suspension of leucoquinizarin (0.63 g, 2.61 mmol) in EtOH (25 mL) under N₂. After 8 h of stirring at reflux temperature, the reaction mixture was stirred at RT another 14 h. The EtOH was removed in vacuo and the remaining residue was added to ice-cold brine. The precipitated solid was isolated by filtration and lyophilised. The dark blue solid was flash chromatographed in a short column by first using CH₂Cl₂to remove non-polar side-products, then by increasing the polarity gradually removing more side-products and finally by using CH₂Cl₂/CH₃OH (4:1), obtaining the desired crude components. The crude solid was flash chromatographed using CH₂Cl₂/CH₃OH/NH₃(94:6:0.75) as eluent. To obtain pure components a final purification was made by preparative TLC using CH₂Cl₂/CH₃OH/NH₃(94:6:0.75) as eluent. HAQ22 and HAQ24 were afforded as dark-blue solids (0.12 g, 10%) and (0.16 g, 12%) respectively.

HAQ22 δ_H(250 MHz; CDCl₃); 1.5 (m, 2H, CH₂CH₂CH₂), 1.8 (m, 1H, 1×CH₂CH₂CH), 2.0 (m, 1H, 1×CH₂CH₂CH), 2.2 (t, 1H, 1×NCH₂CH₂), 2.3 (s, 6H, 2×NCH₃), 2.5 (d, 1H, 1×NCH₂CH), 2.6-2.8 (m, 7H, 2×NCH₂CH₂N, 1×NCH₂CH and OH), 3.4 (m, 4H, 2×H₂NCH₂CH₂N), 3.9 (m, 1H, CH₂CHOH), 7.1 (d, 2H, C(2)H, C(3)H), 7.62 (m, 2H, C(6)H, C(7)H), 8.32 (m, 2H, C(5)H, C)8)H), 10.75 (t, 2H, C(1)NH) and 11 (t, 1H, C(4)NH); d(62.9 MHz; CDCl₃); 21.18, 31.47, 39.72, 41.13, 45.72, 53.06, 56.05, 58.63, 60.06, 65.99, 110.15, 123.55, 126.16, 131.97, 134.48, 145.78 and 182.41; FAB MS, m/z(M+H)⁺ 437; IR ν_mac/cm⁻¹; 3450 (broad, OH), 3220 (NH), 3020 (Ar—CH), 2960-2800 (CH₂—CH₃), 1650, 1625, 1575, 1480, 1380 and 1220.

HAQ24 δ_H(250 MHz; CDCl₃); 1.5-1.9 (m, 10H, 8×ring-H and 2×OH), 2.1 (m, 2H, 2×ring-H), 2.25 (m, 2H, 2×ring-H), 2.5 (d, 2H, 2×ring-H), 2.75-2.95 (m, 6H, 4×ring-H and 2×HNCH₂CH₂N), 3.5 (q, 4H, 2×HNCH₂CH₂N), 3.95 (m, 2H, 2×CH₂CHOH), 7.2 (s, 2H, C(2)H and C(3)H), 7.65 (m, 2H, C(6)H and C(7)H), 8.35 (m, 2H, C(5)H and C)8)H), and 11.0 (t, 2H, C(1)NH and C(4)NH); FAB MS, m/z (M+H)⁺493.

The following are examples of the invention and comparative examples. The reaction schemes are shown in schemes 2 (FIG. 1) and 3 (FIG. 2). In these schemes, step (I) is the addition of one amino alkyl amino side chain carried out in pyridine at 90° C. for 30min-1 hr; step (II) is chlorination carried out using (Ph)₃PCClY and CH₂Cl₂and at reflux for 3-1 Oh using ethereal HCl. In scheme 3, step (Ill) is a further side chain linking step carried out at reflux in ethanol; and step IV is chlorination of the side chain hydroxyl groups carried out in pyridine at 30-60° C. for 2 to 5 h, using ethereal HCl.

Example 3
1,4-Bis-{[2-(3-hydroxymethylpiperidine-1-yl)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione (HAQ70)

[1-(2-aminoethyl)-piperidin-3-yl]methanol (1.9 g, 12 mmol) in EtOH (2 mL) was added to a stirred suspension of 5,8-dihydroxyleucoquinizarin (272 mg, 1 mmol) in EtOH (15 mL) under N₂. After 7 h of stirring at reflux temperature, the reaction mixture was cooled down to RT and stirred for another 16 h. The EtOH was removed in vacuo and the remaining residue was added to ice-cold brine. The precipitated solid was isolated by filtration and lyophilised. The dark blue solid was flash chromatographed using CH₂Cl₂followed by gradual increase in polarity to CH₂Cl₂/CH₃OH (4:1). Subsequently, the crude product was flash chromatographed using CH₂Cl₂/CH₃OH/NH₃(94:6:0.75) as eluent. The title compound was afforded as dark-blue solid (115.9 mg, 21%). M.p. 180-183° C.; δ_H(250 MHz; CDCl₃); 1.1-1.95 (m, 12H, 2×OH and 10×ring-H), 2.25 (m, 4H, 4×ring-H ), 2.7 (t, 6H, 2×HNCH₂CH₂N and 2×ring-H), 2.95 (2×d, 2H, 2×ring-H), 3.5 (m, 4H, 2×HNCH₂CH₂), 3.65 (m, 4H, 2×CHCH₂OH), 7.1 (s, 2H, C(2)H and C(3)H), 7.2 (s, 2H, C(6)H and C(7)H), 10.5 (t, 2H, C(1)NH and C(4)NH) and 13.6 (s, 2H, C(5)OH and C(8)OH); δ_C(62.9 MHz; CDCl₃); FAB MS, m/z(M+H)³⁰ 553; IR ν_max/cm⁻¹; 3400 (OH), 3225 (NH), 3100 (Ar—CH), 2960-2800 (CH₂, CH₃), 1650, 1625, 1575, 1480, 1370 and 1225; Anal. calcd for C₃₀H₄₀N₄O₆: C, 65.20; H, 5.47; N, 10.14. Found: C, 65.16; H, 5.49; N, 9.93.

Example 4
1,4-Bis-{[2-(3-hydroxymethylpiperidine-1-yl)ethyl]amino}-anthracene-9,10-dione (HAQ38)

Although not shown in scheme 3, the method follows that of HAQ70 using leucoquinizarin (0.23 g, 0.95 mmol), [1-(2-aminoethyl)-piperidin-3-yl-]methanol (0.06 g, 3.8 mmol) and ethanol (25 mL). The title compound was yielded as a dark-blue solid (0.15 g, 29%). M.p. 112-114° C.; δ_H(250 MHz; CDCl₃); 1.15 (m, 2H, 2×CH₂CHCH₂OH), 1.5-1.75 (m, 6H, 2×CH₂CH₂CH and 2×CH₂CH₂CH₂), 1.85 (m, 2H, 2×CH₂CH₂CH), 2.3 (m, 4H, 4×ring-H), 2.5 (s (broad), 4H, 2×ring-H, 2×OH), 2.65 (t, 4H, J=5 Hz, 2×HNCH₂CH₂N), 2.75 (2×d, 2H, J=3 Hz and 12 Hz, 2×ring-H), 3.42 (q, 4H, J=5 Hz, 2×HNCH₂CH₂N), 3.55 (2×d, 2H, J=5 Hz and 12 Hz, 2×CHCH₂OH), 3.7 (2×d, 2H, J=9 Hz and 12 Hz, 2×CHCH₂OH), 7.1 (s, 2H, C(2)H and C(3)H), 7.6 (m, 2H, C(6)H and C(7)H), 8.3 (m, 2H, C(5)H, C(8)H) and 10.75 (t, 2H, C(1)NH and C(4)NH); δ_C(62.9 MHz; CDCl₃); 24.08, 26.92, 37.90, 40.52, 54.43, 56.95, 57.75, 65.70, 109.89, 123.87, 126.13, 132.09, 134.46, 146.6 and 182.36; FAB MS, m/z(M+H)⁺ 521; IR ν_max/cm⁻¹; 3400 (OH), 3090 (Ar—CH), 2960-2800 (CH₂, CH₃), 1650, 1585, 1550, 1520, 1265, 1175 and 1125; Anal. calcd for C₃₀H₄₀N₄O₄: C, 69.21; H, 7.74; N, 10.76. Found: C, 69.22; H, 7.88; N, 10.69.

Ipso Substitution of Fluorides of 1,4-Difluoro-5,8-dihydroxy-anthraquinone by Diamine
Example 5
1-[(2-Dimethylamino)ethylamino]-4-[2-(3-hydroxymethyl-piperidin-1-yl)ethylamino]-5,8-dihydroxy-anthracene-9,10-dione (HAQ71)

The method follows that of HAQ105 (see Example 7 below) using HAQ107 (200 mg, 0.581 mmol), [1-(2-aminoethyl)-piperidin-3-yl-]methanol (350 mg, 2.215 mmol), pyridine (2 mL), 90° C., 30 min. The product was afforded as a dark blue powder (190 mg, 68%). M.p. 181-183 ° C.; δ_H(250 MHz; CDCl₃); 1.2 (m, 2H, 2×ring-H), 1.6-1.9 (m, 5H, OH and 4×ring-H), 2.18-2.3 (m, 2H, ring-H), 2.35 (s, 6H, 2×NCH₃), 2.6-2.75 (m, 4H, 2×HNCH₂CH₂N), 2.9 (2×d, 1H, 1×ring-H), 3.46 (m, 4H, 2×HNCH₂CH₂N), 3.65 (2×d, 2H, CHCH₂OH), 7.15 (s, 2H, C(2)H and C(3)H), 7.2 (s, 2H, C(6)H and C(7)H), 10.4 (t, 1H, C(1)H), 10.5 (t, 1H, C(4)H), 13.5 (s, 1H, C(8)H) and 13.6 (s, 1H, C(5)H); δ_C(62.9 MHz; CDCl₃); 24.33, 26.92, 38.29, 40.03, 40.87, 45.42, 54.23, 57.03, 58.20, 65.99, 108.97, 115.33, 123.43, 123.65, 124.51, 145.99, 146.09, 155.32, and 184.98; FAB MS, m/z(M+H)⁺ 483; IR ν_max/cm⁻¹; 3425 (OH), 3225 (NH), 3100 (Ar—CH), 2975-2800 (CH₂, CH₃), 1650, 1625, 1575, 1490, 1360 and 1225; Anal. calcd for C₂₆H₃₄N₄O₅: C, 64.71; H, 7.10; N, 11.61. Found: C, 64.81; H, 7.14; N, 11.56.

Example 6
1-[(2-Dimethylamino)ethylamino]-4-[2-(3-hydroxypiperidon-1-yl)ethylamino]-5,8-dihydroxy-anthracene-9,10-dione (HAQ73)

The method follows that of HAQ105 using HAQ107 (120 mg, 0.349 mmol), 1-(2-aminoethyl)-piperidin-3-ol (150 mg, 1.047 mmol), pyridine (1 mL), 30 min, 90° C. The product was afforded as a dark blue powder (95 mg, 58%). M.p. 228-230° C.; δ_H(250 MHz; CDCl₃); 1.55-1.75 (m, 4H, 4×ring-H), 1.83-2.0 (m, 2H, 2×ring-H), 2.28 (2×d, 1H, ring-H), 2.35 (s, 6H, 2×NCH₃), 2.65-2.75 (2×t, 5H, 2×HNCH₂CH₂N and OH), 3.46 (m, 4H, HNCH₂CH₂N), 3.9 (m, 1H, NCH₂CHOH), 7.1 (s, 2H, C(2)H and C(3)H), 7.15 (s, 2H, C(6)H and C(7)H), 10.4 (t, 1H, C(1)H), 10.6 (t, 1H, C(4)H), 13.4 (s, 1H, C(8)H) and 13.5 (s, 1H, C(5)H); δ_C(62.9 MHz; DMSO); 21.48, 31.52, 39.99, 41.26, 45.61, 53.31, 56.14, 58.43, 60.20, 66.03, 109.11, 115.43, 115.48, 123.81, 124.70, 146.29, 155.51, and 185.24; FAB MS, m/z(M+H)⁺ 469; IR ν_max/cm⁻¹; 3425 (OH), 3240 (NH), 3100 (Ar—CH), 2975-2800 (CH₂, CH₃), 1650, 1625, 1575, 1490, 1375 and 1225; Anal. calcd for C₂₈H₃₆N₄O₆: C, 64.09; H, 6.88; N, 11.96. Found: C, 63.88; H, 6.89; N, 11.98.

Example 7
1,4-Bis-{[2-(2-hydroxymethlylpiperidln-1-yl)ethyl]amino}-5,8-dihydroxy-anthracene-9,10-dione (HAQ105)

1,4-Difluoro-5,8-hydroxyanthraquinone (0.17 g, 0.601 mmol) and [1-(2-aminoethyl)-piperidin-2-yl-]methanol (0.95 g, 6.01 mmol) were stirred in pyridine (2 mL) at 90° C. for 1 h. The reaction mixture was added to ice-cold brine and set aside at 4° C. overnight. The precipitated solid was isolated by filtration and lyophilised. The desired product was purified by flash chromatography, initially eluting with CH₂Cl₂/CH₃OH (95:5) to remove non-polar impurities, followed by a gradual increase of CH₃0H to CH₂Cl₂/CH₃OH (85:15). The chromatographed product was then crystallised from CHCl₃affording the title compound HAQ105 as a dark blue powder (0.11 g, 34%). M.p. 208-210° C.; (250 MHz; DMSO); 1.15-1.65 (m, 12H, 2×OH and 10×ring-H), 2.25-2.4 (m, 6H, 6×ring-H), 2.6-2.7 (t,.4H, 2×HNCH₂CH₂N), 2.85 (m, 2H, 2×ring-H), 3.55 (t, 4H, 2×HNCH₂CH₂N), 3.6-3.65 (2×d, 4H, 2×CHCH₂OH), 7.2 (s, 2H, C(2)H and C(3)H), 7.5 (s, 2H, C(6)H and C(7)H), 10.75 (t, 2H, C(1)NH and C(4)NH) and 13.65 (s, 2H, C(5)OH and C(8)OH); δ_C(62.9 MHz; DMSO); 22.55, 24.92, 28.14, 50.99, 52.32, 61.94, 62.42, 107.03, 116.11, 123.89, 125.91, 147.07, 154.46, and 182.88; FAB MS, m/z(M+H)⁺553; IR ν_max/cm⁻¹; 3400 (OH), 3225 (NH), 3100 (Ar—CH), 2960-2800 (CH₂, CH₃), 1650, 1625, 1575, 1480, 1370 and 1225; Anal. calcd for C₃₀H₄₀N₄O₆.1H₂O: C, 63.14; H, 7.24; N, 9.82. Found: C, 63.07; H, 7.49; N, 9.77.

Example 8
1-{[(2-Dimethylamino)ethyl]amino}-4-{[2-(2-hydroxymethylpyrrolidin-1-yl)ethyl]-amino}-5,8-dihydroxy-anthracene-9,10-dione (HAQ110)

The method follows that of HAQ105 using HAQ107 (75 mg, 0.218 mmol), [1-(2-aminoethyl)-pyrrolidin-2-yl-]methanol (650 mg, 4.114 mmol), pyridine (2 mL), 1 h, 90° C. The product HAQ110 was afforded as a dark blue powder (52 mg, 51%). M.p. 202-203° C.; δ_H(250 MHz; DMSO/CDCl₃(1:1)); 1.55-1.9 (m, 4H, 4×ring-H), 2.3 (s, 6H, 2×NCH₃), 2.5-2.55 (m, 1H, 1×ring-H), 3.25-3.4 (m, 7H, 2×HNCH₂CH₂N, 2×ring-H and OH), 3.45-3.6 (m, 6H, 2×HNCH₂CH₂N and NCHCH₂OH), 7.05 (s, 2H, C(2)H and C(3)H), 7.2 (m, 2H, C(6)H and C(7)H), 10.6-10.7 (2×t, 2H, C(1)NH and C(4)NH), and 13.5 (s, 2H, C(5)OH, C(8)OH); δ_C(62.9 MHz; DMSO/CDCl₃(1:1)); 22.71, 27.65, 41.58, 44.83, 53.38, 53.53, 57.54, 64.07, 64.94, 107.10, 114.81, 123.73, 125.00, 125.11, 146.52, 154.36, and 183.17; FAB MS, m/z(M+H)⁺ 469; Anal. calcd for C₂₅H₃₂N₄O₅: C, 64.09; H, 6.88; N, 11.96. Found: C, 63.83; H, 6.99; N, 12.05.

Example 9
1-{[(2-Dimethylamino)ethyl]amino}-4-{[2-(4-hydroxypiperidin-1-yl)ethyl]amino}-5,8-dihydroxy-anthracene-9,10-dione (HAQ111)

The method follows that of HAQ105 using HAQ107 (18 mg, 0.0523 mmol), N-(2-aminoethyl)-piperidin-4-ol (140 mg, 0.97 mmol), pyridine (1 mL), 1 h, 90° C. The product HAQ111 was afforded as a dark blue powder (16.1 mg, 65%). M.p. 231-233° C.; δ_H(250 MHz; DMSO/CDCl₃(1:1)); 1.25-1.8 (m, 6H, 6×ring-H), 2.2-2.25 (m, 2H, 2×ring-H), 2.35 (s, 6H, 2×NCH₃), 2.5-2.8 (m, 5H, 2×HNCH₂CH₂N and 1×OH), 3.5-3.55 (m, 5H, 2×HNCH₂CH₂N and NCH₂CHOH), 7.1 (s, 2H, C(2)H and. C(3)H), 7.25 (m, 2H, C(6)H and C(7)H), 10.65 (t, 2H, C(1)NH and C(4)NH), and 13.65 (s, 2H, C(5)OH, C(8)OH); δ_C(62.9 MHz; DMSO/CDCl₃(1:1)); 27.78, 32.15, 43.72, 49.49, 54.73, 56.39, 65.09, 106.48, 113.66, 122.55, 123.12, 123.39, 144.97, 153.33, 182.56; FAB MS, m/z(M+H)⁺ 469; Anal. calcd for C₂₅H32N₄O₅.1H₂O: C, 61.17; H, 6.84; N, 11.52. Found: C, 61.27; H, 6.64; N, 11.40.

Example 10
1,4-Bis-{[2-(3-hydroxypyrrolidin-1-yl)ethyl]amino}-5,8-dihydroxy-anthracene-9,10-dione (HAQ115)

Although not shown in scheme 3 the method follows that of HAQ105 using 1,4-difluoro-5,8-hydroxy-anthraquinone (75 mg, 0.272 mmol), 1-(2-aminoethyl)-pyrrolidin-3-ol (1 g, 7.7 mmol), pyridine (2 mL), 1 h, 100° C. The product HAQ115 was afforded as a dark blue powder (59.2 mg, 44%). M.p. 197-199° C.; δ_H(250 MHz; DMSO/CDCl₃(1:1)); 1.55-1.65 (m, 2H, 2×ring-H), 2.0-2.1 (m, 2H, 2×ring-H), 2.45-2.9 (m, 10H, 8×ring-H and 2×OH), 2.75-2.85 (t, 4H, 2×HNCH₂CH₂N), 3.55-3.65 (q, 4H, 2×HNCH₂CH₂N), 4.05-4.15 (m, 2H, 2×CH₂CHOH), 7.05 (s, 2H, C(2)H and C(3)H), 7.3 (m, 2H, C(6)H and C(7)H), 10.55 (t, 2H, C(1)NH and C(4)NH), and 13.55 (s, 2H, C(5)OH, C(8)OH); δ_C(62.9 MHz; DMSO/CDCl₃(1:1)); 28.01, 34.73, 41.22, 52.18, 54.42, 62.33, 69.26, 107.01, 115.04, 123.79, 124.90, 146.43, 154.38, and 183.21; FAB MS, m/z(M+H)⁺ 497; Anal. Calcd for C₂₆H₃₂N₄O₆: C, 62.88; H, 6.51; N, 11.28. Found: C, 62.50; H, 6.54; N, 11.00.

Example 11
1,4-Bis-{[2-(4-hydroxypiperidin-1-yl)ethyl]amino}-5,8-dihydroxy-anthracene-9,10-dione (HAQ116)

The method follows that of HAQ105 using 1,4-difluoro-5,8-hydroxy-anthraquinone (45 mg, 0.163 mmol), 1-(2-aminoethyl)-piperidin-4-ol (1 g, 42.6 mmol), pyridine (2 mL), 1 h, 90° C. The product HAQ116 was afforded as a dark blue powder (36.2 mg, 42%). M.p. 241-244° C.; δ_H(250 MHz; CDCl₃); 1.25-1.65 (m, 12H, 12×ring-H), 1.7-2.0 (m, 4H, 4×ring-H), 2.65-3.0 (m, 6H, 2×NCH₂CH₂N and 2×OH), 3.6-3.8 (q, 4H, 2×HNCH₂CH₂N), 4.1 (m, 2H, 2×CH₂CHOH), 7.2 (s, 2H, C(2)H and C(3)H), 7.6 (m, 2H, C(6)H and C(7)H), 10.5 (t, 2H, C(1)NH and C(4)NH), and 13.6 (s, 2H, C(5)OH, C(8)OH); δ_C(62.9 MHz; CDCl₃); 31.67, 39.45, 41.56, 49.56, 54.66, 109.22, 115.40, 124.82, 125.45, 146.39, 154.70, 164.022, and 183.54; FAB MS, m/z(M+H)⁺ 525.

Example 12
1-{[(2-Dimethylamino)ethyl]amino}-4-([2-(3-hydroxypyrrolidin-1-yl)ethyl]amino)-5,8-dihydroxy-anthracene-9,10-dione (HAQ120)

The method follows that of HAQ105 using HAQ107 (45 mg, 0.13 mmol), 1-(2-aminoethyl)-pyrrolidin-3-ol (880 mg, 6.77 mmol), pyridine (1 mL), 30 min, 100° C. The product HAQ120 was afforded as a dark blue powder (24 mg, 41%). M.p. 195-198° C.; δ_H(250 MHz; DMSO/CDCl₃(1:1)); 1.55-1.7 (m, 1H, 1×ring-H), 1.95-2.05 (m, 2H, 2×ring-H), 2.3 (s, 6H, 2×NCH₃), 2.35-2.4 (m, 1H, 1×ring-H), 2.5-2.6 (t, 4H, 2×HNCH2CH₂N), 2.6-2.85 (m, 2H, 1×ring-H and OH), 3.5-3.55 (q, 4H, 2×HNCH₂CH₂N), 4.25 (m, 1H, CH₂CHOH), 7.1 (s, 2H, C(2)H and C(3)H), 7.4 (m, 2H, C(6)H and C(7)H), 10.5 (t, 2H, C(1)NH and C(4)NH), and 13.5 (s, 2H, C(5)OH, C(8)OH); δ_C(62.9 MHz; DMSO/CDCl₃(1:1)); 34.19, 41.22, 52.18, 54.42, 62.33, 69.26, 107.18, 114.78, 123.79, 124.90, 146.90, 154.38, and 183.16; FAB MS, m/z(M+H)⁺ 455.

Example 13
1-{[(2-Dimethylamino)ethyl]amino}-4-{[2-(2-hydroxymethylpiperidin-1-yl)ethyl]-amino}-5,8-dihydroxy-anthracene-9,10-dione (HAQ121)

The method follows that of HAQ105 using HAQ107 (30 mg, 0.0872 mmol), [1-(2-aminoethyl)-piperidin-2-yl-]methanol (700 mg, 4.43 mmol), pyridine (1 mL), 30 min, 100° C. The product HAQ121 was afforded as a dark blue powder (21.3 mg, 51%). M.p. 201-203° C.; δ_H(250 MHz; CDCl₃); 1.7-2.1 (m, 7H, 6×ring-H and OH), 2.6 (s, 6H, 2×NCH₃), 2.8 (m, 2H, 2×ring-H), 2.9 (t, 4H, 2×HNCH₂CH₂N), 2.9-3.1 (m, 1H, 1×NCHCH₂), 3.6-3.65 (t, 4H, 2×HNCH₂CH₂N) 3.9-4.1 (2×, 2H, CHCH₂OH), 7.05 (s, 2H, C(2)H and C(3)H), 7.15 (m, 2H, C(6)H and C(7)H), 10.5 (t, 2H, C(1)NH and C(4)NH), and 13.65 (s, 2H, C(5)OH, C(8)OH); δ_C(62.9 MHz; CDCl₃); 23.06, 23.93, 26.94, 40.57, 41.13, 45.59, 50.42, 51.88, 58.35, 61.44, 62.65, 108.67, 115.43, 123.45, 124.12, 146.11, 155.24, and 184.63; FAB MS, m/z(M+H)⁺ 483; Anal. calcd for C₂₆H₃₄N₄O₅: C, 64.71; H, 7.10; N, 11.61. Found: C, 64.45; H, 6.85; N, 11.79.

Example 14
1,4-Bis-{[2-(2-hydroxymethyl pyrrolidin-1-yl )ethyl]amino}-5,8-dihydroxy-anthracene-9,10-dione (HAQ125)

As shown in scheme 3 the method follows that of HAQ105 using 1,4-difluoro-5,8-hydroxy-anthraquinone (75 mg, 0.272 mmol), [1-(2-aminoethyl)-pyrrolidin-2-yl-]methanol (1.5 g, 10.42 mmol) pyridine (2 mL), 2 h, 100° C. The product HAQ125 was afforded as a dark blue powder (58.1 mg, 41%). M.p. 202-204° C.; δ_H(250 MHz; DMSO/CDCl₃(1:1)); 1.6-1.9 (m, 8H, 8×ring-H), 2.2-2.3 (m, 2H, 2×ring-H), 2.6-2.75 (m, 6H, 2×HNCH₂CH₂N and 2×ring-H), 3.1-3.2 (m, 2H, 2×ring-H), 3.3-3.6 (m, 10H, 2×HNCH₂CH₂N, 2×NCHCH₂OH and 2×OH), 7.1 (s, 2H, C(2)H and C(3)H), 7.5 (m, 2H, C(6)H and C(7)H), 10.7 (t, 2H, C(1)NH and C(4)NH), and 13.55 (s, 2H, C(5)OH, C(8)OH); δ_C(62.9 MHz; DMSO/CDCl₃(1:1)); 22.75, 27.65, 41.59, 53.56, 64.08, 64.96, 107.14, 114.92, 123.79, 125.30, 146.65, 154.37, and 183.15; FAB MS, m/z(M+H)⁺ 525; Anal. calcd for C₂₈H₃₆N₄O₆: C, 64.14; H, 6.87; N, 10.69. Found: C, 64.09; H, 6.98; NV 10.77.

Example 15
1-{[2-(2,6-Dihydroxymethylpiperidine-1-yl)ethyl]-amino}-4-{[(2-Dimethylamino)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione (HAQ143)

The method follows that of HAQ105 using HAQ107 (78 mg, 0.227 mmol), [1-(2-aminoethyl)-piperidin-bis-2,6-yl-]methanol (420 mg, 2.283 mmol), pyridine (2 mL), 30 min, 100° C. The product HAQ143 was afforded as a dark blue powder (63 mg, 54%). M.p. 216-218° C.; δ_H(250 MHz; CDCl₃); 1.4-1.8 (m, 6H, 6×ring-H), 2.35 (s, 6H, 2×NCH₃), 2.65 (t, 2H, 1×HNCH₂CH₂N), 2.75-2.85 (s, 2H, 2×NCHCH₂), 3.0 (t, 2H, HNCH₂CH₂N), 3.35-3.45 (m, 6H, 2×HNCH₂CH₂N and 2×OH), 3.7 (d, 4H, 2×CHCH₂OH) 7.05 (s, 2H, C(2)H and C(3)H), 7.15 (m, 2H, C(6)H and C(7)H), 10.5 (t, 2H, C(1)NH and C(4)NH), and 13.65 (s, 2H, C(5)OH, C(8)OH); δ_C(62.9 MHz; CDC1₃); 20.90, 24.85, 41.27, 42.35, 45.63, 50.50, 58.42, 61.95, 64.60, 107.01, 115.38, 123.89, 124.92, 146.39, 155.85, and 185.21; FAB MS, m/z(M+H)⁺ 513; Anal. calcd for C₂₇H₃₆N₄O₆: C, 63.26; H, 7.08; N, 10.93. Found: C, 62.93; H, 7.12; N, 10.84.

Example 16
1-{[2-(2-Hydroxymethylpyrrolidin-1-yl)ethyl]amino}-anthracene-9,10-dione (HAQ163) (scheme 2)

To [1-(2-aminoethyl)-pyrrolidin-2-yl-]methanol (1.63 g, 11.3 mmol) in pyridine (10 mL) was added 1-chloroanthraquinone (1.37 g, 5.646 mmol), and the mixture was stirred at 65° C. for 24 hours. Pyridine was removed in vacuo and the resulting mixture of oil and solid was dissolved in CH₂Cl₂and washed with H₂O (3×50 mL) to remove any unreacted amine. The separated organic layer was removed in vacuo and the crude product was chromatographed using CH₂Cl₂/CH₃OH (97:3). The desired product was yielded as a red powder (0.21 g, 11%). M.p. 113-115° C.; δ_H(250 MHz; CDCl₃); 1.75-2.0 (m, 4H), 2.25-2.35 (m, 1H), 2.6-2.8 (m, 2H), 3.15-3.3 (m, 2H), 3.35-3.55 (m, 4H), 3.75-3.85 (2×d, 1H), 7.0 (2×d, 1H), 7.45-7.6 (m, 2H), 7.65-7.8 (m, 2H), 8.2 (2×d, H), 8.45 (2×d), and 10.05 (s, 1H, C(1)NH); δ_C(62.9 MHz; CDCl₃); 24.11, 27.15, 41.64, 52.99, 53.82, 62.61, 65.26, 113.15, 115.60, 126.57, 132.86, 133.81, 135.32, 151.49, 183.77, and 184.89; FAB MS, m/z(M+H)⁺351; Anal. calcd for C₂₁H2N₂O₃: C, 71.98; H, 6.33; N, 8.00. Found: C, 71.79; H, 6.13; N, 8.07.

Example 17
1-{[2-(3-Chloropiperidin-1-yl)ethyl]amino}-4-{[(2-dimethylamino)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione (CAQ166M)

Preparation of CAQ166M involved 5 steps, where none of the intermediate products were isolated and purified. The preparation generally follows scheme 5 (FIG. 4) then scheme 3 (FIG. 2).

(i): Boc protection of mono-hydroxylated diamine sidechains

(ii): Mesylation of Boc-protected mono-hydroxylated diamine sidechains

(iii): Chlorination of mono-mesylated diamine sidechains

(iv): Deprotection of Boc group of chlorinated diamine sidechain

(v): Ipso substitution of fluoride of 1-[[2-(dimethylamino)ethyl]amino]-4-fluoro-5,8-dihydroxyanthraquinone by chlorinated diamine

(i) 1-(2-Aminoethyl)-piperidin-3-ol (1 g, 6.94 mmol) and Et₃N (1.16 mL, 8.33 mmol) was stirred together with CH₃OH (10 mL) for 5 min. before Boc₂O (1.82 g, 8.33 mmol), dissolved in CH₃OH (5 mL), was added dropwise over 15-20 min. The reaction mixture was then stirred 20 h at 45° C., before being concentrated in vacuo. The oil was diluted in EtOAc (40 mL) and washed with 2×H₂O (20 mL) and brine (20 mL). The organic phase was dried with magnesium sulphate (MgSO₄), and after filtration was concentrated in vacuo yielding a straw-coloured oil that needed no further purification (1.35 g, 80%).

FAB MS, m/z(M+H)⁺ 245.

(ii) MsCl (420 μL, 5.41 mmol) was added dropwise to an ice-cold solution of the Boc-protected amine (880 mg, 3.61 mmol) and Et₃N (755 μL, 5.41 mmol) in dry CH₂Cl₂(10 mL) under N₂. After the reaction mixture was stirred for 1 h at 0° C., the solution was diluted with cold CH₂Cl₂, washed with ice-cold NaHCO₃and ice-cold brine. The organic phase was dried with MgSO₄, filtered and concentrated in vacuo at room temperature. The mesylated product was afforded as a crude straw-coloured oil (965 mg, 83%). FAB MS, m/z(M+H)⁺ 323.

(iii) Tetra-n-butylammonium chloride (2 g, 7.20 mmol) was added to a stirred solution of the crude mesylate (965 mg, 3.01 mmol) in dry DMF (5 mL). The reaction mixture was heated at 90° C. for 30 min. before DMF was removed in vacuo. The residual oil was taken up in CH₂Cl₂and washed with ice-cold NaHCO₃and ice-cold brine. The organic phase was dried (MgSO₄), filtered and solvent was concentrated in vacuo at room temperature. The crude product was yielded as a straw-yellowish-coloured oil (624 mg, 79%). FAB MS, m/z(M+H)⁺ 263.

(iv) The crude chloride (624 mg, 2.38 mmol) was stirred in 4 M HCl in EtOAc for an hour to remove the Boc group. To the acidic EtOAc solution, cooled in an ice-bath, was slowly added a solution of brine and NH₃(pH=12) until the aqueous phase was pH˜11. The chlorinated diamine was then extracted into the organic phase, which was dried with (MgSO₄). The solvent was removed in vacuo and the crude product was yielded as a brownish oil (175 mg, 45%) that was used directly in the next step. FAB MS, m/z(M+H)⁺163.

(v) A mixture of HAQ107 (36 mg, 0.105 mmol) and the crude Boc-deprotected chloride (175 mg, 1.08 mmol) was reacted in pyridine (2 mL) at RT for 2 h. The reaction mixture was concentrated in vacuo and the crude product was purified by initially eluting with CH₂Cl₂to remove non-polar impurities, followed by a gradual increase of CH₃OH to CH₂Cl₂/CH₃OH (97:3). The chromatographed product was crystallised from CHCl₃. The title compound CAQ166M was yielded as a dark blue solid (35 mg, 60%). M.p. dec.>300° C.; δ_H(250 MHz; CDCl₃); 1.5-1.9 (m, 4H, 4×ring-H), 2.15-2.25 (m, 3H, 3×ring-H), 2.35 (s, 6H, 2×NCH₃), 2.65-2.8 (2×t, 4H, 2×HNCH₂CH₂N), 3.15-3.2 (2×d, 1H, ring-H), 3.45 (t, 4H, 2×HNCH₂CH₂N), 4.1 (m, 1H, CH₂CHCl), 7.05 (s, 2H, C(2)H and C(3)H), 7.15 (s, 2H, C(6)H and C(7)H), 10.45 (t, 2H, C(1)NH and C(4)NH), and 13.5 (s, 2H, C(5)OH, C(8)OH); δ_C(62.9 MHz; CDCl₃); 24.95, 29.65, 34.88, 40.37, 41.26, 45.63, 52.85, 55.78, 56.28, 58.39, 61.43, 109.23, 115.39, 123.64, 123.78, 124.66, 146.05, 146.24, 155.41, 185.28, and 185.35; FAB MS, m/z(M+H)⁺ 487.

Example 18
1-{[2-(4-Chloropiperidin-1-yl)ethyl]amino}-4-{[(2-dimethylamino)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione (CAQ172)

The method follows that of CAQ166M.

(i) 1-(2-Aminoethyl)-piperidin-4-ol (2 g, 13.89 mmol) Et₃N (2.32 mL, 16.65 mmol), CH₃OH (20 mL), Boc₂O (3.63 g, 16.65 mmol), dissolved in CH₃OH (5 mL). The reaction mixture was stirred 18 h. The product was afforded as a strawcoloured oil that needed no further purification (2.35 g, 69%). FAB MS, m/z(M+H)⁺ 245.

(ii) Boc-protected amine (1.70 g, 6.94 mmol), MsCl (810 μL, 10.41 mmol), Et₃N (1.45 mL, 10.41 mmol), dry CH₂Cl₂(20 mL). The mesylated product was afforded as a crude straw-coloured oil (1.39 g, 62%). FAB MS, m/z(M+H)⁺ 323.

(iii) Crude mesylate (1.39 g, 4.29 mmol), tetra-n-butylammonium chloride (2.38 g, 8.58 mmol), dry DMF (10 mL), 120° C., 30 min. The crude chloride was afforded as a straw/yellowish-coloured oil (0.73 g, 65%). FAB MS, m/z(M+H)⁺ 263.

(iv) Crude chloride (0.73 g, 2.78 mmol), 4M HCl EtOAc, 1 h. The crude Boc-deprotected amine was afforded as a brownish oil (160 mg, 35%). FAB MS, m/z(M+H)⁺163.

(v) HAQ107 (36 mg, 0.105 mmol), crude deprotected sidechain (160 mg, 0.98 mmol) pyridine (2 mL), 5 h, 45° C. The title compound (CAQ172) was afforded as a dark blue solid (31.4 mg, 54%). M.p. 200-202° C.; δ_C(250 MHz; CDCl₃); 1.3-1.75 (m, 6H, 6×ring-H), 1.95-2.1 (m, 2H, 2×ring-H), 2.35 (s, 6H, 2×NCH₃), 2.6-2.8 (t, 4H, 2×HNCH₂CH₂N), 3.3 (q, 4H, 2×HNCH₂CH₂N), 3.45 (m, 1H, CH₂CHCl), 7.05 (s, 2H, C(2)H and C(3)H), 7.1 (s, 2H, C(6)H and C(7)H), 10.45 (t, 2H, C(1)NH and C(4)NH), and 13.55 (s, 2H, C(5)OH, C(8)OH); δ_C(62.9 MHz; CDCl₃); 24.06, 34.69, 45.38, 50.79, 56.25, 58.96, 109.09, 115.31, 123.77, 123.95, 124.61, 146.12, 146.22, 155.34, 161.33, and 185.29; FAB MS, m/z(M+H)⁺ 487; Anal. calcd for C₂₅H₃₁ClN₄O₄.2HCl.3H₂O: C, 48.90; H, 6.24; N, 9.12. Found: C, 48.77; H, 6.00; N, 9.10.

Example 19 1-{[2-(2-Chloromethylpyrrolidin-1-yl)ethyl]-amino}-4-{[(2-Dimethylamino)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione (CAQ176M)

The method follows that of CAQ166M.

(i) [1-(2-Aminoethyl)-pyrrolidin-2-yl-]methanol (5 g, 34.72 mmol) Et₃N (5.8 mL, 41.67 mmol), CH₃0H (40 mL), Boc₂O (9.10 g, 41.67 mmol), dissolved in CH₃OH (10 mL). The reaction mixture was stirred 18 h. The product was afforded as a strawcoloured oil that needed no further purification (6.9 g, 82%).

(ii) Boc-protected amine (5.1 g, 20.9 mmol), MsCl (2.43 mL, 31.35 mmol), Et₃N (4.32 mL, 31.35 mmol), dry CH₂Cl₂(50 mL). The mesylated product was afforded as a crude straw-coloured oil (5.63 g, 84%). FAB MS, m/z(M+H)⁺ 245.

(iii) Crude mesylate (5.63 g, 17.48 mmol), tetra-n-butylammonium chloride (9.72 g, 11.26 mmol), dry DMF (30 mL), 90° C., 30 min. The crude chloride was afforded as a straw/yellowish-coloured oil (2.2 g, 48%). FAB MS, m/z(M+H)⁺ 323.

(iv) Crude chloride (2 g, 7.58 mmol), 4M HCl EtOAc, 1 h. The crude Boc-deprotected amine was afforded as a brownish oil (675 mg, 55%). FAB MS, m/z(M+H)⁺ 263.

(v) HAQ107 (95 mg, 0.276 mmol), crude deprotected sidechain (675 mg, 4.15 mmol), pyridine (2 mL), 2 h, 30° C. The title compound CAQ176M was afforded as a dark blue solid (63.8 mg, 41%). FAB MS, m/z(M+H)⁺ 163. M.p. 253-255° C.; δ_H(250 MHz; CDCl₃); 1.5-1.85 (m, 4H, 4×ring-H), 2-2.25 (m, 3H, 3×ring-H), 2.35 (s, 6H, 2×NCH₃), 2.6-2.8 (2×t, 4H, 2×HNCH₂CH₂N), 3.15 (d, 1H, 1×NCHCH₂Cl), 3.4 (m, 5H, 2×HNCH₂CH₂N and 1×NCHCH₂Cl), 7.05 (s, 2H, C(2)H and C(3)H), 7.1 (s, 2H, C(6)H and C(7)H), 10.45 (t, 2H, C(1)NH and C(4)NH), and 13.55 (s, 2H, C(5)OH, C(8)OH); δ_C(62.9 MHz; CDCl₃); 24.93, 34.86, 40.33, 41.19, 45.59, 52.82, 55.78, 56.03, 56.27, 58.35, 61.43, 109.02, 115.40, 123.55, 123.69, 124.49, 146.01, 146.2, 155.32, and 185.11; FAB MS, m/z(M+H)⁺ 487; Anal. calcd for C₂₅H₃₁ClN₄0₄.2HCl.2H₂O: C, 50.38; H, 6.60; N, 9.40. Found: C, 49.81; H, 6.23; N, 9.29.

Example 20
1,4-Bis-{[2-(2-chloromethylpyrrolidin-1-yl)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione (CAQ177M)

The method follows that of CAQ166M.

(i) [1-(2-Aminoethyl)-pyrrolidin-2-yl-]methanol (7 g, 48.61 mmol) Et₃N (8.12 mL, 58.33 mmol), CH₃OH (50 mL), Boc₂O (12.73 g, 58.33 mmol), dissolved in CH₃OH (15 mL). The reaction mixture was stirred 20 h. The product was afforded as a strawcoloured oil that needed no further purification (9.36 g, 79%). FAB MS, m/z(M+H)⁺ 245.

(ii) Boc-protected amine )8 g, 32.8 mmol), MsCl (3.81 mL, 49.2 mmol), Et₃N (6.85 mL, 49.2 mmol), dry CH₂Cl₂(50 mL). The product was afforded as a crude straw-coloured oil (9.06 g, 86%). FAB MS, mlz(M+H)⁺ 323.

(iii) Crude mesylate (9 g, 28 mmol), tetra-n-butylammonium chloride (13.34 g, 42 mmol), dry DMF (50 mL), 90° C., 30 min. The crude product was afforded as a straw-coloured oil (6.78 g, 61%). FAB MS, m/z(M+H)⁺ 263.

(iv) Crude chloride (6.78 g, 25.7 mmol), 4M HCl EtOAc, 1 h. The crude Boc-deprotected amine was afforded as a brownish oil (1.98 g, 47%). FAB MS, m/z(M+H)⁺163.

(v) HAQ107 (125 mg, 0.363 mmol), crude deprotected sidechain (1.98 mg, 12.1 mmol), pyridine (5 mL), 4 h, 30° C. The title compound CAQ177M was yielded as a dark blue solid (88.5 mg, 39%). M.p. dec.>300° C.; δ_H(250 MHz; DMSO); 1.45-1.85 (m, 6H, 6×ring-H), 2.15-2.35 (m, 6H, 6×ring-H), 2.65-2.85 (m, 6H, 2×HNCH₂CH₂N and 2×ring-H), 3.15 (d, 2H, 2×NCHCH₂Cl), 3.45-3.55 (q, 4H, 2×HNCH₂CH₂N), 4.15 (m, 2H, 2×NCHCH₂Cl) 7.05 (s, 2H, C(2)H and C(3)H), 7.15 (s, 2H, C(6)H and C(7)H), 10.45 (t, 2H, C(1)NH and C(4)NH), and 13.45 (s, 2H, C(5)OH, C(8)OH); δ_C(62.9 MHz; DMSO), 28.11, 35.05, 41.78, 50.83, 55.43, 60.52, 65.26, 107.91, 114.93, 124.33, 127.56, 144.33, 156.69, 183.28; FAB MS, m/z(M+H)⁺ 561; Anal. calcd for C₂₈H₃₄Cl₂N₄O₄.2HCl: C, 53.05; H, 5.72; N, 8.84. Found: C, 53.35; H, 5.84; N, 8.72.

Example 21
1-{[2-(3- Chloropyrrolidin-1-yl)ethyl]amino}-4-{[(2-dimethylamino)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione (CAQ188M)

The method follows that of CAQ166M.

(i) 1-(2-Aminoethyl)-pyrrolidin-3-ol (1 g, 7.94 mmol) Et₃N (1.32 mL, 9.52 mmol), CH₃OH (10 mL), Boc₂O (2.08 g, 9.52 mmol), dissolved in CH₃OH (5 mL). The reaction mixture was stirred 16 h. The product was afforded as a strawcoloured oil that needed no further purification (1.54 g, 86%). FAB MS, mn/z(M+H)⁺ 230.

(ii) Boc-protected amine (960 mg, 4.16 mmol), MsCl (483 μL, 6.24 mmol), Et₃N (868 μL, 6.24 mmol), dry CH₂Cl₂(10 mL). The crude product was afforded as a straw-coloured oil (1 g, 78%). FAB MS, m/z(M+H)⁺ 308.

(iii) Crude mesylate (1 g, 3.24 mmol), tetra-n-butylammonium chloride (1.35 g, 4.86 mmol), dry DMF (10 mL), 100° C., 30 min. The product was afforded as a straw-/yellowish coloured oil (705 mg, 81%). FAB MS, m/z(M+H)⁺ 248.

(iv) Crude chloride (705 mg, 2.88 mmol), 4M HCl EtOAc, 1 h. The crude Boc-deprotected amine was afforded as a brownish oil (124 mg, 30%). FAB MS, m/z(M+H)⁺148.

(v) HAQ107 (50 mg, 0.18 mmol), crude deprotected sidechain (124 mg, 0.86 mmol) pyridine (2 mL), 2 hours, 60° C. The product CAQ188M was afforded as a dark blue powder (15 mg, 15%). M.p. dec.>300° C.; δ_H(250 MHz; DMSO/CDCl₃(1:1)); 1.8-2.15 (m, 2H, 2×ring-H), 2.3 (m, 1H, ring-H), 2.35 (s, 6H, 2×NCH₃), 2.85-3.05 (m, 7H), 3.8-4.1 (m, 5H), 7.1 (s, 2H, C(2)H and C(3)H), 7.2 (s, 2H, C(6)H and C(7)H), 10.45 (t, 2H, C(1)NH and C(4)NH), and 13.55 (s, 2H, C(5)OH, C(8)OH); δ_C(62.9 MHz; CDCl₃); 37.19, 42.31, 52.01, 55.05, 62.05, 68.95, 107.65, 114.40, 124.91, 125.15, 146.09, 154.74, 184.44; FAB MS, m/z(M+H)⁺ 473; Anal. calcd for C₂₄H₂₉ClN₄O₄.2HCl.4H₂O: C, 46.65; H, 6.04; N, 9.07. Found: C, 47.10; H, 5.80; N, 9.07.

Chlorination of Hydroxylated Anthraquinone Using Ph₃P—CCl₄Complex
Example 22
1,4-Bis-{[2-(3-chloromethylpiperidin-1-yl)ethyl]amino}-anthracene-9,10-dione (CAQ39)

Ph₃P (377.7 mg, 1.44 mmol) and CCl₄(425 μL, 432 mmol) were stirred for 15 min. before it was added dropwise to a stirred solution of HAQ38 (125 mg, 0.24 mmol) in dry CH₂Cl₂(5 mL) under N₂at reflux temperature. The reaction mixture was kept at reflux temperature for 4 h before it was cooled down to RT. Ethereal HCl was added to the solution and after 1 h of stirring, the precipitated solid was filtered off. To remove excess of Ph₃P and Ph₃PO, the precipitated solid was dissolved in warm CH₃OH (10 mL). While stirring the dark blue solution at reflux, a mixture of EtOAc and EtOH (1:1) was added until precipitation of solid was observed. The solution was set aside for 1 h before the precipitated product was isolated by filtration; the excess Ph₃P and Ph₃PO remained in the EtOAc/EtOH solution. The title compound was afforded as a dark blue solid (98.3 mg, 65%). M.p. dec.>300° C.; δ_H(250 MHz; CDCl₃); 1.1 (m, 2H, 2×CH₂CHCH₂Cl), 1.75 (m, 6H, CH₂CH₂CH and 2×CH₂CH₂CH₂), 2.05 (m, 4H, ring-H), 2.18 (3×d, 2H, J=3 Hz and 10 Hz, 2×NCH₂CH₂), 2.73 (t, 4H, J=9 Hz, 2×HNCH₂CH₂N), 2.8 (m, 2H, ring-H), 2.96 (2×d, 2H, 2×NCH₂CH), 3.5 (m, 8H, 2×CHCH₂Cl and 2×HNCH₂CH₂N), 7.25 (s, 2H, C(2)H and C(3)H), 7.7 (m, 2H, C(6)H and C(7)H), 8.3 (m, 2H, C(5)H, C(8)H) and 10.75 (t, 2H, C(1)NH and C(4)NH); δ_C(62.9 MHz; CDCl₃); 24.42, 28.34, 38.47, 40.56, 48.14, 54.22, 57.52, 57.60, 110.30, 123.52, 126.12, 132.01, 134.56, 145.82 and 182.65; FAB MS, m/z(M+H)⁺ 557; IR ν_max/cm⁻¹; 3400 (NH), 3090, 2960-2825 (CH₂, CH₃), 1650, 1600, 1585, 1525, 1275, 1025 and 740; Anal. calcd for C₃₀H₃₈Cl₂N₄0₂.2HCl.2H₂O: C, 54.06; H, 6.65; N, 8.41. Found: C, 54.07; H, 6.27; N, 8.14.

Example 23
1-{[2-(3-Chloropiperidin-1-yi)ethyl]amino}-4-{[(2-dimethylamino)ethyl]amino}-anthracene-9,10-dione (CAQ46M)

The method follows that of CAQ39 using HAQ22 (167 mg, 0.384 mmol), Ph₃P (302 mg, 0.152 mmol), CCl₄(333 μL, 3.456 mmol) and dry CH₂Cl₂(2 mL). The reaction was stopped after 6 hours of reflux. The title compound was afforded as a dark blue solid (131 mg, 75%). δ_H(250 MHz; CDC1₃); 1.4-1.8 (m, 4H, CH₂CH₂CH₂and CH₂CH₂CH), 2.2 (3×d, 1H, J=3 Hz and 11 Hz, 1×NCH₂CH₂), 2.25 (d, 1H, J=11 Hz, 1×NCH₂CH), 2.35 (s, 6H, 2×NCH₃), 2.7 (t, 2H, J=7 Hz, HNCH₂CH₂N), 2.75 (t, 2H, J=7 Hz, HNCH₂CH₂N), 2.9 (m, 1H, 1×NCH₂CH₂), 3.15 (2×d, 1H, J=3 Hz and 11 Hz, 1×NCH₂CH), 3.5 (m, 4H, 2×HNCH₂CH₂N), 4.05 (m, 1H, CH₂CHCl), 7.1 (d, 2H, C(2)H, C(3)H), 7.6 (m, 2H, C(6)H, C(7)H), 8.3 (m, 2H, C(5)H, C(8)H), and 10.75 (t, 2H, C(1)NH and C(4)NH); δ_C(62.9 MHz; CDCl₃); 24.50, 34.95, 40.42, 41.02, 45.65, 52.99, 55.88, 56.85, 58.55, 61.61, 110.23, 123.41, 126.12, 132.03, 134.48, 145.73 and 182.62; FAB MS, m/z(M+H)⁺ 455.

Example 24
1,4-Bis-{[2-(3-chloromethylpiperidin-1-yl)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione (CAQ74)

The method follows that of CAQ39 using HAQ70 (142 mg, 0.26 mmol), Ph₃P (404 mg, 1.54 mmol), CCl₄(447 μL, 4.63 mmol) and 5 mL CHCl₃/CH₃CN (4:1) as solvent. The reaction was stopped after 5 hours of reflux. The title compound was yielded as a dark blue solid (117 mg, 68%). M.p. dec.>300° C.; δ_H(250 MHz; CDCl₃/D₂O (10: 1)); 1.4 (m, 2H, 2×CH₂CHCH₂OH), 1.85-2.3 (m, 8H, 8×ring-H), 3.0 (m, 4H, ring-H), 3.4-3.8 (m, 16H, 2×HNCH₂CH₂N, 2×HNCH₂CH₂N] and 8×ring-H), 6.96 (s, 2H, C(2)H and C(3)H) and 7 (s, 2H, C(6)H and C(7)H); δ_C(62.9 MHz; CDCl₃/D₂O (10:1)), 27.99, 38.45, 40.03, 48.97, 56.36, 58.40, 111.43, 117.38, 126.78, 127.37, 148.32, 156.32, and 187.18; FAB MS, m/z(M+H)⁺ 589; Anal. calcd for C₃₀H₃₈Cl₂N₄O₄.2HCl.2H₂O: C, 51.59; H, 6.35; N, 8.02. Found: C, 51.49; H, 6.14; N, 8.22.

Example 25
1-{[2-(2-Chloromethylpiperidin-1-yl)ethyl]amino}-4-{[(2-dimethylamino)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione (CAQ75)

The method follows that of CAQ39 using HAQ71 (48 mg, 0.1 mmol), Ph₃P (78.7 mg, 0.3 mmol), CCl₄(300 μL, 3.11 mmol) and dry CH₂Cl₂(10 mL). The reaction was stopped after 5 hours of reflux. The product (CAQ75) was afforded as a dark blue powder (46.3 mg, 81%). M.p. dec.>300° C.; δ_H(250 MHz; DMSO:CDCl₃(1:1)); 1.3-1.45 (m, 3H, 3×ring-H), 1.45-1.6 (m, 2H, 2×ring-H), 2.35 (s, 6H, 2×NCH₃), 2.9-3 (m, 6H), 3-3.2 (m, 2H), 3.9 (m, 4H), 4.1 (d, 2H, CHCH₂Cl), 7.15 (s, 2H, C(2)H and C(3)H), 7.6 (s, 2H, C(6)H and C(7)H), 10.45 (t, 2H, C(1)NH and C(4)NH), and 13.35 (s, 2H, C(5)OH, C(8)OH); δ_C(62.9 MHz; CDCl₃); 22.45, 25.33, 41.70, 42.56, 54.63, 54.95, 61.95, 107.94, 114.43, 124.41, 125.03, 145.03, 145.81, 155.05, and 184.03; FAB MS, m/z(M+H)⁺ 501; Anal. calcd for C₂₆H₃₃ClN₄O₄.2HCl.2H₂O: C, 51.20; H, 6.44; N, 9.19. Found: C, 51.30; H, 6.18; N, 9.01.

Example 26
1-{[2-(2-Chloromethylpiperidin-1-yl)ethyl]-amino}-4-{[(2-dimethylamino)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione (CAQ183M)

The method follows that of CAQ39 using HAQ121 (105 mg, 0.21 mmol), Ph₃P (165.23 mg, 0.63 mmol), CCl₄(500 μL, 5.25 mmol) and dry CH₂Cl₂(10 mL). The reaction was stopped after 3 hours of reflux. The product (CAQ183M) was afforded as a dark blue powder (88.8 mg, 73%). M.p. 233-235° C.; δ_H(250 MHz; CDCl₃); 1.55-2.1 (m, 8H, 8×ring-H), 2.8 (s, 6H, 2×NCH₃), 3.2-3.35 (m, 4H), 3.6-3.75 (m, 2H), 3.9-4.15 (m, 4H), 4.2 (m, 1H), 7.15 (s, 2H, C(2)H and C(3)H), 7.6 (s, 2H, C(6)H and C(7)H), 10.45 (t, 2H, C(1)NH and C(4)NH), and 13.35 (s, 2H, C(5)OH, C(8)OH); δ_C(62.9 MHz; CDCl₃); 21.60, 26.03, 36.70, 42.21, 51.73, 54.85, 62.25, 108.34, 114.23, 124.81, 124.93, 145.83, 145.90, 154.65, and 184.20; FAB MS, m/z(M+H)⁺ 501; Anal. calcd for C₂₆H₃₃ClN₄O₄.2HCl.3H₂O: C, 49.73; H, 6.58; N, 8.92. Found: C, 50.09; H, 6.27; N, 8.96.

Example 27
1-{[2-(2,6-Dichloromethylpiperidin-1-yl)ethyl]-amino}-4-{[(2-dimethylami no)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione (CAQ187M)

The method follows that of CAQ39 using HAQ143 (14 mg, 0.0273 mmol), Ph₃P (43 mg, 0.164 mmol), CCl₄(79 μL, 0.82 mmol) and dry CH₂Cl₂(5 mL). The reaction was stopped after 5 hours of reflux. The product (CAQ187M) was afforded as a dark blue powder (12.2 mg, 81%). δ_H(250 MHz; CDCl₃); 1.3-1.65 (m, 6H, 6×ring-H), 2.35 (s, 6H, 2×NCH₃), 2.7 (t, 2H, 1×HNCH₂CH₂N), 2.8-2.9 (m, 2H, 2×NCHCH₂), 3.0 (t, 2H, 1×HNCH₂CH₂N), 3.35-3.45 (q, 4H, 2×HNCH₂CH₂N), 3.8-3.9 (d, 4H, 2×CHCH₂Cl) 7.1 (s, 2H, C(2)H and C(3)H), 7.15 (m, 2H, C(6)H and C(7)H), 10.55 (t, 2H, C(1)NH and C(4)NH), and 13.65 (s, 2H, C(5)OH, C(8)OH); FAB MS, m/z(M+H)⁺ 549.

Example 28
1,4-Bis-{[2-(2-chloromethylpiperidin-1-yl)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione (CAQ190M)

The method follows that of CAQ39 using HAQ105 (60 mg, 0.109 mmol), Ph₃P (171.5 mg, 0.654 mmol), CCl₄(190 μL, 1.96 mmol) and dry CH₂Cl₂(5 mL). The reaction was stopped after 5 hours of reflux. The product (CAQ190M) was afforded as a dark blue powder (49.8 mg, 78%). M.p. decompose>300° C.; δ_H(250 MHz; DMSO); 1.5-1.65 (m, 4H, 4×ring-H), 1.75-2.15 (m, 10H, 10×ring-H), 3.4-3.8 (m, 8H, 2×HNCH₂CH₂N and 4×ring-H), 4-4.1 (q, 4H, 2×HNCH₂CH₂N), 4.15 (2×d, 4H, 2×NCHCH₂Cl) 7.2 (s, 2H, C(2)H and C(3)H), 7.65 (s, 2H, C(6)H and C(7)H), 10.45 (t, 2H, C(1)NH and C(4)NH), and 13.45 (s, 2H, C(5)OH, C(8)OH); δ_C(62.9 MHz; DMSO), 26.11, 37.05, 42.78, 50.78, 51.83, 60.32, 62.26, 108.41, 114.33, 125.00, 126.56, 145.93, 154.69, 184.28; FAB MS, m/z(M+H)⁺ 589.

Example 29
1-{[2-(2-chloromethylpyrrolidin-1-yl)ethyl]amino}-anthracene-9,10-dione (CAQ191M)

The method follows that of CAQ39 using HAQ163 (115 mg, 0.329 mmol), Ph3P (260 mg, 0.99 mmol), CCl4 (100 μL, 9.86 mmol) and dry CH2Cl2 (5 mL). The reaction was stopped after 3 hours of reflux. The product (CAQ191M) was afforded as a orange powder (91.9 mg, 69%). M.p. 250-253° C.; δH (250 MHz; CDCl3); 1.6-2.1 (m, 4H), 2.15-2.35 (m, 1H), 2.65-2.75 (m, 2H), 3.05-3.15 (m, 2H), 3.4-3.6 (m, 4H), 3.8 (2×d, 1H), 7.2 (2×d, 1H), 7.45-7.65 (m, 2H), 7.75-7.95 (m, 2H), 8.1-8.3 (m, 2H), and 9.95 (s, 1H, C(1)NH); δC (62.9 MHz; CDCl3); FAB MS, m/z(M+H⁺ 351; Anal. calcd for C₂₈H₃₆N₄O₆: C, 62.23; H, 5.47; N, 6.91. Found: C, 62.15; H, 5.11; N, 6.77.

N-oxide Derivatisation (Scheme 4, FIG. 3)
Example 30
1-{[2-(2-Hydroxymethylpyrrolidin-1-yl-N-oxide)ethyl]amino}-4-{[(2-dimethylamino-N-oxide)ethyl]amino}-5,8-dihydroxy-anthracene-9,10-dione (HAQ132N)

The oxidising agent m-chloroperoxy benzoic acid acid m-CPBA (25 mg, 0.145 mmol) dissolved in dry dimethyldioxirine DCM (1 mL) was added dropwise to a stirred solution of HAQ110 (20 mg, 0.043 mmol) in dry CH₂Cl₂(5 mL) under N₂. After 15 minutes of stirring at −10° C. (acetone-ice bath), the reaction was stirred 3 h at 4° C. The solution was then diluted with hexane and after 2 h, the precipitated solid was filtered off and washed successively with ice-cold hexane, ether, CH₂Cl₂and EtOAc. The crude product HAQ132N was afforded as a crude dark blue solid (15.5 mg, 73%). Anal. calcd for C₂₅H₃₂N₄O₇: C, 59.99; H. 6.44; N, 11.19. Found: C, 52.94; H, 6.15; N, 10.01.

Example 31
1-{[2-(3-Chloropiperidin-1-yl-N-oxide)ethyl]amino}-4-{[(2-dimethylamino-N-oxide)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione (CAQ167MN)

The method follows that of HAQ132N using CAQ166M (19 mg, 0.0391 mmol), m-CPBA (21.6 mg, 0.125 mmol), dry CH₂Cl₂(5 mL). The product CAQ167MN was afforded as a crude dark blue solid (12.5 mg, 62%). Anal. calcd for C₂₅H₃₁ClN₄O₆: C, 57.86; H, 6.02; N, 10.8. Found: C, 54.82; H, 4.93; N, 7.43.

Example 32
1-{[2-(4-Chloropiperidin-1-yle-N-oxide)ethyl]amino}-4-{[(2-dimethylamino-N-oxide)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione (CAQ179N)

The method follows that of HAQ132N using CAQ172 (17 mg, 0.035 mmol), m-CPBA (21.5 mg, 0.119 mmol), dry CH₂Cl₂(5 mL). The product CAQ179N was afforded as a crude dark blue solid (15.5 mg, 86%). Anal. calcd for C₂₅H₃₁ClN₄O₆: C, 57.86; H, 6.02; N, 10.80. Found. C, 54.62; H, 4.84; N, 6.80.

Example 33
1-{[2-(2-Chloromethylpyrrolidin-1-yl-N-oxide)ethyl]amino}-4-{[(2-dimethylamino-N-oxide)ethy]amino}-5,8-dihydroxyanthracene-9,10-dione (CAQ181MN)

The method follows that of HAQ132N using CAQ176M (48.3 mg, 0.0994 mmol), m-CPBA (58.3 mg, 0.338 mmol), dry CH₂Cl₂(10 mL). The product CAQ181MN was afforded as a crude dark blue solid (46.2 mg, 90%). Anal. calcd for C₂₅H₃₁ClN₄O₆: C, 57.86; H, 6.02; N, 10.80. Found: C, 54.66; H, 4.51; N, 6.81.

Example 34
1,4-Bis-{[2-(2-chloromethylpiperidin-1-yl-N-oxide)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione (CAQ192MN)

The method follows that of HAQ132N using CAQ190M (11 mg, 0.0187 mmol), m-CPBA (12.9 mg, 0.075 mmol), dry CH₂Cl₂(5 mL). The product CAQ192MN was afforded as a crude dark blue solid (10.2 mg, 83%). Anal. calcd for C₃₀H₃₈Cl₂N₄O₆: C, 57.97; H, 6.16; N, 9.01. Found: C, 55.46; H, 4.49; N, 6.93.

Comparative Example 1
1,4-Bis-{[2-(piperidin-1-yl)ethyl]amino}-5,8-dihydroxy-anthracene-9,10-dione (HAQ145)

The method follows that of HAQ105 using 1,4-Difluoro-5,8-hydroxy-anthraquinone (50 mg, 0.181 mmol), 1-(2-aminoethyl)-piperidine (232 mg, 1.81 mmol), pyridine (1 mL), 30 min, 100° C. The product HAQ145 was afforded as a dark blue powder (31.7 mg, 35%). M.p. 219-221° C.; δ_H(250 MHz; CDCl₃); 1.45-1.55 (m, 4H, 4×ring-H), 1.55-1.7 (m, 8H, 8×ring-H), 2.55 (m, 8H, 2×NCH₂H₂), 2.65 (t, 4H, 2×HNCH₂CH₂N), 3.55 (q, 4H, 2×HNCH₂CH₂N), 7.05 (s, 2H, C(2)H and C(3)H), 7.15 (s, 2H, C(6)H and C(7)H), 10.5 (t, 2H, C(1)NH and C(4)NH), and 13.65 (s, 2H, C(5)OH, C(8)OH); δ_C(62.9 MHz; CDCl₃; 24.37, 26.09, 40.72, 54.64, 57.63, 109.21, 115.48, 123.63, 124.63, 146.31, 155.46, and 185.35; FAB MS, m/z(M+H)⁺ 493; Anal. calcd for C₂₈H₃₆N₄O₆.1.5H₂O: C, 64.66; H, 7.27; N, 10.78. Found: C, 64.70; H, 7.55; N, 10.67.

Comparative Example 2
1-{[(2-Dimethylamino)ethyl]amino}-4-{[2-(piperidin-1-yl)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione (HAQ148)

The method follows that of HAQ105 using HAQ107 (62 mg, 0.18 mmol), 1-(2-aminoethyl)-piperidine (250 mg, 1.953 mmol), pyridine (1 mL), 30 min, 100° C. The product HAQ148 was afforded as a dark blue powder (42.9 mg, 65%). M.p. 220-223° C.; δ_H(250 MHz; CDCl₃); 1.45-1.7 (m, 6H, 6×ring-H), 2.35 (s, 6H, 2×NCH₃), 2.55 (m, 4H, 2×NCH₂CH₂), 2.75 (2×t, 4H, 2×HNCH₂CH₂N), 3.5(q, 4H, 2×HNCH₂CH₂N), 7.05 (s, 2H, C(2)H and C(3)H), 7.1 (m, 2H, C(6)H and C(7)H), 10.45 (t, 2H, C(1)NH and C(4)NH), and 13.55 (s, 2H, C(5)OH, C(8)OH); δ_H(62.9 MHz; CDCl₃); 24.28, 25.93, 40.51, 41.24, 45.63, 54.62, 57.50, 58.39, 109.23, 115.41, 123.73, 123.99, 124.75, 146.24, 155.44, and 185.41; FAB MS, m/z(M+H)⁺ 453.

Biological Evaluation
Cytotoxicity of Substituted pyrollidinyl- and piperidinyl-alkylaminoanthraquinones

Table 1 compares the activity of compounds possessing a side chain hydroxyl moiety (HAQ71, HAQ73, HAQ111) with HAQ148 and shows that the hydroxyl group is essential to the nM activity of these piperidinyl/pyrollidinyl substituted alkylaminoanthraquinones (see Table 1).

The non-symmetrically substituted compounds (HAQ71, HAQ73, HAQ110, HAQ111, HAQ121 and CAQ75) have similar potencies against the A2780 wildtype cell line.

Substitution of OH with Cl (compare HAQ71 with CAQ75) results in a >10-fold decrease in cytotoxicity although the enhanced activity against the resistant cells is still maintained for CAQ75 (RF=0.4). Furthermore CAQ75 was shown to be a significantly more potent in the A2780 cell line than the symmetrical chloropropyl congener CAQ74, which further emphasises the importance of the non-symmetrical substitution of these 1,4-disubstituted alkylaminoanthraquinones.

The chloromethyl piperidinyl and pyrollidinyl 1,4 disubstituted alkylaminoanthraquinones showed low cross resistance in a doxorubicin resistant (2780AD) and cisplatin resistant (A2780/cp70) ovarian carcinoma cell lines (Table 2). Hydroxyl and chloro substituted compounds show significant antitumour activity in human xenografted ovarian cancers in vivo in mice (Table 3).

In Vivo Studies

Compound HAQ71, with the lowest RF (0.2) of the panel of compounds tested against the cisplatin resistant cells and its chloropropyl-substituted analogue, was selected for in vivo investigation in mice using tumour A2780 and A2780/cp70 cells grown as xenografts in mice. Significant anticancer activity in vivo of HAQ71 and CAQ75 was shown; both compounds exerting similar tumour growth delay in human xenografted wild-type and acquired cisplatin resistant tumours (A2780/cp70). The latter is very refractory to cisplatin and other covalent binding agents and has been characterised with elevated levels of glutathione, alterations in drug uptake/efflux and DNA repair mechanisms including mismatch repair deficiency.

Cytotoxicity In Vitro of Di-N-Oxides of aminoanthraquinones and chloroalkylaminoanthraquinones.

The cytotoxicity of the novel di-N-oxides of CAQs and non-covalent binding aminoanthraquinones was investigated in the ovarian carcinoma wild type cell line A2780, and the adriamycin resistant 2780AD and cisplatin resistant 2780CP cell lines.

All di-N-oxides were considerably less cytotoxic than their parent cytotoxic agents with ICso values in the ZLM range. The order of cytotoxicity of all agents tested was HAQ132N (>100 μm)<CAQ179N (46 μM)<CAQ181MN (38 μM)<CAQ167MN (36 μM)<CAQ192MN (8 μM). The cytotoxicity ratio (CR=IC₅₀of alkylaminoanthraquinone-di-N-oxide/IC₅₀alkylaminoanthraquinone) was used to calculate relative activities. Three di-N-oxides, HAQ132N, CAQ181MN and CAQ167MN, were more than 1000-fold less cytotoxic than their amine counterparts in the A2780 cell line. The CAQs were approx. 40-100 fold less cytotoxic than their amine counterparts. The order of CR was found to be AQ4N (17,000)>>HAQ132N (1,450)>>CAQ181MN (1293)>CAQ179N (80)>CAQ167MN (4167)>CAQ192MN (44).

N-oxides of selected compounds had very low cytotoxicity (Table 4) and have potential as bioreductive prodrugs.

TABLE 1

Inhibition of cell growth (IC50, nM) by

1,4-disubstituted aminoanthraquinones

Compound
A2780

HAQ71
8.4

HAQ73
10.8

HAQ111
6.1

HAQ148
>1,000

TABLE 2

Inhibition of cell growth by 1,4-disubstituted aminoanthraquinones

A2780
2780AD

A2780/cp70

Anthraquinone
nM
nM
RF
nM
RF

HAQ163
>1,000
>1,000
—
>1,000
—

HAQ148
>1,000
>1,000
—
>1,000
—

CAQ191M
>1,000
>1,000
—
>1,000
—

CAQ177M
428
782
1.8
442
1

CAQ190M
178
>1,000
—
>1,000
—

CAQ187M
24
106
4.4
357
14.9

CAQ183M
40
91
2.3
69
1.7

CAQ176M
29
292
10.1
79
2.7

CAQ166M
9
75
8.3
63
7

CAQ188M
68
342
5.0
96
1.4

CAQ172
576
426
0.7
535
0.9

CAQ74
>1,000
>1,000
—
>1,000
—

HAQ = hydroxy deriv;

CAQ = chloro deriv;

M = mustard

All compounds are R and S mixtures unless otherwise stated.

All compounds are mixed side chains (non-symmetrical) unless otherwise stated

IC₅₀is the concentration of drug (nM) required to inhibit cell growth by 50%.

A2780 is the wild type ovarian cell line; A2780/cp70 cisplatin and 2780AD adriamycin resistant variants.

RF = resistance factor (IC₅₀in resistant cell line/IC₅₀in parent cell line).

TABLE 3

Effect of compound HAQ71 and CAQ75 on doubling time (days) of a

human ovarian tumour xenograft (A2780) and a cisplatin resistant

variant (A2780/cp70).

tumour
Untreated
HAQ71
CAQ75

A2780
2.94 ± 0.34
5.23 ± 0.39 (177%)
5.83 ± 0.40 (198%)

A2780/cp70
2.52 ± 0.17
4.33 ± 0.16 (172%)
4.18 ± 0.27 (166%)

HAQ = hydroxy deriv;

CAQ = chloro deriv

( ) = percent increase in median life span - calculated as time taken for tumour to reach twice its initial volume.

Compound HAQ71 dosed at 20 mg/kg and compound CAQ75 at 16 mg/kg both i.p.

TABLE 4

Growth Inhibition (IC₅₀) of Di-N-oxides of Aminoanthraquinones and

Chloroalkylaminoanthraquinones Against Ovarian Cancer Cell Line

A2780

Compound
[nM]

HAQ110
69

HAQ132N
>100,000

CR
>1,449

CAQ172
5.765e+09

CAQ179N

CR

CAQ166M
9.357e+09

CAQ167MN

CR

CAQ176M
2.938e+10

CAQ181MN

CR

CAQ190M
178790044

CAQ192MN

CR

AQ4
6

AQ4N
>100,000

CR
>16,667

HAQ = hydroxy deriv;

CAQ = chloro deriv;

N = N-oxide;

M = mustard;

MN = N-oxide of mustard

CR = cytotoxicity ratio (IC₅₀of amine/IC₅₀of N-oxide).

Number	Date	Country	Kind
0329820.5	Dec 2003	GB	national
0330011.8	Dec 2003	GB	national

Antharquinone Compounds as Anti Cancer Compounds

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