Total Synthesis, Cytotoxic Effects of Damnacanthal, Nordamnacanthal and Related Anthraquinone Analogues

Naturally occurring anthraquinones, damnacanthal (1) and nordamnacanthal (2) were synthesized with modified reaction steps and investigated for their cytotoxicity against the MCF-7 and K-562 cancer cell lines, respectively. Intermediate analogues 2-bromomethyl-1,3-dimethoxyanthraquinone (5, IC50 = 5.70 ± 0.21 and 8.50 ± 1.18 μg/mL), 2-hydroxymethyl-1,3-dimethoxyanthraquinone (6, IC50 = 12.10 ± 0.14 and 14.00 ± 2.13), 2-formyl-1,3-dimethoxyantharquinone (7, IC50 = 13.10 ± 1.02 and 14.80 ± 0.74), 1,3-dimethoxy-2-methylanthraquinone (4, IC50 = 9.40 ± 3.51 and 28.40 ± 2.33), and 1,3-dihydroxy-2-methylanthraquinone (3, IC50 = 25.60 ± 0.42 and 28.40 ± 0.79) also exhibited moderate cytotoxicity against MCF-7 and K-562 cancer cell lines, respectively. Other structurally related compounds like 1,3-dihydroxyanthraquinone (13a, IC50 = 19.70 ± 0.35 and 14.50 ± 1.28), 1,3-dimethoxyanthraquinone (13b, IC50 = 6.50 ± 0.66 and 5.90 ± 0.95) were also showed good cytotoxicity. The target compound damnacanthal (1) was found to be the most cytotoxic against the MCF-7 and K-562 cancer cell lines, with IC50 values of 3.80 ± 0.57 and 5.50 ± 1.26, respectively. The structures of all compounds were elucidated with the help of detailed spectroscopic techniques.

The synthesis of nordamnacanthal (2) was accomplished by first acetylating the precursor compound 3 with acetic anhydride and potassium carbonate to afford the monoacetylated intermediate 8.
The synthesis of damnacanthal (1) was accomplished by first acetylated the precursor compound 3 with (CH 3 ) 2 SO 4 and potassium carbonate to afforded monoacetylated derivative 8. Upon methylation of compound 8 with K 2 CO 3 /CH 3 I compound 9 was obtained, which then brominated with NBS to yield 1-methoxy-3-aectoxy-2-bromomethyl-1-methoxyathraquinone (10) [25,32]. The bromo derivative 10 was converted into ethoxymethyl derivative 11 by dissolving it in a mixture of aq. NaOH/ethanol and followed by reflux in acidic media. Compound 11 was acetylated using acetic anhydride and K 2 CO 3 to afforded 12, which on oxidation with PCC in CH 2 Cl 2 to give damnacanthal (1) in good yield. Thus the synthesis of damnacanthal (1) was achieved through modified steps as shown in Scheme 2.   For the SARs additional anthraquinone analogous 13a-h were synthesized by Friedel-Crafts acylation of phthalic anhydride with resorcinol or catechol to yielded respective anthraquinones [29,30] as shown in Figure 2. All compounds showed significant in vitro cytotoxicity against two cancer cell lines, indicating that anthraquinone is an interesting class of compounds for cancer therapy. The compound damnacanthal (1) contains methoxy, formyl and hydroxyl groups at the 1, 2 and 3 positions that might be important for the cytotoxicity since the standard drug doxorubicin [33] also possesses an anthraquinone moiety. However, nordamnacanthal (2), 3, 8, 13a, 13c, 13f and 13h exhibited less cytotoxic effects on both the MCF-7 and K-562 cell lines, suggesting that a protected OH group at position 1 and 3 increase the solubility and might make it easy for the compounds to diffuse across the cellular membrane as investigated by a theoretical study of 188 drug-like compounds by MI-QSAR analysis [34,35], while for poorly soluble drugs dissolution could be the rate limiting step in the absorption process. The methylated compound 13b (IC 50 = 24.25 ± 2.46 μM and 22.01 ± 3.54 μM) showed the stronger cytotoxicity than 13a (IC 50 = 82.08 ± 1.46 and 60.42 ± 5.33 μM) and 13c (IC 50 = 87.80 ± 2.01 and 64.96 ± 1.57μM). Other derivatives 13e (IC 50 = 43.64 ± 2.12 and 46.61 ± 12.37) and 2-hydroxymethylanthraquinone (13d) (IC 50 = 49.58 ± 12.61and 50.84±14.12) showed poor cytotoxicity as shown in Table 1. However, previously synthesized compounds 13f-h with mono or dihydroxyl groups at the 1 and 2 positions exhibited moderate cytotoxic activity [36]. Further derivatives as exemplified by compounds 13c-h indicated that this series of compounds only exhibited moderate activities not exceeding that of damnacanthal (1), suggesting that functional groups such as formyl at C-2, methoxy at C-1, hydroxyl groups at C-3 (damnacanthal) and bromomethyl at C-2 are important pharmacophores for the anticancer activity. However, the active compound 5 ((IC 50 = 15.83 ± 0.58 and 23.61 ± 3.28 μM) might involve another mechanism like perfusion. The overall cytotoxic compounds (1, 5, 13b) showed more selectivity towards cancer cell lines because of their side chains and might fulfill the Lipinski rule-of-5 and drug-like properties.

General
Melting points were determined on a XSP-12 500X hot-stage melting point apparatus, and are uncorrected. UV spectra were recorded on a CARY 100 Conc. UV-visible spectrophotometer in MeOH. IR spectra were recorded on a Perkin-Elmer RXI Fourier Transform Infrared spectrometer (FTIR) as KBr discs. Mass spectra were measured on a Finnigan Mat SSQ 710 Spectrometer with ionization induced by electron impact at 70 eV. NMR spectra were recorded in CDCl 3 and acetone-d 6 using a Varian 500 MHz NMR spectrometer. Column chromatography was performed on silica gel (60 Merck 9385, 230-400 mesh, ASTM). The molecular structure of the intermediate 1, (5) was determined by X-ray crystallography. The data were collected at 100K using a Bruker APEXII with a CCD area-detector X-ray diffractometer. The structure was solved by direct method with SHELXS97 program and refined on F2 by full-matrix least-squares methods with anisotropic non-hydrogen atoms. The compound crystallizes in the monoclinic P2(1)/c space group with the crystallographic detail given in Table 2. The molecule exists as a planar molecule with r.m.s deviation of 0.018Å (Figure 1).

General Procedure for Anthraquinone Synthesis
A mixture of anhydrous aluminium chloride (30 g, 225.0 mmol) and sodium chloride (12 g, 205.0 mmole) were melted at 125-130 °C. Phthalic anhydride (6.7 g, 45.0 mmole) and 2-methyl resorcinol (40.5 mmole) were mixed well and added slowly into the molten mixture of aluminum chloride and sodium chloride. The reaction temperature was raised till to 165-175 °C and maintained for 45-60 min. Another portion of anhydrous aluminum chloride (30 g, 225.0 mmole) was added into the hot mixture of the reactants [28]. After being cooled, the deep red solid product was decomposed by adding a mixture of ice water (250 mL) and conc. hydrochloric acid (250 mL). The crude mixture was dissolved in distilled water and organic layer was extracted with ethyl acetate, washed with brine and dried over anhydrous sodium sulfate. The crude products were purified by flash silica gel column chromatography with elution of the ethyl acetate /hexane as yellow needles.   (2). Compound 7 (150 mg, 0.5 mmole) was suspended in dry CH 2 Cl 2 (10 mL), stirred for 10 min. at rt and anhydrous AlCl 3 (500 mg, 3.7 mmole) was added and stirred for 1h and then reflux for 4-6 h. The reaction mixture was cooled and added mixture of ice HCl (250 mL) and water (250 mL). The products was extracted by EtOAc and purified by CC and followed by recrystallized from a mixture of MeOH/EtOAc as orange crystals. Yield 28%; m.p.  (1). Compound 12 (200 mg 0.56 mmole) was dissolved in dry dichloromethane (30 mL) and PCC (125 × 2 mg, 1.39 mmole) was introduced slowly in a stirred solution. The reaction mixture was stirred at room temperature for 4-6 h. A black brown solid was obtained, dissolved in aqueous solution and extracted with EtOAc, solvent removed in vacuo on a rotary evaporator and dried over anhydrous sodium sulfate. The product was purified by column chromatography using EtOAc/hexane as solvent system. The product was obtained as orange crystals.

X-ray Crystallography of Compound 5
A single crystal of compound 5 was obtained by slow evaporation at room temperature from a mixture of ethyl acetate-MeOH (drops). The crystal structure was solved by direct method with SHELXS97 programe and refined on F 2 by full-matrix least-squares methods with anisotropic non-hydrogen bond atoms. The compound crystallized as a monoclinic P2(1)/c space group with the crystallographic data is given in Table 2. The molecule exists as a planar molecule with r.m.s deviation of 0.018 Å (Figure 1). There is no evidence of any hydrogen bonding interaction or π-π stacking present in the crystal packing of the compound.

Preparation of Compounds
The compounds were dissolved in dimethylsulfoxide (DMSO, Sigma, St. Louis., MO, USA) to get a stock solution of 1 mg/mL and stored at 4 °C.

Preparation of Cell Lines
Adherent human estrogen dependent breast carcinoma MCF-7 and suspension human chronic myelogenic leukemia K-562 cell lines were obtained from ATCC, USA. Both MCF-7 and K562 were cultured in RPMI-1640 supplemented with 10% FBS and incubated at 37 °C, 5% CO 2 and 90% humidity throughout the study. Cell viability was assessed by trypan blue exclusion method where both MCF-7 and K562 cell lines were collected, washed with PBS and mixed with 1:1 ratio of trypan blue dye and the cell number and viability were evaluated using haemacytometer under inverted light microscope. Only cell with viability higher than 95% will be subjected to MTT cell viability assay.

Cell Viability Assay
The effect of all compounds on cell viability of MCF-7 and K-562 cell lines were determined using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [37]. MCF-7 cell were seeded on a 96 well microtiter plate with each well containing 200 µL of culture media with 10% of FBS per well at a density of 1 × 10 5 cells/mL and incubated overnight at 37 °C, 5% CO 2 at humidified atmosphere of 95% air. When cells were reached 70% confluence then subjected to the cell viability assay. Medium containing the seeded MCF-7 cell was discarded. Treatment of both cultures were carried out by first adding 100 µL of RPMI-1640 medium supplemented with 10% of FBS into all wells except row A of the 96 well plates (TPP, Zollstrasse, Trasadingen, Switzerland). Then, 100 µL of each diluted compound (60 µg/mL) was added into row A and row B. A series of two-fold dilutions of compounds were carried out from row B to row G to give concentrations of 30, 15, 7.5, 3.75, 1.375 and 0.687 µg/mL, while row H was left as untreated control cell. For K-562 culture plate, 100 µL of cells (2 × 10 5 cells/mL) was added into all wells. For MCF-7 culture plate, 100 µL of RPMI-1640 medium was added into all wells. The plates were incubated in 37 °C, 5% CO 2 and 90% humidity incubator for 72 h. After incubation period, 20 µL of MTT (Sigma, 5 mg/mL) was added into all wells and the plates were further incubated for 4 h. Then, 170 µL of supernatant was aspirated from every well plate. The plates containing K-562 culture were centrifuged at 200 × g for 5 min prior to medium aspiration. The resulting formazan crystals in each well were solubilized by 100 µL of DMSO (Fisher Scientific, Waltham, MA, USA) followed by incubation for 20 min. Finally, the plates were read at 570 nm and 630 nm as reference wavelength by using µQuant ELISA Reader (Bio-tech Instruments, Winooski, VT, USA). The results of the compound-treated cells were compared with the standard doxorubicin. Each compound and control was assayed in triplicates in three independent experiments. The percentage of inhibitions were calculated by using Graph pad and expressed in µg/mL or µM and listed in Table 1.

Conclusions
The synthesis of damnacanthal (1) and nordamnacanthal (2) was achieved. The key difference from the previously synthesis was the conversion of hydroxymethylanthraquinones 7 or 12 into the corresponding aldehyde, which was achieved by the use of optimized mild conditions with PCC as oxidant. The synthesis in good yield of 2-ethoxymethyl-3-hydroxy-1-methoxyanthraquinone (11) was achieved through a short sequence. The structure activity relationship suggested that methoxy, formyl, bromomethyl and hydroxyl groups are important for the cytotoxicity and selectivity in these substituted anthraquinones. These side chain modifications give an idea for further efforts to increase the therapeutic potential of this class of compounds.